An Intranasal Delivery Method for Novel Peptide Therapeutics Designed to treat Major Depressive Disorder

Transcription

1 An Intranasal Delivery Method for Novel Peptide Therapeutics Designed to treat Major Depressive Disorder by Virginia Joan Margaret Brown A thesis submitted in conformity with the requirements for the degree of Masters of Science Department of Physiology University of Toronto Copyright by Virginia Brown, 2013

2 An Intranasal Delivery Method for Novel Therapeutics designed to treat Major Depressive Disorder Abstract Virginia Brown Masters of Science Department of Physiology University of Toronto 2013 A problem in designing drugs that act upon the central nervous system is developing effective delivery methods. Major depressive disorder (MDD) affects 12% of men and 20% of women in the United States, and treatment options are often inadequate. In patients, the interaction between dopamine D1 and D2 receptors is correlated with major depressive disorder. A small peptide that disrupts this interaction can be delivered to brain areas using intranasal delivery. The D1-D2 interfering peptide has an antidepressant effect comparable to imipramine in the forced swimming test (FST), a test for antidepressant efficacy. At doses greater than 5.75 mg/kg, the D1-D2 interfering peptide has antidepressant action in the FST for 2 hours after intranasal administration. The D1-D2 interfering peptide disrupts the D1-D2 receptor interaction in the PFC after intranasal administration. This study provides preclinical support for intranasal administration of the D1-D2 interfering peptide as a new treatment option for MDD. ii

3 Acknowledgments I would like to thank my supervisor, Dr. Fang Liu, for her guidance and support over the last months. Her encouragement and positive attitude have made working in the lab a pleasure. Her scientific guidance over the course of this project as well as her personal guidance has been invaluable. I would also like to thank my Masters committee members, Dr. Paul Fletcher and Dr. Paul Frankland. Their expertise, positive encouragement and guidance significantly contributed to my learning throughout this project and to its overall success. Impel NeuroPharma, the company that developed the POD used throughout my study, provided training and important input into the development of the protocol we used to administer substances intranasally. I am grateful to them, especially to John Hokeman, for their patience and encouragement. Finally, I would like to thank my wonderful family, friends and roommates for their encouragement, understanding and support throughout the last two years. iii

4 Table of Contents Acknowledgments... iii Table of Contents... iv List of Tables... vi List of Figures... vii List of Abbreviations... ix 1 Introduction Dopamine neurotransmission in the mammalian brain Dopaminergic pathways in the mammalian CNS Heterodimerization of Dopamine Receptors Major Depressive Disorder Epidemiology of MDD Symptoms and clinical presentation of MDD Treatments for MDD Neurobiological changes and pathophysiology of MDD Preclinical models of MDD Intranasal delivery to the CNS Mechanisms of intranasal delivery to the CNS Experimental considerations for successful intranasal delivery to the CNS Rationale Hypothesis Materials and Methods Animals Intranasal administration procedures Intranasal administration using the POD Verification of POD delivery to the olfactory epithelium Substances injected intranasally Intra-peritoneal injection procedures Immunofluorescence and confocal microscopy Tissue fixation and storage Immunofluorescent staining procedures The Forced Swimming Test FST Procedure FST behavioral scoring method FST experiments: experimental design Effect of the D1-D2 interfering peptide in the FST Effect of the D1-D2-FLAG interfering peptide in the FST Efficacy of the D1-D2 interfering peptide at various intranasal doses Duration of behavioral effect of D1-D2 interfering peptide in the FST Locomotor activity test Co-immunoprecipitation and western blots Tissue Collection Co-Immunoprecipitation of D1 receptor by anti-d2dr Western Blots Results iv

5 3.1 Experiment 1: The POD preferentially deposits substances on the olfactory epithelium within the rat nasal cavity Experiment 2: The D1-D2-FLAG interfering peptide can be detected in the prefrontal cortex after intranasal administration Experiment 3: Intranasal administration of the D1-D2 interfering peptide has an antidepressant effect in the forced swimming test The D1-D2 Interfering Peptide has an Anti-Immobility Effect in the FST The D1-D2-FLAG interfering peptide has an anti-immobility effect in the FST after intranasal administration Experiment 4: Efficacy of the D1-D2 interfering peptide at various intranasal doses D1-D2 interfering peptide dose: 4.0nmol/g (13.72 mg/kg) D1-D2 interfering peptide dose: 2.0nmol/g (6.86 mg/kg) D1-D2 interfering peptide dose: 1.67nmol/g (5.75 mg/kg) D1-D2 interfering peptide dose: 1.0nmol/g (3.43 mg/kg) Experiment 5: Duration of the behavioral effect of the D1-D2 interfering peptide Behavioral Effect in FST 2 hours after intranasal administration Behavioral Effect in FST 3 hours after intranasal administration Behavioral effect in the FST 4 hours after intranasal administration Experiment 6: The D1-D2 interfering peptide does not increase locomotor activity Overall locomotor activity Effect of time on locomotor activity during 30-minute test Experiment 7: Intranasal administration of the D1-D2 interfering peptide disrupts the interaction between dopamine D1 and D2 receptors in the PFC Experiment 8: The D1-D2 interfering peptide does not change the expression of dopamine D1 or D2 receptors in the PFC Expression of Dopamine D1 receptors in the PFC after intranasal administration of the D1-D2 interfering peptide Expression of Dopamine D2 receptors in the PFC after intranasal administration of the D1-D2 interfering peptide Discussion Overall Findings The POD delivers biologically active peptides to the CNS Mechanism of transport to the CNS after intranasal administration The D1-D2 interfering peptide is effective at intranasal doses 5.75 mg/kg for up to 2 hours after intranasal administration Possible neurobiological mechanisms of the D1-D2 interfering peptide s antidepressant effect Limitations of the FST as a preclinical test for antidepressant efficacy The D1-D2 interfering peptide, TAT-peptide and imipramine significantly decrease locomotor activity Future Directions References Appendix 1: Sufficient intranasal D1-D2 interfering peptide dose to produce antidepressant effect in the Forced Swimming Test (Calculation) v

6 List of Tables Table 2-1 Efficacy of the D1-D2 interfering peptide at various doses: overall experimental design and Treatment Groups Table 2-2 Duration of the anti-immobility effect of the D1-D2 interfering peptide: treatment groups and overall experimental design vi

7 List of Figures Figure 1-1 Dopamine receptors: structure and function... 9 Figure 1-2 Schematic Representation of D1-D2R receptor interaction and activation of intracellular signalling pathways Figure 1-3 Previous findings demonstrating the role of the D1-D2R interaction in MDD Figure 2-1 Pressurized Olfactory Device (POD) for intranasal administration: apparatus Figure 2-2 Overall experimental procedure for FST Figure 2-3 Representative photographs of behaviors exhibited during the FST Figure 3-1 Representative images of deposition of Mark-It Blue tissue marker deposition after correct POD administration Figure 3-2 Immunofluorescent staining for anti-flag antibodies is visible in PFC slices of animals who were administered TAT-D1-D2-FLAG-IPep (A) but not those who were administered saline (B) Figure 3-3 The D1-D2 interfering peptide has an antidepressant effect in the FST when administered intranasally Figure 3-4 The D1-D2-FLAG interfering peptide has an anti-immobility effect in the FST Figure 3-5 The D1-D2 interfering peptide and D1-D2-FLAG tagged interfering peptide have similar behavioral effects in the FST Figure 3-6 The D1-D2 interfering peptide has an anti-immobility effect in the FST at an intranasal dose of 4.0nmol/g Figure 3-7 The D1-D2 interfering peptide has an anti-immobility effect in the FST at an intranasal dose of 2.0nmol/g Figure 3-9 The D1-D2 interfering peptide has an anti-immobility effect in the FST at an intranasal dose of 1.67 nmol/g Figure 3-8 The D1-D2 interfering peptide does not have an anti-immobility effect in the FST at an intranasal dose of 1.0nmol/g Figure 3-10 Efficacy of the D1-D2 interfering peptide at various doses in the FST: summary of findings Figure 3-12 The D1-D2 interfering peptide has an anti-immobility effect in the FST 2 hours after intranasal administration vii

8 Figure 3-11 The D1-D2 Interfering Peptide does not have an anti-immobility effect in the FST 3 hours after intranasal administration Figure 3-13 The D1-D2 interfering peptide does not have an anti-immobility effect in the FST 4 hours after intranasal administration Figure 3-14 The D1-D2 interfering peptide no longer has a behavioral effect in the FST 3 hours after it is administered via intranasal injections Figure 3-15 The D1-D2 interfering peptide does not increase locomotor activity during a 30- minute open field test Figure 3-16 The D1-D2 interfering peptide decreases overall locomotor activity but does not change the activity pattern during a 30-minute open field test Figure 3-17 Co-Immunoprecipitation of D1 by anti-d2r is reduced in the PFC of animals who received intransal injections of TAT-D1-D2-IPep (Dose: 1.67nmol/g) Figure 3-18 Intranasal administration of the D1-D2 interfering peptide does not change the expression of the dopamine D1 receptor in the PFC Figure 3-19 Intranasal administration of the D1-D2 interfering peptide does not change the expression of the dopamine D2 Receptor in the PFC Figure 3-20 Representative immunoblot of α-tubulin expression in rat PFC tissue viii

9 List of Abbreviations 5-HT serotonin AC adenyl cyclase ADHD attention deficit hyperactivity disorder ANOVA Analysis of Variance anti α-tubulin immunoglobulin against α-tubulin protein anti-cy2 immunoglobulin conjugated to cyanine 2 fluorescent dye anti-d1dr immunoglobulin against dopamine D1 receptor anti-d2dr immunoglobulin against dopamine D2 receptor ATP adenosine triphosphate BDNF brain derived neurotrophic factor CaMKII calmodulin kinase II camp cyclic adenosine monophosphate cdna complementary DNA (deoxyribonucleic acid) CNS central nervous system CSF cerebrospinal fluid C-terminal carboxy-terminal of protein D1 dopamine D1 receptor D1-D2 dopamine D1-D2 receptor interaction D2 dopamine D2 receptor D2L dopamine D2 receptor - long isoform D2S dopamine D2 receptor - short isoform D3 dopamine D3 receptor D4 dopamine D4 receptor D5 dopamine D5 receptor DA dopamine DAT dopamine transporter protein DDC DOPA decarboxylase DSM-IV-TR Diagnostic and Statistical Manual, 4th edition, text revision (2000) FLAG FLAG octapeptide (protein tag) FST forced swimming test GABA gamma-aminobutyric acid GABAAR gamma-aminobutyric acid receptor type A Gi/o G protein, α subunit type i/o GPCR G protein coupled receptor Gq G protein, α subunit type q Gs GSK-3β G protein, α subunit type s Glycogen Synthase Kinase 3β GTP guanine triphosphate Gα G protein, α subunit ix

10 HIV1 human immunodeficiency virus type 1 IGF-1 insulin-like growth factor 1 IP intraperitoneal injection IP3 inositol triphosphate kd kilodalton L-DOPA L-3,4-dihydroxyphenylalanine LH learned helplessness MAO monomaine oxidase MAOI monoamine oxidase inhibitors MCI mild cognitive impairment MDD major depressive disorder mg/kg milligrams per kilogram mrna messenger RNA (ribonucleic acid) MSN medium spiny neuron NAc nucleus accumbens NE norepinephrine NGF nerve growth factor NMDARs n-methyl-d-aspartate receptors nmol/g nanomoles per gram OEC olfactory ensheathing cell ORN olfactory receptor neuron PBS phosphate buffered saline PD Parkinson's Disease PFA 4 % paraformaldehyde PFC prefrontal cortex PLC phospholipase C POD pressurized olfactory device RGP regulators of G proteins SNpc substantia nigra pars compacta SSRI selective serotonin reuptake inhibitor STAR*D sequenced treatment alternatives to relieve depression clinical trial TAT membrane permeable protein from HIV1 TAT-D1-D2-FLAG-Ipep TAT-linked membrane permeable D1-D2 interfering peptide with c- terminal 8-amino acid FLAG tag TAT-D1-D2-Ipep TAT-linked membrane permeable D1-D2 interfering peptide TAT-Pep 9-amino acid membrane permeable peptide fragment from HIV1 TAT protein TH tyrosine hydroxylase TrkB tyrosine receptor kinase type B VMAT2 vesicular monoamine transporter 2 VTA ventral tegmental area x

11 1 1 Introduction The neurotransmitter dopamine is involved in many processes within the brain, including motor control, cognition, reward, emotion and pleasure. Dopamine exerts its effects through five unique dopamine receptors, termed D1 through D5. These receptors are G-protein coupled receptors (GPCRs) that contain seven trans-membrane domains and initiate intracellular signaling cascades. 4 In addition to existing as unique receptors, dopamine receptors can also couple with other proteins and receptors to form functional heterodimers that activate signaling cascades, independent from those activated by each component receptor. 5,6 Recently, scientific evidence has shown that these heterodimers can play a pathological role in the progression of psychiatric conditions. 7 Dopamine D1 and D2 receptors couple in this manner and are thought to play a role in psychiatric conditions such as Major Depressive Disorder (MDD). 3,8 Our laboratory has found a pathophysiological role for the dopamine D1-D2 heterodimer in MDD. 3 MDD is a common, serious psychiatric condition that accounts for 4.4% of total global disease burden 9 and is often left undiagnosed and untreated in patients Furthermore, many patients do not respond to available pharmacological or psychological treatment for MDD with over 50% of patients not responding to first-line pharmacological treatment. 13,14 Pei et al 3 demonstrated that the D1-D2 heterodimer is up-regulated in the striatum of patients with MDD. Disrupting this interaction using a membrane permeable peptide (the D1-D2 interfering peptide) had an antidepressant effect in the Forced Swimming Test (FST) and the Learned Helplessness (LH) task, two strongly validated preclinical tests for antidepressant efficacy. 3 These results are promising, but lack clinical validity since invasive, direct administration methods were used to deliver the peptide to the prefrontal cortex (PFC). In order for the D1-D2 interfering peptide to

12 2 become a clinically relevant antidepressant treatment, a less invasive, clinically applicable method of drug delivery must be developed. The purpose of this project is to test whether we can effectively administer the D1-D2 interfering peptide to the brain using intranasal delivery. Intranasal delivery is clinically applicable, offers a direct pathway to the brain and is a non-invasive method to target therapeutics to the central nervous system (CNS). A number of proteins including insulin and nerve growth factor have been delivered to the CNS intranasally, both in animals and in humans. 15 The goals of this project are to (1) confirm that the D1-D2 interfering peptide is able to disrupt the interaction between D1 and D2 when administered intranasally, (2) test whether the D1-D2 peptide has an antidepressant effect in the FST when administered intranasally, and (3) further investigate the pharmacological properties of the D1-D2 interfering peptide. In the introduction, I will briefly review the scientific literature relating the role of dopamine in cognitive and behavioral processes within the mammalian brain as well as currently held hypotheses about dopamine receptor heterodimerization and its role in psychiatric conditions (Section 1.1). I will focus specifically on the Dopamine D1 and D2 receptor-receptor interaction our laboratory has previously identified (Section 1.1.3). Next, the etiology, symptoms, and neurobiology of MDD will be reviewed, along with currently available antidepressant treatment options and their efficacy in treating this disorder (Section 1.2). I will also discuss preclinical models of depression, and their strengths and weaknesses for identifying new therapeutics for this complex psychiatric disorder (Section 1.2.5). Finally, I will discuss the evidence supporting the use of intranasal administration methods to target therapeutic substances to the CNS (Section 1.3).

