Download File - Joris Vangeneugden

The role of serotonin in post-traumatic stress disorder
1. Background
Experiencing intense terror as during combat situations or sexual violation can have devastating
effects on a person’s mental sanity. Flashbacks, often expressed in nightmares, confront the person
with the traumatic event otherwise avoided as much as possible in both actions and thoughts. A
general hyperarousal debilitates the person and seriously constrains the outlook on the future. The
combined symptomatology is referred to as post-traumatic stress disorder, which has a lifetime
prevalence of 5-8% globally (Kessler et al., 2005) and up to 35% in post-war countries (Priebe et al.,
2010). Although not everybody experiencing the same traumatic event will eventually develop PTSD,
conversely interpersonal differences in perceiving the level of the trauma make it considerably hard
to study PTSD in experimentally reduced settings. This differential susceptibility to stressors is
governed by epigenetic mechanisms such as DNA methylation that regulate the impact of traumatic
stress on gene expression and hence brain functioning (Mehta et al., 2013).
The mechanism of learned fear allows animals to generate adaptive responses to situations
that threaten their safety on the basis of previous experiences. These responses take place,
depending on time scale at the neurochemical (e.g. serotonin, catecholamines etc.), neuroendocrine
(e.g. HPA-axis releasing corticotropin releasing hormones) and/or neuroanatomical level (e.g.
hippocampus, amygdala etc.) (Sherin and Nemeroff, 2011). Based on simple classical conditioning
principles, neutral stimuli repeatedly presented in close space-time association with the adverse
event will eventually suffice in eliciting a conditioned fear response. New, non-identical but similar
stimuli will not trigger the conditioned response in healthy subjects, but do so in subjects suffering
from PTSD (Pitman et al., 2012). It is believed that faulty pattern separation processes, resulting in
over-generalizing (Kheirbek et al., 2012) lay at the root of this pathological reaction. In such a
scenario e.g. the smell of a summer BBQ is not distinguished anymore from the smell of burning
human flesh during combat hence triggering the same aberrant behavior.
Using generalization gradient techniques this process can be studied at the behavioral level
(Honig and Urcuoli, 1981). Responses to stimuli parametrically varied in similarity to an aversively
conditioned stimulus (CS+) are compared and typically a gradient is observed, i.e. less fear response
with more dissimilar stimuli. This process of pattern separation correlates with activity in the human
hippocampal dentate gyrus (DG) and cornu ammounis (CA3) region, as revealed by fMRI (Bakker et
al., 2008; Lissek et al., 2014). Hippocampal DG is known for its characteristic encoding of experiences
in memory engrams, i.e. configurations of cells within a microcircuit representing specific memories
(Liu et al., 2012; Redondo et al., 2014).
Together with the olfactory bulb, the hippocampus retains the remarkable capacity of
generating new neurons and implementing them into existing neural circuitry. This phenomenon,
called adult neurogenesis, results from the presence of a specific neurogenic zone, i.e. the subgranular zone of the dentate gyrus (van Praag et al., 2002). Ablation of adult hippocampal
neurogenesis leads to impaired pattern separation in rodents respectively (Tronel et al., 2010) while
increasing neurogenesis results in improved discriminability between two highly similar contexts
(Sahay et al., 2011). The brain’s neurogenic capacity is influenced by a number of factors. While
stress reduces hippocampal neurogenesis (McEwen, 2001), environmental enrichment, exercise
(Kempermann et al., 1997), learning (Leuner et al., 2004) and antidepressant drugs like selective
serotonin reuptake inhibitors (SSRI) and tricyclic antidepressants (TCA) (Malberg et al., 2000;
Santarelli et al., 2003) facilitate proliferation of adult-born neurons in DG (Dranovsky and Hen, 2006).
Analysis of immunostained hippocampal tissue for neural progenitor cells (NPC) from patients with
major depressive disorder (MDD) either treated with antidepressants or left untreated clearly
showed a marked increase in the amount of NPC in the former group (Boldrini et al., 2009).
The serotonergic system originates from the dorsal raphe nucleus and innervates a
multitude of subcortical and cortical structures (Lesch and Waider, 2012) with prominent connectivity
to the prefrontal cortex in the latter group (Raghanti et al., 2008). Selective activation of this
serotonergic pathway in a transgene mouse (ePet1Cre) using optical stimulation of cells in the DRN
genetically transfected with a light-sensitive cation channel ChannelRhodopsin2 (ChR2) dramatically
improved sensory discrimination performance in an olfactory Go/No Go task (Liu et al., 2014). Lightinduced upregulation of serotonin resulted in reaching a stable performance plateau of 90% 17 times
faster than during no stimulation, mainly achieved by a marked reduction in false alarm responses.
