Orexin and Sleep. Team: A Little Bit of Leptin

Orexin and Sleep Team: A Little Bit of Leptin

Intro to Orexin 1997 -Scripps Research Institute gene expression in the hypothalamus Found gene clone 35 - expression limited to the lateral hypothalamus NTs in area resembled secretin - Hypocretin Today the gene responsible for prepro-orexin is named HCRT 1998 University of Texas Transgenic cell lines to discover orphan receptors When administered centrally induces eating Nutritional state influences production Named after Greek word orexis which means appetite

Synthesis Translation of the HCRT gene produces a hypothalamic neuropeptide precursor protein called Prepro-orexin. Prepro-orexin is cleaved into two orexin proteins Both are linear peptides that form 2 alpha helices Orexin A Orexin B -Chain length 33 amino acids -Chain length of 28 amino acids -4 sulfur cysteine residues -OX2R -OX1R

Orexin peptide and receptors EXCITATORY neuropeptides!! Orexin receptors are found on several monoaminergic and cholinergic neurons in the brain.

Intracellular pathways

So what does Orexin do? It depends: Where the orexin receptors location? What systems is Orexin activating?

Orexin receptors are ubiquitous!! RE AL H TE N!!! And

Orexin receptors in the periphery Kidneys Pituitary Gland Adrenal Glands Pancreas Gastrointestinal tract And in rodents: Lungs, Testes, and Ovaries

Orexin and its main role Key modulator: sleep and wakefulness cycle

Receptors OX2R Maintenance of a stronger wakefulness state OX1R and OX2R Contribute to inhibition of REM sleep

Receptor-deficient mice Narcolepsy: sleep disorder characterized by excessive sleepiness, sleep paralysis, hallucinations, and sometimes loss of muscle control Boundary between sleep and awake states are blurred, causing some attributes of one state to appear in the other How important is orexin? OX1R-/- show little abnormality in sleep and wakefulness OX2R-/- show clear narcoleptic symptoms Orexin-/- (both) show a severely narcoleptic phenotype

Receptor-deficient mice Wild-type (control) OX1R-deficient OX2R-deficient

Orexin, glucose, and obesity Hypothalamic orexin system is also critical in the regulation of energy and glucose metabolism Orexin-/- and OX2R-/- mice on high-fat diets Developed glucose intolerance, severe obesity, and insulin resistance Narcolepsy due to orexin-deficiency also showed glucose intolerance and obesity Transgenic mice overexpressing orexin on high-fat diets Did not develop abnormal weight gain or glucose intolerance

Orexin levels In the cerebrospinal fluid (CSF), orexin levels fluctuate daily Increased during awake-active phase Decreased during sleep-resting phase Changes in levels support activities while in that phase Orexin helps us stay awake, allowing us to focus and be awake in the active phase - especially true when seeking for food After enough food is consumed, orexin lowers

Orexin levels, cont These orexin-level changes are suggested to be a cause of insomnia Extremely low CSF orexin levels found in narcoleptic patients However, narcolepsy is not insomnia! More research is needed regarding this relationship Transgenic mice overexpressing orexin Displayed reduced quality of sleep Reduced sleep quality is significant because insomnia is a sleep disorder where falling and staying asleep is difficult. Too much orexin leads to being more awake (unable to sleep or unrestful sleep = symptoms of insomnia)

Orexin-A And its relationship to blood glucose and the endocrine pancreas

Orexin (Hypocretin) Neuropeptide Plays role in regulation of energy balances Two forms: Orexin-A and B OXA binds to OXR1 and 2 OXB mainly to OXR2 CNS - Found in lateral hypothalamic area (LHA)

Orexin-A (OXA) is also found in the endocrine pancreas What causes its release? What is the functional significance of islet OXA?

OXA release from islets Effects of depolarizing stimuli on OXA release from islets isolated from the rat pancreas Ouedraogo et al., Diabetes, 2003

Extracellular glucose modulates OXA release Effects of glucose on IR-OXA, IR-glucagon, & IR-insulin release from islets isolated from the rat pancreas Ouedraogo et al., Diabetes, 2003

OXA affects pancreatic hormone secretion Effect of OXA on glucagon secretion from isolated pancreatic islets Ouedraogo et al., Diabetes, 2003

OXA affects pancreatic hormone secretion Effect of OXA on insulin secretion from isolated pancreatic islets Ouedraogo et al., Diabetes, 2003

