A THALAMOCORTICAL BRAINSTEM CIRCUIT MODULATES RESILIENCE TO DEPRESSION. Heankel Cantu Oliveros NEUR 300

Transcription

1 A THALAMOCORTICAL BRAINSTEM CIRCUIT MODULATES RESILIENCE TO DEPRESSION Heankel Cantu Oliveros NEUR 300

2 1 SPECIFIC AIMS It is estimated that 1 in 5 adults in the United States will experience one episode of major depression disorder (MDD) in their life with a high probability of relapse (Hirschfeld, 2012); still some people will rarely or never experience depression (Southwick and Charney, 2012). What are the individual differences that make some of us resilient to develop trauma-related mood disorders? It is known that subordinate animals are more resilient to develop depression-like behaviors after social defeat stress (Larrieu et al., 2017). In addition, subordinates are less prone than dominant conspecifics to show a loneliness-like state (Matthews et al., 2016). These studies suggest that a mechanism of resilience to depression may be developed in animals from a lower social status, but the exact neural circuit dynamics involved in both social hierarchy and depression remain elusive. Previously, it was shown that stress-induced stimulation of the serotonergic neurons in the dorsal raphe nucleus (DRN) is blocked by the medial prefrontal cortex (mpfc) to prevent the adverse effects of the stress response (Amat et al., 2005a). Neurons in the mpfc, regulated by the thalamus, have also being implicated in the establishment of social hierarchies (Zhou et al., 2017). Our long-term goal is to understand how this thalamocortical brainstem circuitry mediates both social and motivational states. In this project, we aim to study the functional connectivity of this thalamocortical brainstem circuitry and its mediation in stress-induced depression. Our hypothesis is that, during distress, the overall activity of this circuitry will lower the activity of serotonergic neurons in the DRN of subordinates, making them resilient to depression. Our proposition is consistent with experiments showing that the mpfc regulates the activation of DRN 5-HT during controllable stress and, unlike uncontrollable stress, prevents depressionlike behavior (Amat et al., 2005a). Understanding how social status could modulate this cortical circuitry to prevent depression is fundamental to develop circuit-based medications to treat and prevent the relapse of major depression disorder in vulnerable populations. Aim 1: Determining the functional connectivity between dmpfc and DRN GABAergic neurons. Much research has focused on the ventromedial PFC (vmpfc), but previous studies have demonstrated that the dorsomedial prefrontal cortex (dmpfc) projects to the DRN and mediates both social and motivational states (Kumar et al., 2013; Zhou et al., 2017). Based on vmpfc studies (Challis et al., 2014), our working hypothesis is that most dmpfc projections to the DRN target GABAergic neurons, which in turn regulate the release of 5-HT. To test this idea, we will 1) identify the co-distribution of dmpfc terminals and GABA neurons in the DRN and 2) characterize the postsynaptic response of GABA neurons by photostimulating dmpfc terminals. These experiments will be accomplished by expressing fluorescent markers and performing optogenetic-assisted electrophysiology. These findings will provide a mechanism by which dmpfc projections regulate 5-HT in the DRN. Aim 2: Measuring synaptic transmission in the thalamocortical brainstem circuit. We are interested in studying stress-induced depression and the activity of the thalamocortical brainstem circuit of resilient mice. Our working hypothesis is that, during stress, subordinate mice will have a higher activation of this circuit, which will decrease 5-HT release in DRN and prevent stress-induced depression. We will use fiber photometry with calcium imaging to measure the simultaneous neural activity in this circuit in freely moving mice undergoing stress. These findings will provide information on how resiliency is mediated by the coordinated activity of these brain regions. Aim 3: Testing the causal role of the thalamocortical brainstem circuit in the development of depression-like behavior. If there is a causal relationship between the thalamocortical brainstem circuit and the resilience of subordinate mice to depression, then the manipulation of this system should shed light on its regulation of motivational states. Optogenetics will be used again to stimulate or inhibit neurons in the thalamocortical brainstem circuit pathway in distressed mice. Our working hypothesis is that for all mice, regardless of their social hierarchy, photo stimulation of this circuitry during distress will increase their resilience to depression. To our knowledge, this is the first attempt to directly investigate the neural circuits regulating both social and motivational states. This will have an important impact in understanding the neural basis for the individual differences to cope with stressful situations in order to treat disabling mood disorders.