Cross-talk between the DRN and the lateral habenula (LHb) which in turn inhibits the action of
dopamine neurons (Hikosaka, 2010) could explain these findings.
2. Purpose
Combined these findings point toward the necessity and the malleability of DG engrams firstly in
establishing mnemonic information such as a contextual fear memory and secondly in changing the
weight of this engram by a potential serotonergic network involving DRN, DG and prefrontal cortex.
In addition, the importance of memory transfers from sub-cortical to other neocortical structures
(Squire and Wixted, 2011) for long-term storage (Arruda-Carvalho et al., 2014) should not be
underestimated. The famous patient H.M. manifested complete anterograde amnesia while showing
relatively spared retrograde amnesia, indicating that recollection of past events is characterized by
the coordinated reactivation of cells distributed across the neocortex and engaged at the time of
encoding. Within the auditory system, associative fear memories can be supported by a merely
neocortical microcircuit of disinhibition (Letzkus et al., 2011). During the acquisition phase, layer 1
interneurons activated by cholinergic basal forebrain afferents inhibit layer 2/3 parvalbumin
interneurons resulting in disinhibition of pyramidal neurons. Optogenetic stimulation of this latter
group of interneurons considerably abolishes conditioned fear memory.
We speculate that the release of serotonin by the DRN increases hippocampal
neurogenesis, allowing for better pattern separation between the original stimulus eliciting the fear
responses and neutral stimuli similar but not identical to this original stimulus. This would result in
less over-generalization and reduced PTSD symptomatology. Providing the transient character of
memory storage from subcortical to cortical structures (Arruda-Carvalho et al., 2014; Vangeneugden
et al., 2015), together with the evidence of cortical microcircuits subserving fear memories (Letzkus et
al., 2011), we will also investigate the involvement of auditory cortex over time to the process of
pattern separation and potential modulation of this microcircuit by serotonin.
Building forth on the idea that faulty pattern separation lays at the root of PTSD our goal is to
utilize a six-pronged approach in order to dissect the underlying neural circuitry and formulate
clinically relevant intervention strategies. We are particularly interested in the role of serotonin and
the DRN in mediating the functional properties of this network. Due to considerable flexibility in
experimentation and previously acquired knowledge we will use the auditory system of regular
Bl6/C57 and transgene mice (ePet1Cre) as the sensory modality of choice.
3. Relevance
PTSD, the most prevalent anxiety disorder, was for long conceptualized merely in psychological
terms, i.e. as a blend of intrusive memories of a traumatic event, avoidance of reminders of it,
emotional numbing and hyper arousal (Pitman et al., 2012). Gradually more research and insight into
the underlying neurobiological mechanisms in PTSD have emerged, however these studies mainly
focused on prefrontal cortex, amygdala and dorsal anterior cingulate cortex. More recently, evidence
for the involvement of the hippocampus with its remarkable capacity to generate new neurons,
functionally relevant in separating between highly similar events has been postulated (Kheirbek et al.,
2012).
Observational studies have demonstrated that the serotonergic system impinges on
hippocampal neurogenesis (Dranovsky and Hen, 2006;Boldrini et al., 2009) however how this may
relate to altered pattern separation is still a matter of debate. In this project we wish to elucidate the
role of the serotonergic system on hippocampal neurogenesis, pattern separation and the potential
involvement of other brain structures influenced by serotonin release, such as the prefrontal and
auditory cortices, in the development of PTSD.
These results could potentially yield improvement in the therapy of PTSD in human patients,
by combining more cognitive psychotherapeutic interventions, inspired to increase pattern
separation, with a pharmacological tailored level of serotonin, using SSRIs, medication mainly used to
treat major depressive disorders.
