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The Neuropeptide story…

Explore the neuropeptide story of Galanin, discovered in 1978 by Professor Viktor Mutt. Galanin is involved in various functions such as anxiety, depression, feeding, and more, mainly expressed in brain regions like the amygdala and hypothalamus. Learn about Galanin receptors GALR1 and GALR2, their roles, and experimental evidence linking Galanin to exercise effects. Dive into studies like Holmes et al. (2006, 2003) investigating the effects of exercise and antidepressant therapy on Galanin gene expression in the Locus Coeruleus, shedding light on neuroendocrine responses and behavior.

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The Neuropeptide story…

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  1. The Neuropeptide story… Galanin

  2. Outline • Neuropeptide Reminder • Galanin & Receptors • Holmes et al. 2006 • Holmes et al. 2003 • Main: Zhao et al. 2013 • Discussion

  3. Nestler et al., 2001

  4. Levitan & Kaczmarek,1997 Nolte 5th ed. 2002

  5. Nestler et al., 2009

  6. Galanin • 29 amino-acid neuropeptide • 1978 • Professor Viktor Mutt and colleagues • Porcine intestinal extracts

  7. Galanin • Expressed widely in brain, spinal cord, and gut. • Involved in: • Nociception • Anxiety • Depression • Waking and sleep regulation • Cognition • Feeding • Regulation of mood • Regulation of blood pressure • Roles in development • Seizures • Sexual behavior • Alzheimer’s

  8. Galanin • Cortico-limbic brain regions: • Amygdala • Hippocampus • Septum • Hypothalamus • Often Co-expressed • Norepinephrine (LC) • Serotonin (DRN) • Dopamine (VTA) • Acetylcholine

  9. Galanin Receptors • GALR1 • Gi/Go • Inhibits adenylylcyclase • Close voltage gated Calcium channels • Expression • Olfactory tract • Amygdala • Hypothalamus • Thalamus • Hippocampus • Spinal trigeminal nucleus • Spinal cord • Dorsal root ganglia

  10. Galanin Receptors • GALR2 • Gq • Phospholipase C / Protein Kinase C • Expression widespread, similar to GALR1 • GALR3 • Not as much known • Ligand with high specificity for GALR3 hard to find

  11. Experimental evidence exists that suggests Galanin increases after exercise • Anxiolytic/Anxiogenic debate of Galanin • Could be due to GALR1/GALR2 localization and opposite effects.

  12. Holmes 2006 Introduction • Locus Coeruleus • Nucleus in the Pons • Principle site of NE synthesis • Outputs to: • Amygdala & Hippocampus • Brain stem & Spinal cord • Cerebellum • Cerebral cortex • Hypothalamus • Thalamus • Ventral tegmental area

  13. Holmes 2006 Introduction • Locus Coeruleus • Arousal and sleep-wake cycle • Attention and memory • Behavioral flexibility, behavioral inhibition, and stress (psychological) • Cognitive control • Emotions • Neuroplasticity

  14. Holmes 2006 Introduction • Clomipramine • Tricyclic antidepressant • Block SERT & NET • Negligible affinity for DAT

  15. Clomipramine Action Nestler et al., 2009

  16. Holmes 2006 Introduction • Goals • Test exercise effects on GAL gene expression • Examine the effect of antidepressant therapy on GAL gene expression in the LC

  17. Holmes 2006 • Hypothesized that chronic clomipramine treatment would elevate GAL mRNA in the LC similar to exercise effects.

  18. Holmes 2006 Methods • Male Sprague-Dawley Rats • Variables: • Wheels and Locked Wheels • Saline or Clomipramine treatment • 10 mg/kg/day, IP injection • Daily injection for 21 days • In situ hybridization to measure prepro-GAL mRNA levels

  19. Holmes 2006 Results

  20. Holmes 2006 Results

  21. Holmes 2006 Results • Findings: • Significant correlation between running distance and prepro-GAL mRNA • Exercise induced upregulation of prepro-GAL mRNA in the Locus Coeruleus • Elevated GAL gene expression in the LC after chronic clomipramine treatment • Negative interaction (exercise & clomipramine), suggesting altered gene expression through distinct and possibly interfering processes

  22. Holmes 2006 Results • What is next? • By what mechanisms do exercise and chronic clomipramine treatment alter GAL gene expression?