13 3 1.1 Dopamine neurotransmission in the mammalian brain Dopamine is a catecholamine neurotransmitter synthesized from the amino acid tyrosine. 16 Discovered in the mid-20 th century, 17,18 dopamine and its role in the central nervous system have been the subject of extensive scientific investigation. It is involved in a wide variety of cognitive and behavioral processes in the brain, such as reward seeking and motivation, voluntary motor movement, emotional and cognitive processing, attention, and working memory. It also plays a role in numerous neurological and psychiatric illnesses, including but not limited to Parkinson s Disease 19, Huntington s Disease 20, schizophrenia 21, major depression 22, and addiction 23,24. For example, Parkinson s Disease (PD) is a neurodegenerative disorder that occurs due to a loss of dopaminergic neurons in the substantia nigra (SN) and dopaminergic innervations to the striatum, a brain area involved in voluntary motor movements. 19 Dopamine itself is a small organic compound made up of a benzene ring, an amine group attached to a 3-carbon chain, and two hydroxyl groups. 25 In the central nervous system, dopamine is synthesized from the amino acid tyrosine in neurons containing the enzymes necessary for this conversion. These neurons originate in three distinct areas: the substantia nigra pars compacta (SNpc), the ventral tegmental area (VTA) and the arcuate nucleus of the hypothalamus. 16 Briefly, the amino acid tyrosine is converted into L-DOPA by the enzyme tyrosine hydroxylase (TH), the rate-limiting enzyme in the production of dopamine. 16 L-DOPA is converted into dopamine by DOPA decarboxylase (DDC). 16 Once synthesized, dopamine is transported from the cytosol into synaptic vesicles by the vesicular monoamine transporter 2 (VMAT2), from where it is released into the synaptic cleft when dopamine neurons fire. 4 Once released into the synaptic cleft, it binds to and activates dopamine receptors on the post-synaptic (and pre-synaptic) membranes. Subsequently, it can be transported back into the presynaptic dopaminergic neuron by the dopamine transporter protein (DAT) for re-use or degradation. 16

14 4 Dopamine is broken down into inactive metabolites by a sequence of reactions catalyzed by monoamine oxidases (MAOs) resulting in the production of homovanillic acid, which is released into the cerebrospinal fluid (CSF) as metabolic waste. Dopamine also serves as the precursor for norepinephrine, as the two neurotransmitters differ by the addition of one β-hydroxyl group, a reaction catalyzed by the enzyme dopamine β-hydroxylase. 16 Presynaptic neurons that produce and release dopamine originate from distinct areas in the basal ganglia and innervate cortical areas, the hippocampus and limbic cortex, and brain areas related to movement and endocrine function (see Section 1.1.2). 4 Like many neurotransmitters, dopamine exerts its effects in the brain through specific receptors termed D1 through D5, G-protein coupled receptors that each have specific downstream effects in cells that effect complex intracellular signaling pathways. 26,27 Dopamine receptors can form functional interactions with different types of dopamine receptors 3,5,28 while also interacting with other types of neurotransmitter receptors and a variety of other proteins to facilitate cross-talk between neurotransmitter systems (Section 1.1.3). Unlike other neurotransmitters such as glutamate and GABA, dopamine is not considered an excitatory or an inhibitory neurotransmitter, as the ultimate effect of dopamine on a given neuronal population depends on the type of dopamine receptor and the ultimate effect of the intracellular signaling cascade that is activated Dopamine receptors and their intracellular effects As stated above, dopamine exerts its intracellular effects through five distinct receptors, D1 through D5. These receptors belong to the guanine nucleotide-blinding (G-protein) coupled receptors superfamily. G-proteins are signal transducers that mediate the transduction of intracellular signals for a vast number of endocrine, neurotransmitter, autocrine and paracrine compounds. There are four main types of G proteins, G s, G t, G i and G o, each consisting of three

15 5 subunits (Gα, Gβ and Gγ). 32 There are 20 known Gα, 6 Gβ and 11 Gγ subunits, and G-proteins are typically named by the identity of their α-subunit. 32,33 As a result of their heterogeneity, a large number of GPCRs can form with resultant activation of vastly different intracellular signaling pathways. 32 Dopamine receptors, like all GPCRs, exert their downstream signaling effects by activating (or inhibiting) intracellular second messenger cascades. 25,34 In the case of dopamine receptors, the receptor itself contains 7 transmembrane domains, and is coupled to G protein subunits on the intracellular side (See Figure 1-1). 4,25 After dopamine binds to its binding site, the G-protein becomes activated and causes downstream intracellular effects, which underlie the cognitive and behavioral changes mediated by dopamine in the brain. 35 The existence of dopamine receptors was first proposed in the 1970s when Kebabian and Greengard published evidence for a dopamine-selective adenyl cyclase 36 (AC, an enzyme that converts ATP to camp, a potent second-messenger signaling molecule). 4 After the initial discovery of camp-coupled dopamine receptors, Spano et al 37 demonstrated that dopamine receptors exist in two groups, one that is positively coupled to camp production and one that is not. 4 Based on their opposing effects on camp signaling, these receptors types were named D1 (camp-activating) and D2 (camp-inhibiting). 26 Quickly, the hypothesis that dopaminergic signaling was mediated by two dopamine receptors with opposing effects was proven to be an oversimplification, 35 as the advent of molecular biology and genetic cloning techniques allowed for the identification of D1 and D2 cdna 38 and three additional distinct dopamine receptors activated by dopamine: D3 39, D4 40 and D5 41. Currently, dopaminergic pathways in the CNS are thought to be mediated by these five dopamine receptors, that are separated into two families based on their effects on camp: D1-like and D2-like. 4

16 D1-like dopamine receptors The D1-like dopamine receptor family is comprised of dopamine D1 and D5 (formerly D1B) receptors. 41 When agonists bind to these receptors they activate the G s/α family of GPCRs. Activation of G s/α results in activation of AC, and camp production. 25 D1-like receptors can also couple to G olf/α (also stimulating AC and camp production) in specific brain areas (caudate, nucleus accumbens (NAc) and olfactory tubercle). 42 The genes for both D1 and D5 receptors do not contain introns in their coding sequences, and thus there are no splice variants of D1 or D5. 34,43 Some pharmacological differences exist between D1 and D5 receptors, as the D5 receptor is more pharmacologically sensitive to dopamine than D1 receptor. 34 D1 receptors are the most common dopamine receptors in the CNS, and are highly expressed in post-synaptic targets of the mesocortical, mesolimbic and nigrostriatal dopamine pathways (see Section 1.1.2), including the striatum, NAc, amygdala and PFC. 25,44,45 D5 receptors are expressed at lower levels in the PFC, the cingulate cortex, substantia nigra, hypothalamus, hippocampus and dentate gyrus D1 and D5 receptors are co-expressed in pyramidal neurons of the prefrontal, premotor, and cingulate cortices and the dentate gyrus. 48,49 Unlike the D2-like dopamine receptors, D1-like receptors are, for the most part, expressed mostly in the post-synaptic membrane of neurons receiving dopaminergic input 4,25, although recent evidence suggests that D5 is expressed presynaptically in the basolateral amygdala and other brain structures. 49 Thus, it is probable that dopamine D1-type receptors are responsible for mediating the diverse effects of dopamine on its post-synaptic cellular targets. For example, dopaminergic signaling through D1-type receptors in the PFC is critical to working memory processes 50 and to the occurrence of motor movements gated by basal ganglia circuits. 51

17 D2-like dopamine receptors The D2-like family of dopamine receptors includes dopamine D2, D3 and D4 receptors. Originally identified as those receptors that were not coupled to AC activation and camp production 26, they have since been found to play complex roles in various intracellular signaling, cognitive and behavioral processes. When the dopamine D2, D3 and D4 receptors were cloned in the early 1990s, it emerged that inhibition of AC was a general property of the D2-like receptors, although the degree to which AC is inhibited varies by receptor subtype. 25 This property of D2-like dopamine receptors is mediated by coupling to the Gα i/o GPCR subunit, which inhibits AC function. 32,52 Dopamine D2-type receptors also activate intracellular signaling cascades independently of G-protein activation. For example, the D2 receptor complexes with the regulatory protein β- arrestin, protein phosphotase 2A and Akt ( a serine/threonine kinase), and this pathway regulates the function of Glycogen synthase kinase 3β (GSK-3β). 53,54 This signaling pathway typically takes longer to become active, and stays active for a much longer period of time than the G- protein mediated pathways. GSK-3β is also involved in signaling pathways activated by other neurotransmitters, such as serotonin. The D2 receptor involvement in GSK-3β function may represent a point where signaling from numerous neurotransmitters is integrated. 4,54 Furthermore, β-arrestin plays a role in GPCR desensitization and regulation, and thus, dopaminergic signaling through D2-like receptors may also be involved in dopamine receptor desensitization processes. 4 Unlike the D1-type dopamine receptors, the D2-like receptor coding genes contain intron sequences, allowing for the generation of receptor splice variants. 43 The most widely studied of these splice variants are the D2S (short) and D2L (long) dopamine D2 receptors, generated by alternative splicing of an 87-base pair exon between introns 4 and 5 of the D2 receptor gene. 55,56 As a result, the D2L receptor isoform contains a 29-amino acid sequence in the third intracellular

18 8 loop that is missing in the D2S receptor isoform. 55,56 Interestingly, these isoforms localize differently within the CNS, with the D2L receptor isoform predominantly located in postsynaptic targets of dopamine pathways and the D2S isoform located presynaptically, in dopaminergic neurons. 57 D2-like receptors are expressed presynaptically, indicating that they can act as autoreceptors, providing an important negative feedback mechanism by modulating dopamine synthesis, neuronal firing rate and dopamine release in response to extracellular dopamine levels. 4,25 The D2S receptor isoform, and not the D2L receptor isoform, of the dopamine D2 receptor is likely at least partially responsible for this autoregulation, as generation of a D2L -/- transgenic model did not affect the ability of dopaminergic neurons to auto regulate. 45,57,58 Dopamine D2 receptors are found in various brain areas, including the striatum, NAc, substantia nigra, VTA, hypothalamus, cortical areas including the PFC and the hippocampus. 39,59,60 D3 dopamine receptors are more limited in their expression than D2s, and are mostly expressed in the limbic areas. 60,61 D4 receptors have the lowest expression of all dopamine receptor subtypes, but are expressed in the frontal cortex, amygdala, hippocampus, hypothalamus and other brain areas. 62, Regulation of dopamine receptors After activation of dopamine receptors and activation of intracellular signaling cascades, (e.g. camp in the case of D1 receptors), the downstream events initiated in the cell occur regardless of whether dopamine remains bound to its receptor. 4 To control these intracellular signaling events, dopamine receptors are regulated through a number of mechanisms including G-protein regulatory proteins (RGP family), phosphorylation of intracellular loops, receptor sensitization and desensitization to agonist binding, and receptor internalization. 5,25,32 The

19 9 Figure 1-1 Dopamine receptors: structure and function D1-type and D2-type Dopamine receptors act on adenyl cyclase (AC) in opposing ways. D1-type receptors activate AC via coupling with G s/olf, while D2-type dopamine receptors inhibit AC via coupling with G i/o G-proteins. All dopamine receptors contain 7 trans-membrane domains, an extracellular N-terminus and intracellular C-terminal tail. D2-like receptors have shorter C-terminal cytosolic tails and a larger third intracellular loops. Receptor function is modulated in part by phosphorylation sites on intracellular loops and C-terminal tails, which can mediate receptor desensitization and endocytosis. Figure Prepared with help from S.Chen, Liu Lab (2011).

20 10 regulators of G-protein family (RGP) typically increase the rate of G-protein GTP hydrolysis, decreasing the amount of time the proteins spend active, modulating the efficacy of G-protein mediated signaling. 64 For example, activation of protein kinase A by camp elevations in response to D1-type activation will phosphorylate residues on the C-terminal tail of D1 receptors, and initiate the recruitment of adaptor proteins (typically, β-arrestins) that prevent further G-protein activation. 65 Arrestins can also recruit proteins such as clatherin and β-adaptin that mediate receptor endocytosis, which can reduce signaling in response to high extracellular levels of dopamine. 66 Dopamine receptors are also regulated by their interactions with other proteins and other transmembrane receptors (see Section 1.1.3), which can change the receptor s affinity for agonists and the intracellular pathways activated by each component receptor Dopaminergic pathways in the mammalian CNS Although neurons producing dopamine are relatively few in number in the brain, they project extensively to numerous cortical and subcortical structures. There are four main dopaminergic pathways in the CNS: the nigrostriatal, mesocortical, mesolimbic and tuberoinfundibular. 25 Each pathway plays an important role in the functions of its target areas and creates a complex system of dopamine-modulated circuits within the brain. The functions of the mesolimibic (Section ) and mesocortical (Section ) dopaminergic pathways will be the focus of this review, as these are the most relevant to this project and to the pathogenesis of MDD. The role of the nigrostriatal pathway in motor behavior will be briefly discussed (Section ). The tuberoinfundibular pathway, in which dopamine functions as a neuroendocrine hormone to inhibit prolactin secretion from the anterior pituitary 16, will not be reviewed here.

21 The nigrostriatal dopamine pathway The nigrostriatal pathway originates in the SNpc and projects to the striatum. 16,67 The striatum plays a major role in the gating of motor movements, and the vast majority of the neurons originating from the striatum are GABA-ergic (inhibitory) Medium Spiny Neurons (MSNs). 16 Striatal neuronal firing activity has opposing functions: depending on the area that the neurons project to, it can control both the direct (favoring movement) and indirect (favoring no movement) basal ganglia circuits. 68 D1, D2 and D3 receptors are all involved in the effects of dopamine on motor movements. 4 When dopamine D1 receptors on MSNs are activated and converge with cortical premotor inputs, the firing of MSN projections disinhibits the thalamus, favoring the behavioral occurrence of that movement. 25,51 The role of D2 and D3 receptors in the gating of locomotor activity is more complex than that of D1 receptors, as they function both as presynaptic auto-receptors (decrease dopamine release when activated) and post-synaptic receptors. 4,25 In PD, the dopaminergic neurons originating in the SNpc are gradually lost, gradually reducing dopaminergic input to the striatum, resulting in the symptoms of stiffness and reduced movement in PD. 16, The mesolimbic dopamine pathway The mesolimbic dopamine pathway originates in a second brain area containing dopaminergic neurons, the VTA. 69 This pathway projects predominantly the NAc (also known as the ventral striatum). 70 The NAc is highly interconnected with other limbic areas including the amygdala, cingulate cortex, parahippocampal gyrus, hippocampal formation, anterior thalamic nuclei and NAc. 16 The amygdala, the area most involved with fear and emotional experience,

22 12 sends neuronal projections to the hypothalamus, which, among other functions, is thought to regulate the physiological and endocrine changes associated with emotional states. 16 Animal models of disrupted mesolimbic dopaminergic input to the limbic areas and amygdala have demonstrated that dopamine transmission in the amygdala is associated with the acquisition of Pavlovian-conditioned fear responses. 71 Both D1-type and D2-type dopamine receptors seem to play a role in fear conditioning. Briefly, D1/D5 antagonists diminish conditioned fear responses 69, and D1 agonists potentiate them 72, indicating that dopamine signaling via D1 receptors potentiates fear responses in the amygdala. Paradoxically, both D2- type agonists and antagonists, impair fear conditioning and recall of emotional memory. 71 Dopaminergic input to the NAc is highly involved in behavioral reinforcement mechanisms and reward-dependent learning. 16 Most addictive recreational drugs such as amphetamine, cocaine and nicotine, along with naturally rewarding experiences such as food and social interactions increase the levels of dopamine in the NAc at the terminals of dopaminergic projections originating from the VTA. 73 Evidence from rodents, non-human primates and human neuroimaging studies strongly suggest that the mesolimbic system is involved in cue association to positive rewards, and the reinforcement of behaviors that result in acquisition of a reward. 74 The increased levels of dopamine in the NAc are thought to contribute to the strong reinforcing and addictive properties of recreational drugs and other substances Di Chiara et al 75 found that drugs with aversive effects reduced dopamine release in the NAc, implying that dopamine release is correlated with hedonistic, reinforcing events. In fact, many recreational drugs enhance dopamine neurotransmission, either by blocking DAT (cocaine and amphetamine), enhancing release of dopamine through pre-synaptic modulation (nicotine) or inhibiting inhibitory, GABA-ergic neurons that suppress dopaminergic neurons in the VTA (muopioid agonists). 77 Due to the involvement of the dopaminergic system in the CNS response to

23 13 recreational drugs and other hedonistic experiences, dopaminergic system dysfunction is thought to play a major role in the pathophysiological changes that accompany addiction. 23,24 The reward circuit modulated by dopamine neurotransmission has also been implicated in the pathogenesis of MDD and other psychiatric conditions. This is not surprising, given the symptoms of anhedonia, lack of motivation present in MDD and the role of the mesolimibic pathway in reward-based learning, motivation and emotional processing. 22,78 Dopamine signaling in the VTA NAc mesolimbic reward circuit modulates motivation for rewards and pleasure, implying that these common symptoms of MDD could be due to pathological changes in this circuit. 79,80 Interestingly, dopamine does not, as was initially suggested, code for pleasure in the mesolimibic reward pathway. 77,81 Studies in mice missing tyrosine hydroxylase (the rate-limiting enzyme in dopamine synthesis) show that these mice still have hedonic preferences, preferring sweetened water over unsweetened. 77,82 At the same time, dopamine-deficient mice did not seek rewards during reward-directed tasks, that is, although they enjoyed the reward, they did not seek it out. 83 These studies support the hypothesis that dopamine in the NAc is required for wanting a reward, but not for liking it, that is, dopamine signaling encodes incentive salience, leading to the modification of behavior in order to obtain the reward. 77,84,85 The large number of studies investigating mesolimbic dopaminergic signaling strongly suggest that dopamine is involved in motivation, reward-based learning and emotional processing in the limbic areas, and that dysfunction within the mesolimbic system could lead to addiction, MDD and other psychiatric illnesses.