4. Research strategy
4a Overview
In order to examine the role of serotonin in PTSD we will apply a number of state-of-the-art
neuromodulatory interventions at different nodes within the underlying neural circuitry whilst
meticulously observing behavioral changes using auditory fear conditioning protocols. After
validating our behavioral assays as good proxies for studying PTSD in a mouse model, we will use
genetic and pharmacological interventions to up- or down-regulate serotonin release within the
circuitry. In order to know where to impinge in the circuitry we will also perform structural
connectivity mapping. Once we have determined the key players within the circuit and have provided
evidence for their behavioral involvement in either facilitating or buffering PTSD symptomatology, we
will use optogenetic interventions to intervene in a more acute manner in the serotonergic system.
These behavioral observations will be supplemented with electrophysiological recordings of action
potentials from different nodes in awake behaving mice. To investigate possible functional
differences within DRN serotonergic cell populations we will utilize the very new and innovative
technique of in-vivo fluorescence microendoscopy. This technique measures fluorescence excitation
elicited by underlying neural activity and is able to capture > 75 cells simultaneously in awake and
freely moving mice. Finally we will investigate in more detail the large inter-subject variability in
developing PTSD by looking at the promising avenue of epigenetic differences, particularly in two
recently discovered genes via an unique opto-epi-genetic approach. Using optogenetics we will be
able to selectively activate these genes for a specific duration whilst documenting potential
behavioral changes.
The behavioral paradigms will be based on existing human protocols (Lissek et al., 2014), but
adapted for the mouse. In a first approach we will associate a tone of a particular frequency at either
side of a range of frequencies (5 – 15 kHz) with a mild electric shock (1 sec, .6 mA; cf. previously
published fear conditioning studies, e.g. Letzkus et al., 2011) (see 1a. Fear
generalization/discrimination test [FREQ-DISC]). The other tones will not be associated with the
shock. As an operant for fear/anxiety we will use freezing behavior, again cf. previous studies (Letzkus
et al., 2011; Wolff et al., 2014). Prey animals (mice) that experience fear will resort to feigning being
death as a final escape mechanism. The amount of freezing to other non-associated tones will provide
a measurement for generalization/separation. In a second behavioral paradigm we will employ an
auditory fear conditioning task containing a spatial component. In an open field arena all four
quadrants will be associated with a tone of particular frequency however only one quadrant will be
paired with a mild foot shock. We will investigate the time spent in the four quadrants as a measure
of spatial discrimination (see 1b. Frequency-location coupling fear conditioning test [FREQ-LOC]).
In our genetic and pharmacological approach we will utilize genetic ablation via cre-inducible
caspase injections and chemogenic activation via DREADD technology. Both techniques are explained
more in detail further in the project. We will also infuse fenfluramine, a serotonin agonist, and
clozapine, a serotonin antagonist, directly in the DRN and its projection areas of standard Bl6/C57 or
transgene ePet1Cer mice and monitor behavioral changes in the behavioral paradigms (see 3. Genetic
and pharmacological interventions).
Optogenetically, we will target the DRN and the projections of the DRN to other structures,
such as DG, prefrontal and auditory cortices, using ChannelRhodopsin2 (ChR2), an artificial light-gated
cation channel (‘genetics’) that pumps sodium into the cell when hit with blue light (‘optics’). This
then causes depolarization of the cell’s membrane and gives rise to action potentials. We will monitor
these neural manipulations on both behavior (see 4a. Optogenetic interventions: behavior) and
electrophysiological signatures (see 4b. Optogenetic interventions: electrophysiology). As the DRN
contains multiple cell types and consensus over the functional properties of these different cell
populations and their microcircuitry has not yet been reached (Liu et al., 2014; McDevitt et al., 2014)
we will first apply a general approach, targeting all DRN cells and all projections to the
abovementioned brain areas (ChR2-CaMKII virus in standard Bl6/C57 mice). In a second phase we will
employ a cre-dependent ChR2 virus (dio-ChR2 in the transgene ePET1Cre mouse strain) in order to
specifically target serotonergic cells only and their respective projections, again to these
abovementioned brain areas.
The exact projection areas from the DRN to other regions will be determined in a separate
group of ePET1Cre mice injected with virus in the DRN. Fluorescence imaging of the acquired postmortem slices will specifically instruct us which projection areas to target (see 2. Structural
connectivity).
To investigate potentially different functional serotonergic cell populations within the DRN,
e.g. projecting to the different regions, we will employ the innovative method of microendoscopy,
allowing epi-fluorescence imaging of hundred of cells simultaneously in the transgene ePET1Cre
mouse injected with a fluorescent marker (GCaMP6). A miniature microscope can be installed on the
head of a mouse that is allowed to roam freely (see 5. In-vivo fluorescence microendoscopy).
