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Anxiety – behavioural state arising in anticipation of potential threat -Protective role by preparing an organism for potential danger -Becomes pathological.

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Presentation on theme: "Anxiety – behavioural state arising in anticipation of potential threat -Protective role by preparing an organism for potential danger -Becomes pathological."— Presentation transcript:

1 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

2 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

3 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

4 BRAINSTEM Serotonin (5HT)-producing neurons 9 clusters (nuclei) B1-B9

5 BRAINSTEM Serotonin (5HT)-producing neurons 9 clusters (nuclei) B1-B9 Autonomic body physiology modulation of breathing, body temperature blood pressure heart rate learning and memory sensorimotor gating modulation of mood states

6 BRAINSTEM Serotonin (5HT)-producing neurons 9 clusters (nuclei) B1-B9 Serotonergic neurons are implicated in a range of clinical disorders: Sudden infant death syndrome Fetal alcohol syndrome Autism Anxiety Depression Addiction and Compulsive behaviors Autonomic body physiology modulation of breathing, body temperature blood pressure heart rate learning and memory sensorimotor gating modulation of mood states

7 Serotonergic neurons have different functional properties Message: there are many subtypes of serotonergic neurons different Anatomical locations different Projection patterns different Cell morphologies different Physiological properties

8 Which serotonin neurons underlie which behaviors and physiological processes? ? breathing heart rate blood pressure learning and memory sensorimotor gating anxiety aggression

9 breathing heart rate blood pressure learning and memory sensorimotor gating anxiety aggression Defining serotonergic neurons by gene expression pattern during early development ?

10 Serotonergic progenitors dorsal view r1 r4 r7 embryonic hindbrain Serotonergic progenitors Serotonergic neuron progenitors are located in embryonic hindbrain

11 Serotonergic progenitors dorsal view embryonic hindbrain r1 r2 r3 r4 r5 r6 r7 Embryonic hindbrain is composed of morphologically distinct structures called rhombomeres

12 r1 r2 r3 r4 r5 r6 r7 embryonic hindbrain Hoxa-4 Hoxb-4 Hoxd-4 Hoxd-3 Hoxb3 Hoxa-3 Hoxb-2Hoxa-2 Hoxb-1Hoxa-1 Elk-L3Elf-2Elk-L Ebk Sek-4Sek-3Sek-2Sek-1 Krox-20Kreisler Adapted from Lumsden and Krumlauf, 1996 rhombomeres Serotonergic progenitors Rhombomeres are molecularly distinct structures expressing different sets of genes

13 dorsal view embryonic hindbrain Serotonergic progenitors Rhombomeres are molecularly distinct structures expressing different sets of genes Message: serotonergic neuron progenitors in different rhombomeres have different gene expression pattern

14 dorsal view Serotonergic progenitors embryonic hindbrain Question: How the embryonic gene expression patterns are related to serotonergic neuron function?

15 loxP OFF BAP Dual recombinase-mediated gene activation FRT effector

16 loxP OFF BAP FRT effector Cre excision BAP effector FRT OFF Dual recombinase-mediated gene activation

17 loxP OFF BAP FRT effector Flp excision BAP effector loxP OFF Dual recombinase-mediated gene activation

18 loxP OFF BAP FRT effector BAP effector ON FlpCre Dual recombinase-mediated gene activation

19 loxP OFF BAP FRT effector BAP effector ON GeneB::Flp GeneA::Cre cre Flp Dual recombinase-mediated gene activation

20 loxP OFF BAP FRT effector BAP effector ON GeneB::Flp GeneA::Cre Effector ON Dual recombinase-mediated gene activation

21 r1 r2 r3 r4 r5 r6 r7 ON Pet1-Flpe Serotonergic-specific Flpe Rhombomere-specific Cre lines GFP OFF ON F P FRT GFP-TOX GFP FRT rosa26 CAG promoter Flpe FF Cre P P loxP

22 r1 r2 r3 r4 r5 r6 r7 ON Pet1-Flpe Serotonergic-specific Flpe Rhombomere- specific Cre lines GFP OFF ON F P FRT GFP-TOX GFP FRT rosa26 CAG promoter Flpe FF Cre P P loxP

23 Jensen et al, Nature Neuroscience 2008 Genetic fate map of serotonergic neurons

24 contributions from a single rhombomere Jensen et al, Nature Neuroscience 2008 Genetic fate map of serotonergic neurons