24 The mesocortical dopamine pathway The mesocortical dopamine pathway consists of dopaminergic neurons originating in the VTA and projecting to the PFC, insular cortex and cingulate cortex. 86 The PFC is highly involved in higher-order cognitive processing including motivation, planning, attention to salient stimuli, decision making, behavioral flexibility, and working memory. Working memory is conceptualized as the manipulation of a number of items in short-term memory storage in order to effectively plan and organize future thought or actions. 86 Early findings by Brozoski et al 87 demonstrated that depletion of dopamine in the PFC of monkeys produced cognitive and working memory deficits comparable to those observed when the frontal lobes were completely removed. Subsequent research into the role of dopaminergic neurotransmission in working memory indicated that an optimal range of dopaminergic signaling in the PFC existed, where too little or too much signaling through D1 receptors increased errors in the radial arm maze and other working memory tests mediated by the PFC. 50,88,89 The PFC also modulates behavioral flexibility, or the ability to alter behavior in response to changing environmental conditions. A common test for behavioral flexibility and set-shifting is the Wisconsin Card Sort Task, which requires the human or animal to disregard a previously beneficial strategy (e.g. sort cards by shape) and engage in a novel one (e.g. sort cards by color) to obtain a reward. 50,86 Patients with damage to the dorsolateral PFC are unable to alter their sorting strategy when they are required to organize cards by another dimension, a finding replicated in non-human primate 90,91 and rodent versions of dimensional set-shifting tasks and reversal learning (where the animal must discriminate between two or more stimuli, only one of which is relevant to reinforcement) tasks. 50,86 In microdialysis studies that measured PFC dopamine levels in freely-behaving rats during a set-shifting task, dopamine levels in the PFC

25 15 increased when the rat had to shift to a different rule in conflict with the first, indicating a role for DA signaling in behavioral flexibility. 50,95 Both attention deficit hyperactivity disorder (ADHD) and schizophrenia patients show marked impairments in set-shifting, 96,97 and both disorders are associated with various changes in the mesocortical dopaminergic pathway. 98 Pharmacological treatments for ADHD such as methylphenidate, which increases mesocortical dopamine transmission, are able to decrease impairments in set shifting seen in patients. 95 These clinical findings suggest that dopaminergic systems are involved in modulating behavioral flexibility, but the exact mechanisms through which this modulation occurs remain unknown. The PFC is also very important in decision making processes, specifically when weighing the advantages and disadvantages of a given choice. Bechara et al 99 demonstrated that patients with damage to the ventromedial PFC were impaired on behavioral tasks designed to simulate real-life decisions, and the uncertainty and rewards involved. 98 In rodents, one can model this cost/benefit decision making by manipulating the cost (i.e. increasing the delay to reward delivery, increasing the amount of physical activity required, or making reward delivery probabilistic) of a reward (typically more, or better, food). 98 These different forms of cost/benefit decision making are regulated by anatomically-distinct regions of the PFC, and all are sensitive to manipulations in dopamine PFC levels. 100,101 Both D1-type and D2-type dopamine receptors seems to be implicated in cost/benefit decision making paradigms, but they seem to play a complex role, with their specific function dependent on PFC area and the type of cost/benefit decision being made. 50,102,103 Dopaminergic signaling in the PFC occurs through both D1-type and D2-type dopamine receptors. In both rodent and monkey PFC, the distribution of D1 receptor messenger RNA (mrna) is significantly greater than the other dopamine receptor subtypes. 104 Both D1 and D2

26 16 receptors are found on excitatory, glutamatergic pyramidal neurons and non-pyramidal, GABAergic interneurons in the PFC. 86,105,106 In fact, a subset of layer V pyramidal neurons (approximately 25 %) 107 as well as non-pyramidal PFC neurons express both D1 and D2 receptors, indicating that these receptors may co-localize within these cells. 8,105,106 Beyond cellular and sub-cellular expression patterns of dopamine receptors in the CNS, little is currently understood regarding how intracellular signaling pathways activated by dopamine receptors eventually modulate the higher-order cognitive processes mediated by the PFC Heterodimerization of Dopamine Receptors After the cloning of the five distinct dopamine receptor subtypes in the early 1990s, structural, pharmacological and biochemical studies suggested that each receptor had unique properties, although they fell into the two previously described families of dopamine receptors (D1-like and D2-like). 4 Over the last 20 years, it has become apparent that dopamine receptors function both as independent entities and form heterodimers with members of the same family and with structurally divergent families of receptors. 5 The pharmacological and functional profiles of dopamine receptor heterodimers are often very different from that of the component receptors and these are thought to contribute to the numerous heterogeneous functions of dopaminergic signaling in the CNS. 5,6 Dopamine receptors have been shown to form heterodimers through direct protein-protein interactions between D1 and D2 receptors, 3, D1 and D3 receptors in the striatum 111,112, D2 and D5 receptors 109,113, D1 receptors and NMDA receptors (NMDARs) 30,114 and D5 and GABA-A receptors 115, among other transmembrane and cytoplasmic proteins. All dopamine receptors subtypes form non-obligatory heterodimers, that is, dimerization is not necessary in order for the receptor to function. 4 However, a large degree of complexity in

27 17 the signaling effects of dopamine receptors results from their ability to heterodimerize. 5 For example, dopamine receptor heteromers could create novel ligand binding sites, activation of one or both component receptors could initiate different intracellular signaling pathways than those initiated by the component receptors, or a synergistic increase in signaling could occur when both agonists are present. 5 The D1-D2 receptor interaction and the activation of independent intracellular signaling pathways that occurs will be the focus of this section, as this interaction is implicated in the pathogenesis of MDD 3 and is the target of the D1-D2 interfering peptide used in this project The Dopamine D1-D2 Receptor Interaction An interaction between the dopamine D1 and D2 receptors was first investigated because of the observation that a D1-like receptor could activate Inisitol Phosphate 3 (IP3) production (leading to increases in intracellular calcium) in various brain regions including striatum, hippocampus and cortex. 116,117 An interaction between dopamine D1 and D2 receptor was proposed because of the observations that the presence of calcium signaling activated by D1 was absent in D1-transfected cells, and present in cells transfected with both D1 and D2 receptors. 2 Research from our laboratory identified the specific regions through which D1 and D2 receptors interact as a 15 amino acid sequence within the 30-amino acid insert in the third intracellular loop of the D2L receptor isoform, and the D1 intracellular C-terminal tail. 3 This finding provided indirect evidence that the D1-D2 receptor interaction occurs in post-synaptic membranes, as both D1 and D2L dopamine receptors are generally localized to post-synaptic areas. 25,57

28 Figure 1-2 Schematic representation of D1-D2R receptor interaction and activation of intracellular signaling pathways. (A) Activation of D1 or D2 when those receptors are in complex is thought to activate PLC, resulting in release of calcium from the endoplasmic reticulum and subsequent activation of CamKII. 1,2 (B) The D1-D2 interfering peptide (TAT-D1-D2-IPep) disrupts the interaction between D1 and D2L receptors, resulting in disruption in the G q -mediated downstream signaling pathways. 3 (A) and (B) prepared with help from S.Chen, Liu Lab. 18

29 19 Research by George and colleagues 2,118 confirmed the interaction between D1 and D2 using co-immunoprecipitation and through fluorescence resonance energy transfer (FRET) techniques. They also demonstrated that the D1-D2 receptor complex induces intracellular calcium via a G q GPCR-dependent pathway in the striatum. 119 In the G q -pathway, Phospholipase C (PLC) becomes activated by G q, resulting in an increase in inisitol triphosphate (IP3) which then causes activation of downstream molecules resulting in increased calcium concentration in the cytoplasm (Figure 1-2A). 32,119 The colocalization of D1 and D2 receptors seems to occur in a number of brain regions including the dorsal and ventral striatum, and the PFC Although the intracellular pathway activated by the D1-D2 receptor heterodimer is characterized, the physiological relevance of this interaction and its role in neurological and psychiatric illnesses is not yet clear. Recently, our laboratory demonstrated that the D1-D2 interaction may have a role in the pathogenesis of MDD. Most importantly, the D1-D2 receptor interaction was up-regulated in post-mortem samples from the striatum of patients with MDD, implying that this interaction may be disrupted in this illness. 3 Uncoupling the D1-D2 receptor interaction using a small, membrane permeable peptide (TAT-D1-D2-IPep) results in an antidepressant effect in animal model of depression (Figure 1-2B, Figure 1-3). 3 This study suggests that the D1-D2 receptor interaction may play a role in the pathogenesis of MDD, and warrants further investigation into possible therapies based on disrupting this interaction. 1.2 Major Depressive Disorder MDD is the most common psychiatric illness in the world, with the lifetime incidence in the United States 12% in men and 20% in women. 120 Despite its prevalence, many patients who have MDD are not adequately treated with current antidepressant therapies. Since the discovery of the first antidepressant compounds over 50 years ago, much progress has been made

30 20 investigating the neurobiological changes underlying MDD. 121 Although numerous hypotheses attempt to explain the neurobiological and pathological changes underlying the clinical presentation of MDD, no unitary hypothesis explaining all the pathological changes and the complex symptoms observed in MDD exists. Here, the epidemiology (Section 1.2.1) and symptoms of MDD (Section 1.2.2) will be reviewed, along with currently available treatments and their efficacy in treating MDD (Section 1.2.3). Next, a number of different hypotheses regarding the pathophysiology underlying MDD will be explored (Section 1.2.4). Since the treatment in our current investigation targets a protein-protein interaction between D1 and D2 dopamine receptors, the evidence for the involvement of the dopaminergic system in MDD will be reviewed (Section ). To conclude, the use of preclinical models to model MDD and test new antidepressant treatments will be discussed (Section 1.2.5) Epidemiology of MDD Depression is a common psychiatric illness characterized by 2 or more weeks of a distinct change in mood, sadness and/or constant irritability, as well as feelings of hopelessness and loss of interest in pleasurable activities. 122 The lifetime incidence of depression in the United States is 12% in men and 20% in women 120. In fact, women are 70% more likely than men to experience depression in their lifetimes. 123,124 Although depression is closely related to the normal emotions of sadness, it often does not regress when the external cause of these emotions dissipates, and can be disproportionate to their cause. 78 Often, episodes of depression will recur two or more times and become classified as MDD. 122 At its most severe, MDD can lead to suicide attempts, which can result in loss of life or significant disability. MDD is responsible for 4.4 % of the worldwide disease burden and, is the leading cause of disability worldwide when considering total years lost to disability. 125 Depression often goes

31 21 undiagnosed and untreated because of the prevalent societal stigma associated with psychiatric conditions and seeking treatment for these conditions. According to statistics from the National Institute of Mental Health, only 57% of patients with MDD in the United States are receiving any kind of treatment for the disorder, and only 19% of patients with MDD are receiving adequate treatment. 120,126 Additionally, MDD can often occur in conjunction with other serious illnesses such as cancer, chronic pain, epilepsy and cardiovascular disease. 9,120, When this is the case, both MDD and the co-morbid illness are adversely affected, as treatment outcomes in patients who have diabetes, epilepsy or ischemic heart disease along with MDD have poorer outcomes than those without MDD. 130 Overwhelmingly, epidemiological data regarding the prevalence of MDD indicates that it is extremely common, often undiagnosed and, in the majority of cases, not adequately treated Symptoms and clinical presentation of MDD The Diagnostics and Statistical Manual of Mental Disorders IV (DSM IV-TR) 122 criteria for MDD are the most commonly used criteria for MDD diagnosis. A single depressive episode is categorized by the presence of five or more of the following symptoms during a two-week period where at least one of the symptoms is either depressed mood most of the day, nearly every day, or loss of interest or pleasure in almost all activities. 122,131 Recurrent MDD occurs when a patient experiences two or more major depressive episodes, with a symptom-free period of two or more months separating them. Other symptoms include a change of more than 5% in body weight over the course of 1 month, insomnia or hypersomnia, psychomotor agitation or retardation, persistent fatigue and loss of energy, feelings of worthlessness or guilt, diminished ability to think or concentrate, indecisiveness, and/or recurrent thoughts of death or

32 22 suicide. 8,122,131 These symptoms can cause significant impairment in the patient s social, occupation and other functioning. MDD and its symptoms are often variable in both clinical presentation and severity, which may contribute to the large number of patients left undiagnosed and untreated Treatments for MDD Patients diagnosed with MDD are typically treated with pharmaceutical agents that increase the amount of monoaminergic neurotransmitters at the synapse. Currently, the firstline pharmaceutical therapy for MDD is a class of drugs termed selective serotonin re-uptake inhibitors (SSRIs). 78 When given to patients, these drugs produce an increase in the neurotransmitter serotonin in the brain by inhibiting its re-uptake into the presynpatic neurons from where it was released. 9 In the CNS, serotonergic neurons project to numerous cortical and subcortical areas from the brainstem raphe nuclei and are involved in regulation of mood, appetite, sleeping behavior, learning and memory. 78 Serotonin act through serotonin receptors, of which there are seven families that have diverse effects in cells. 16 SSRIs quickly increases the total amount of serotonin available at serotonergic synapses and, as such, increase the amount of serotonin-mediated neurotransmission in the brain. 132 The side-effects of SSRIs are often apparent almost immediately after the initiation of treatment, while any therapeutic antidepressant effect from these medications takes approximately three weeks to become apparent in patients. 78 The delay in onset of any therapeutic effect suggests that the ability to increase serotonin at the synapse may not be the only mechanism through which SSRIs have an antidepressant effect and that they may be mediating other, longer-term effects in the CNS responsible for its therapeutic efficacy. 8,133

33 23 In a large clinical trial for antidepressant efficacy, the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) trial, it was shown that only % of patients with MDD adequately responded to first line treatment with citalopram, an SSRI medication. 134 If, after a number of weeks of citalopram or other SSRI treatment, little or no improvement on a standardized rating scale such as the Hamilton Depression Rating Scale 135, is observed, patients can be treated by increasing the SSRI dose (if side effects are tolerable), switched to a new SSRI, or started on another antidepressant along with the original SSRI. 9 Studies have also indicated that SSRI efficacy is correlated with the severity of depression when treatment is initiated, implying that SSRIs are more effective for patients with severe depression and may not be significantly more effective than placebo for patients with mild or moderate depression. 136,137 For patients who do not respond to SSRIs, pharmaceutical treatment alternatives include SNRIs (Selective norepinephrine reuptake inhibitors), Triple reuptake inhibitors (inhibit reuptake of serotonin, dopamine and norepinephrine) 138, tricyclic antidepressants such as imipramine, and monoamine oxidase inhibitors (MAOIs). MAOIs exert their antidepressant effect by blocking the breakdown of monoaminergic neurotransmitters (serotonin, norepinephrine and dopamine) and are effective in the treatment of MDD, but also have strong and often intolerable side effects. 139,140 A final, invasive option in the case of severe, unremitting MDD is electro-convulsive therapy, which remains the most effective treatment for severe, unremitting depression. 139, Efficacy of current antidepressant treatments: The STAR*D trial The STAR*D trial 134 is a large clinical trial designed to investigate remission rates after antidepressant treatment in a large and generalizable sample of patients. In the first level of the trail, all patients enrolled were treated with citalopram (an SSRI) as a first-line treatment, with

34 24 their depressive symptoms evaluated every 2 weeks after initiation of treatment. 134,142 Between 28 and 33 % of patients treated with citalopram achieved remission within 12 to 14 weeks of treatment. 134 In Level 2 of the STAR*D trial, those patients who did not respond to citalopram treatment after 14 weeks were given the choice between pharmacotherapy augmentation, psychotherapy or switching to a different SSRI medication for 12 weeks. 14 Of the patients in Level 2, approximately 30% achieved remission of symptoms within 12 weeks, with no significant differences in remission rates with any of the treatment strategies employed. 14,142 In Levels 3 and 4 of the trial, patients who had not responded to antidepressant treatments or psychotherapy in Level 1 or 2 were treated with alternative pharmacotherapies, including tricyclic antidepressants. In Level 3 and 4 of the STAR*D trial, the remission rats dropped substantially, ranging from 12 % to 25%, depending on the treatment used. 142 In all, only 67% of patients originally enrolled in the STAR*D achieved remission of their depressive symptoms. 142 The STAR*D trial reveals that almost one third of patients with unipolar depression do not respond to multiple trials of SSRI and other antidepressant treatments. After the first two levels of the trial, patients were much less likely to respond to further pharmaceutical treatment trials, indicating the importance of achieving a treatment response with the first few antidepressants prescribed to patients. 142 The STAR*D trial also provides a strong rationale for further investigation into new antidepressant therapies that could help treat MDD in the subset of patients who currently do not respond to available antidepressant treatments.