Providing that not all individuals experiencing extreme stressors will eventually develop PTSD
and otherwise not everybody who suffers from PTSD has experienced extreme traumatic events, a
considerable amount of intersubject variability could be explained by epigenetic differences. Using an
innovative combination of epigenetics and optogenetics (see 6. Opto-epi-genetic approach) we will
upregulate two genes in particular, DUSP22 and NINJ2, inspired by previous research in the
laboratory (Hammels et al., 2015). We will look at possible differences between optogenetically
upregulated and control mice in our different behavioral paradigms.
4b Detailed
1. Validation of behavioral paradigms (fear conditioning)
The first objective will focus on the development and validation of two new behavioral
contextual fear conditioning tests allowing measurement of the level of generalization between
similar but not identical stimuli.
1a. Fear generalization/discrimination test [FREQ-DISC]
In line with a recent protocol applied in human fMRI (Lissek et al., 2014) we will associate one
tone (e.g. 15 kHz) with a foot shock (CS+) in the training phase, while presenting this tone and
other tones on a parametrically varied axis (steps of 2.5 kHz to 5 kHz: 12.5, 10, 7.5 and 5, i.e. GSs
or generalization stimuli) without an aversive event during this test phase. As it has been shown
that mice can significantly discriminate between tones differing only 2% (de Hoz and Nelken,
2014) we should consider piloting and hence adjusting the frequency range. The amount of
freezing behavior is considered as an anxiety operant. We expect our behavioral data to fall along
an ascending line going from little (furthest GS-CS+ distance) to maximal freezing (closest GS-CS+
distance). The steepness of this line will be indicative of the level of generalization versus
separation: steep, shallow and flat lines will point toward high, low and no perceptual
discriminability respectively.
In another version of this test we will present auditory sweeps, again associating one
sweep, e.g. from 5 to 15 kHz with a shock (CS+) and using other sweeps, e.g. 7.5 to 12.5 kHz, 12.5
to 7.5 kHz or 15 to 5 kHz (all with the same duration) as GSs. This version of the test is inspired
by the hypothesis that stimulus complexity determines the engagement of the auditory cortex
(LeDoux, 2000).
1b. Frequency-location coupling fear conditioning test [FREQ-LOC]
Although it has been postulated that the neural algorithms for navigation in real and mental
space are fundamentally the same (Buzsaki and Moser, 2013) activity in the hippocampusentorhinal cortex system has often been considered in spatial terms only. To corroborate the two
previous tests we will devise a frequency-location test consisting of a wide open field grid
divided into quadrants, each associated with the presentation of a tone (3/4 CSs-), but only one
quadrant paired with an electrical shock (1 CS+). The level of similarity between CS+ en CSs-, i.e.
complexity, can be adjusted based on a quick pilot test. We will present different versions of this
test, each time with different similarity values between conditions, e.g. Q = quadrant: Q1-3 = 10
and Q4 = 15 or Q1-3 = 14 and Q4 = 15, all values in Hz). The operant here would be time spend in
these quadrants, an active instrumental behavior.
2. Structural connectivity
The DRN is one of many moderate-size clusters along the midline of the brainstem and harbors
the somata of most serotonergic neurons in the brain (Jacobs and Azmitia, 1992). It receives
input from a multitude of regions, notably the hypothalamus, cortex, basal ganglia and midbrain.
Considerable hyper-direct inputs from PFC and basal ganglia are present (Pollak Dorocic et al.,
2014).
DRN efferents mainly target serotonin-containing cells of the ventral midbrain, lateral
hypothalamus, midline thalamus, amygdala, striatum and most of the cortex (Vertes et al., 2010)
however these projections also include dopaminergic, GABAergic and nonserotonergic glutamate
fibers, yielding caution in extrapolating functionality from connectivity (McDevitt et al., 2014). In
order to accurately discern the contributions of the different nodes within the serotonergic
circuitry, detailed information on the serotonergic fibers per se is necessary. The ePet1Cre
transgene mouse line with expression of cre-recombinase only in serotonin-producing cells
permits exact tagging of serotonergic efferents only. Injection of a cre-dependent fluorescent dye
(similar as the optogenetic virus, i.e. AAV2.CaMKIIa.hChR2(H134R)-eYFP.WPRE.hGH) in the DRN
followed by careful whole-brain histological analysis will allow us to discern the relevant
targets for subsequent neural modulation (up- or down-regulating serotonin) via light
stimulation.
