Anxiety disorders - abundant and costly 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 anxiety is emotional state arising when you anticipate threat and it has protective function, but when it is exaggerated it causes mental disorders. Anxiety disorders are common and costly, but our understanding of the neural substrates underlying anxiety still remain elusive.

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 today I will present two neural substrates that we have found to be influential to anxiety behaviours in mice. one is a neural substrate defined by gene expression in developing embryo, and the other is 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 First part of my talk is about a group of serotonergic neurons that we found important for anxiety behaviour and about how those neurons are defined by gene expression pattern in the embryo. And I would like to start by a brief introduction to serotonergic neurons.

Serotonin (5HT)-producing serotonergic neurons are neurons that produce neurotransmitter, serotonin and they are found in clusters of neurons in the brainstem known as B1 to B9 nuclei as shown in this sagittal section of rodent brain. Serotonin (5HT)-producing neurons 9 clusters (nuclei) B1-B9 BRAINSTEM

Serotonin (5HT)-producing Autonomic body physiology modulation of breathing, body temperature blood pressure heart rate learning and memory sensorimotor gating modulation of mood states serotonergic neurons have been implicated in different physiological processes and behaviours. They have been implicated in some of the most basic body physiology such as breathing and body temperature control, and they also modulate some of cognitive behaviors such as learning and memory, and emotional behaviors by modulating mood state. Serotonin (5HT)-producing neurons 9 clusters (nuclei) B1-B9 BRAINSTEM

Serotonin (5HT)-producing Serotonergic neurons are implicated in a range of clinical disorders: Autonomic body physiology modulation 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 Serotonergic neurons have been implicated in a range of clinical disorders. They have implicated in early childhood development disorder such as sudden infant death syndrome and autism, as well as adult psychiatric disorder such as anxiety and depression. Serotonin (5HT)-producing neurons 9 clusters (nuclei) B1-B9 BRAINSTEM

Message: there are many subtypes of serotonergic neurons Serotonergic neurons have different functional properties different Anatomical locations different Projection patterns different Cell morphologies different Physiological properties as reflected in their diverse functions, serotonergic neurons are different in their properties. They have different anatomical locations, different xxx. so while they all produce serotonin, message is that there are many different subtypes of serotonergic neurons. 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 ? And one of main question in serotonergic neuron biology is how these different types of serotonergic neurons are specified and which serotonin neurons types underlie which behaviors and physiological processes.

? Defining serotonergic neurons by gene expression pattern during early development breathing heart rate blood pressure learning and memory sensorimotor gating anxiety aggression ? We have been interested in addressing this question by defining serotonergic neurons based on gene expression pattern in the developing embryo, and let me explain what I actually mean by gene expression pattern in the developing embryo.

Serotonergic neuron progenitors are located in embryonic hindbrain Serotonergic progenitors Serotonergic progenitors r4 This cartoon image represent a dorsal view of midgestation embryo at the level of hindbrain. Progenitors that later develop into serotonerigc neurons are located in the embryonic hindbrain. and serotonergic neuron progenitors are shown in these two blue stripes. r7 dorsal view

Embryonic hindbrain is composed of morphologically distinct structures called rhombomeres Serotonergic progenitors r4 r5 embryonic hindbrain is a very interesting structure. It is composed of morphologically distinct segments called rhombomeres, and there are actuarially 7 rhombomeres from rhombomere 1 to 7. r6 r7 dorsal view

Rhombomeres are molecularly distinct structures expressing different sets of genes embryonic hindbrain rhombomeres Serotonergic progenitors r4 r5 rhombomeres are not only morphologically distinct but also molecularly distinct. As each of these rhombomeres express different sets of genes. r6 r7 Elf-2 Elk-L Ebk Hoxa-4 Hoxb-4 Hoxd-4 Hoxd-3 Hoxb3 Hoxa-3 Hoxb-2 Hoxa-2 Hoxb-1 Hoxa-1 Elk-L3 Sek-4 Sek-3 Sek-2 Sek-1 Krox-20 Kreisler 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 this means that serotonergic neuron progenitors in different rhombomeres also have different gene expression pattern, therefore they are genetically distinct. dorsal view

embryonic hindbrain Serotonergic progenitors this raises very important question about the functional significance of gene expression pattern in the serotonergic progenitors. how this gene expression pattern in the developing embryo are related to serotonergic neurons function in the adult brain? Question: How the embryonic gene expression patterns are related to serotonergic neuron function? dorsal view