OXA modulates plasma glucose OXA Saline Effect of OXA on blood glucose levels Ouedraogo et al., Diabetes, 2003

OXA and OXR1 in pancreatic islets Both beta and alpha cells display OXA and OXR1 immunoreactivity Ouedraogo et al., Diabetes, 2003

What do we know based on the data? OXA is released from pancreatic islet cells Glucose levels regulate OXA release OXA modulates pancreatic hormone secretion and blood glucose levels

Implications OXA may function as a hormone and neuromodulator OXA may play a role in the physiological control of blood glucose

A Decade of Orexin Hypocretin and Addiction: Where Are We Now? Top 10 hits: A review of key research indicating how orexin functions Serendipity happens! James et al 2017

A Decade of Orexin Hypocretin and Addiction: Where Are We Now? Methods: Self Administration/Intracranial Self Stimulation Models Different reinforcement schedules. Trained to turn a wheel to first deliver electrical current shows greater degree of effort and motivation to obtain stimulus Reward functions specifically associated with orexin neurons in LH. Stress-, arousal-, wake promoting functions are associated with orexin neurons in DMH and PFA Classic Operant Conditioning models show direct evidence that orexin signaling is directly involved in drug seeking behavior. James et al 2017

A Decade of Orexin Hypocretin and Addiction: Where Are We Now? James et al, 2017

A Decade of Orexin Hypocretin and Addiction: Where Are We Now? Motivational Activation Hypothesis Orexins primarily mediate behavior under situations of high motivational relevance (hunger), exposure to threats (stress) or reward opportunities. Behavioral functions of orexins reflect an integrated function that translates motivation into adaptive behaviors Orexin system drives externally motivated drug-seeking behavior. James et al 2017

A Decade of Orexin Hypocretin and Addiction: Where Are We Now? From A Different Perspective: Behavioral Economics Model The language of BE: Work, Demand, Budget, Labor The language of Psychology: Reinforcement, Stimulus Response How much are subjects willing to work for the reward? Which comes first, the reinforcing properties of a drug or the motivation to obtain and consume it? Using techniques from other disciplines can help us understand more. James et al, 2017

Interaction Between Orexin Neurons and Brain Reward Circuitry James et. al. 2017

Interaction Between Orexin Neurons and Brain Reward Circuitry Orexin Functional Heterogeneity: Medial/Lateral Dichotomy in Function James et. al. 2017

Interaction Between Orexin Neurons and Brain Reward Circuitry Examination of: - afferent and efferent projection patterns of orexin neurons - orexin subpopulations: selective participation in behavioral and physiological functions Orexin Functional Heterogeneity: Medial/Lateral Dichotomy in Function James et. al. 2017

Orexin Afferent Projections Exploring inputs to orexin neurons in the VTA * How is this achieved? Method: Retrograde Tracing Ex. rabies virus, pseudorabies virus, fluoro-gold mpfc - medial prefrontal cortex LS - lateral septum NAcSh - nucleus accumbens shell VP - ventral pallidum Amy - amygdala BNST - bed nucleus of the stria terminalis Arc - arcuate nucleus VTA - ventral tegmental area MRN - medial raphe nucleus James et. al. 2017

Orexin Afferent Projections Exploring inputs to orexin neurons in the VTA * How is this achieved? Method: Retrograde Tracing Ex. rabies virus, pseudorabies virus, fluoro-gold Sakurai et. al (2005) used tetanus toxin to identify: Arc mpfc Amy (basolateral & medial) BNST basal forebrain PVN MRN mpfc - medial prefrontal cortex LS - lateral septum NAcSh - nucleus accumbens shell VP - ventral pallidum Amy - amygdala BNST - bed nucleus of the stria terminalis Arc - arcuate nucleus VTA - ventral tegmental area MRN - medial raphe nucleus James et. al. 2017

Orexin Afferent Projections Exploring inputs to orexin neurons in the VTA * How is this achieved? Method: Retrograde Tracing Ex. rabies virus, pseudorabies virus, fluoro-gold Sakurai et. al (2005) used tetanus toxin to identify: Arc mpfc Amy (basolateral & medial) BNST basal forebrain PVN MRN Yoshida et. al (2006) used CTb to identify: NAc Amy (central) LS (dorsolateral) VTA VP Hypothalamus (ventromedial & anterior) Confirmed inputs ID d by Sakurai & ID d strong projections from more regions mpfc - medial prefrontal cortex LS - lateral septum NAcSh - nucleus accumbens shell VP - ventral pallidum CTb - cholera toxin B subunit Amy - amygdala BNST - bed nucleus of the stria terminalis Arc - arcuate nucleus VTA - ventral tegmental area MRN - medial raphe nucleus James et. al. 2017