3. Genetic and pharmacological interventions
A first step into elucidating the role of serotonin in contextual fear conditioning would be to
de/in-crease serotonin first at the origin itself (DRN) and in a later phase specifically at the
efferent projection targets from the DRN (see ‘2. Structural connectivity’) and measure its effects
on behavior (see ‘1. Validation of behavioral paradigms’). To achieve these goals we will employ a
three-pronged approach: (a) genetic ablation of serotonergic cells specifically using caspase
injections, (b) chemogenic activation of the serotonergic system specifically using DREADD
methodology and (c) pharmacological infusions. The first two approaches can be applied to
study more long-term effects of the serotonergic system, while the last approach is more acute in
nature. The read-out will be the same as under phase 1, i.e. freezing (FREQ-DISC) and avoidance
(FREQ-LOC) behavior. As we speculate more involvement of the auditory cortex in the sweepsounds version of the FREQ-DISC we will also concentrate on this task version specifically, having
in effect 3 different behavioral tasks.
The caspase vector (AAV-flex-taCasp3-TEVp), a cre-dependent virus will only ablate
serotonergic cells over time in the ePet1Cre mouse line (Yang et al., 2013). Three weeks should
suffice to completely deplete serotonergic production by 5HT-cells in the DRN. On the other
hand, DREADD vectors (designer receptors exclusively activated by designer drugs) can be
selectively expressed in serotonergic cells only using a cre-inducible viral construct, AAV-hSynDIO-hM3D(Gq)-mCherry (Jennings et al., 2015) injected specifically in the DRN. The artificial
receptor can then be activated by injecting an inert molecule, clozapine-N-oxide (CNO),
upregulating serotonin for as long as the CNO is active (Alexander et al., 2009). Finally, we will
infuse the serotonin agonist fenfluramine and antagonist clozapine at these different locations
by implanting bilateral cannulae over DRN, DG, PFC and primary auditory cortex (A1). This can
be achieved in regular Bl6/C57 mice. Potential differences comparing drug infusions at different
target locations will allow us to better guide our light fibers in the subsequent, optogenetic, part
of the project.
In relation to the FREQ-DISC test we are interested whether we would observe a change
in steepness of the slope, in relation to the FREQ-LOC we are interested whether mice will avoid
the CS+ quadrant more, even with highly similar stimulus conditions. Compared to the genetic
ablation and the chemogenic activation technique, infusions of the pharmacological agents will
learn us more on the acute effects of serotonin in these tasks.
Given the involvement of A1 in sensory processing, a crucial control will be comparing
performances between easy (highly dissimilar) and difficult (highly similar) stimulus conditions
before (baseline) and after our genetic and pharmacological interventions. In line with the
hypothesis of pattern separation, one would only expect differences to baseline behavior in the
difficult but not the easy stimulus conditions (Alonso et al., 2012). Applying a similar logic to
behavioral performance in the FREQ-DISC test one would expect to see deviation from the
baseline mainly for the stimulus conditions mostly resembling CS+.
4a. Optogenetic interventions: behavior
To further investigate a more acute role of serotonin in the process of pattern separation in
PTSD and to explore the direct functionality of the different nodes within this circuitry, we will
employ an optogenetic stimulation strategy in our ePet1Cre transgene mice. Cre-dependent virus
to stimulate serotonergic cells (upregulation) within the DRN and specifically at the different
target locations, e.g. DRN-DG, DRN-PFC or DRN-A1, will be achieved by using
AAV5.EF1a.DIO.hChR2(H134R)-eYFP.WPRE.hGH. Vice versa, silencing (downregulation) at the
same locations will be achieved by using AAV5.CBA.Flex.Arch-GFP.WPRE.SV40. In contrast to
pharmacological interventions, optogenetics allows for millisecond temporal precision and thus
does not evoke massive (short- or long-term) restructuring within the network (e.g. plasticity
mechanisms). Furthermore this technique allows testing functional connectivity between
different brain regions and double fusion injections (ChR2 and Arch) will result in on/off
transistor modality (Yizhar et al., 2011).
Light fibers will be implanted bilateral in the DRN and at the different locations. Shining light
over the target locations allows for modulating only the projections from the DRN to that target
without interfering with the other nodes in the network. This entails the most accurate and pure
examination of the circuitry. Similar to our pharmacological interventions we will measure the
effect of in/de-creasing serotonin at these different locations on the different behavioral tests.