Dual recombinase-mediated gene activation loxP loxP FRT FRT BAP effector OFF To address this question, we used recombinase-based gene activation strategy where removal of transcriptional stop sequence by recombinase trigger downstream gene expression. Usually it is done by cre recombinase alone, but it turns out that you can improve selectivity of this approach by having two stop sequences, one flanked by loxP and the other flanked by Frt.

Dual recombinase-mediated gene activation loxP loxP FRT FRT BAP effector OFF excision Cre FRT FRT BAP effector OFF Since there are two stop sequences between the promoter and effector genes, the excision of this first stop by cre is not enough for gene activation. And the same is for Flp.

Dual recombinase-mediated gene activation loxP loxP FRT FRT BAP effector OFF excision Flp loxP loxP BAP effector OFF And the same is for Flp.

Dual recombinase-mediated gene activation loxP loxP FRT FRT BAP effector OFF Cre Flp BAP effector ON You need to excise out both stop sequences by both cre and Flp reaction.

Dual recombinase-mediated gene activation loxP loxP FRT FRT BAP effector OFF GeneA::Cre GeneB::Flp BAP effector ON Now, you can imagine that if cre is expressed by driver gene A and Flp is expressed by driver gene B, cre Flp

Dual recombinase-mediated gene activation loxP loxP FRT FRT BAP effector OFF GeneA::Cre GeneB::Flp BAP effector ON effector gene will be expressed only at the intersectinoal territory of these two genes. Effector ON

ON Flpe Cre Rhombomere-specific Cre lines Pet1-Flpe rosa26 FRT FRT loxP loxP rosa26 CAG promoter GFP F F P P OFF Flpe Cre rosa26 GFP CAG promoter F P GFP-TOX ON r1 r2 r3 r4 Rhombomere-specific Cre lines r5 we used this intersectional approach to gain a selective genetic access to serotonergic neuron progenitors in different rhombomeres. And we achieved this by combining serotonergic neurons specific Flp with a series of rhombomere specific cre lines. r6 r7 ON Pet1-Flpe Serotonergic-specific Flpe

ON Flpe Cre Rhombomere-specific Cre lines Pet1-Flpe rosa26 FRT FRT loxP loxP rosa26 CAG promoter GFP F F P P OFF Flpe Cre rosa26 GFP CAG promoter F P GFP-TOX ON r1 r2 r3 r4 r5 Rhombomere-specific Cre lines Using this approach, we first ask where serotonergic neurons originating from different rhombomeres settle in the adult brain. To address this question, we expressed GFP as a lineage tracer in serotonergic neurons in different rhomobomere and studied where individual serotonergic neurons settle in the adult brain. This is one example of the study. This is a coronal section image taken at the medullary rap he region. Green neurons are the serotonergic neurons originated from rhombomere 5 and the red neurons are serotonergic neurons originated from rhombomere 6 and 7. r6 r7 ON Pet1-Flpe Serotonergic-specific Flpe

Genetic fate map of serotonergic neurons By applying the same approach with different rhombomeres, we establish the fate map of serotonergic neurons based on their gene expression pattern in the developing embryo. This is the summary of the finding. Serotonergic progenitors in different rhombomeres are shown in different colors in this left panel, and serotonergic neurons originating from each are shown in the matching color. For example, the blue x neuron here is from r1 and orange circle neurons are from r2. Jensen et al, Nature Neuroscience 2008

Genetic fate map of serotonergic neurons contributions from a single rhombomere We found in some cases, anatomically defined nuclei receive contribution from a single rhomomere Jensen et al, Nature Neuroscience 2008

Genetic fate map of serotonergic neurons contributions from a single rhombomere contributions from multiple rhombomeres contributions from multiple rhombomeres , but in other cases such as B9, 8, and 5, the anatomically defined nuclei receive contribution from multiple rhombomeres. Jensen et al, Nature Neuroscience 2008

Genetic Fate map = Classical, Anatomical Map Message #1: Genetic Fate map = Classical, Anatomical Map contributions from a single rhombomere contributions from multiple rhombomeres contributions from multiple rhombomeres The study provided two messages. First, the genetic fate map, based on developmental gene expression histories, is very different for the classical, anatomical map.