35 Neurobiological changes and pathophysiology of MDD Currently, there is no unitary hypothesis that explains the various pathological and neurobiological changes that occur in MDD. What remains clear, however, is that MDD is a heterogeneous disorder with complex pathological mechanisms that vary considerably between individuals affected by the disease. In human neuroimaging studies, the brain regions that are consistently found to be involved in MDD are the PFC, the cingulate cortex (area Cg25), the hippocampus and the amygdala. 79,143,144 These findings are consistent at both the structural level, where magnetic resonance imaging (MRI) data and other neuroimaging data suggests decreased hippocampal and PFC volume in depressed patients, and the functional level, where functional MRI (fmri) and positron emission tomography studies suggest abnormal connectivity and decreased functionality of limbic, cingulate and prefrontal areas in depression. 143 The monoamine and catecholamine neurotransmitters, serotonin, dopamine and norepinephrine innervate these areas through extensive axonal projections from the dorsal raphe nucleus (serotonin), VTA (dopamine) and locus coeruleus (norepinephrine). 16,73 Disruptions in these systems are thought to contribute to the pathogenesis of MDD (Section ). More recent investigations into the neurobiological mechanisms behind MDD have also demonstrated that neurotrophic factors such as Brain Derived Neurotrophic Factor (BDNF) also play a large role in MDD (Section ). The evidence implicating dopamine in the pathogenesis of MDD will be reviewed, as the D1-D2 interfering peptide used in this project specifically targets the heterodimerization of two dopamine receptors (Section ). Although these theories of MDD all attempt to explain the complex symptoms of the disease, it is currently unclear how neurobiological, genetic, societal and environmental factors result in the complex and variable clinical presentation of MDD.

36 The Monoaminergic deficiency hypothesis of depression The most widely accepted hypothesis of the neurobiological basis of depression is the monoamine deficiency hypothesis. This hypothesis states that depressive symptoms occur due to a decrease in the amount of monoaminergic neurotransmitters in brain areas implicated in MDD such as the prefrontal and limbic cortex. 78,138 The hypothesis was first proposed in the 1960s because of the antidepressant actions of two structurally unrelated compounds (tricyclic compounds and MAOIs). Both tricyclic compounds and MAOIs were found to increase overall levels of monoamines in the brain, thereby increasing mood in patients being treated with them. 145 The early MAOI type antidepressants inhibited MAOs, enzymes that break down serotonin, dopamine or norepinephrine in the presynaptic neuron, rendering these neurotransmitters inactive. 78 Due to their action on MAOs, MAOIs increase the available stores of monoamines in the CNS, which is the proposed mechanism for their antidepressant effect and the basis of the monoaminergic hypothesis of MDD. There is extensive clinical and pre-clinical evidence that suggests that serotonin, dopamine and norepinephrine are highly involved in the neurobiology of MDD, and that a sustained deficiency in any one could result in depression. 78,138 The clinical efficacy of SSRIs, SNRIs, and triple reuptake inhibitors, which all increase the availability of monoaminergic neurotransmitters in the synapse, supports the monoaminergic hypothesis of MDD pathophysiology. Although these drugs all produce immediate, substantial increases in serotonin and other monoamines in the brain any antidepressant effect takes a number of weeks to become apparent. 9,134 Thus. it seems probable that the longer-term, antidepressant effects of SSRIs and other antidepressants are due to adaptive responses in the brain secondary to the effects of increasing monoaminergic neurotransmission. 133 Furthermore, most antidepressant therapies

37 27 currently in use are based on increasing monoamine levels, and are only effective in approximately 50% of patients with MDD. 121 It is probable that a more complex pathological mechanism underlies MDD than the monoaminergic deficiency hypothesis. Human dietary studies suggest that depleting tryptophan stores (rate-limiting for synthesis of serotonin in the brain) or depleting TH (required for catecholamine synthesis) does not cause depressive symptoms in healthy subjects, but can cause relapse in patients previously treated for MDD. 146 Furthermore, post-mortem studies on human brain tissue from patients with MDD have not consistently shown decreases in brain monoamine levels. 79,147,148 These findings suggest that monoamines and catecholamine neurotransmitter levels play an important role in MDD, but depleting these neurotransmitters alone may not be sufficient to cause MDD. 78 Thus, the monoaminergic deficiency hypothesis of MDD may be too simplistic to explain the pathological changes and clinical presentation of MDD Role of dopamine in the pathophysiology of MDD The majority of theories regarding the neurobiology of MDD focus on the role of disruptions in serotonin and norepinephrine neurotransmitter systems. Disruptions in brain dopamine levels, as well as changes in dopaminergic neurotransmission have also been identified as factors contributing to in the neurobiology of MDD, and are the target of a number of clinically effective antidepressants. 8 For example, nomifensine and bupropion block the reuptake of norepinephrine and dopamine and are both effective antidepressants when used alone or in conjunction with other antidepressant treatments. 149 The involvement of dopamine in MDD is also supported by clinical studies, as the turnover rate of dopamine, as measured by CSF or plasma levels of dopamine metabolite homovanillic acid, is decreased in patients with MDD compared with controls. 22,150

38 28 Many of the common clinical symptoms of MDD, such as anhedonia, loss of ability to concentrate, flattened affect and motor changes are present in disorders in which dopaminergic signaling is disrupted, such as PD and schizophrenia. 151 Cognitive and behavioral functions modulated by the mesolimibic and mesocortical pathways such as emotional processing, planning, motivation and other executive functions, can be impaired in patients with MDD, implying that dopaminergic signaling in areas such as the cingulate, prefrontal and limbic cortices may be disrupted in MDD. 80,152 Preclinical models of depression have also been used to study the role of dopamine in MDD. In the LH model of depression, dopamine levels are reduced in the caudate nucleus and the NAc of animals with MDD, and depressive-like behavior can be prevented by treatment with a dopamine agonist prior to the behavioral task. 22,153,154 In the FST, a commonly used test for antidepressant efficacy, dopamine agonists and DAT blockers tend to increase mobility, indicating that these substances have antidepressant-like effects. 22,155 Furthermore, dopamine D1 and D2 receptor antagonists can inhibit the effects of antidepressants in the chronic unconditioned stress model of MDD. 156 It is apparent that dopamine is implicated in the pathology of MDD, but given the complexities of dopaminergic signaling and its role in prefrontal and limbic processes, we do not have a complete understanding of these mechanisms. Our laboratory has recently shown that heterodimerization of the dopamine D1 and D2 receptors is up-regulated in patients with MDD, and disrupting this interaction has an antidepressant effect in preclinical models of depression (See Figure 1-3 for a summary of these findings) 3,8 Despite these promising findings, the mechanism by which the D1-D2 receptor interaction is involved in the neurobiology of depression is unclear. Overall, it is clear that dopamine and dopaminergic signaling is disrupted in MDD, and may be involved in the pathogenesis of the disorder,

39 Figure 1-3 Previous findings demonstrating the role of the D1-D2R interaction in MDD. (A) The D1-D2 interaction is significantly increased in post-mortem samples from patients with MDD as assessed by Co-immunoprecipitation of D1 by anti-d2r. (B) A 15-maino acid, membrane permeable peptide capable of disrupting the interaction between D1-D2LR (TAT-D2 -Il ) has an anti-immobility effect in the FST when infused directly into the PFC. (C) Co-imunoprecipitation of D1 by anti-d2r is significantly decreased in the PFC after infusion with the D1-D2 Interfering peptide (TAT-D2- Il ). Figures prepared by Pei et al. (2010) and used by F.Liu in conference presentations. This data was also published, in different figures, in Nature Medicine 16, (2010). 3 29

40 30 although we currently do not have a complete understanding, or a unitary hypothesis, to explain dopamine s role in this complex illness The Neurotrophic Hypothesis of Depression Neurotrophic factors such as Brain Derived Neurotrophic Factor (BDNF) are expressed in the brain and act through transmembrane receptors (tropomyosin receptor kinase B receptors (TrkB)) to promote neuronal survival. 157 The neurotrophic hypothesis of MDD postulates that in MDD, expression of neurotrophic factors such as BDNF is altered in the brain. These alterations in BDNF expression may be caused by chronic stress often associated with MDD 78, and result in decreases in BDNF, which may account for its expression in brain areas involved in MDD pathogenesis. 157,158 This hypothesis is supported by the observed decrease in adult neurogenesis markers in the hippocampus and the PFC of patients with MDD who had committed suicide Antidepressants may cause long-term changes in BDNF expression as post-mortem and serum BDNF levels increased in the hippocampus and cortex after long-term antidepressant use compared with patients not taking antidepressants. 162 Rodent models of MDD have provided contradictory evidence about the role of BDNF and other neurotrophic factors in MDD. Infusing BDNF into the hippocampus and surrounding brain areas has an antidepressant effect in rodent models of depression. 163,164 Diverse antidepressant pharmacological agents increase signaling pathways activated by the BDNF membrane receptor, TrkB, in a BDNF-mediated manner. 165,166 However, male mice who were knockouts for the BDNF membrane receptor, TrkB, did not exhibit endogenous depressive behavior in a number of preclinical tests for depression and anxiety, 167 and hippocampal BDNF infusions in male rats did not prevent learned helplessness behaviors. 168 Interestingly, BDNF infusion directly into the VTA and NAc, two areas highly involved in the mesolimibic and

41 31 mesocortical dopamine pathways, had a pro-depression effect and enhanced social aversion behaviors in mice. 169,170 Since BDNF works to promote neuronal survival (among many other functions) in areas where it is expressed, it is likely that its role in the neurobiology of MDD depends on the brain area where it is elevated, and its underlying functions. As such, considering it as an antidepressant in the classical sense oversimplifies its role in the neurobiology of MDD Preclinical models of MDD Clinical studies investigating the pathological mechanisms in MDD can only provide correlative, and not causative, clues into this complex disorder. New pharmacological treatment options for MDD must be validated using preclinical models of MDD to ensure their efficacy and safety before being tested in the clinical setting. However, accurately modeling a complex, multi-faceted disease like MDD with unknown neurobiological mechanisms in a laboratory preclinical animal model is a challenging task. As such, the development of reliable animal models that model one symptom of MDD (versus attempting to model the spectrum of human MDD symptoms) has been more useful in the laboratory setting. 171 A number of criteria for the validity of animal models of MDD have been proposed. These include strong predictive validity (i.e. all antidepressants that are clinically effective produce a similar antidepressant-like effect in the behavioral model), that the behavioral output of the model is reliable within and between laboratories and that a similarity between the behavioral output in animals and clinical symptoms of depression is present In general, animals must be exposed to some type of stress, either acutely or over a sustained period, in order to produce depressive-like symptoms. 171 In animals, chronic stress can result in helplessness (inability and unwillingness to escape from a stressful situation), anhedonia (lack of

42 32 interest in otherwise pleasurable activities), or social aversion (avoidance of other animals). 79,171 These behaviors resemble specific human symptoms of depression, albeit in a simplified manner. Although a number of well-validated models of depression exist including social aversion tests, and chronic unconditioned stress paradigms, 171,172 only the FST and the LH task will be discussed here, as the D1-D2 interfering peptide used in this study was shown to have an antidepressant effect using these models. The FST is a two-day acute test of antidepressant efficacy developed by Porsolt et al The FST is not considered a chronic model of depression, as it is an acute test used to screen substances that may act as antidepressants. 171 Briefly, the animal is placed in an inescapable plexiglass cylinder for 15 minutes before being given a treatment intervention. The next day, the animal is replaced in the cylinder for a 5 minute period, and its behavior is scored. An animal that didn t receive an antidepressant treatment will display behavioral despair or helplessness and will assume a floating, immobility posture for the majority of the 5-minute test. 176,177 On the other hand, an animal that receives pharmacological agent with antidepressant properties will remain active and try to escape for the majority of the 5-minute test (this is referred to as an antiimmobility effect). 175,176 The FST is a useful test because it has high predictive validity, as all antidepressants currently used in the clinic have an anti-immobility effect in the FST. 176,178 On the other hand, it is not considered an animal model of MDD because it bears little resemblance to the etiology and symptoms of MDD in patients. Another widely used depression model is the LH task, a 5-day test in which the animal is exposed to inescapable shock. Subsequently, its passive response to subsequent shocks (from which the animal can escape) is measured. 179,180 This model is thought to have clinical relevance and etiological validity since evidence exists indicating that stressful life events perceived as uncontrollable (such as death of a loved one and romantic breakups) are major predictors of

43 33 MDD onset and severity This clinical finding can be replicated in rodents by controlling the onset and duration of uncontrollable, aversive events, as is done in the LH paradigm. 183,184 The LH model also has pharmacological validity, as administration of substances that act as antidepressants over the 5-day task results in an increase in the animal s escape attempts compared to untreated animals. 3,180, Thus, the LH model is a useful model of both helplessness in human MDD and has pharmacological validity as a test for novel antidepressant therapies. 1.3 Intranasal delivery to the CNS The nasal anatomy, both in humans and rodents, contains a number of features that make it attractive as a delivery pathway for proteins and peptides targeted to the CNS. 15,188 A number of properties of the vasculature in the CNS limit the entry of molecules, ions, pathogens and toxins into the CSF, and are together called the Blood Brain Barrier (BBB). 189 Tight control of the extracellular environment in the CNS provided by the BBB is required to maintain proper neuronal function and prevent injury within the CNS. At the same time, the BBB makes it difficult to effectively deliver therapeutic substances unable to cross it, such as foreign peptides and proteins, to the CNS and the brain. 189 A number of features of the nasal anatomy make intranasal administration of peptides and proteins an effective way to bypass the BBB and specifically deliver these substances to the CNS. 15,188 A number of protein and peptide therapies have been effectively delivered to the CNS using the intranasal pathway. For example, in recent years, intranasal administration of insulin has been shown to slow memory impairments in rodent models of Alzheimer s disease (AD) A recent meta-analysis suggested an overall beneficial effect of intranasal insulin on cognitive functions in human trials of intranasal insulin delivery in healthy patients, patients with mild

44 34 cognitive impairment (MCI) and those with AD, with few detectable side effects and low systemic levels of insulin. 193 Other protein and peptide therapies that have been successfully delivered to the CNS after intranasal delivery include nerve growth factor (NGF) 194,195 other neurotrophic factors such as BDNF 196, Insulin-like Growth factor 1 (IGF-1) 197,198 and numerous other proteins and drugs (reviewed in Dhuria et al 15 ). Furthermore, a TAT-linked membrane permeable peptide similar in size to the D1-D2 interfering peptide was effectively delivered to the CNS using the intranasal approach. 196 In fact, the authors 196 found that less than 10% of the intravenous dose administered intranasally resulted in equivalent brain concentrations of their 22-amino acid TAT-linked peptide. The success of intranasal insulin delivery to the CNS, along with other proteins and peptides that were delivered successfully to the CNS after intranasal delivery, suggests that a direct pathway exists between the nasal olfactory epithelium and the central nervous system Mechanisms of intranasal delivery to the CNS Both the human and the rodent nasal anatomy have several features that make it conducive to drug transport to the CNS while minimizing systemic exposure to the drug (see Figure 1-5). The olfactory nerve pathways that connect the olfactory sensing region of the nasal cavity to the olfactory bulbs and other CNS areas are important for intranasal drug delivery to the CNS. For example, a fluorescently-labeled 3 kilodalton (kd) Dextran allowed visualization of the olfactory pathway after intranasal administration, and demonstrated that the Dextran was transported to the olfactory bulbs along olfactory nerve pathways in approximately 15 minutes. 199 Olfactory receptor neurons (ORNs) are responsible for conveying information about odors to the CNS. 16 ORNs are bipolar cells whose cell bodies are located within the olfactory