Given our more cognitive approach into PTSD, i.e. operationalizing this anxiety disorder
in terms of faulty pattern separation process, we will not focus primarily on the considerable
DRN-amygdala projections. However based on point ‘2. Structural connectivity’, light fibers can
be implanted over the amygdala to examine behavioral consequences. A similar logic can be
applied to the projections from DRN to LHb. If warranted based on our pharmacology, we will
incorporate this structure in our light-induced neuromodulation experiments.
In all our genetic, pharmacological and optogenetic interventions, control conditions consist of
sham injections of either a similar construct but without the cre-dependent cassette (i.e.
AAV2.CaMKIIa.hChR2(H134R)-eYFP.WPRE.hGH and AAV2.CAMKII.ArchT.GFP.WPRE.SV40) or
saline in different ePet1Cre mice.
4b. Optogenetic interventions: electrophysiology
Based on the results from the behavioral observations with optogenetic stimulation, we will
concentrate on the most interesting efferent projections from the DRN and on DRN proper.
Optrodes with multiple contact points for registering extracellular activity and an
attached light fiber along the shank will be inserted into DRN and one target region. We will
register single- and multi-unit and LFP activity extracellularly. Recordings without light
stimulation will yield information about the connectivity pattern (Liebe et al., 2012), while
recordings with light stimulation will reveal the causative relationship between these two
regions. These recordings will first be made under general anesthesia but also in awake mice
engaged in our behavioral tests.
5. In-vivo fluorescence microendoscopy
Using a commercially available endoscopic microscopy method (Inscopix Inc.) for imaging invivo epi-fluorescence from DRN cells (deep structures) in the ePet1Cre mouse line transfected
with a genetically encoded calcium indicator (GECI; e.g. GCaMP6), a proxy for neural activity, we
will be able to visualize and document the activity of hundred cells simultaneously while the mice
are engaged in the different behavioral tasks. This will allow us to dissociate possibly between
different serotonergic cell populations in the DRN. The primary applicant of this project has
gained experience with this innovative technique as a visiting scholar at UNC Chapel Hill in the
laboratory of Garret Stuber (see Jennings et al., 2015).
6. Opto-epi-genetic approach
Not everybody exposed to a traumatic stressor will eventually develop PTSD, conversely, not
everybody that has developed PTSD has experienced traumatic stressors of the same magnitude.
Large between-subject differences in susceptibility exist and it is believed that DNA methylation
plays an important part in this process (Hammels et al., 2015). Epigenetic changes in DUSP22
(dual specificity phosphatase 22) and NINJ2 (ninjurin 2) genes could potentially differentiate
between trauma-susceptible and trauma-resilient subjects.
Within this project we are interested in the potential effect of epigenetic states in
resilience/susceptibility to traumatic stressors. Based on a highly innovative method
(Konermann et al., 2013; Nihongaki et al., 2015), which allows optical control over epigenetic
states, we will target the abovementioned genes (DUSP22 and NINJ2) using the CRISPR
(clustered regularly interspaced palindromic repeats)-Cas9 (CRISPR-associated) system, allowing
spatiotemporal gene regulation by light delivery. We will monitor the effects of this opto-epiintervention on proneness to extreme stressors and subsequent development of PTSD
symptomatology compared to non-genetically altered mice, subjected to the same behavioral
paradigms. The CRISPR-Cas-procedure is a two-part genome-editing tool, comprising a single
guide RNA (sgRNA) that directs the Cas9 null mutant endonuclease to cause transcriptional
activation.
4c Coherence
All experiments are aimed at unraveling the role of serotonin in PTSD and fit nicely together into a
coherent project. Before advancing to the initial aim of this endeavor, i.e. the optogenetic and optoepi-genetic neuromodulation, we first need to work on two fronts.
Firstly, we need to validate our behavioral paradigms, the FREQ-DISC and FREQ-LOC tasks
(see 1. Validation of behavioral paradigms). This can be achieved in a standard mouse strain (Bl6/C57
mice), however considering that we also need to disentangle specifically the serotonergic projections
from the DRN it would be opportunistic (cf. Reduction) to use the mice from the behavioral validation
protocol also for structural connectivity mapping. Therefore it is more opportunistic to run the
behavioral protocols in ePet1Cre mice. We have worked out two different tasks in order to be able to
select the most promising task for the remainder of this project.