3 2 Significance The World Health Organization ranked depression as the most challenging disease worldwide with respect to time living with impairment (Murray CJL, 1996). There is mounting evidence showing that traumatic life events make individuals prone to depression (Johnson and Sarason, 1978; Reiche et al., 2004; Pittenger and Duman, 2007), still some people will rarely or never experience depression presumably because of their ability to cope with such traumatic experiences (Southwick and Charney, 2012). This evidence indicates that there are higher order control processes (potentially occurring in the prefrontal cortex) that might counteract the adverse results of stress. Instead of targeting this type of higher order cortical regions, most antidepressants target the raphe nucleus (e.g., SSRIs) (Lindsley, 2012). Despite antidepressants being effective on approximately 30% of patients, they have a delayed onset and most cases do not show remission (Fava and Rush, 2006; Rush et al., 2006; Howland, 2008). On the other hand, current research is pointing toward the use of NMDA receptor antagonists, like ketamine, in the treatment of depression (Kantrowitz et al., 2015). These drugs predominantly act on the forebrain and their onset and antidepressant efficacy are considerably greater than their SSRIs counterparts (Thakurta et al., 2012; Browne and Lucki, 2013). By studying a cortical circuitry mediating stress-induced depression, we expect to contribute to the knowledge needed for the development of more efficient and fast acting antidepressants. This is especially relevant in the treatment of depressive patients under suicidal risk. It is well known that SSRIs medications require a time frame of 4 to 6 weeks in order to show significant antidepressant results. This delayed action may be too late in the treatment of patients with high suicidal risk. Targeting the glutamatergic system via NMDAR antagonists is emerging as a breakthrough in the fast treatment of suicidal patients with resistant depression (Thakurta et al., 2012; Browne and Lucki, 2013; Price et al., 2014). Innovation Multiple studies have tried to dissect the brain circuitry involved in motivation, emotion, and depression (Krishnan and Nestler, 2008; Belzung et al., 2014), but a shortcoming on these studies is the isolation of such circuits with respect to other brain regions regulating other apparently unrelated behaviors. For instance, despite the fact that there is evidence showing a relationship between social hierarchy and depression (Matthews et al.; Larrieu et al., 2017), as far as we know, the concrete interaction between these circuits has not been studied yet. The proposed research is innovative, in our opinion, because it will investigate a circuit involved in both social behavior and depression. Given the gender-specific differences in the incidence of social and mood disorders (women being twice as likely to develop depression than men) (Li et al., 2016), it is possible to find a cortical circuit for sexually dimorphic depression behavior in our experiments. Our findings will contribute to the knowledge needed to develop more efficient and gender-specific pharmacological options in the treatment of depression.

4 3 Background A Prefrontal Cortex Brainstem Circuitry Regulates the Stress Response The ability of individuals to cope with traumatic events determines the impact of such events in their system (Krishnan et al., 2007). Despite the fact that the stress response is at a great extent automatic, mounting evidence suggests that there are cognitive factors regulating this unconscious response (Amat et al., 2005b). The prefrontal cortex, one of the executive centers of the brain, and the dorsal raphé nucleus, a region from which the serotonergic system diffuses, are the two major candidates regulating the cognitive and automatic biological responses to stress. The most striving evidence of this hypothesis comes from experiments where animals are exposed to escapable or unescapable stressors (footshocks) (Seligman and Maier, 1967). Animals in the uncontrollable shock group show an increase in ulceration and depression-like behavior such as weight loss and disruption of sleep. More importantly, a number of these animals fails to escape when facing escapable shocks. This stress-induced behavior is known as learned helplessness. In mice exposed to an escapable shock, however, activation of DRN 5-HT neurons is inhibited by the mpfc, so the negative consequences of uncontrollable distress are prevented (Amat et al., 2005b). Similarly, there is evidence of prefrontal cortex brainstem neuronal projections that modulate decision-making and motivational states when facing stressful situations (Warden et al., 2012). As these studies show, the prefrontal cortex brainstem circuitry is fundamental in the study of the conscious and unconscious brain regions regulating the proclivity of individuals to cope with traumatic events. In Aim 1, we will characterize this type of functional connection between the dorsal mpfc and the DRN. The Antidepressant Glutamatergic System Multiple studies dissecting the circuitry in the mpfc support the idea that there are abundant glutamatergic projections regulating the DRN in social and motivational states (Warden et al., 2012; Challis et al., 2014). This glutamatergic system is now being investigated as an effective approach to treat depression, particularly by ketamine, a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist (Kantrowitz et al., 2015). Moreover, there is evidence that the antidepressant effects of ketamine involve AMPA-receptor-mediated stimulation in the mpfc to regulate the release of 5-HT in the DRN (Fukumoto et al., 2016). Therefore, as proposed in Aim 1, it is worth using optogenetic-assisted electrophysiology to characterize the glutamatergic input from the dmpfc into the DRN in order to understand the mechanisms underlying this antidepressant response. Rodent Models of Social Hierarchy Social hierarchy is a common phenomenon observed from tiny social animals like ants to complex societies like humans (Midgley, 1984). Social rank is a major factor determining the access of individuals to resources such as food, territory, and mates. In humans, socioeconomic status works in a similar way by defining the occupation, income, and life-style of people. Not surprisingly, social status has a major