25 contributions from a single rhombomere contributions from multiple rhombomeres contributions from multiple rhombomeres Genetic fate map of serotonergic neurons Jensen et al, Nature Neuroscience 2008

26 contributions from a single rhombomere Message #1: Genetic Fate map = Classical, Anatomical Map contributions from multiple rhombomeres contributions from multiple rhombomeres

27 contributions from a single rhombomere Message #1: Message #2: provide framework to study the function of genetically defined serotonergic neuron subtypes Genetic Fate map = Classical, Anatomical Map contributions from multiple rhombomeres contributions from multiple rhombomeres

28 Question: How the embryonic gene expression patterns are related to serotonergic neuron function?

29 GOAL - Silence individual serotonergic neuron subtypes to determine their function loxP BAP FRT GFP-TOX

30 GOAL - Silence individual serotonergic neuron subtypes to determine their function loxP BAP FRT GFP-TOX Tetanus toxin light chain : blocks neurotransmitter release Tetanus toxin light chain : blocks neurotransmitter release

31 FIRST - silence all serotonergic neurons to reveal the full extent of attainable phenotypes loxP BAP FRT GFP-TOX

32 anxiety-related behaviors contextual learning sensorimotor gating Assayed a set of 3 behaviors known to be modulated by serotonin (5-HT): 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

33 24hrs repeat x3 at 2.5 min intervals assay fear-induced freezing Contextual Fear Conditioning Paradigm Can they learn to associate an aversive stimulus to a particular cage context?

34 24hrs repeat x3 at 2.5 min intervals assay fear-induced freezing p<.044 enhanced fear induced freezing behavior Contextual Fear Conditioning Paradigm

35 24hrs repeat x3 at 2.5 min intervals assay fear-induced freezing p<.044 No difference tripleControl Activity in the altered context No difference “silenced” mice show enhanced contextual learning and memory enhanced fear induced freezing behavior

36 Elevated zero maze (advancement over Plus maze) Open field test Light-dark exploration test 3 tests of anxiety-related behaviors Mice with higher level of anxiety-related behavior spend more time here. innate fear of open areas vs. desire to explore novel environments

37 Light-dark exploration test Elevated zero maze Open field test n=34n=32 *p< 0.03 A 2-factor (genotype x sex) multivariate analysis of variance (MANOVA) was used. F(1,62)=4.81, p<.032 *

38 anxiety-related behaviors contextual learning sensorimotor gating “silenced” mice (ePet1::Flpe, hßact::Cre, RC::PFtox)

39 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 prepulse TIME Stimulus intensity 85 dB Stimulus intensity pulse TIME Startle response 120 dB % inhibition : indication of sensorimotor gating

40 Prepulse Inhibition (PPI) test Mean percentage Inhibition of Startle Response “Silenced” mice exhibited less of a startle in presence of a prepulse control “silenced” - Pet1Flpe, hßact-cre, RC::PFtox P<.023 “Silenced” mice show enhanced sensorimotor gating. background white noise P<.007

41 anxiety-related behaviors contextual learning sensorimotor gating “silenced” all serotonergic neurons

42 anxiety-related behaviors contextual learning sensorimotor gating “silenced” all serotonergic neurons Do serotonergic neurons with different embryonic origins serve different functions?

43 r1-derived 5-HT neurons TOX in All 5-HT neurons What if we “ silence ” just the r1-derived 5-HT neurons? ??? anxiety-related behaviors contextual learning sensorimotor gating

44 silence all 5-HT neurons r1-derived anxiety-related behaviors contextual learning sensorimotor gating

45 no difference silence all 5-HT neurons r1-derived no difference anxiety-related behaviors contextual learning sensorimotor gating

46 silence all 5-HT neurons r1-derived anxiety-related behaviors contextual learning sensorimotor gating no difference Anxiety- related behaviors R1-derived 5-HT neurons may modulate anxiety-related behaviors

47 silence all 5-HT neurons r1-derived anxiety-related behaviors contextual learning sensorimotor gating no difference Modulated more by r2- and/or r3-derived 5-HT neurons? Anxiety- related behaviors Contextual learning Sensorimotor gating

48 silence all 5-HT neurons r1-derived anxiety-related behaviors no difference Modulated more by r2- and/or r3-derived 5-HT neurons? r2-derived r3-derived r5-derived in progress contextual learning sensorimotor gating

