Transcript Luke Fuhrman - USD Biology

The Neuropeptide story…
Galanin
Outline
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Neuropeptide Reminder
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Galanin & Receptors
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Holmes et al. 2006
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Holmes et al. 2003
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Main: Zhao et al. 2013
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Discussion
Nestler et al., 2001
Levitan &
Kaczmarek,1997
Nolte 5th ed. 2002
Nestler et al., 2009
Galanin
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29 amino-acid
neuropeptide
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1978
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Professor Viktor Mutt and
colleagues
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Porcine intestinal
extracts
Galanin
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Expressed widely in brain, spinal cord, and gut.
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Involved in:
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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
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Cortico-limbic brain regions:
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Amygdala
Hippocampus
Septum
Hypothalamus
Often Co-expressed
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Norepinephrine (LC)
Serotonin (DRN)
Dopamine (VTA)
Acetylcholine
Galanin Receptors
 GALR1
○ 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
 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
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Experimental evidence exists that
suggests Galanin increases after
exercise
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Anxiolytic/Anxiogenic debate of Galanin
 Could be due to GALR1/GALR2 localization
and opposite effects.
Holmes 2006 Introduction
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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
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Locus Coeruleus
 Arousal and sleep-wake cycle
 Attention and memory
 Behavioral flexibility, behavioral inhibition,
and stress (psychological)
 Cognitive control
 Emotions
 Neuroplasticity
Holmes 2006 Introduction
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Clomipramine
 Tricyclic antidepressant
○ Block SERT & NET
○ Negligible affinity for DAT
Clomipramine Action
Nestler et al., 2009
Holmes 2006 Introduction
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Goals
 Test exercise effects on GAL gene
expression
 Examine the effect of antidepressant therapy
on GAL gene expression in the LC
Holmes 2006
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Hypothesized that chronic clomipramine
treatment would elevate GAL mRNA in
the LC similar to exercise effects.
Holmes 2006 Methods
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Male Sprague-Dawley Rats
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Variables:
 Wheels and Locked Wheels
 Saline or Clomipramine treatment
○ 10 mg/kg/day, IP injection
○ Daily injection for 21 days
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In situ hybridization to measure preproGAL mRNA levels
Holmes 2006 Results
Holmes 2006 Results
Holmes 2006 Results
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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
Holmes 2006 Results
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What is next?
 By what mechanisms do exercise and
chronic clomipramine treatment alter GAL
gene expression?
Holmes 2003 Introduction
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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
Holmes 2003 Methods
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C57BL/6J mice
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GALR1 -/- knockout (also had +/-)
 Mice are viable, reproduce, and develop normally
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RT-PCR confirmation
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4 week acclimation
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3-5 per cage
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2 Cohorts
Holmes 2003 Methods
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Sensory, motor, and neurological assessment
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Elevated Plus-Maze
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Light/dark exploration test
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Emergence test
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Open field test
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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
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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
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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)
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Bailey et al. 2007
 GALR2 -/- display anxiogenic like behavior specific to the elevated plus-
maze
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GALR1 is Gi.
GALR2 is Gq.
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By what mechanism?
Holmes 2003 Discussion
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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
Zhao 2013 Introduction
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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
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NAc involved in:
 Motivation
 Pleasure
 Reward
 Reinforcement
 Addiction
○ Lesser involvement
 Fear
 Impulsivity
 Placebo effect
Zhao 2013 Introduction
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Fluoxetine
 Anitdepressant
 Selective Serotonin Reuptake Inhibitor
(SSRI)
 Act on SERT
Zhao 2013 Introduction
Zhao 2013 Introduction
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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
Zhao 2013 Methods
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C57BL/6J male mice
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4 per cage
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1 week acclimation
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Restraint stress for 6 hours daily
Zhao 2013 Methods
Open Field Test
 Forced Swim Test
 White-Black box shuttle
 Conditioned Place Preference
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 Morphine hydrochloride and Fluoxetine
dissolved in 0.9% saline
 Two morphine hydrochloride dosages used:
○ 3 mg/kg
○ 20 mg/kg
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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
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CRS may dapen DA release following morphine in Nac
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Flu has no effect on the expression of TRH2 and 5-HT1b
receptor suggests that GALR2 may have little impact on 5HT system in Nac
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Following Flu treatment, GALR2 may enhance α2aadrenceptor gene expression
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The mRNA levels of galanin, GALR1, and TH were
enhanced following stress, suggesting activation of the
galanin and NE systems in NAc
Take Home
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The possibility that:
 “A GALR1 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 (GalR2)
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-R1 Receptor
Null Mutant Mice Display Increased Anxiety-Like Behavior Specific to the Elevated PlusMaze. Neuropsychopharmacology, 28, 1031-1044.
Holmes, P., Yoo, H., & Dishman, R. (2006). Voluntary exercise and clomipramine treatment elevate preprogalanin mRNA levels in the locus coeruleus in rats. Neuroscience Letters,(408), 1-4.
Levitan and Kaczmarek (1997) The Neuron 2nd ed. Oxford: New York
Nestler E.J., Hyman, S.E., & Malenka, R.C. (2001). Molecular Neurophamacology. A Foundation for
Clinical Neuroscience. McGraw Hill: New York.
Nestler E.J., Hyman, S.E., & Malenka, R.C. (2009). Molecular Neurophamacology. A Foundation for
Clinical Neuroscience. McGraw Hill: New York.
Nolte J (2002) The Human Brain: An Introduction to its Functional Anatomy 2nd 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-R1
and –R2 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
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D2 receptors can be located on the presynaptic DA terminal or soma/dendrite (on
DA cell bodies); also located on postsynaptic targets.
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D2 receptors have two isoforms – D2 short (presynaptic) & D2 long (postsynaptic)
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The presynaptic D2 receptor serves as an autoreceptor to reduce the release of
dopamine.
 Has comparable affinity for DA to postsynaptic D2 receptors (although affinity
for specific exogenous ligands is usually higher in D2S)
 Both D2S & D2L are Gi-protein coupled (inhibit cAMP)

Activation of the presynaptic Gi coupled D2 receptor can reduce dopamine
release by:
- opening of K+ channels (somatodendrites) causing hyperpolarizing
- inhibition of voltage-gated Ca2+ channels (projection terminals) to
prevent vesicle docking and transmitter release.
Gi -coupled a2 Receptors as autoreceptors to reduce NE release
On soma/dendrite
- Opens K+ channels causing
hyperpolarization
On presynaptic terminal
- Inhibit voltage-gated
Ca2+ 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