Orexin Afferent Projections Further exploration: Which inputs are important for reward seeking? mpfc - medial prefrontal cortex LS - lateral septum NAcSh - nucleus accumbens shell VP - ventral pallidum CTb - cholera toxin B subunit Amy - amygdala BNST - bed nucleus of the stria terminalis Arc - arcuate nucleus VTA - ventral tegmental area MRN - medial raphe nucleus James et. al. 2017

Orexin Afferent Projections Further exploration: Which inputs are important for reward seeking? Case Study 1: Examined: Fos expression during expression of cocaine CPP Results: 1) neurons in rostral LS & ventral BNST activated during cocaine CPP 2) septum-lh pathway disconnection reduced expression of cocaine CPP mpfc - medial prefrontal cortex LS - lateral septum NAcSh - nucleus accumbens shell VP - ventral pallidum CTb - cholera toxin B subunit Fos - early gene product indicating neuronal stimulation CPP - conditioned placed preference Amy - amygdala BNST - bed nucleus of the stria terminalis Arc - arcuate nucleus VTA - ventral tegmental area MRN - medial raphe nucleus LH - lateral hypothalamus James et. al. 2017

Orexin Afferent Projections Further exploration: Functional role of orexin afferents in reward seeking? Case Study 2: Examined: NAcSh shell activation & deactivation Results: 1) inactivation of region induces Fos expression in LH orexin field Promotes alcohol seeking & feeding behavior 2) Cocaine exposure excitatory drive from glutamatergic neurons to orexin neurons mpfc - medial prefrontal cortex LS - lateral septum NAcSh - nucleus accumbens shell VP - ventral pallidum CTb - cholera toxin B subunit Fos - early gene product indicating neuronal stimulation CPP - conditioned placed preference Amy - amygdala BNST - bed nucleus of the stria terminalis Arc - arcuate nucleus VTA - ventral tegmental area MRN - medial raphe nucleus LH - lateral hypothalamus James et. al. 2017

Orexin Efferent Projections Exploring dispersal pathways of orexin * How is this achieved? Method: Anterograde Tracing Ex. GFP, genetic (HSV) & molecular tracers (WGA) PL - prelimbic cortex IC - insular cortex NAc - Nulceus accumbens PVT - anterior paraventricular BNST - bed nucleus of the stria terminalis VTA - ventral tegmental area NI - nucleus incertus thalamus James et. al. 2017

Orexin Efferent Projections Exploring dispersal pathways of orexin * How is this achieved? Method: Anterograde Tracing Ex. GFP, genetic (HSV) & molecular tracers (WGA) Experimental Findings: - Input of orexin to VTA via: - Ox1R & Ox2R - nonsynaptic en passant fibers/nonsynapsing terminals - Orexin in VTA: - depolarizes DA neurons - facilitates glutamatergic inputs increase DA release in prefrontal cortex and ventral striatum PL - prelimbic cortex IC - insular cortex NAc - Nulceus accumbens PVT - anterior paraventricular BNST - bed nucleus of the stria terminalis VTA - ventral tegmental area NI - nucleus incertus thalamus Ox(1,2)R - James et. al. 2017

Orexin, AMPA, NMDA, & DA Shin & Rehermann 2006

Orexin, AMPA, NMDA, & DA Take away: VTA NMDA is important for reward learning, AMPA mediates expression of cocaine seeking from previously learned cocaine cues Shin & Rehermann 2006

Functional Heterogeneity of Orexin Neurons Exploring: how the orexin system participates in such diverse, contradictory functions? Hypothesis: Orexin neurons in Lateral Hypothalamus (LH) = reward motivation, orexin neurons in DMH and perifornical area (PFA) = arousal and stress. Basis: 1) LH orexin neurons were Fos-activated by cues associated with food, morphine, and cocaine 2) PFA-DMH orexin neurons preferentially activated by arousal/anxiety inducing footshock stimulation Findings: Mild stress = Fos expression PFA/DMH, LH Naloxone-induced morphine withdrawal DMH/PFA, LH Anxiogenic doses of nicotine seeking Fos in PFA and LH, but NOT DMH Alcohol seeking induced by baclofen/muscimol infusions in NAc = Fos in PFA and LH, but NOT DMH James et. al. 2017