5 4 influence on health (Smith, 1998). Recent research on rodents is shedding light on the neurobiological basis of social hierarchy. In natural conditions, rodents show a socially dominant conduct characterized by aggressive behavior to access food, mark territory, reproduce, and avoid labor (Wang et al., 2014). The paradigm that allows scientist to draw these observations relies on the tendency of high ranks to win when facing social conflict (Hand, 1986). The tube test mimics this situation by creating a nonviolent conflict where rodents meet half way a narrow tube (Lindzey et al., 1961). They have to either push or retreat in order for both of them to exit the tube. By testing all mice from the same social group in a round-robin arrangement, mice can be classified as dominants or subordinates. Such rodent models of social hierarchy allow us to study diseases, such as depression, in animals of high and low ranks. These models also shed light on the potential individual differences that make animals more resilient or vulnerable to disease (Wang et al., 2014), as we will study on Aim 2. Resilient Subordinates Subordinate mice are resilient to display depression-like behaviors following chronic stress (Matthews et al., 2016; Larrieu et al., 2017). Nuclear magnetic resonance spectroscopy analysis showed that the metabolic profile in the nucleus accumbens (NAc), a limbic brain region receiving projections from the mpfc and the DRN, relates to both social hierarchy and susceptibility to depression after chronic stress (Larrieu et al., 2017). Before being exposed to stressful conditions, subordinates had lower levels of metabolites such as glutamate in the NAc. After social defeat stress, unlike dominant mice, subordinates displayed an increase in NAc metabolic profile. This metabolic difference across social ranks predicted the ability of subordinates to adapt to stress and prevent depression-like behavior. Another study supporting the resilient profile of subordinates focused on the DRN. In this case, subordinate mice were more resilient to display a loneliness-like state produced by optical inhibition of DRN neurons (Matthews et al., 2016). Despite the fact that the mechanisms controlling these stress-induced adaptations in subordinates remain unknown, these studies provide evidence that, as we propose in this project, there are neurobiological markers that predict the ability of individuals to cope with trauma. A Thalamocortical Circuitry Regulates Social Hierarchy The establishment of a dominant role in a social hierarchy is known to be related to a neural process defined by the experience of winning when competing against conspecifics (Zhou et al., 2017). Specifically, dominating others or being subordinated by others remodels a circuit between the medial dorsal thalamus (MDT) and the dorsal medial prefrontal cortex (dmpfc). Given that the development of social hierarchy reshapes the connectivity of the MDT-dmPFC circuitry, it is plausible that cortical neurons in the MDTdmPFC circuitry are diverse across individuals from different social hierarchies. These variations in cortical circuitries between dominants and subordinates could explain the tendency of subordinate mice to be more

