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Anxiety – behavioural state arising in anticipation of potential threat Protective role by preparing an organism for potential danger Becomes pathological when exaggerated or uncontrollable Anxiety disorders - abundant and costly
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Anxiety – behavioural state arising in anticipation of potential threat • Protective role by preparing an organism for potential danger • Becomes pathological when exaggerated or uncontrollable • Anxiety disorders - abundant and costly • 20% of the general population suffer from at least one form of anxiety disorder at some point of their life • Financial cost of more than 65 billion dollars in Canada alone
Neural substrates underlying anxiety 1. A neural substrate defined by gene expression pattern in the developing embryo 2. A neural substrate defined by connectivity in the adult brain
Neural substrates underlying anxiety • 1. A neural substrate defined by gene expression pattern in the developing embryo • Genetically distinct subset of serotonergic neurons • 2. A neural substrate defined by connectivity in the adult brain - Hippocampus to amygdala connection
Serotonin (5HT)-producing neurons 9 clusters (nuclei) B1-B9 BRAINSTEM
Autonomic body physiologymodulation of breathing, body temperature blood pressure heart rate • learning and memory • sensorimotor gating • modulation of mood states Serotonin (5HT)-producing neurons 9 clusters (nuclei) B1-B9 BRAINSTEM
Serotonergic neurons are implicated in a range of clinical disorders: • Autonomic body physiologymodulation of breathing, body temperature blood pressure heart rate • learning and memory • sensorimotor gating • modulation of mood states • Sudden infant death syndrome • Fetal alcohol syndrome • Autism • Anxiety • Depression • Addiction and Compulsive behaviors Serotonin (5HT)-producing neurons 9 clusters (nuclei) B1-B9 BRAINSTEM
Serotonergic neurons have different functional properties • different Anatomical locations • different Projection patterns • different Cell morphologies • different Physiological properties Message:there are many subtypes of serotonergic neurons
Which serotonin neurons underlie which behaviors and physiological processes? • breathing • heart rate • blood pressure • learning and memory • sensorimotor gating • anxiety • aggression ?
Defining serotonergic neurons by gene expression pattern during early development • breathing • heart rate • blood pressure • learning and memory • sensorimotor gating • anxiety • aggression ?
Serotonergic neuron progenitors are located in embryonic hindbrain r1 embryonic hindbrain Serotonergic progenitors Serotonergic progenitors r4 r7 dorsal view
Embryonic hindbrain is composed of morphologically distinct structures called rhombomeres r1 r2 r3 embryonic hindbrain Serotonergic progenitors r4 r5 r6 r7 dorsal view
Rhombomeres are molecularly distinct structures expressing different sets of genes r1 r2 r3 embryonic hindbrain rhombomeres r4 Serotonergic progenitors r5 r6 r7 Ebk Elf-2 Elk-L Sek-4 Sek-3 Sek-2 Sek-1 Elk-L3 Hoxb3 Hoxa-4 Hoxb-2 Hoxa-2 Hoxb-4 Hoxd-4 Hoxa-3 Hoxb-1 Hoxa-1 Hoxd-3 Kreisler Krox-20 Adapted from Lumsden and Krumlauf, 1996
Rhombomeres are molecularly distinct structures expressing different sets of genes Message: serotonergic neuron progenitors in different rhombomeres have different gene expression pattern embryonic hindbrain Serotonergic progenitors dorsal view
embryonic hindbrain Serotonergic progenitors Question: How the embryonic gene expression patterns are related to serotonergic neuron function? dorsal view
Dual recombinase-mediated gene activation loxP loxP FRT FRT effector BAP OFF
Dual recombinase-mediated gene activation loxP loxP FRT FRT effector BAP OFF excision Cre FRT FRT BAP effector OFF
Dual recombinase-mediated gene activation loxP loxP FRT FRT effector BAP OFF excision Flp loxP loxP OFF effector BAP
Dual recombinase-mediated gene activation loxP loxP FRT FRT effector BAP OFF Cre Flp effector ON BAP
Dual recombinase-mediated gene activation loxP loxP FRT FRT effector BAP OFF GeneB::Flp GeneA::Cre effector ON BAP cre Flp
Dual recombinase-mediated gene activation loxP loxP FRT FRT effector BAP OFF GeneB::Flp GeneA::Cre effector ON BAP Effector ON
GFP GFP Pet1-Flpe ON Serotonergic-specific Flpe FRT FRT loxP loxP rosa26 CAG promoter F F P OFF P Flpe Cre rosa26 ON GFP-TOX CAG promoter F P r1 r2 r3 r4 Rhombomere-specific Cre lines r5 r6 r7
GFP GFP Pet1-Flpe ON Serotonergic-specific Flpe FRT FRT loxP loxP rosa26 CAG promoter F F P OFF P Flpe Cre rosa26 ON GFP-TOX CAG promoter F P r1 r2 r3 r4 r5 Rhombomere-specific Cre lines r6 r7
Genetic fate map of serotonergic neurons Jensen et al, Nature Neuroscience 2008
contributions from a single rhombomere Genetic fate map of serotonergic neurons Jensen et al, Nature Neuroscience 2008
contributions from a single rhombomere contributions frommultiple rhombomeres contributions frommultiple rhombomeres Genetic fate map of serotonergic neurons Jensen et al, Nature Neuroscience 2008
contributions from a single rhombomere contributions frommultiple rhombomeres contributions frommultiple rhombomeres Message #1: Genetic Fate map = Classical, Anatomical Map
contributions from a single rhombomere contributions frommultiple rhombomeres contributions frommultiple rhombomeres Message #1: Genetic Fate map = Classical, Anatomical Map Message #2: provide framework to study the function of genetically defined serotonergic neuron subtypes
Question: How the embryonic gene expression patterns are related to serotonergic neuron function?