45 35 epithelium, with chemoreceptor-containing dendrites extending into the nasal mucosal layer and axons travelling via the cribiform plate and olfactory nerve bundle into the CNS and olfactory bulb. 200 The cribiform plate of the ethmoid bone contains many small perforations that allow ORN axons to extend into the CNS, effectively bypassing the BBB. 15,200 Extracellular channels between the olfactory ensheathing cells (OECs) (which protect the axonal projections of the ORNs) and the ORN axons allow drugs and peptides administered intranasally to access the CSF and brain directly. 15,188 Intracellular transport mechanisms along the ORN axons may be important for certain substances 201,202 but are not currently thought to be the predominant mode of transportation into the CNS. This is because most intranasal delivery studies, particularly of proteins and peptides, have demonstrated rapid transport from the olfactory epithelium and nasal cavity into the CNS, suggesting that these substances are transported extracellularly via the channels between OECs and ORNs In addition, intracellular transport of intranasally administered substances requires uptake of the substance into the ORNs, necessitating receptor-mediated transport mechanisms, or the ability of the substance to cross the phospholipid bilayer, which cannot account for the large variety of drugs, proteins and peptides that have successfully been delivered to the CNS using the intranasal route. 15 Other potential transport mechanisms from the nasal cavity to the CNS include transport via the trigeminal nerve pathways and via vascular pathways. The trigeminal nerve innervates the respiratory and olfactory epithelium of the nose and enters the CNS in the brainstem. 200 It is possible that substances administered to the nasal cavity are also transported to the CNS via this pathway, as this has been demonstrated for radioactively-labeled IGF-1 and other proteins and peptides. 203,206,207 Secondly, the nasal passages are highly vascularized structures, and intranasally administered substances can be absorbed into the bloodstream through the

46 36 endothelial cells making up the capillary wall. 15 In order for successful delivery to the CNS to occur after absorption into the systemic circulation, substances must cross the BBB. This approach is not thought to mediate the transport of proteins and peptides to the CNS after intranasal delivery, because they lack the ability to cross the BBB in appreciable amounts Experimental considerations for successful intranasal delivery to the CNS For successful delivery of proteins and peptides to the CNS using the olfactory pathway, experimental considerations including intranasal administration technique, head position and drug formulation must be taken into account. The vast majority of preclinical studies involving intranasal administration to rodents have administered substances intranasally in anaesthetized animals. To optimize delivery to the CNS, the substance being administered must reach the olfactory epithelium and upper third of the nasal cavity, and different head positions can alter absorption of substances into the bloodstream and CSF. 208 In anaesthetized rodents, a number of studies have demonstrated that intranasal administration targeted to the CNS can be achieved via the insertion of flexible tubing into the nostrils, localizing delivery to the olfactory epithelium and surrounding tissue The Pressurized Olfactory Device (POD) used in our present study combines delivery to the olfactory epithelium using flexible tubing and aerosolized delivery to deliver substances preferentially to the olfactory epithelium and surrounding tissue, favoring delivery to the CNS. 210, Rationale After the discovery of the antidepressant effects of MAOIs and tricyclic antidepressants nearly 60 years ago 9, there has been extensive investigation into the pathogenesis of MDD in the

47 37 human brain and new pharmacological approaches to treating it. Most effective antidepressant therapies target the monoaminergic (serotonin, norepinephrine and dopamine) neurotransmitter systems. A disadvantage of these therapies is that they broadly increase monoaminergic signaling, resulting in a number of unpleasant side effects in patients. These often appear immediately, a couple of weeks before any appreciable benefit of the antidepressant becomes apparent. 9 As many as 50% of patients do not respond to current antidepressant treatments, 121 necessitating new and more effective therapeutic options for this disorder. New approaches to treating MDD, and the development of new therapies targeting specific pathological changes occurring in MDD, are necessary in order to advance the treatment as well as our scientific understanding of this illness. Peptides specifically designed to disrupt pathological interactions between neurotransmitter receptors are promising therapeutics for psychiatric and neurological disorders because they allow for specific targeting of these pathological interactions. 8 They disrupt the pathological interaction between the two proteins without having an effect on either receptor s independent function, minimizing the likelihood of side-effects. Our laboratory has demonstrated that an interfering peptide specifically designed to disrupt the dopamine D1-D2 receptor heterodimer has a significant antidepressant effect in the FST and the LH task, a preclinical model of depression, when given directly to the PFC of rats. 3 Pei et al 3 also demonstrated that the interaction between dopamine D1 and D2L receptors is significantly increased in post-mortem striatal samples from patients with MDD compared with controls. This finding suggests that the efficacy of the D1-D2 interfering peptide in preclinical models of depression is relevant to the clinical pathogenesis of this disorder, warranting further investigation into its pharmacological and biochemical functions.

48 38 Although the previous findings from our laboratory are promising, they are not yet clinically applicable, as the administration methods (direct microinjections to the brain and intracerebral ventricular (ICV) injections) Pei et al 3 used are extremely invasive and not feasible in the clinical setting. For this interfering peptide, and any other peptide that has a therapeutic effect in animal models of psychiatric or neurological conditions, to translate to the clinical setting a non-invasive, clinically applicable method of administration must be tested and developed. A major challenge in the development of novel therapies for psychiatric or neurological disorders is successfully and non-invasively delivering them to the central nervous system, without substantial accumulation of these therapies in the systemic circulation. 15 In the last 20 years, intranasal delivery of peptide and proteins targeted to the CNS has been extensively studied (for review, see Dhuria et al.) 15 This relatively non-invasive approach exploits the weakened blood-brain barrier at the olfactory epithelium to deliver therapeutic substances such as peptides and proteins to the central nervous system. 192,195,196 Many substances, including TATlinked membrane permeable peptides 212, insulin 190,192, IGF and NGF 195, have been successfully delivered to the CNS using the intranasal route. The POD used to administer the D1-D2 interfering peptide intranasally is designed to preferentially deposit substances on the olfactory epithelium within the nasal cavity, favoring absorption of substances into the CNS. Studies on intranasal delivery to the CNS have also indicated that the intranasal pathway preferentially delivers substances to anterior brain regions such as the olfactory bulbs, PFC and adjacent areas. 15 Since many patients suffering from MDD do not respond to current antidepressant therapies, or cannot tolerate these therapies because of aversive side effects, newer and better therapeutic options must be investigated. For this novel therapeutic approach targeting the D1-

49 39 D2 interaction in MDD to become a relevant treatment, translational studies in rodents must be carried out investigating non-invasive methods to deliver the D1-D2 interfering peptide to the CNS. This project is designed to test whether the D1-D2 interfering peptide can be successfully delivered to the central nervous system, and the PFC in particular, using the intranasal approach and whether it will have an antidepressant effect in the FST after intranasal delivery. We will also investigate the pharmacological properties of the D1-D2 interfering peptide, including the intranasal doses required to observe an antidepressant effect in the FST and the amount of time it remains biologically active in the body. Overall, this project will indicate whether the intranasal pathway is a viable method to deliver peptides like the D1-D2 interfering peptide to the CNS, and better inform whether the D1-D2 interfering peptide is suitable for further development as a novel treatment for depression. 1.5 Hypothesis Based on our laboratory s previous findings 3 that the D1-D2 interfering peptide, when infused directly into the PFC, has an antidepressant effect in the FST and the LH task, 3 we hypothesize that it will have this same effect after intranasal delivery. In order for this hypothesis to be correct, a number of criteria must be met. First, we hypothesize that the POD used in this study preferentially deposits substances on the olfactory epithelium, favoring uptake into the CNS via olfactory nerve pathways, and that after intranasal delivery, we will be able to visualize a FLAG-tagged D1-D2 interfering peptide in the PFC. Second, the efficiency of delivery of peptides and protein therapies to the CNS is largely unknown, but some studies have suggested that between 1 and 5% of the dose delivered

50 40 intranasally reaches the CNS and anterior brain areas. 15 Based on these estimates, we hypothesize that the D1-D2 interfering peptide will be effective in the FST at a dose 100-fold larger than that delivered directly to the PFC. Third, since we currently do not have any information about the efficacy of the D1-D2 interfering peptide after intranasal administration, we will also test whether it is effective at doses higher or lower than our original 100-fold dose. We predict that at doses 100-fold or greater than those given directly to the PFC in the previous study, the D1-D2 interfering peptide will have an antidepressant effect in the FST. Along these same lines, we will investigate the length of time the D1-D2 interfering peptide remains biologically active in the body, as we are unsure about how long it remains stable in the CNS once it is administered intranasally. Finally, the D1-D2 interfering peptide displayed an antidepressant effect when it was delivered directly to the PFC, but not to other brain areas such as the NAc and hippocampus 3. We hypothesize that if the D1-D2 interfering peptide has an antidepressant effect after intranasal administration, it will be due, at least in part, to the ability of the D1-D2 interfering peptide to disrupt the D1-D2 receptor-receptor interaction in the PFC. Taken together, these experiments will further our understanding of the intranasal delivery route for small interfering peptides as well as reveal whether the D1-D2 interfering peptide is a promising novel therapeutic for the treatment of MDD.

51 41 2 Materials and Methods 2.1 Animals Adult Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were used in all experiments. Rats were pair-housed at a constant temperature (20-23 C) on a 12-hour light/dark cycle (light on at 8:00AM) with unrestricted food and water. After arriving at the facility, rats were given 1 week to acclimatize before being subjected to behavioral testing and injections. All rats weighed between 300 and 350g when they underwent behavioral testing. All experimental procedures were approved by the Animal Care Committee at the Centre for Addiction and Mental Health (Toronto, ON). 2.2 Intranasal administration procedures Intranasal administration using the POD All animals were anaesthetized using 5% isoflurane, an inhalant anesthetic (Benson Medical Industries, Inc.) for 3-4 minutes. Rats were then placed in a supine position and dosed with the POD developed by Impel NeuroPharma (Seattle, Washington). When dosing the animals, the POD tip (with the relevant dose) was inserted approximately 8-10mm into the rat s nostril, angled towards the olfactory epithelium (towards the top of the head) and the propellant can was fired for 1 second. 2 seconds later, the POD tip was slowly removed. The propellant can and the POD tip were attached by a 30cm-long piece of plastic tubing (see Figure 2-1). This allowed for maximum maneuverability of the POD tip in the nose. This protocol was adapted from the POD administration procedure originally developed by Impel NeuroPharma. 213

52 42 After intranasal administration animals were replaced in the anesthetic chamber in the supine position for 4 minutes at decreasing isoflurane concentrations. This step allows for maximum absorption of the peptide dose, while decreasing sneezing and other behaviors that would result in expulsion of the substance from the nose. Animals were then replaced in their home cages and returned to their housing rooms once they had recovered from the anesthesia and had regained complete locomotor control. Rats that bled after intranasal administration were eliminated from the study. The time of injection was recorded as the time at which the animals woke up from the anesthesia and regained locomotor control Verification of POD delivery to the olfactory epithelium In order to verify that the POD deposits substances preferentially onto the olfactory epithelium, we gave intranasal injections (IN) of Richard Allen Scientific Mark-It Blue Dye (5000BL, Thermo Scientific). The Mark-It blue dye allowed us to visualize where within the rat nose the POD was preferentially depositing substances. Using the POD, we gave 10uL per nostril using the administration method outlined in Section 2.2a). After POD administration, animals were immediately sacrificed using trans-cardiac perfusion of 1X Phosphate Buffered saline (ph=7.4). The nasal anatomy was then dissected and examined for traces of Mark-IT Blue Dye. After dissection, the nasal anatomy was photographed for qualitative evaluation of the deposition of substances by the POD Substances injected intranasally In each experiment, animals received the same total number of intranasal injections (IN) (3-4) in alternating nostrils over a 24-hour period. Animals received either filtered saline (0.9% 9- NaCl, 8-12 L/injection), the D1-D2 interfering peptide (TAT-D1-D2-IPep) (8-12µL, 50mM);

53 Figure 2-1 Pressurized Olfactory Device (POD) for intranasal administration: apparatus (A) POD apparatus: the Propellant can is attached to the administration tip with a 30cm long piece of plastic tubing. This allows for maneuverability when inserting the administration tip into the nose and administering the intranasal dose. (B) Optimal position of the administration tip within the nose for preferential dose deposition onto the olfactory epithelium. 43

54 44 a amino acid membrane permeable TAT peptide (TAT-Pep) (8-12 µl, 50mM) from the Human Immunodeficiency Virus 1(HIV1) TAT protein 214 ; or D1-D2-FLAG tagged interfering peptide (TAT-D1-D2-FLAG-IPep) (8-12µL, 50mM). The 9 amino acid TAT-peptide sequence from the HIV1 TAT protein (YGRKKQRRR) 214 rendered all peptides used in these studies cellpermeable. All peptides were custom synthesized by Gen Script (New Jersey, USA) and/or Biomatik, Inc (Cambridge, Ontario) and had purity levels between 95 and 99%. All peptides were dissolved in filtered saline at a concentration of 50mM and stored at -80 C. 2.3 Intra-peritoneal injection procedures We administered imipramine hydrochloride (15mg/mL, Sigma-Aldrich) at a dose of 15mg/kg using intra-peritoneal (IP) injections into the abdominal cavity. To control for anesthetic exposure, these animals were also anaesthetized before administration of IP injections. Rats were anaesthetized in an induction chamber using 5% isoflurane. Rats were placed in a supine position and given imipramine via IP injections. After the injection, animals were replaced in the induction chamber at decreasing isoflurane concentrations for 4 minutes. Animals were allowed to recover in their home cages and the time of injection was recorded as the time when the rats regained locomotor control. 2.4 Immunofluorescence and confocal microscopy Tissue fixation and storage The purpose of this study was to test whether a biologically active peptide that has an anti-immobility effect in the FST, can be visualized in the PFC and after intranasal administration. To do this, we used a peptide with the same sequence as the D1-D2 interfering

55 45 peptide 3, but with an 8-amino acid FLAG-tag (Sequence: DYKDDDDK) 215 fused to the C- terminal of the peptide. This modification allowed us to use immunofluorescent (see section 2.4.1) methods to detect it in the brain after intranasal administration and completion of the FST. 216 For this experiment, a small number of animals were sacrificed by transcardiac perfusion. After being anaesthetized for 4 minutes in an induction chamber with 5% isoflurane, animals were perfused transcardially with 60mL phosphate buffered saline (PBS, ph =7.4) followed by 60mL 4% Paraformaldehyde ( 4% PFA, in PBS). Subsequently, whole brains were dissected and stored in 4% PFA overnight. The next day, brains were transferred to a 20% sucrose cryoprotection solution for approximately 48 hours. The tissue was then stored at -80 C for subsequent use Immunofluorescent staining procedures Rats that had completed the FST and were assigned to the TAT-D1-D2-FLAG-IPep or saline treatment groups were sacrificed, their tissue fixed and collected as described in Section The olfactory bulbs and PFC of 2 brains from each condition (TAT-D1-D2-FLAG-IPep and saline) were cut into 12 M sections using a cryostat. After slicing, we blocked non-specific antibody interactions using 5% donkey serum for 1 hour, before staining overnight with an anti- FLAG monoclonal antibody (mouse monoclonal, M2, Sigma-Aldrich). Sections were then incubated with a secondary immunofluorescent antibody (donkey anti-mouse Cy2-conjugated antibody, Jackson Immuno Research Laboratories, Inc.) before being counter-stained with NeuroTrace 530/615 red fluorescent Nissl Stain (Molecular Probes, Invitrogen). Sections were mounted on slices with PureGold anti-fade mounting reagent (Molecular Probes, Invitrogen.) and stored at 4 C.