  23. Holmes 2003 Introduction • Goals • Study the role of the glananin GALR1 receptor subtype in relation to anxiety-like behavior • See if the array of tests produce differences in neuroendocrine responses

  24. Holmes 2003 Methods • C57BL/6J mice • GALR1 -/- knockout (also had +/-) • Mice are viable, reproduce, and develop normally • RT-PCR confirmation • 4 week acclimation • 3-5 per cage • 2 Cohorts

  25. Holmes 2003 Methods • Sensory, motor, and neurological assessment • Elevated Plus-Maze • Light/dark exploration test • Emergence test • Open field test • Neuroendocrine Responses – Plasma Assays

  26. Holmes 2003 – Elevated Plus-Maze

  27. Holmes 2003 – Light/Dark Exploration Test

  28. Holmes 2003 – Emergence Test

  29. Holmes 2003 – Open Field Test

  30. Holmes 2003 – Neuroendocrine Responses

  31. Holmes 2003 Results • Findings • Significant anxiogenic behavior on Elevated Plus-Maze. • Significant increase in ACTH on Elevated Plus-Maze compared to other tests. • Could be evidence for “higher stress test” compared to other tests. • Neuropeptides require high neuronal firing frequency for release. • Not pertinent at lower levels of activity. • Probably not a false positive. Supported by similar findings in independent cohorts.

  32. Holmes 2003 Discussion • Galanin reduces the firing rate and hyperpolarizes noradrenergic neurons of the locus coeruleus, and inhibits the release of norepinephrine from terminals located in the cortex (Xu et al. 1998) • Bailey et al. 2007 • GALR2 -/- display anxiogenic like behavior specific to the elevated plus-maze • GALR1 is Gi. • GALR2 is Gq. • By what mechanism?

  33. Holmes 2003 Discussion • Author hypothesis: • GALR1 mediates galanin’s inhibitory effects on noradrenergic neuronal activation, whereas GALR2, probably located presynaptically, may regulate norepinephrine release in the forebrain areas

  34. Zhao 2013 Introduction • Nucleus Accumbens (Core & Shell) • A part of the Ventral Striatum  Basal Ganglia • Common Neurotransmitters in the NAc • Dopamine • GABA • Glutamate • Serotonin • Glucocorticoids • Outputs to: • Rest of Basal Ganglia • Globus Pallidus • VTA • Substantia Nigra • Reticular Formation of the Pons

  35. Zhao 2013 Introduction • NAc involved in: • Motivation • Pleasure • Reward • Reinforcement • Addiction • Lesser involvement • Fear • Impulsivity • Placebo effect

  36. Zhao 2013 Introduction • Fluoxetine • Anitdepressant • Selective Serotonin Reuptake Inhibitor (SSRI) • Act on SERT

  37. Zhao 2013 Introduction

  38. Zhao 2013 Introduction • Goals • Quantify mRNA expression of Galanin and its receptors in the Nac (implicated in anxiety, depression, and addiction) • Effects of chronic restraint on morphine-induced conditioned place preference and sensitization • With and without fluoxetine • To understand the effects of Galanin system on monoamine neurotransmitters, check levels of rate-limiting enzymes and autoreceptors

  39. Zhao 2013 Methods • C57BL/6J male mice • 4 per cage • 1 week acclimation • Restraint stress for 6 hours daily

  40. Zhao 2013 Methods • Open Field Test • Forced Swim Test • White-Black box shuttle • Conditioned Place Preference • Morphine hydrochloride and Fluoxetine dissolved in 0.9% saline • Two morphine hydrochloride dosages used: • 3 mg/kg • 20 mg/kg • RT-PCR to test monoamine changes

  41. Zhao 2013 Methods

  42. Zhao 2013 Methods

  43. Zhao 2013 Results

  44. Zhao 2013 Results

  45. Zhao 2013 Results

  46. Zhao 2013 Results

  47. Zhao 2013 Results

  48. Zhao 2013 Results

  49. Zhao 2013 Results

  50. Zhao 2013 Results • CRS may dapen DA release following morphine in Nac • Flu has no effect on the expression of TRH2 and 5-HT1b receptor suggests that GALR2 may have little impact on 5-HT system in Nac • Following Flu treatment, GALR2 may enhance α2a-adrenceptor gene expression • The mRNA levels of galanin, GALR1, and TH were enhanced following stress, suggesting activation of the galanin and NE systems in NAc

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