6 5 resilient to depression after facing chronic stress. This is the reason why we will target the MDT-dmPFC circuitry in our experiments. Learned helplessness: an animal model of depression Resilience-related research relies on animal models that provide some neurobiological understanding of clinical studies (Krishnan and Nestler, 2008). In order for preclinical models of depression to be useful in humans, they must result in depression-like behavior caused by similar factors that trigger human depression, and they should be sensitive to antidepressant compounds known to work on human patients (Krishnan and Nestler, 2008). Learned helplessness is a preclinical model of depression in which animals are exposed to random and unescapable stress (e.g. foot shocks in a cage) (Seligman and Maier, 1967). After this treatment, 1) a consistent portion of animals develop coping maladaptation for aversive but escapable conditions (Overmier and Seligman, 1967); 2) more than 80% of susceptible animals continue helpless for at least one week; 3) helplessness can be reverted by antidepressants which show the pharmacological validity of this model (Chourbaji et al., 2005). We have chosen learned helplessness as a model in this study because it is easier to apply to female mice, unlike social defeat stress which is just starting to emerge as a model in female animals (Takahashi et al., 2017). Finally, a mouse model is ideal in this project because it will allow us to use Cre-dependent genetic identification and manipulation of the thalamocortical brainstem circuit in question. Experimental Design Approach Aim 1: Determining the Functional Connectivity Between dmpfc and DRN GABAergic Neurons Our objective is to examine the functional connectivity between the dmpfc and the DRN. We focus on the dmpfc instead of the entire mpfc because the dorsomedial prefrontal cortex presumably modulates both social hierarchy and motivational states (Amat et al., 2005b; Zhou et al., 2017). In this section of the experiment, we will test the working hypothesis that glutamatergic projections from the dmpfc activate DRN GABAergic neurons. We will test our hypothesis by using conditional fluorescent markers to colocalize dmpfc terminals and GABAergic neurons. Then we will use optogenetic-assisted electrophysiology to characterize the synaptic current in the dmpfc-drn GABA circuitry. After completing this aim, we expect to understand the mechanisms in this top-down circuitry regulating motivational states. In particular, we anticipate expanding our knowledge on how AMPA receptors mediate these synapses, which is relevant to understand how ketamine-induced AMPA receptors might work as antidepressants.

7 6 Experiment 1.1: Investigating the local co-distribution of excitatory dmpfc terminals and GABAergic neurons in the DRN To investigate the projections from dmpfc neurons to the DRN, we will first express a Cre-dependent adeno-associated virus (AAV) vector coding for a egfp-tagged synaptic protein Synaptophysin (SynP)(Veerakumar et al., 2014). This vector will be stereotaxically injected in CaMKIIa-Cre mice, so only excitatory neurons in the dmpfc will be infected (fig. 1 A) (Lee et al., 2003). Then we will analyze the distribution of glutamatergic dmpfc terminals by imaging SynP-eGFP fluorescence in the DRN. Next, we will determine whether these terminals project preferentially to GABAergic or 5-HT neurons, another major subtype of neurons in the DRN, by visualizing the SynP-eGFP efferents with genetically labeled 5-HT neurons (Pet1-tdTomato) and GABA-labeled neurons (GAD2-tdTomato) (figure 1 B) (Challis et al., 2013). These mice will be obtained from Jackson Laboratory (Bar Harbor, ME). For our tracing experiments, we will use one-year old male mice bred onto a C57BL/6 background. For 5-HT labelling, we will obtain BAC transgenic Pet1-Cre mice (B6.Cg-Tg(Fev-cre)1Esd/J; JAX stock number )(Scott et al., 2005); for GABA-labeled neurons we will buy GAD2-Cre mice (Gad2tm2(cre)Zjh/J; JAX stock number ) (Taniguchi et al., 2011). We estimate to use six to eight mice per group (Challis et al., 2014). Once we collect tissue slices (30µm) at similar anterior-posterior levels, we will compare the relative luminous intensities corresponding to glutamate (E), 5-HT, and GABA neurons to define the distribution of these synapses in the DRN (fig. 1 B). We expect to observe a higher correlation in the relative intensity of SynP-eGFP (E) and GAD2-tdTomato (GABA) neurons in these regions than in the ones corresponding to 5-HT neurons, meaning that most cortical projections synapse onto GABAergic neurons in the DRN. Figure 1. Expression and distribution of GABAergic neurons and dmpfc terminals in the DRN. A) Schematic of the stereotaxic injection and expression of AAV- CaMKIIa-Cre- SynP-eGFP in the dmpfc. Excitatory terminals projecting to the DRN are expected to show egfp when analyzed in coronal sections. B) Hypothetical overlap of sections expressing egfp-labeled dmpfc terminals (green), Pet1-tdTomato-labeled 5-HT neurons (orange), and GAD2-tdTomato-labeled GABA neurons (red) in the DRN. Graphics were modified from Challis et al. (2014). Experiment 1.2: Characterizing the Postsynaptic Response in GABAergic Neurons in the DRN by Optogenetic-Assisted Electrophysiology In order to establish how the dmpfc drives synaptic activity on GABAergic neurons in the DRN, we will start by injecting the channelrhodopsin-2 gene with the enhanced yellow fluorescent protein (ChR2 EYFP) under the CaMKIIa promoter into the dmpfc (Tsien et al., 1996; Ji and Neugebauer, 2012). The control