56 46 Sections were visualized and imaged using a Zeiss LSM 510 confocal microscope. Images of PFC coronal sections in both conditions were taken under 25X magnification. Cy2 immunofluorescence was imaged using an argon laser with maximum excitation at 488nm. To detect the NeuroTrace 530/615 stain, we used a helium 1 laser with maximum excitation at 530 nm. Images were overlaid using Image J software. 2.5 The Forced Swimming Test FST Procedure The FST is an acute test for antidepressant efficacy originally developed by Porsolt et al. in On the first day of the test, animals undergo a training session where they are placed in an inescapable plexiglass cylinder for 15 minutes. The plexiglass cylinders were 60cm high and 20cm in diameter. The plexiglass cylinders were filled to a height of 40 cm with water at a temperature of 25 +/- 0.2 C, which was changed between each animal (See Figure 2-3 for a picture of the FST cylinder). In accordance with the dosing schedule established in the literature and used previously 3, subjects were given the treatment intervention three times after the training and testing sessions: 30 minutes after the 15-minute training session, 5 hours after training and 1 hour before undergoing the FST. 24 hours after the training session and 1 hour after the last behavioral intervention, the rat was replaced in the same cylinder for 5 minutes. The session was video recorded and scored blindly at a later date. After both the training and test FST sessions, rats were towel-dried and placed in a heated cage for a minimum of 15 minutes. See Figure 2-2 for a detailed schematic of the FST experimental design.

57 Figure 2-2 Overall experimental procedure for FST. See section for detailed description of the FST experimental procedure 47

58 FST behavioral scoring method The 5-minute session of the FST was video recorded and animal behavior during the FST was scored at a later date after the experimenter was blinded to the treatment groups. All FST videos were scored by the same experimenter who had been blinded to the experimental conditions of the animals. Behaviors were scored in five-second bins with the predominant behavior (immobility, swimming, climbing, diving) in each 5-second period recorded for a total of 60 behavioral counts. The animal s activity during the FST was segregated into four behaviors, in keeping with the literature and previous studies 3,174,176 : immobility, which consisted of the animal only making those movements necessary to keep its nose above the water; swimming, consisting of active movement of the forepaws and legs and movement around the cylinder; climbing, consisting of vigorous movement of the forepaws along the sides of the cylinder, as if trying to climb out of it; and diving, when the animal entered head-first into the water and spent a minimum of 2 seconds completely submerged. Figure 2-3A-D displays representative photographs of each of these behaviors. Mean immobility counts across all groups were analyzed by 1-way independent groups analysis of variance (ANOVA). Post-hoc Newman-Keuls multiple comparisons tests were used to evaluate differences across individual groups, as necessary. In some cases, other behaviors (swimming, diving and climbing) were also analyzed using 1-way independent groups ANOVAs, followed by post-hoc Newman-Keuls multiple comparisons tests to evaluate differences between treatment groups. Data were analyzed using Prism (GraphPad Software, Inc.).

59 Figure 2-3 Representative photographs of behaviors exhibited during the FST. Top L to Bottom R: immobility, or passive floating with nose out of the water, swimming, actively moving forepaws and legs to remain afloat, climbing, vigorous movement of forepaws and legs in tandem, as if to climb up the walls of the FST cylinder, and diving, head-first submersion of the entire body in the FST cylinder. 49

60 FST experiments: experimental design Effect of the D1-D2 interfering peptide in the FST We tested whether the D1-D2 interfering peptide would have an anti-immobility effect in the FST when administered at a dose 100-fold that which was administered directly to the PFC 3 (for detailed calculations of the D1-D2 interfering peptide intranasal dose, see Appendix 1.) Animals were randomly assigned into four treatment groups: TAT-D1-D2-IPep (IN, 1.67nmol/g, n=7), TAT-Pep (IN, 1.67nmol/g, n=6), saline (IN, 1.67nmol/g, n=6), and imipramine (IP, 15mg/kg, n=6). This group was included in order to confirm that the 9-amino acid, membrane permeable TAT peptide fragment did not have any behavioral effect in the FST. We followed the 2-day FST protocol and behavioral analysis procedure outlined in Section 2.5. These treatments were given 30 minutes after the 15-minute FST training session, 5 hours later and 1 hour before the 5-minute FST test. The 5-minute FST behavioral tests were video-recorded, scored and analyzed as described in Section Effect of the D1-D2-FLAG interfering peptide in the FST The purpose of this study was to test whether a biologically active peptide that can be visualized in the PFC areas after intranasal administration also has an anti-immobility effect in the FST. To do this, we synthesized a peptide with the same sequence as the D1-D2 interfering peptide 3, but with an 8-amino acid FLAG-tag (Sequence: DYKDDDDK) 215 fused to the C- terminal of the peptide, tested whether it had an anti-immobility effect in the FST and whether we could visualize it in the PFC. To test whether the D1-D2-FLAG interfering peptide had an anti-immobility effect in the FST (for detailed procedure, see Section 2.5), animals were randomly assigned into four treatment groups: TAT-D1-D2-IPep-FLAG (IN, 1.67nmol/g, n=4), TAT-Pep (IN, 1.67nmol/g,

61 51 n=4), saline (IN, 1.67nmol/g, n=5), and imipramine (IP, 15mg/kg, n=4). We followed the 2-day FST protocol and behavioral analysis procedure outlined in Section 2.5. The 5-minute FST behavioral tests were video-recorded, scored and analyzed as outlined in Section Efficacy of the D1-D2 interfering peptide at various intranasal doses In order to better understand the pharmacological effects of the D1-D2 interfering peptide upon intranasal administration, information regarding its efficacy at various doses is required. To address this, we varied the dose of the D1-D2 interfering peptide and analyzed their antiimmobility effects in the FST. We used saline and imipramine groups as a negative and positive control, respectively. We followed the general FST design and behavioral analysis procedure outlined in Section 2.5. Table 2-1 details the treatment groups, doses and the overall experimental design employed in this study. Immobility counts during the 5-minute FST in each treatment dose was compared with saline and imipramine groups using 1-way independent groups ANOVAs, followed by post-hoc Newman-Keuls multiple comparisons tests. From our results, we identified the minimum dose of the D1-D2 interfering peptide with the maximal behavioral effect as 1.67nmol/g, which we used in all subsequent experiments. In order to control for any behavioral effect of the TAT-peptide, we included two treatment groups that received TAT-pep at a dose of 1.67 nmol/g and 2.0nmol/g in order to test whether the TAT-peptide s effect in the FST changes if the dose is increased.

62 52 Treatment saline Volume- Controlled Doses, nmol/g (mg/kg), number of animals per group n=4 n=8 (same group as 1.0nmol/g) n=6 n=8 (same group as 2.0nmol/g) Imipramine 15mg/kg, IP n=3 n=8 (same group as 1.0nmol/g) n=6 n=8 ( same group as 2.0nmol/g) TAT-Pep n=7 2.0 nmol/g TAT-D1-D2- IPep 4.0nmol/g (13.72mg /kg) n=3 2.0nmol/g (6.86mg/ kg) n=7 n=6 1.67nmol /g 1.67nmol/g (5.75mg/ kg) n=7 1 nmol/g (3.45mg/k g) n=7 Table 2-1 Efficacy of the D1-D2 interfering peptide at various doses: overall experimental design and Treatment Groups.

63 Duration of behavioral effect of D1-D2 interfering peptide in the FST It is unclear how long the D1-D2 interfering peptide remains active in the CNS after intranasal administration. To test this, we compared the anti-immobility effect of the D1-D2 interfering peptide in the FST at 2, 3 and 4 hours after intranasal administration. We used the general FST protocol and analysis procedure outlined in Section 2.5. The overall experimental design and treatment groups are shown in Table 2-2. Each animal in this study received intranasal or IP injections 30 min and 5 hrs after the FST training session while the time point of the last intranasal injection was increased to 2, 3 and 4 hours before the 5-minute FST session. The amount of immobility behavior in treatment groups was compared using 1-way independent groups ANOVA at each time point (2h, 3h and 4h). Saline and imipramine groups were compared to at all time points (2h, 3h, and 4h). 2.7 Locomotor activity test We tested whether the increased mobility we observed in the FST after intranasal administration was due to the antidepressant effect of the D1-D2 interfering peptide or due to an overall increase in locomotor activity. To do this, we tested whether the D1-D2 interfering peptide had an effect on activity during a 30-minute locomotor activity test after intranasal administration. 25 rats that had already been exposed to the FST were used in this experiment. Rats were given TAT-D1-D2-IPep (IN, 2.0nmol/g, n=5); TAT-D1-D2-IPep (IN, 1.67nmol/g, n=5); saline (IN, n=5); TAT-Pep (IN 2.0nmol/g, n=5); or 15mg/kg imipramine (IP, n=5) three times before the open field test: 24 hours before the test, 19 hours before the test, and one hour before testing. This dosing schedule was identical to that used in the forced swimming

64 54 Treatment Saline Imipramine Time point before FST, number of animals/group 1 hour before FST Volume-Controlled (10ul/dose) n=6 1 hour before FST 15mg/kg, IP n=6 TAT-Pep (1.67nmol/g) 1 hour before FST n=7 2 hours before FST n=6 3 hours before FST n=6 4 hours before FST n=6 TAT-D1-D2- IPep (1.67nmol/g) 1 hour before FST n=6 2 hours before FST n=6 3 hours before FST n=6 4 hours before FST n=6 Table 2-2 Duration of the anti-immobility effect of the D1-D2 interfering peptide: treatment groups and overall experimental design

65 55 experiments (see Section 2.5.1). Animals were kept in their original treatment groups, for example if an animal had been assigned to the saline treatment group during the FST, it remained in the saline group during the locomotor experiments. To record locomotor activity, rats were placed in a custom-made locomotor activity recording apparatus. Each animal was placed in a 20cm high, 20 cm wide and 30 cm long cage (standard housing cage) in the locomotor apparatus for 30 minutes in a dark room. Rats had not previously been exposed to the testing room or to the locomotor activity boxes. An array of 11 infrared photocells was placed along the long axis of the cages. Interruption of infrared beams (Beam Breaks) was used as a measure of locomotor activity. The total amount of locomotor activity was recorded in 5 minute intervals and for the entire 30 minute session. Total activity data were analyzed via 1-way independent groups ANOVA according to the locomotor measure followed by Newman-Keuls multiple comparisons tests, using Prism Software (GraphPad Software, Inc.). The locomotor activity at various intervals during the test was analyzed by 2- way independent factors ANOVA with treatment group (saline, imipramine, TAT-Pep and TAT- D1-D2-IPep) and Time Point (5min, 10min, 15min, 20min, 25min, 30min) as main factors. 2.9 Co-immunoprecipitation and western blots Tissue Collection Animals were sacrificed the day they completed the FST or, if applicable, the open field test. Animals were anaesthetized for 3 minutes with 5% isoflurane and were decapitated. Their brains were quickly removed and relevant tissue areas were dissected on ice. These areas included the olfactory bulbs, PFC, striatum, hippocampus and VTA. Brain tissue was stored at - 80 C for subsequent use in biochemical assays.

66 Co-Immunoprecipitation of D1 receptor by anti-d2dr Since the peptide we are testing in the FST is designed to disrupt the interaction between the D1 and D2 receptors, we investigated the co-immunoprecipitation of the D1 receptor by an antibody against the D2 receptor in the PFC of animals who had received intranasal injections of the D1-D2 interfering peptide or saline. We compared animals who had been given intranasal injections of TAT-D1-D2-IPep (IN, 1.67nmol/g, n=3), or saline (IN, n=3) and had been exposed to the FST and the open field test. For the co-immunoprecipitation, solubilized proteins from the PFC and striatum (500 µg) from each animal were incubated with 1ug goat polyclonal anti-d2dr (N-19, Santa Cruz Biotechnology) and protein A/G PLUS agarose beads (Santa Cruz Biotechnologies) overnight. A control sample was incubated with polyclonal Goat IgG (Sigma-Aldrich) to confirm the absence of non-specific immunoprecipitation. After incubation, the immunoprecipitated proteins were washed and incubated with SDS sample buffer (BioRad, Inc.) at 37 C for 40 minutes before being separated from the Protein A/G PLUS-agarose beads using centrifugation. Immunoprecipitated proteins were then subjected to separation using 10% SDS-Page gels, transferred onto nitrocellulose membranes and immunoblotted overnight using anti-d1dr (D187, Sigma-Aldrich). Each immunoblot included samples from saline and TAT-D1-D2-IPep treatment groups, along with a control sample incubated with goat IgG (Sigma-Aldrich) and 75µg of tissue-extracted input protein from PFC tissue. After overnight incubation, secondary antibodies conjugated with horseradish peroxidase were applied to the blots for approximately two hours. After washing, immunoblots were developed with ECL reagent (GE Healthcare, Inc.) and imaged using a BioRad ChemiDoc MP system (BioRad Technologies, Inc.). To quantify the expression of protein, we conducted

67 57 densitometry analyses using ImageLab software (BioRad Technologies, Inc.). Densitometry data were analyzed using two-tailed, unpaired Student s t-tests (Prism Software, GraphPad, Inc) Western Blots We compared the expression of the D1 and D2 receptors using western blots in animals given intranasal injections of TAT-D1-D2-IPep (IN, 1.67nmol/g, n=3) or saline (IN, n=3). All groups were given intranasal injections and exposed to the FST and open field tests. 80 µg of solubilized protein extracts from the PFC, in SDS sample buffer (BioRad Technologies, Inc.) were denatured by boiling for 5 minutes before being separated by 10% SDS page gel. After transfer onto nitrocellulose membranes, we immunoblotted for D1 and D2 receptors with monoclonal anti-d1dr (rat IgG, D187, Sigma-Aldrich) and monoclonal anti-d2dr (Mouse IgG, B-10, Santa Cruz Biotechnologies). In order to confirm that the total amount of protein in each sample were equal, we separated 20µg solubilized protein extracts with 10% SDS page gel and immunoblotted using monoclonal anti-α-tubulin (Mouse IgG, DM1A clone, Sigma- Aldrich). After overnight incubation, immunoblots were incubated for 2 hours with species specific Horseradish Peroxidase conjugated secondary antibodies. After washing, immunoblots were developed with ECL reagent (GE HealthCare, Ltd.) and visualized using a BioRad ChemiDoc MP system (BioRad Technologies, Inc). To quantify the expression of protein, we conducted densitometry analyses using ImageLab software (BioRad Technologies, Inc). Densitometry data were analyzed with two-tailed, unpaired Student s T-tests using Prism Software (GraphPad Software, Inc.).

68 58 3 Results 3.1 Experiment 1: The POD preferentially deposits substances on the olfactory epithelium within the rat nasal cavity In order to verify that the POD deposits substances preferentially onto the olfactory epithelium, we administered intranasal injections of Richard Allen Scientific Mark-It Blue Dye using the POD. The Mark-It dye allowed us to visualize where the POD was preferentially depositing substances within the rat nasal cavity. It also allowed us to optimize our POD delivery protocol (see Section 2.2.1) in order to maximize deposition of substances on the olfactory epithelium and surrounding tissue. We assessed the deposition of the Mark-It dye after delivery using the POD by grossly dissecting the nasal anatomy. Figure 3-1A and B show representative images of Mark-It dye deposition after POD delivery on the olfactory epithelium within the rats nasal cavity (A) and visible through the cribiform plate (B), the porous bone through which the olfactory axons travel to the olfactory bulb and CNS. 15 As the images show, the POD preferentially deposited the Mark-It Dye on the olfactory epithelium, favoring transport mechanisms that deliver substances to the CNS. 3.2 Experiment 2: The D1-D2-FLAG interfering peptide can be detected in the prefrontal cortex after intranasal administration After confirming that the POD preferentially deposits substances on the olfactory epithelium and olfactory sensory areas of the rat s nasal anatomy, we next assessed whether a TAT-linked, membrane-permeable peptide would reach the CNS, and the PFC in particular, after

69 Figure 3-1 Representative images of deposition of Mark-It Blue tissue marker deposition after correct POD administration (A) Deposition of Blue Mark-It dye on olfactory epithelium after intranasal administration using the POD following the protocol outlined in Section Animal s nasal anatomy was dissected through the midline in order to visualize both the left and right nasal cavities. (B) Blue Mark-It Dye is visible through the cribiform plate (bone separating the nasal olfactory epithelium from the CNS, CSF and olfactory bulbs. The cribiform plate contains perforations through which the olfactory receptor neuron (ORN) axons travel to the olfactory bulb and CNS. Representative images shown. 59

70 60 administration with the POD. We focused on detecting the peptide in the PFC because Pei et al s study 3 showed that the D1-D2 interfering peptide only had an antidepressant effect in the FST when it was administered directly to this brain area. 3 After staining selected sections of the PFC from animals that received TAT-D1-D2-FLAG-IPep (1.67nmol/g, n=2) and saline (n=2) with anti-flag antibodies, followed by a Cy2-conjugated fluorescent secondary antibody, we visualized the resulting immunofluorescence using a confocal microscope. Figure 3-2A-B shows representative images of prefrontal cortical slices from individual animals who received intranasal injections of TAT-D1-D2-IPep (A) or saline (B). We detected the presence of a TAT-D1-D2-IPep peptide in the PFC, as the Cy-2- conjugated immunofluorescent signal was visible in slices from animals that had received TAT- D1-D2-FLAG-IPep intranasal injections and not visible in the PFC of animals that received saline injections. Immunofluorescence was visible through the anterior PFC coronal sections, with no extreme variations in staining in dorsal, ventral medial or lateral areas. The results of this experiment suggest that intranasal administration is a viable method to deliver membranepermeable peptides to the PFC. 3.3 Experiment 3: Intranasal administration of the D1-D2 interfering peptide has an antidepressant effect in the forced swimming test Although the experiment outlined in Section 3.2 allowed us confirm the presence of the D1-D2-FLAG interfering peptide in the PFC after intranasal administration, it did not indicate whether the accumulation of the peptide in the PFC was sufficient to produce a behavioral antidepressant effect in the FST. Thus, it remained unclear whether intranasal administration resulted in sufficient accumulation of the D1-D2 interfering peptide in the PFC to produce an observable behavioral effect in the FST.