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

With this new genetic fate map of serotonergic neurons, we returned to the original question and ask how the embryonic gene expression pattern relate to serotonergic neurone function in the adult.. 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 BAP GFP-TOX and to address this question, instead of expressing GFP, we express tetanus toxin light chain in different sub lineage of serotonergic neurons using the same intersectional approach

Tetanus toxin light chain : GOAL - Silence individual serotonergic neuron subtypes to determine their function loxP loxP FRT FRT BAP GFP-TOX Tetanus toxin light chain : blocks neurotransmitter release and to address this question, instead of expressing GFP, we express tetanus toxin light chain in different sub lineage of serotonergic neurons using the same intersectional approach

FIRST - silence all serotonergic neurons to reveal the full extent of attainable phenotypes loxP loxP FRT FRT BAP GFP-TOX While our ultimate goal wast to silence individual SN subtypes, we first started by silencing the entire SN so we understand a full spectrum of attainable phenotypes.

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

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

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

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

(advancement over Plus maze) 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 Elevated zero maze Open field test Light-dark exploration test * *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

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

% inhibition sensorimotor gating: a neurological gating process that prevents distracting sensory inputs from generating unnecessary motor outputs operationally measured by inhibition of the acoustic startle response after a leading (prepulse) stimulus prepulse TIME Stimulus intensity 85 dB pulse Startle response 120 dB % inhibition : indication of sensorimotor gating regulation is disrupted in Schizophrenia and Huntington disease

“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 Startle Response Inhibition of background white noise control “silenced” - Pet1Flpe, hßact-cre, RC::PFtox

“silenced” all serotonergic neurons contextual learning anxiety-related behaviors sensorimotor gating So from different behavioral tests, we learned that silencing the entire serotonergic neurons affect mouse behaviors a lot. Silencing serotonergic neurons reduced the anxiety related behaviors and enhanced the contextual learning ability and sensorimotor gating.

“silenced” all serotonergic neurons contextual learning anxiety-related behaviors sensorimotor gating Do serotonergic neurons with different embryonic origins serve different functions? Now the question is whether serotonergic neurons with different embryonic origin have different functions in modulating these behaviors.

? ? ? 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 As a first step to address this question, we expressed the tetanus toxin only in the rhomobomere 1 derived serotonergic neurons and test the mice for these three behaviors in the same set of experiments we used so far.

silence all 5-HT neurons r1-derived contextual learning anxiety-related behaviors sensorimotor gating silence all 5-HT neurons r1-derived And we found that silencing r1-derived serotonergic neurons influenced the anxiety-related behaviors. For example, in the open field test, r1 silenced mice spend much more time in the open area.

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 But very interestingly, it did not change their contextual learning and sensorimotor gating.

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 This result really provide an interesting possibility that, while the anxiety related behavior may be modulated by the r1 derived s.n.,

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 the contextual learning and sensorimotor gating may be modulated more by r2 and r3-derived s.ns. Contextual learning Sensorimotor gating

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 We are now in the process of analyzing the result of silencing r2,3, and 5 derived serotonergic neurons. So the study is still in progress.

GOAL - Assigning specific serotonergic neuron subtypes to specific GOAL - Assigning specific serotonergic neuron subtypes to specific behaviors contextual learning anxiety-related behaviors sensorimotor gating X serotonergic neuron subtype ? ? And I hope that in the end, we can assign specific serotonergic neuron subtypes to specific behaviors, so we can tell this blue x neurons are important for the anxiety related behaviors, and orange neurons …

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 The first part I have presented a subtype of serotonergic neurons that we found to be important for anxiety behaviours and how they are defined by gene expression pattern in the developing embryo. In the second part of my talk, I will change a gear and talk about what we learned about anxiety circuits by tapping into the neural connection between two limbic structures: amygdala and the hippocampus.