8 7 group will follow the same treatment, except that their vector will not contain the ChR2 coding part. Four weeks after, we expect that ChR2-EYFP will be richly expressed in the dmpfc, so we will prepare slices of the DRN to perform whole-cell patch clamp electrophysiology and record from labeled GAD2 + GABAergic neurons. Then we will stimulate the dmpfc terminals in slices with blue light (470 nm) to produce inward currents in transduced dmpfc neurons when doing voltage-clamp recordings at a holding potential of 70 mv in DRN-GABAergic cells (fig. 2 A). We will generate successions of brief pulses (0.5Hz to 25Hz,10mW, 1 to 10 ms) of blue light to elicit action potentials in ChR2-expressing neurons during clamp recordings. If the pattern of action potential firing follows the illumination frequency, then we can confirm the functionality of expressed ChR2 and test whether there are pulse-locked excitatory postsynaptic current (EPSC) responses in the dmpfc. Laser + NBQX Figure 2. Photostimulation of dmpfc terminals and the hypothetical synchronized EPSCs in DRN GABAergic neurons. A) Schematic of labeled dmpfc terminals, 5-HT, and GABAergic neurons in the DRN undergoing single-unit electrophysiological recordings during light-stimulation. B) Proposed relative mean voltage-clamp measurements of spontaneous, light elicited, and NBQX inhibited EPSCs. C) Putative voltage-clamp recording from GAD2-tdTomato cell during brief light pulses to activate ChR2-dmPFC endings (470 nm, 10mW, 1Hz, 10ms per pulse for all graphics shown). Blue rectangles represent photo- pulses. Black rectangles mark spontaneous EPSCs. D) Expected trace of synchronized EPSCs to a higher frequency (20Hz) light stimulation sequence from a GAD2-tdTomato cell. E) EPSCs from a Pet1-tdTomato neuron are hypothesized to lack a time-locked response during photostimulation. Figure from Challis et al. (2014). After verifying the functionality of expressed ChR2 in the dmpfc, we will investigate how these EPSC events are mediated by ionotropic glutamate receptors (iglurs). In order to study iglurs, we will use iglur -antagonists. We expect that in the presence of the AMPA/kainate receptor antagonist NBQX (the putative NMDR-antagonist-induced receptor involved in antidepressant effects), the EPSC responses will vanish (fig. 2 B). This result would support our hypothesis that the dmpfcàdrn pathway is modulated by AMPA receptors. Moreover, we will compare spontaneous events and EPSCs elicited by photostimulation in order to support the conclusion that the light-evoked responses from the dmpfc-terminals are different from random vesicle release (fig. 2 C). We expect that GABAergic neurons will show a higher percent of postsynaptic response compared to 5-HT neurons (fig. 2 C-E). This result would be consistent with studies