71 Figure 3-2 Immunofluorescent staining for anti-flag antibodies is visible in PFC slices of animals who were administered TAT-D1-D2-FLAG-IPep (A) but not those who were administered saline (B). A) 1,2: Representative images of PFC sections of two separate animals who received TAT-D1-D2-FLAG- Ipep (1.67nmol/g) intranasally. B) 1,2 Representative images of PFC sections from two separate animals who received intranasal injections of Saline. Representative fluorescent images taken with a Zeiss LM confocal microscope (25X magnification ) of anti-flag (Cy2 secondary antibodies) and NeuroTrace 530/615 neuronal cell body stain. Representative images shown. 61

72 The D1-D2 Interfering Peptide has an Anti-Immobility Effect in the FST We compared immobility, swimming and diving behaviors during the 5-minute FST across four treatment groups: animals that received saline (IN, n=6, Volume-controlled) imipramine (IP, n=6, 15mg/kg), TAT-D1-D2-IPep (IN, n=7, 1.67nmol/g), or TAT-Pep (IN, n=6, 1.67nmol/g). The D1-D2 interfering peptide had a significant anti-immobility effect in the FST that was comparable to that of imipramine and significantly greater than that of saline or TAT-Pep. A 1- way Independent Groups ANOVA revealed a significant difference in immobility behavior between all groups: TAT-D1-D2-IPep, saline, imipramine and TAT-Pep (n=6-7 per group, F(3,21)=12.25, p<0.0001). Post-Hoc Newman-Keuls multiple comparisons tests showed significant differences when comparing TAT-D1-D2-IPep and saline (p<0.01), TAT-D1-D2- IPep and TAT-Pep (p<0.01), imipramine and saline (p<0.001) and imipramine and TAT-Pep (p<0.001). No significant differences were present between imipramine and TAT-D1-D2-IPep groups (p>0.05) and saline and TAT-Pep groups (p>0.05). For the swimming behavior, a 1-way Independent Groups ANOVA revealed a significant difference between all treatment groups, (F(3,21)=12.57 p<0.0001). Post-hoc Newman Keuls multiple comparisons tests revealed significant differences between TAT-D1-D2-IPep and saline (p<0.01), TAT-D1-D2-IPep and TAT-Pep (p<0.01), imipramine and saline (p<0.001) and imipramine and TAT-Pep (p<0.001). No significant differences were present between imipramine and TAT-D1-D2-IPep (p>0.05) and saline and TAT-Pep (p>0.05) treatment groups. There was no significant difference in diving behavior between saline, imipramine, TAT-D1-D2- IPep and TAT-Pep groups (F(3,21)=0.99, p>0.05). Data is represented graphically in Figure 3-3.

73 Figure 3-3 The D1-D2 interfering peptide has an antidepressant effect in the FST when administered intranasally. After intranasal administration using the POD, the D1-D2 interfering peptide (dose = 1.67nmol/g) significantly decreases immobility and increases swimming behavior in the FST. Data for each standard behavior (immobility, swimming, diving) analyzed via 1-way independent groups ANOVA followed by Newman Keuls post-hoc multiple comparisons tests. ** p<0.01, *** p<0.01 compared to saline; ^^ p<0.01, ^^^ p<0.001 compared to TAT-Pep. No significant difference was observed between behavior in animals who received TAT-D1-D2-Ipep or imipramine. Error bars represent SEM. 63

74 The D1-D2-FLAG interfering peptide has an anti-immobility effect in the FST after intranasal administration We tested whether the D1-D2 FLAG peptide produced an anti-immobility effect in the FST, and whether we could detect it in the PFC of animals who had been exposed to the FST(see Section 3.2). We hypothesized that, at a sufficient dose, the TAT-linked D1-D2 interfering peptide with a FLAG tag on the C-terminal end (TAT-D1-D2-FLAG-IPep) would have an antiimmobility effect in the FST comparable to that of imipramine and the D1-D2 interfering peptide. The D1-D2-FLAG interfering peptide had a significant anti-immobility effect in the FST that was comparable to imipramine and significantly greater than that of saline and TAT- Pep. A 1-way independent groups ANOVA of immobility behavior across treatment groups demonstrated a significant difference between all groups (saline, imipramine, TAT-D1-D2-IPep and TAT-Pep, n=4-5 per group, F(3,14)=7.746, p<0.01). Post-hoc Newman-Keuls multiple comparisons tests revealed significant differences between imipramine and saline groups (n=4-5, p<0.01), imipramine and TAT-Pep groups (n=4-5, p<0.05), TAT-D1-D2-FLAG-IPep and saline groups (n=4-5, p<0.01) and TAT-D1-D2-FLAG-IPep and TAT-Pep groups (n=4-5, p<0.05). There was no significant differences between TAT-Pep and saline groups (p>0.05) or imipramine and TAT-D1-D2-IPep groups (p>0.05). Data is represented graphically in Figure 3-4. To investigate whether the behavior during the 5-minute FST was significantly different in animals that received the D1-D2-FLAG peptide and those that received the D1-D2 peptide, we compared immobility, swimming and diving behavior in these two groups. The D1-D2 interfering peptide and the D1-D2-FLAG tagged interfering peptide had indistinguishable

75 Figure 3-4 The D1-D2-FLAG interfering peptide has an anti-immobility effect in the FST. The D1-D2-FLAG interfering peptide significantly decreases immobility behaviors in the FST after intranasal administration. TAT-D1-D2-FLAG-IPep and TAT-Pep administered intranasally at a dose of 1.67nmol/g. Data analyzed via 1-way independent groups ANOVA followed by Newman Keuls Post-Hoc Multiple Comparisons Tests. ** p<0.01, *** p<0.01 compared to saline; ^^ p<0.01, ^^^ p<0.001 compared to TAT-Pep. No significant difference was observed between behavior in animals who received saline or imipramine. Error bars represent SEM. Numbers within bars represent number of animals per group. 65

76 Figure 3-5 The D1-D2 interfering peptide and D1-D2-FLAG tagged interfering peptide have similar behavioral effects in the FST. We compared immobility, swimming and diving behavior in treatment groups that that received TAT-D1-D2- IPep (see Section 3.3.1) and TAT-D1-D2-FLAG-IP (see Section 3.3.2). Two-tailed, unpaired student s t-tests revealed no significant differences in immobility (p=0.83), swimming (p=0.73) or diving (p=0.18) behaviors between these groups, indicating that the D1-D2 interfering peptide and the D1-D2-FLAG interfering peptide s effect in the FST are indistinguishable. Error bars represent SEM. 66

77 67 behavioral effects during the 5-minute FST. Two-tailed, unpaired Student s T-tests revealed no significant differences between immobility, swimming or diving behavior in animals who received TAT-D1-D2-IPep or TAT-D1-D2-FLAG-IPep (n=4-7 per group, Immobility, t(9)=0.21, p=0.83; Swimming, t(9)=0.32, p=0.74 ; Diving t(9)=1.447, p=0.18).(figure 3-5). 3.4 Experiment 4: Efficacy of the D1-D2 interfering peptide at various intranasal doses The D1-D2 interfering peptide has an antidepressant effect in the FST when given intranasally at a dose of 1.67nmol/g (5.75mg/kg) (Section , Figure 3-3.). The 1.67nmol/g dose administered intranasally represents an estimated 100-fold increase over the dose given directly to the PFC (5nmol/injection). 3 Although this particular intranasal dose can produce an antidepressant effect in the FST, the behavioral effects of the D1-D2 interfering peptide at doses higher or lower than 1.67nmol/g (5.75 mg/kg) remains unknown. To investigate the antidepressant effects of the D1-D2 interfering peptide at various intranasal doses, we varied its dose, exposed animals to the 2-day FST, and analyzed the resulting immobility behavior in the treatment groups (see Section and Table 2-1 for detailed experimental design). We hypothesized that at intranasal doses larger than 1.67nmol/g, the D1-D2 interfering peptide would have a significant anti-immobility effect in the FST D1-D2 interfering peptide dose: 4.0nmol/g (13.72 mg/kg) At a dose of 4.0nmol/g, the D1-D2 interfering peptide had an anti-immobility effect in the FST comparable to that of imipramine and significantly greater than that of saline. We compared immobility behavior during the 5-minute FST in rats that received saline (IN, n=3), imipramine (IP, 15mg/kg, n=3) and TAT-D1-D2-IPep (IN, n=3, 4.0 nmol/g). A 1-way

78 68 independent groups ANOVA revealed a significant difference between all groups (F(2,6) = 9.207, p<0.01). Post-hoc Newman-Keuls multiple comparisons tests showed significant differences between TAT-D1-D2-IPep and saline (p<0.05) and saline and imipramine (p<0.05). No significant difference was present between TAT-D1-D2-IPep and imipramine groups (Figure 3-6, 3-10) D1-D2 interfering peptide dose: 2.0nmol/g (6.86 mg/kg) At an intranasal dose of 2.0nmol/g, the D1-D2 interfering peptide had an anti-immobility effect in the FST comparable to that of imipramine and significantly greater than that of saline. We compared immobility behavior during the 5-minute FST in rats that received saline (IN, n=8), imipramine (IP, 15 mg/kg, n=8), TAT-D1-D2-IPep (IN, 2nmol/g, n=8), and TAT-Pep (IN, 2nmol/g, n=7). A 1-way independent groups ANOVA revealed a significant difference between immobility behavior in all groups ( F(3,27)=6.836, p<0.01). Post-hoc Newman-Keuls Multiple comparisons tests revealed significant differences between TAT-D1-D2-IPep and saline (p<0.01), imipramine and saline (p<0.01) and imipramine and TAT-Pep (p <0.05). There were no significant differences between saline and TAT-Pep (p>0.05), imipramine and TAT-D1-D2- IPep (P>0.05) and TAT-Pep and TAT-D1-D2-IPep (p>0.05). Immobility behavioral counts are represented graphically in Figure 3-7 and summarized in Figure D1-D2 interfering peptide dose: 1.67nmol/g (5.75 mg/kg) For the purposes of comparison, data relating to immobility behavior in rats that received our original intranasal dose of 1.67nmol/g (5.57mg/kg) is included here (for complete analysis, see Section ). Briefly, a 1-way independent groups ANOVA revealed a significant

79 Figure 3-6 The D1-D2 interfering peptide has an anti-immobility effect in the FST at an intranasal dose of 4.0nmol/g Immobility behavioral of animals who received intranasal injections of TAT-D1-D2-IPep (dose = 4nmol/g) was compared to those who received saline or IP injections of imipramine. Immobility behavior between groups compared by 1-way independent groups ANOVA followed by post-hoc Newman Keuls multiple comparisons tests. * p<0.05 compared to saline. Error bars represent SEM. Numbers within bars represent number of animals per group. 69

80 Figure 3-7 The D1-D2 interfering peptide has an anti-immobility effect in the FST at an intranasal dose of 2.0nmol/g Immobility behavioral of animals who received intranasal injections of TAT-D1-D2-IPep (dose = 2nmol/g) was compared to those who received saline, TAT-Pep or IP injections of imipramine. Immobility behavior between groups compared by 1-way Independent Groups ANOVA followed by post-hoc Newman-Keuls multiple comparisons tests. ** p<0.01 compared to saline, ^ p<0.05 compared to TAT-Pep. Error Bars Represent SEM. Numbers within bars represent number of animals per group. 70

81 71 difference between groups who received saline (IN, n=6), imipramine (IP, 15mg/kg, n=6), TAT- D1-D2-IPep (IN, 1.67nmol/g, n=7) and TAT-Pep (IN, 1.67nmol/g, n=6), (n=6-7, F(3,21)=12.25, p<0.001). Post-Hoc Newman-Keuls multiple comparisons tests revealed significant differences when comparing TAT-D1-D2-IPep and saline (p<0.01), TAT-D1-D2-IPep and TAT-Pep (p<0.01), imipramine and saline (p<0.001) and imipramine and TAT-Pep (p<0.001). No significant differences were found between imipramine and TAT-D1-D2-IPep (p>0.05) and saline and TAT-Pep (p>0.05). Data is represented graphically in Figure 3-3, 3-8 and summarized in Figure D1-D2 interfering peptide dose: 1.0nmol/g (3.43 mg/kg) At an intranasal dose of 1.0nmol/g, the D1-D2 interfering peptide did not have an antiimmobility effect in the FST, as it was not significantly different from that of saline, and significantly less than that of imipramine. We compared immobility behavior during the 5- minute FST in rats that received saline (IN, n=8), imipramine (IP, 15 mg/kg, n=8) and TAT-D1- D2-IPep (IN, n=6, 1.0 nmol/g). A 1-way independent groups ANOVA revealed a significant difference between all groups (n=6-8 per group, F(2,19)=9.653, p<0.01). Post-hoc Newman- Keuls multiple comparisons tests revealed significant differences between imipramine and saline (P <0.001) and imipramine and TAT-D1-D2-IPep (P <0.05). No significant difference was found between saline and TAT-D1-D2-IPep (p>0.05). Data is represented graphically in Figure 3.9 and summarized in Figure 3.10.

