The Neuropeptide story Galanin Outline Neuropeptide Reminder Galanin

The Neuropeptide story… Galanin

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

Nestler et al. , 2001

Levitan & Kaczmarek, 1997 Nolte 5 th ed. 2002

Nestler et al. , 2009

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

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

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

Galanin Receptors GALR 1 ○ Gi/Go ○ Inhibits adenylyl cyclase Close voltage gated Calcium channels ○ Expression Olfactory tract Amygdala Hypothalamus Thalamus Hippocampus Spinal trigeminal nucleus Spinal cord Dorsal root ganglia

Galanin Receptors GALR 2 ○ Gq ○ Phospholipase C / Protein Kinase C ○ Expression widespread, similar to GALR 1 GALR 3 ○ Not as much known ○ Ligand with high specificity for GALR 3 hard to find

Experimental evidence exists that suggests Galanin increases after exercise Anxiolytic/Anxiogenic debate of Galanin Could be due to GALR 1/GALR 2 localization and opposite effects.

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

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

Holmes 2006 Introduction Clomipramine Tricyclic antidepressant ○ Block SERT & NET ○ Negligible affinity for DAT

Clomipramine Action Nestler et al. , 2009

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

Holmes 2006 Hypothesized that chronic clomipramine treatment would elevate GAL m. RNA in the LC similar to exercise effects.

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 m. RNA levels

Holmes 2006 Results

Holmes 2006 Results

Holmes 2006 Results Findings: Significant correlation between running distance and prepro-GAL m. RNA Exercise induced upregulation of prepro-GAL m. RNA 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

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

Holmes 2003 Introduction Goals Study the role of the glananin GALR 1 receptor subtype in relation to anxiety-like behavior See if the array of tests produce differences in neuroendocrine responses

Holmes 2003 Methods C 57 BL/6 J mice GALR 1 -/- knockout (also had +/-) Mice are viable, reproduce, and develop normally RT-PCR confirmation 4 week acclimation 3 -5 per cage 2 Cohorts

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

Holmes 2003 – Elevated Plus-Maze

Holmes 2003 – Light/Dark Exploration Test

Holmes 2003 – Emergence Test

Holmes 2003 – Open Field Test

Holmes 2003 – Neuroendocrine Responses

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.

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 GALR 2 -/- display anxiogenic like behavior specific to the elevated plus- maze GALR 1 is Gi. GALR 2 is Gq. By what mechanism?

Holmes 2003 Discussion Author hypothesis: GALR 1 mediates galanin’s inhibitory effects on noradrenergic neuronal activation, whereas GALR 2, probably located presynaptically, may regulate norepinephrine release in the forebrain areas

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

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

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

Zhao 2013 Introduction

Zhao 2013 Introduction Goals Quantify m. RNA 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

Zhao 2013 Methods C 57 BL/6 J male mice 4 per cage 1 week acclimation Restraint stress for 6 hours daily

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

Zhao 2013 Methods

Zhao 2013 Methods

Zhao 2013 Results

Zhao 2013 Results

Zhao 2013 Results

Zhao 2013 Results

Zhao 2013 Results

Zhao 2013 Results

Zhao 2013 Results

Zhao 2013 Results CRS may dapen DA release following morphine in Nac Flu has no effect on the expression of TRH 2 and 5 -HT 1 b receptor suggests that GALR 2 may have little impact on 5 HT system in Nac Following Flu treatment, GALR 2 may enhance α 2 aadrenceptor gene expression The m. RNA levels of galanin, GALR 1, and TH were enhanced following stress, suggesting activation of the galanin and NE systems in NAc

Take Home The possibility that: “A GALR 1 agonist could exert anxiolytic effects under conditions of extreme or persistent negative affect, as in anxiety disorders and depression, while remaining silent in everyday situations where normal responses to mild stressors are desirable” Holmes et al. 2003

Sources Bailey, K. , Pavlova, M. , Rohde, A. , Hohmann, J. , & Crawley, J. (2007). Galanin receptor subtype 2 (Gal. R 2) null mutant mice display an anxiogenic-like phenotype specific to the elevated plusmaze. Pharmacology Biochemistry and Behavior, 86, 8 -20. Holmes, A. , Kinney, J. , Wrenn, C. , Li, Q. , Yang, R. , Ma, L. , . . . Crawley, J. (2003). Galanin GAL-R 1 Receptor Null Mutant Mice Display Increased Anxiety-Like Behavior Specific to the Elevated Plus. Maze. Neuropsychopharmacology, 28, 1031 -1044. Holmes, P. , Yoo, H. , & Dishman, R. (2006). Voluntary exercise and clomipramine treatment elevate preprogalanin m. RNA levels in the locus coeruleus in rats. Neuroscience Letters, (408), 1 -4. Levitan and Kaczmarek (1997) The Neuron 2 nd ed. Oxford: New York Nestler E. J. , Hyman, S. E. , & Malenka, R. C. (2001). Molecular Neurophamacology. A Foundation for Clinical Neuroscience. Mc. Graw Hill: New York. Nestler E. J. , Hyman, S. E. , & Malenka, R. C. (2009). Molecular Neurophamacology. A Foundation for Clinical Neuroscience. Mc. Graw Hill: New York. Nolte J (2002) The Human Brain: An Introduction to its Functional Anatomy 2 nd ed. Elsevier: Mosby, Inc. Xu ZQ, Shi TJ, Hokfelt T (1998). Galanin/GMAP- and NPY-like immunoreactivities in locus coeruleus and noradrenergic nerve terminals in the hippocampal formation and cortex with notes on the galanin-R 1 and –R 2 receptors. J Comp Neurol 392: 227 -251. Zhao, X. , Seese, R. , Yun, K. , Peng, T. , & Wang, Z. (2013). The role of galanin system in modulating depression, anxiety, and addiction-like behaviors after chronic restraint stress. Neuroscience, 246, 8293.

Tryptophan tryptophan hydroxylase (TPH) 5 -Hydroxytryptophan aromatic amino acid decarboxylase (AADC) 5 -Hydroxytryptamine (Serotonin)

Tyrosine tyrosine hydroxylase (TH) DOPA aromatic amino acid decarboxylase (AADC) (dopa decarboxylase) Dopamine dopamine b- hydroxylase (DBH) Norepinephrine phenylethanolamine-Nmethyl-transferase (PNMT) Epinephrine Dopaminergic neurons Adrenergic neurons

Ø D 2 receptors can be located on the presynaptic DA terminal or soma/dendrite (on DA cell bodies); also located on postsynaptic targets. Ø D 2 receptors have two isoforms – D 2 short (presynaptic) & D 2 long (postsynaptic) Ø The presynaptic D 2 receptor serves as an autoreceptor to reduce the release of dopamine. Ø Has comparable affinity for DA to postsynaptic D 2 receptors (although affinity for specific exogenous ligands is usually higher in D 2 S) Ø Both D 2 S & D 2 L are Gi-protein coupled (inhibit c. AMP) Ø Activation of the presynaptic Gi coupled D 2 receptor can reduce dopamine release by: - opening of K+ channels (somatodendrites) causing hyperpolarizing - inhibition of voltage-gated Ca 2+ channels (projection terminals) to prevent vesicle docking and transmitter release.

Gi -coupled a 2 Receptors as autoreceptors to reduce NE release On soma/dendrite - Opens K+ channels causing hyperpolarization On presynaptic terminal - Inhibit voltage-gated Ca 2+ channels - Inhibit NE synthesis by reducing PKA-dependent phosphorylation of TH NE binding site (increases affinity for NE, in turn increases end-product inhibition of TH) Nestler et al. , 2001