9 8 pointing toward the mpfc as an inhibitory input to the DRN-5-HT release (Amat et al., 2005b), and we would clarify that these terminals are AMPA-dependent acting on GABAergic neurons. Potential Problems and Alternative Solutions Spread of virus to other mpfc subdivisions: In order to properly localize and manipulate glutamatergicdmpfc terminals in the DRN, it is necessary to ensure that our stereotaxic injections are accurate and that the virus is not extended over the other mpfc subdivisions. In this case, we are trying to target the dmpfc, so the risk of extending over the vmpfc is low. After obtaining brain sections, we will confirm our sites of injection and, if the virus significantly extends over other mpfc areas, we will have to add controls where we specifically label vmpfc neurons in order to distinguish them from dmpfc terminals. Aim 2: Measuring Synaptic Transmission in The Thalamocortical Brainstem Circuit Experiment 2.1: Viral Expression of Calcium Indicators in the MDT-dmPFC -DRN Circuit After understanding the single cell dynamics between the dmpfc and the DRN, we will investigate the unresolved question of how the information transmitted in the MDT-dmPFC -DRN circuit varies among social hierarchies and mice resilient to stress-induced depression. Our goal here is to express genetically encoded calcium indicators to then study the dynamics between the neural population modulating social hierarchy (MDTàdmPFC) and the neurons underlying motivational states (dmpfcàdrn). In order to record real-time neuronal activity in freely moving animals treated with inescapable electric shocks, we will perform independent-fiber photometry (FIP) (Kim et al., 2016). Fiber photometry requires the use of a genetic Ca +2 sensor, in this case GCaMP6f, which will emit green fluorescence when bound to Ca +2 and excited with 490nm light (Chen et al., 2013). An increase in fluorescence will indicate the generation of an action potential because Ca +2 enters into the neuron, binds to GCaMP6f, and produces more fluorescence emission. This light emission is subsequently detected at axon terminals in the brain regions of interest (dmpfc and DRN). As shown by other studies, fiber photometry using calcium indicators is a reliable marker of real-time neural activity (Lerner et al., 2015). To perform independent-fiber photometry (Kim et al., 2016), we will 1) bilaterally inject a recombinant AAV-cre ( nl) driven under a CaMKIIa promoter to target glutamatergic neurons in the dmpfc [anteroposterior (AP), +1.9 mm; mediolateral (ML), 0.25 mm; dorsoventral (DV), 2.0 mm from bregma (Adhikari et al., 2015)] and 2) inject AAV-DIO- GCaMP6f driven under a Gad2-promoter (250 nl) into the DRN (AP: -4.10; ML: 1.25; DV: -2.90; at a 20 angle from the right side (Matthews et al.)) of previously classified dominant or subordinate GAD2-Cre mice (8 mice per social rank and per sex group). This injection will ensure the selective expression of cre recombinase GCaMP6f in GABAergic cell bodies in the dmpfcàdrn neurons (fig 3. A). Next, in order to measure multisynaptic neural dynamics between

10 9 the MDTàdmPFC and the dmpfcàdrn circuitry of mice, we will bilaterally inject a retrograde viral vector CAV-cre (250 nl) in the dmpfc and AAV-DIO- GCaMP6f in the MDT (AP: -1.50, ML: ±0.4, DV: 3.35 mm from bregma (Zhou et al., 2017)). The retrograde viral vector carrying cre will ensure that GCaMP6f is only expressed in dmpfc-projecting MDT neurons. This viral strategy will allow us to record activity from dmpfcàgabaergic neurons in the DRN, as well as from MDTàdmPFC neurons in the dmpfc. Experiment 2.2: Fiber Photometry in the MDT-dmPFC-DRN Circuit of Dominant/Subordinate Freely Moving Mice During Inescapable Shocks Learned helplessness paradigm First, we will classify mice as dominants or subordinates by applying the tube test paradigm as described by Wang et al., (2011). Second, following Berton et al., (2007), we will expose each mouse, from both subordinate and dominant groups, to inescapable shocks (IS) for two consecutive days. Neural activity in these mice will be recorded using fiber photometry (set up explained below) and they will receive 120 shocks randomly distributed over 1 hr (0.50 ma, 5 s duration; average intershock interval of 30 s) in a box through the grid floor. Animals receiving IS will be part of the experimental group (n=8 females and males) whereas animals spending time in the box without IS will be the controls (n=8). After two consecutive treatments, all mice will be tested for 15 escape trials. In this case, a footshock of the same intensity and duration as before will be administered to each mouse, but they will have the option to avoid it by getting into an un-electrified adjacent chamber. The latency time will be recorded for mice that successfully escape whereas failure will be noted for animals that did not escape within a 25s range. The average IS-escape latency in seconds will be evaluated between IS and No-IS groups, as well as within subordinate and dominant mice. Fiber Photometry Set Up After four to six weeks, our vectors will be expressed in regions of interest and we will proceed to do optic fiber implantation above the dmpfc and the DRN. We will measure fluorescence from these regions using optical fibers to photoactivate and record the fluorescence emission coming from MDTàdmPFC terminals and the fluorescence from cell bodies in the glutamatergic-dmpfc à DRN-GABAergic neurons in the DRN (Gunaydin et al., 2014). One fiber optic cannula will be implanted over the dmpfc and another one over the DRN through the same hole drilled for viral injections (fig. 3 B). Following Lerner et al., (2015), the light emission from GCaMP6f will be regulated by modulating the intensity used for the excitation light. This will allow us to control the fluorescence signal in order to recover the baseline response and to obtain the emission generated by neural activity corresponding to the response to footshocks.