82 Figure 3-8 The D1-D2 interfering peptide has an anti-immobility effect in the FST at an intranasal dose of 1.67 nmol/g. Immobility behavioral of animals who received intranasal injections of TAT-D1-D2-IPep (dose = 1.67nmol/g) was compared to those who received saline, TAT-Pep or IP injections of imipramine. Immobility behavior between groups compared by 1-way independent groups ANOVA followed by post-hoc Newman-Keuls multiple comparisons tests. *** p<0.001, ** p<0.01 compared to saline, ^^^ p<0.001, ^^ p<0.01 compared to TAT-Pep. Error bars represent SEM. Numbers within bars represent number of animals per group. 72

83 Figure 3-9 The D1-D2 interfering peptide does not have an anti-immobility effect in the FST at an intranasal dose of 1.0nmol/g Immobility behavioral of animals who received intranasal injections of TAT-D1-D2-IPep (dose = 1nmol/g) was compared to those who received saline or IP injections of imipramine. Immobility behavior between groups compared by 1-way independent groups ANOVA followed by post-hoc Newman-Keuls multiple comparisons tests *** p<0.001 compared to saline, # p<0.05 compared to imipramine. Error bars represent SEM. Numbers within bars represent number of animals per group. 73

84 Figure 3-10 Efficacy of the D1-D2 interfering peptide at various doses in the FST: summary of findings. Data Points in imipramine and saline group are combined from experiments outlined in Sections For each dose, complete results represented graphically in Figure Data at each dose compared using 1-way independent groups ANOVA. *** p <0.001, ** p<0.01, * p<0.01 compared to Saline, # p<0.05 compared to imipramine. Numbers below data points are number of animals per group. Error bars represent SEM. Data from treatment groups who received TAT-Pep excluded from figure for the purposes of clarity (Figure 3-7, 3-8). 74

85 Experiment 5: Duration of the behavioral effect of the D1-D2 interfering peptide We investigated the length of time that the D1-D2 interfering peptide remains behaviorally active (has a detectable anti-immobility effect in the FST) after intranasal administration. To do this, we increased the amount of time between the last intranasal injection of either the D1-D2 interfering peptide or the 9-amino acid TAT peptide and the beginning of the FST to 2, 3 or 4 hours. Overall results at all time points are summarized in Figure Behavioral Effect in FST 2 hours after intranasal administration The D1-D2 interfering peptide had a significant anti-immobility effect in the FST two hours after the final intranasal injection. We compared immobility behavior during the FST in rats that had received saline (IN, 1 hr before FST, n=6), imipramine (15mg/kg, IP, 1 hr before FST, n=6), TAT-D1-D2-IPep (1.67nmol/g, IN 2 hrs before FST, n=6) or TAT-Pep (1.67nmol/g, IN, 2 hrs before FST, n=5). A 1-way independent groups ANOVA revealed a significant difference between all groups (n=5-6 per group, F(3,19)=5.399, p<0.01). Post-Hoc Newman- Keuls multiple comparisons tests revealed a significant difference between TAT-D1-D2-IPep and saline (p<0.05), TAT-D1-D2-IPep and TAT-Pep (p<0.05), imipramine and saline (p<0.05) and imipramine and TAT-Pep (p<0.05). No significant differences exist between imipramine and TAT-D1-D2-IPep groups (p>0.05) or TAT-Pep and saline groups (p>0.05). Data is represented graphically in Figure 3-11 and summarized in Figure Behavioral Effect in FST 3 hours after intranasal administration Three hours after the last intranasal injection, the D1-D2 interfering peptide did not have a significant anti-immobility effect in the FST. We compared immobility behavior during the 5-

86 76 minute FST in rats that had received saline (IN, 1 hr before FST, n=6), imipramine (15mg/kg, IP, 1 hr before FST, n=6), TAT-D1-D2-IPep (1.67nmol/g, IN, 3 hrs before FST, n=7) or TAT- Pep (1.67nmol/g, IN, 3 hrs before FST, n=5). A 1-way independent groups ANOVA revealed a significant difference between immobility behaviors across all groups (n=5-6 per group, F(3,20)=4.669, p<0.01). Post-Hoc Newman-Keuls Multiple Comparisons Tests indicated significant differences between imipramine and saline groups (p<0.05), imipramine and TAT- Pep groups (p<0.05) and imipramine and TAT-D1-D2-IPep groups (p<0.05) groups. No significant differences were present between TAT-D1-D2-IPep and saline groups (p>0.05), TAT-D1-D2-Ipep and TAT-Pep groups (p>0.05) or TAT-Pep and saline groups (p>0.05). Data is represented graphically in Figure 3-12 and summarized in Figure Behavioral effect in the FST 4 hours after intranasal administration Four hours after the last intranasal injection, the D1-D2 interfering peptide did not have a significant anti-immobility effect in the FST. We compared immobility behavior during the FST in rats that had received injections of saline (IN, 1 hr before FST, n=6), imipramine (15mg/kg, IP, 1 hr before FST, n=6), TAT-D1-D2-IPep (1.67nmol/g, IN, 4 hrs before FST, n=6) or TAT- Pep (1.67nmol/g, IN, 3 hrs before FST, n=6). A 1-way independent groups ANOVA revealed a significant difference between immobility behaviors across all groups (n=5-6 per group, F(3,19)=3.727, p<0.05). Post-Hoc Newman-Keuls multiple comparisons tests indicated significant differences between imipramine and saline groups (p<0.05), imipramine and TAT-

87 Figure 3-11 The D1-D2 interfering peptide has an anti-immobility effect in the FST 2 hours after intranasal administration. Saline and imipramine were administered via IN and IP injections, respectively, 1 hour before the FST. TAT-D1- D2-IPep and TAT-Pep administered via IN injections 2 hours before the FST. Immobility behavioral counts analyzed by 1 way independent groups ANOVA (p<0.01) followed by post hoc Newman-Keuls multiple comparisons tests. * p<0.05 compared with saline, ^ p<0.05 compared with TAT-Pep. Error bars represent SEM. Numbers within bars represent number of animals per group. 77

88 Figure 3-12 The D1-D2 Interfering Peptide does not have an anti-immobility effect in the FST 3 hours after intranasal administration. Saline and imipramine were administered via IN and IP injections, respectively, 1 hour before the FST. TAT-D1-D2- IPep and TAT-Pep administered via IN injections 3 hours before the FST. Immobility behavioral counts analyzed by 1 way independent groups ANOVA (p<0.01) followed by post hoc Newman-Keuls multiple comparisons tests. * p<0.05 compared with saline, ^ p<0.05 compared with TAT-Pep, # p<0.05 compared with imipramine. Error bars represent SEM. 78

89 Figure 3-13 The D1-D2 interfering peptide does not have an anti-immobility effect in the FST 4 hours after intranasal administration Saline and Imipramine were administered via IN and IP injections, respectively, 1 hour before the FST. TAT-D1-D2- IPep and TAT-Pep administered via IN injections 4 hours before the FST. Immobility behavioral counts analyzed by 1 way independent groups ANOVA (p<0.05) followed by post hoc Newman-Keuls multiple comparisons tests. * p<0.05 compared with saline, ^ p<0.05 compared with TAT-Pep, # p<0.05 compared with imipramine. Error bars represent SEM. Numbers within bars represent number of animals per group. 79

90 Figure 3-14 The D1-D2 interfering peptide no longer has a behavioral effect in the FST 3 hours after it is administered via intranasal injections. Summarized data from Figures 3-11 to 3-13 and Figure 3-3. Immobility data from our original time point of 1 hour after intranasal administration is included for the purposes of comparison. Imipramine and saline treatment groups received IP and IN injections, respectively, 1 hour before the FST. Immobility behavioral data from each time point (1,2,3 and 4 hrs) analyzed via 1-way independent groups ANOVA followed by Newman Keuls post hoc tests. *** p<0.001, * p<0.05 compared to saline ^^ p<0.01, ^ p<0.05 compared to TAT-Peptide # p<0.05 compared to imipramine. Numbers below data points represent number of animals per group. Error bars represent SEM. 80

91 81 Pep groups (p<0.05) and imipramine and TAT-D1-D2-IPep groups (p<0.05). No significant differences were present between TAT-D1-D2-IPep and saline (p>0.05), TAT-D1-D2-Ipep and TAT-Pep (p>0.05) or TAT-Pep and saline (p>0.05). Data is represented graphically in Figure 3-13 and summarized in Figure Experiment 6: The D1-D2 interfering peptide does not increase locomotor activity In order to investigate whether the anti-immobility effect of the D1-D2 interfering peptide in the FST was due to its specific anti-depressant effects, we examined the effect of the D1-D2 interfering peptide on locomotor activity. To test this, we compared the amount of locomotor activity during a 30-minute test in animals given intranasal injections of saline (n=5), TAT-Pep (IN, 2nmol/g, n=5), TAT-D1-D2-IPep (IN, 2nmol/g, n=5) or imipramine (IP, 15mg/kg, n=5) Overall locomotor activity Animals in the D1-D2 interfering peptide, TAT-peptide or imipramine treatment groups had significantly lower overall locomotor activity during the 30-minute test than animals in the saline group. A 1-way independent groups ANOVA of locomotor activity (as measured by Beam Breaks) revealed a significant difference between all four treatment groups (n=5 per group, F(3,16)=9.775 p<0.001). Post-Hoc Newman-Keuls multiple comparisons tests revealed significant differences between TAT-D1-D2-IPep and saline groups (p<0.001), TAT-Pep and saline groups (p<0.01), and imipramine and saline groups (p<0.001). No significant difference was present between imipramine, TAT-Pep or TAT-D1-D2-IPep groups (p>0.05 for all comparisons). Data is represented graphically in Figure 3-15A.

92 82 to test whether the D1-D2 interfering peptide s effect on locomotor activity was consistent across different doses, we compared the locomotor activity of animals in treatment groups that received the D1-D2 interfering peptide at two doses: 2.0 nmol/g (n=5) and 1.67nmol/g (n=5). A twotailed, unpaired student s t-test revealed no significant differences between overall locomotor activity in these groups (n=5 per group, t(8)=1.203, p>0.05) (Figure 3-15B) Effect of time on locomotor activity during 30-minute test We examined locomotor activity across the treatment groups during the open-field test in 5-minute intervals. There was a significant difference in the amount of locomotor activity across different treatment groups, as well as the amount at different time points in the test. However, all treatment groups displayed the same pattern of locomotor activity (highest at the beginning of the test) (Figure 3-16). Locomotor activity at various time course data was analyzed by 2-way Independent Groups ANOVA, with treatment groups (saline, imipramine, TAT-D1-D2-IPep or TAT-Pep) and time point (5min, 10min, 15min, 20min, 25min, 30min) as independent factors. The analysis revealed a significant main effect of treatment groups (F(3,96)= 20.24, p<0.001) and time point (F(5,96)= 30.66, p<0.001). The interaction between treatment group and time point was not significant (F(15,96)=1.06, p>0.05).

93 Figure 3-15 The D1-D2 interfering peptide does not increase locomotor activity during a 30-minute open field test. (A) Total locomotor activity during a 30-minute open field test. Locomotor activity was assessed during a 30- minute open field test in a novel environment, and measured by the amount of movement around the testing chamber (Beam Breaks). Animals were given intranasal injections of saline, TAT-D1-D2-IPep or TAT Pep (2.0nmol/g) or IP injections of imipramine 24 hours, 19hours and 1 hour before the open field test. Locomotor activity data was analyzed by 1-way independent groups ANOVA (p<0.001) followed by post-hoc Newman-Keuls multiple comparisons tests. *** p<0.001, ** p<0.01, compared to saline. (B) The TAT-D1-D2-IPep has a similar effect in the open field test at 2.0nmol/g and 1.67nmol/g. Differences in locomotor activity produced by TAT-D1- D2-IPep dose evaluated by unpaired, two-tailed Student s t-test (p>0.05). 83

94 Figure 3-16 The D1-D2 interfering peptide decreases overall locomotor activity but does not change the activity pattern during a 30-minute open field test. Each Point on the test represents total number of beam breaks during the previous 5-minutes of the open field test. Data across treatment group and time points analyzed using 2-way independent groups ANOVA with time point and treatment group as independent factors. There was a significant main effect for time course (p<0.001) and treatment group (p<0.001) but no significant interaction between the two factors. n=5 animals per group. TAT-D1-D2-Ipep and TAT-Pep were given at an intranasal dose of 2.0nmol/g. Error bars represent SEM. 84

95 Experiment 7: Intranasal administration of the D1-D2 interfering peptide disrupts the interaction between dopamine D1 and D2 receptors in the PFC The D1-D2 interfering peptide is designed to disrupt the interaction between the Dopamine D1 receptor and the Dopamine D2-Long Receptor isoform by interacting with a 15- amino acid segment in the third intracellular loop of the D2L receptor. 3 In previous studies from our laboratory, administering this peptide directly to the PFC decreased the interaction between dopamine D1 and D2 receptors in the PFC (as assessed by co-immunoprecipitation) and had an anti-immobility effect in the FST (see Figure 1-4). 3 The D1-D2 interfering peptide also had a significant antidepressant effect in the FST after intranasal administration (Figure 3-3, 3-10), thus we examined whether there was a concurrent decrease in the D1-D2 dopamine receptor interaction in the PFC after intranasal administration of the D1-D2 interfering peptide. A representative immunoblot of D1R (anti-d1dr) immunoprecipitated by anti-d2dr is shown in Figure 3-17A. Densitometry analysis of immunoblots revealed that the detectable interaction between the Dopamine D1 and D2 receptors was significantly reduced in prefrontal tissue from animals that received TAT-D1-D2-IPep compared with animals who received saline (Student s t-test, n=3 per group, t(4)=3.872, p=0.018) (Figure 3-17B). 3.8 Experiment 8: The D1-D2 interfering peptide does not change the expression of dopamine D1 or D2 receptors in the PFC After intranasal administration, the D1-D2 interfering peptide can disrupt the interaction between Dopamine D1 and D2 receptors (Figure 3-17). We tested if this disruption is due to the D1-D2 interfering peptide s pharmacological effect or due to a change in the expression of either

96 Figure 3-17 Co-Immunoprecipitation of D1 by anti-d2r is reduced in the PFC of animals who received intranasal injections of TAT-D1-D2-IPep (Dose: 1.67nmol/g) (A) Representative immunoblot of anti-d2dr immunoprecipitated tissue from the PFC of rats that received intranasal injections of saline (n=3) or TAT-D1-D2-Ipep (IN, 1.67nmol/g, n=3). Input lane: 75 µg solubilized PFC tissue, IgG: tissue incubated with non-specific immunoglobulin antibody. (B) The interaction between D1 and D2R is significantly reduced in the PFC of animals who received TAT-D1-D2-IPep. The interaction between D1 and D2R quantified by densitometry analysis of immunoblots. All samples were standardized to control (saline) samples and analyzed by two-tailed, unpaired student s T-test (n=3 per group=0.018). 86

97 87 the dopamine D1 or D2 receptor proteins. To do this, we evaluated the expression of D1 and D2 receptors in the PFC of animals that were given intranasal injections of TAT-D1-D2-IPep (n=3) or saline (n=3) using Western Blot analysis Expression of Dopamine D1 receptors in the PFC after intranasal administration of the D1-D2 interfering peptide We compared prefrontal expression of D1 in the same subsample of animals used in the co-immunoprecipitation experiment (Section 3.8). The Western Blot analysis showed no change in the overall expression of the D1 dopamine receptor in the PFC after intranasal administration of the D1-D2 interfering peptide (IN, 1.67nmol/g, n=3) compared with saline (IN, n=3) (Student s t-test, t(4)=0.167, p>0.05), Figure 3-18A-B Expression of Dopamine D2 receptors in the PFC after intranasal administration of the D1-D2 interfering peptide We compared prefrontal expression of D2 in the same subsample of animals used in the co-immunoprecipitation analysis described in Section 3.7. We found no change in the overall expression of D2 dopamine receptors in the PFC after intranasal administration of the D1-D2 interfering peptide (IN, 1.67nmol/g, n=3) compared with saline (IN, n=3) (Student s t-test, t(4)=0.009, p>0.05), Figure 3-19A-B. We also performed an immunoblot for α-tubulin (Figure 3-20) to verify that the amount of protein in each sample was equivalent.

98 Figure 3-18 Intranasal administration of the D1-D2 interfering peptide does not change the expression of the dopamine D1 receptor in the PFC. (A) Representative immunoblot of D2R in PFC tissue from animals who received intranasal injections of TAT-D1- D2-IPep (IN, 1.67nmol/g, n=3) or saline (IN, n=3). (B) Expression of D2 is unchanged in animals who received intranasal injections of TAT-D1-D2-IPep compared with those who received Saline (Control). Data quantified using densitometry and analyzed using unpaired, two-tailed student s t-test (n=3 per group, p>0.05). Error bars represent SEM. 88

99 Figure 3-19 Intranasal administration of the D1-D2 interfering peptide does not change the expression of the dopamine D2 Receptor in the PFC (A) Representative Immunoblot of D1 in PFC tissue from animals who received intranasal injections of TAT-D1- D2-IPep (IN, 1.67nmol/g, n=3) or saline (IN, 1.67nmol/g, n=3). (B) Expression of D2 is unchanged in animals who received intranasal injections of TAT-D1-D2-IPep compared with those who received saline (control). Data quantified using densitometry and analyzed using unpaired, two-tailed student s t-test (n=3 per group, p>0.05). Error bars represent SEM. 89

100 Figure 3-20 Representative immunoblot of α-tubulin expression in rat PFC tissue. Rats received intranasal injections of saline (IN, n=3) or TAT-D1-D2-IPep (IN, 1.67nmol/g, n=3). 20 µg protein from PFC of each animal resolved using SDS page and immunoblotted with anti- α-tubulin. α-tubulin levels were used as a loading control for experiments assessing expression of D1 or D2R in the PFC. 90