49 anxiety-related behaviors contextual learning sensorimotor gating X serotonergic neuron subtype ? ? GOAL - Assigning specific serotonergic neuron subtypes to specific behaviors

50 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

51 Amygdala and Anxiety c-Fos induction upon exposure to anxiogenic stimuli (Hale et al., 2006; Knapska et al., 2007) Stimulating the BLA increases anxiety : GABA antagonist bicucullin infusion into the BLA (Sajdyk et al., 1999, 2002; Spiga et al., 2006) Suppressing the BLA decrease anxiety : glutamate blocker infusion into the BLA (Sajdyk et al., 1997)

52 Hippocampus and Anxiety Sustained anxiety activates the vHPC : Brain imaging with human and rhesus monkeys (Oler et al., 2010; Hasler et al., 2007) Stimulating the vHPC increases anxiety : GABA antagonist Bicuculline or picrotoxin infusion (Rezvanfard et al. 2009; Bast T et al., 2001) Suppressing the vHPC reduces anxiety : GABA agonist infusion or mechanical lesion (Kjelstrup et al., 2002; Bannerman et al., 2003; Mchugh et al., 2004; Trent et al., 2010; Mceown et al. 2010)

53 Amygdala and HPC are reciprocally connected Basolateral AmygdalaVentral hippocampus

54 Amygdala and HPC likely coordinate with each other to control anxiety behaviours Q. How does the neural communication between the two structures contributes to anxiety?

55 Optogenetic manipulation

56 Behavioural consequence of activating the vHPC to BLA inputs AAV-hSyn-ChR2-YFP 473nm blue laser

57 Which subregions of the vHPC project to the BLA? Cholera toxin B chain (CTB)

58 Ventral CA1, subiculum, lateral entorhinal cortex project to the basolateral amygdala intermediate ventral dorsal

59 AAV-hSyn-ChR2-YFP mPFC Hypothalamus BLA

60 AAV-hSyn-ChR2-YFP mPFC Hypothalamus BLA 200um optic fiber delivering 473nm laser

61 Optogenetic stimulation of vHPC axon terminals at the basolateral amygdala N= 12 Mice were tested in 4 consecutive sessions of 5 min EPM (OFF/ON/OFF/ON)

62 Acute stimulation of vHPC terminals at the BLA induced anxiety behaviors in the EPM

63

64 Optogenetic stimulation of vHPC axon terminals at the basolateral amygdala

65 Summary A neural substrate defined by gene expression pattern in the developing embryo - Genetically distinct subtype of 5-HT neurons derived by rhombomere 1 A neural substrate defined by connectivity in the adult brain -vHPC to BLA connection -Acute stimulation of the vHPC to BLA inputs is sufficient to induce anxiety behaviours in mice Genetic approaches to link neurons to behaviors

66 Canadian Foundation Fighting Blindness National Institute of Heath Part 1. Natural Sciences and Engineering Research Council of Canada University of Toronto Shadi Bakir Robin Nguyen Wendy Xin Harvard University Susan Dymecki Wade Reghre Patricia Jensen Jia Jia Mai University of Memphis Melloni Cook Part 2.

67 Cre loxP DNA target excision Site specific DNA excision by recombinase

68 Flp FRT DNA target excision Site specific DNA excision by recombinase

69 Recombinase-mediated gene activation loxP OFF BAP effector Transcriptional STOP sequences : Broadly Active Promoter

70 Recombinase-mediated gene activation loxP OFF BAP effector excision ON BAP effector Cre

71 Activity modulator (silence/activate neuron activity) Ablator (kill neurons) Fluorescent reporter (label neurons)

72 embryonic hindbrain Serotonergic progenitors Where do serotonergic neurons originating from different rhombomeres settle in the adult brain? ? dorsal view

73 Hippocampus and Anxiety Sustained anxiety activates the vHPC : Brain imaging with human and rhesus monkeys (Oler et al., 2010; Hasler et al., 2007) Stimulating the vHPC increases anxiety : GABA antagonist Bicuculline or picrotoxin infusion (Rezvanfard et al. 2009; Bast T et al., 2001) Suppressing the vHPC reduces anxiety : GABA agonist infusion or mechanical lesion (Kjelstrup et al., 2002; Bannerman et al., 2003; Mchugh et al., 2004; Trent et al., 2010; Mceown et al. 2010) Spatial learning Anxiety


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