11 10 The two LEDs excitation intensities in our experiment will be 490nm blue and 405nm violet, Thor Labs M490F1 and M405F1. In addition, each LED will be fixed to 30µW, the metal optical implant ferrules will be cleaned with isopropanol before starting the experiment, and they will be attached through a zirconia sleeve. Finally, the fluorescence signal will be recorded via the patch cord and collimator, passed through a filter (Thor Labs, MF525-39), and collected in a femtowatt photoreceiver (Newport, Model 2151) using a lens (Edmund Optics, Cat. No ) (fig. 3 C). The cannulas will be ordered from Doric Lenses and they will be made using 0.48 NA 400µM BFH fiber, non- fluorescent epoxy, and metal 2.5mm ferrules in order to decrease autofluorescence artifacts and to optimize fluorescence recordings (Lerner et al., 2015). Figure 3. Scheme of viral injections, fiber optic implantation, and fiber photometry set up. A) Areas of injection for the dmpfcàdrn circuitry. B) Implantation of fiber optic above the DRN. C) Set up for the equipment used in fiber photometry. The light intensities and respective filters are shown. Mice will receive footshocks during recordings. Image modified from (Lerner et al., 2015). We will further filter the fluorescence signal following Lerner et al. (2015), and then we will consider the footshock times fed into a real-time processor from the behavioral cage. A custom script on MATLAB will be used to analyze the output data. In this analysis, we will apply a least-squares linear fit to the low (405nm) intensity to align it to the high (490nm) signal. Next, we will calculate the ΔF/F time series: ((490nm signal - fitted 405nm signal) / fitted 405nm signal), and we will create a histogram considering the timestamps from the footshock cage. Expected Outcomes Learned helplessness In conformity with previous research (Matthews et al.; Larrieu et al., 2017), we expect that subordinate mice will have a lower deficit in IS-escape latency compared to dominant mice. That is, subordinate mice

12 11 will cope better with stress-induced depression, supporting our hypothesis that subordinates are resilient to stress-induced depression. Fiber photometry Based on in-vivo fiber photometry studies in resilient and susceptible mice (Muir et al., 2017), we expect two results. First, during shocks, the neural activity in the MDTà dmpfcàdrn-gabaergic neurons will be higher for subordinate mice and will be associated with resiliency to stress-induced depression, supporting our hypothesis that higher neural activity in this circuit allows animals to cope better with stressinduced depression. Second, a significant synchronization in the recordings from MDTàdmPFC and dmpfcàdrn-gabaergic neurons will suggest that the DRN activity is driven by cortical neurons targeted by the MDT, supporting our hypothesis that this circuitry mediates both social and motivational states. Potential Problems and Alternative Solutions Learned Helplessness It is assumed that the neural activity recorded during this paradigm corresponds to a motivational state, but a potential confounding variable is pain from footshocks. A solution is to add a second behavioral task that does not rely on pain such as despair-based models (e.g., forced-swimming test and tail suspension test) (Krishnan and Nestler, 2011). Having two behavioral tests would further support our conclusions about depression-like behavior and the potential neural activity associated with resiliency. Viral expression of calcium indicators A potential challenge in our methods is the specific cell-type targeting that we require in both the dmpfc and the DRN. If our Cre recombinase method fails by showing cross-reaction, we will have to use multifeature targeting via intron engineering and specialized viruses (INTRSECT) (Fenno et al., 2014). INTRSECT reduces cross-reactivity by using Cre- and Flp-dependent constructs to achieve conditional cell-type fluorophore expression. Therefore, we could use Flp- constructs when targeting DRN-GABAergic neurons in order to increase both the expression and accuracy of our calcium indicators. Aim 3: Testing the Causal Role of the Thalamocortical Brainstem Circuit in the Development of Depression-Like Behavior Experiment 3.1: Optogenetic Activation of MDTà dmpfcà DRN-GABAergic Circuitry to Test its Mediation in Resiliency to Depression To test for a causal connection between the MDTà dmpfcàdrn-gabaergic circuit activation and resiliency to depression-like behavior in mice, we will combine ChR2-mediated photostimulation with inescapable shock trials. The reasoning is that if DRN GABAergic activation by MDTàdmPFC neurons is sufficient to produce the resiliency to depression-like behavior observed after treating mice with inescapable shocks, then its photostimulation during IS will prevent depression-like behavior in mice. To