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) Both amygdala and hippocampus activity have shown strong association with anxiety. In both rodent and primate, activation of amygdala increases anxiety and inhibition of amygdala decrease anxiety.

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) Similarly, activation of hippocampus increase anxiety and its inhibition decrease anxiety

Amygdala and HPC are reciprocally connected And these two structures are connected to each other. Basolateral nuclei of amygdala send glutamatergic projection to the ventral portion of the hippocampus. Particularly to the CA1 and subiculum of the ventral hippocampus. The ventral hippocampus project back to the basolateral amygdala Basolateral Amygdala Ventral hippocampus

Amygdala and HPC likely coordinate with each other to control anxiety behaviours Despite this strong association between two structures and anxiety, and their reciprocal connection, little has been studied how the communication between the amygala and hippocampus contribute to anxiety behaviours. Q. How does the neural communication between the two structures contributes to anxiety?

Optogenetic manipulation To better understand the role of the communication between the structures in anxiety, we manipulated selectively the hippocampal inputs arriving at the amygala using optogenetic approaches.

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

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

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

AAV-hSyn-ChR2-YFP BLA mPFC Hypothalamus

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

Optogenetic stimulation of vHPC axon terminals at the basolateral amygdala And our genetic tool kit is continuously growing. Some of them are already available, others are in development. You can silence neuron reversibly Mice were tested in 4 consecutive sessions of 5 min EPM (OFF/ON/OFF/ON)

And our genetic tool kit is continuously growing And our genetic tool kit is continuously growing. Some of them are already available, others are in development. You can silence neuron reversibly Acute stimulation of vHPC terminals at the BLA induced anxiety behaviors in the EPM

And our genetic tool kit is continuously growing And our genetic tool kit is continuously growing. Some of them are already available, others are in development. You can silence neuron reversibly Acute stimulation of vHPC terminals at the BLA induced anxiety behaviors in the EPM

Optogenetic stimulation of vHPC axon terminals at the basolateral amygdala And our genetic tool kit is continuously growing. Some of them are already available, others are in development. You can silence neuron reversibly

Summary Genetic approaches to link neurons to behaviors A neural substrate defined by gene expression pattern in the developing embryo Genetically distinct subtype of 5-HT neurons derived by rhombomere 1 OK, so far I have told you about genetic approaches you are taking to study neuron function, and showed you tools I have made for inhibition neuron activity. Now I am going to talk about experiments where I validated this tool by silencing a well defined circuit concerning motor behaviors. 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

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

Site specific DNA excision by recombinase loxP loxP DNA target excision Cre To achieve this, we employed DNA recombinase technology. DNA recombinase technology takes advantage of this simple dna excision reaction catalyzed by the enzyme, recombinase. There is for example, DNA recombinase Cre, which recognize two loxp sites and excise out the intervening DNA sequences.

Site specific DNA excision by recombinase FRT FRT DNA target excision Flp And there is another recombinase named Flp, which recognize two FRT sites and do the same thing.

Recombinase-mediated gene activation loxP loxP BAP effector OFF : Broadly Active Promoter Transcriptional STOP sequences And we can use this simple excision reaction to activation gene expression. And to do this you need three things. First, broadly active promoter, and second effector molecule, and between the promoter and effector sequences, you have this big red transcriptional stop sequences flanked by two loxp sites. This transcriptional stop sequences block RNA polymerases from passing through, so the gene expression is now off.

Recombinase-mediated gene activation loxP loxP BAP effector OFF excision Cre BAP effector ON But when you excise out the stop sequences by cre reaction. Now expression of effector is turned on.

Activity modulator (silence/activate neuron activity) And depending on the types of effector molecules you deliver, you can achieve different things. The approach is very versatile and open up many interesting possibility. Activity modulator (silence/activate neuron activity) Ablator (kill neurons) Fluorescent reporter (label neurons)

Where do serotonergic neurons originating from different rhombomeres settle in the adult brain? embryonic hindbrain Serotonergic progenitors Before we dive into asking functions, where would these different s.n originating from different rhombomere go and settle in the adult brain? Do they go to all different place or do they mix together and settle in the same area? dorsal view

Hippocampus and Anxiety Spatial learning 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)