GOAL - Silence individual serotonergic neuron subtypes to determine their function loxP loxP FRT FRT GFP-TOX BAP
GOAL - Silence individual serotonergic neuron subtypes to determine their function loxP loxP FRT FRT GFP-TOX BAP Tetanus toxin light chain : blocks neurotransmitter release
FIRST - silence all serotonergic neurons to reveal the full extent of attainable phenotypes loxP loxP FRT FRT GFP-TOX BAP
Assayed a set of 3 behaviors known to be modulated by serotonin (5-HT): contextual learning anxiety-related behaviors sensorimotor gating • contextual fear conditioning • open field • prepulse inhibition of acoustic startle • elevated zero maze • light-dark exploration test Collaborator Melloni Cook Tennessee Mouse Genome Consortium Neuromutagenesis Project
24hrs assay fear-induced freezing repeat x3 at 2.5 min intervals Contextual Fear Conditioning Paradigm Can they learn to associate an aversive stimulus to a particular cage context?
24hrs assay fear-induced freezing repeat x3 at 2.5 min intervals Contextual Fear Conditioning Paradigm enhanced fear induced freezing behavior p<.044
24hrs assay fear-induced freezing repeat x3 at 2.5 min intervals “silenced” mice show enhanced contextual learning and memory No difference No difference Activity in the altered context enhanced fear induced freezing behavior p<.044 Control triple
innate fear of open areas vs. desire to explore novel environments Mice with higher level of anxiety-related behavior spend more time here. Elevated zero maze (advancement over Plus maze) Light-dark exploration test Open field test 3 tests of anxiety-related behaviors
Light-dark exploration test Open field test Elevated zero maze * *p< 0.03 n=34 n=32 A 2-factor (genotype x sex) multivariate analysis of variance (MANOVA) was used. F(1,62)=4.81, p<.032
“silenced” mice (ePet1::Flpe, hßact::Cre, RC::PFtox) contextual learning anxiety-related behaviors sensorimotor gating
120 dB sensorimotor gating: a neurological gating process that prevents distracting sensory inputs from generating unnecessary motor outputs regulation is disrupted in Schizophrenia and Huntington disease operationally measured by inhibition of the acoustic startle response after a leading (prepulse) stimulus % inhibition pulse Startle response : indication of sensorimotor gating 85 dB Stimulus intensity Stimulus intensity prepulse TIME TIME
control “silenced” - Pet1Flpe, hßact-cre, RC::PFtox “Silenced” mice exhibited less of a startle in presence of a prepulse “Silenced” mice show enhanced sensorimotor gating. Prepulse Inhibition (PPI) test P<.007 P<.023 Mean percentage Inhibition of Startle Response background white noise
“silenced” all serotonergic neurons contextual learning anxiety-related behaviors sensorimotor gating
Do serotonergic neurons with different embryonic origins serve different functions? “silenced” all serotonergic neurons contextual learning anxiety-related behaviors sensorimotor gating
What if we “silence” just the r1-derived 5-HT neurons? contextual learning anxiety-related behaviors sensorimotor gating TOX in All 5-HT neurons ? ? ? r1-derived 5-HT neurons
contextual learning anxiety-related behaviors sensorimotor gating silence all 5-HT neurons r1-derived
contextual learning anxiety-related behaviors sensorimotor gating silence all 5-HT neurons r1-derived no difference no difference
R1-derived 5-HT neurons may modulate anxiety-related behaviors contextual learning anxiety-related behaviors sensorimotor gating silence all 5-HT neurons r1-derived no difference no difference Anxiety-related behaviors
Modulated more by r2- and/or r3-derived 5-HT neurons? contextual learning anxiety-related behaviors sensorimotor gating silence all 5-HT neurons r1-derived no difference no difference Anxiety-related behaviors Contextual learning Sensorimotor gating
r2-derived r3-derived r5-derived Modulated more by r2- and/or r3-derived 5-HT neurons? contextual learning anxiety-related behaviors sensorimotor gating silence all 5-HT neurons r1-derived no difference no difference in progress
GOAL - Assigning specific serotonergic neuron subtypes to specific behaviors contextual learning anxiety-related behaviors sensorimotor gating X serotonergic neuron subtype ? ?
Neural substrates underlying anxiety • 1. A neural substrate defined by gene expression pattern in the developing embryo • Genetically distinct subset of serotonergic neurons • 2. A neural substrate defined by connectivity in the adult brain - Hippocampus to amygdala connection