13 12 produce photostimulation in MDTàdmPFCàDRN-GABAergic neurons, ChR2 will be expressed in MDTàdmPFC terminals following the indications in Experiment 2.1, but replacing the calcium indicator by ChR2, leaving dmpfcàdrn neurons intact, and adding only one optic fiber above the dmpfc (n=8 per group). After recovery from surgery, we will photostimulate MDTàdmPFC terminals in mice during IS. Then we will do single trials for escapable shocks and record the latency times for both subordinate and dominant rodents. Controls will express egfp instead of ChR2, and they will be photostimulated during trials as well. In additional trials, we will inject animals with NBQX (200nL) and photostimulate the MDTàdmPFC terminals in mice during IS in order to corroborate that this circuitry s resilient action is AMPA-dependent. Experiment 3.2: Photoinhibition of MDTàdmPFCàDRN-GABAergic Circuitry to Test its Mediation in Vulnerability to Depression To test whether the reduced activity of MDTàdmPFCàDRN-GABAergic neurons increases the vulnerability of mice to stress-induced depression, we will express a hyperpolarizing opsin (NpHR) in a Cre-dependent manner in MDTàdmPFC terminals as described in Experiment 3.1. Based on previous studies (Maier and Watkins, 2005), we postulate that DRN 5-HT release, which is regulated by GABAergic neurons receiving MDT-driven cortical input, is necessary at the time of IS to result in depression-like behavior. Here, we will proceed to test whether optical inhibition of MDTàdmPFC neurons in mice (n=8 per group) during IS facilitates depression in both dominant and subordinate mice. Controls will express egfp instead of NpHR, and they will be photostimulated during trials as well. Expected Outcomes 3.1. By photoactivating the MDTà dmpfc pathway, we expect that cortical neurons will activate their downstream GABAergic targets in the DRN and inhibit 5-HT release during inescapable shocks (Maier and Watkins, 2005). As a result, animals will be less likely to develop depression. In contrast, by adding NBQX, we expect to see a decrease in resiliency, which would support our data from Aim 1 suggesting that the antidepressant action of this circuitry is AMPA-mediated. By dividing mice into subordinates and dominants, we expect to see whether previous social hierarchy experience increases the resiliency result of activating the MDTà dmpfc circuitry. 3.2 In the case of photoinhibition of MDTà dmpfc neurons, we expect that a decrease of MDT-driven cortical activity will lead to a decrease in downstream DRN- GABAergic inhibition. As a result, we expect an increase in serotonin release at the time of IS and subsequently, mice will develop depression-like behavior. This result would support the importance of MDT-driven cortical control in the DRN to prevent the maladaptive results of stress.

14 13 Potential Problems and Alternative Solutions Expression of light sensitive proteins in other areas of the mpfc and DRN As in aim 1, the infection of other mpfc and DRN areas is a risk and we must verify that the light sensitive channels are correctly expressed in our target neurons. In case of cross-infection, we will have to modify our coordinates and/or reduce the amount of virus injected in each region. However, we are confident that performing these experiments to match our objectives is possible because they are standard procedures in the field (Matthews et al.; Kim et al., 2017). Future Directions At the conclusion of this project, we would have 1) determined the functional connectivity between the dmpfc and the DRN, including AMPA-mediated synaptic activity; 2) found evidence that neural activity in the MDT-dmPFC-DRN circuit is higher in resilient subordinate mice and 3) shown that MDT-driven and AMPA-mediated cortical activity allows animals to cope better with stress-induced depression. In future experiments, cytological techniques will be required to study what differences in this circuit (e.g., cell-number, receptors, mrna expression) make subordinates more resilient to depression. Moreover, it is necessary to characterize the molecular mechanism underlying the antidepressant effects of ketamineinduced AMPA receptors in the MDT-dmPFC-DRN circuit. Finally, given the gender-specific differences in the incidence of social and mood disorders, it is possible to find a cortical circuit for sexually dimorphic depression behavior in our experiments (Li et al., 2016). Maybe resiliency in this circuit works differently for females and males. Such type of questions will be addressed after completing this project. Timeline Aim 1 Year 1 Year 2 Year 3 Year 4 Year 5 Aim 2 Aim 3 Experiment A.1.1 Experiment A.1.2 Experiment A.2.1 Experiment A.2.2 Experiment A.3.1 Experiment A.3.2