Secondly, we need more knowledge on the underlying structural network of serotonergic
projections arising from the DRN (see 2. Structural connectivity). This will be achieved by,
simultaneously with the behavioral validation, injecting transgene ePet1Cre mice with cre-dependent
fluorophores. Post-mortem histology will inform us on the most prevalent efferent connections from
the DRN.
The information obtained from these first two components will then be combined with
genetic manipulation tools and pharmacological injections into, first the DRN proper, but then also in
its projection areas, whilst engaging our mice in the different behavioral tasks (see 3. Genetic and
pharmacological interventions). This approach will allow us to get a general idea on the long-term
effects of artificially altered levels of serotonin release.
The optogenetic manipulations, measuring its effects both on behavior (see 4a. Optogenetic
interventions: behavior) and at the neural level (see 4b. Optogenetic interventions: electrophysiology)
will provide evidence for a possibly acute involvement of serotonin in buffering or facilitating PTSD
symptomatology.
To disentangle between potentially different neural cell populations within the DRN we will
apply an innovative imaging technique (see 5. In-vivo microendoscopy) that the primary applicant has
used as a visiting scholar at UNC Chapel Hill (USA).
Finally, motivated by the large individual differences in resilience to extreme stressors it is
believed that DNA methylation and epigenetic changes are important mediators in developing PTSD.
Using optogenetics it is now possible to epigenetically alter expression of certain genes in mice either
by up- or downregulating the transcription of these genes. Also here, we will use the same behavioral
paradigms for testing the effect of theses genes on PTSD (see 6. Opto-epi-genetic approach).
5. References
Alexander GM, Rogan SC, Abbas AL, Armbruster BN, Pei Y, Allen JA, Nonneman RJ, Hartmann J, Moy
SS, Nicolelis MA, McNamara JO, Roth BL. Remote control of neuronal activity in transgenic mice
expressing evolved G protein-coupled receptors. Neuron. 2009;63(1):27-39.
Alonso M, Lepousez G, Sebastien W, Bardy C, Gabellec MM, Torquet N, Lledo PM. Activation of adultborn neurons facilitate learning and memory. Nat Neurosci. 2012;15(6):897-904.
Arruda-Carvalho M, Akers KG, Guskjolen A, Sakaguchi M, Josselyn SA, Frankland PW. Posttraining
ablation of adult-generated olfactory granule cells degrades odor-reward memories. J Neurosci.
2014;34(47):15793-15803.
Bakker A, Kirwan CB, Miller M, Stark CE. Pattern separation in the human hippocampal CA3 and
dentate gyrus. Science. 2008;319(5870):1640-1642.
Boldrini M, Underwood MD, Hen R, Rosoklija GB, Dwork AJ, John Mann J, Arango V. Antidepressants
increase neural progenitor cells in the human hippocampus. Neuropsychopharmacol.
2009;34(11):2376-2389.
Buzsaki G, Moser EI. Memory, navigation and theta rhythm in the hippocampal-entorhinal system.
Nat Neurosci. 2013;16(2):130-138.
de Hoz L, Nelken I. Frequency tuning in the behaving mouse: different bandwidth for discrimination
and generalization. PLoS One. 2014;9(3):e91676.
Dranovsky A, Hen R. Hippocampal neurogenesis: regulation by stress and antidepressants. Biol
Psychiat. 2006;59(12):1136-1143.
Fenno LE, Mattis J, Ramakrishnan C, Hyun M, Lee SY, He M, Tucciarone J, Selimbeyoglu A, Berndt A,
Grosenick L, Zalocusky KA, Bernstein H, Swanson H, Perry C, Diester I, Boyce FM, Bass CE, Neve R,
Huang ZJ, Deisseroth K. Targeting cells with single vectors using multiple-feature Boolean logic. Nat
Meth. 2014;11(7):763-772.
Gray DC, Mahrus S, Wells JA. Activation of specific apoptotic caspases with an engineered smallmolecule-activated protease. Cell. 2010;142:637-646.
Hammels C, Prickaerts J, Kenis G, Vanmierlo T, Fischer M, Steinbusch HW, van Os J, van den Hove DL,
Rutten BP. Differential susceptibility to chronic social defeat stress relates to the number of Dnmt3aimmunoreactive neurons in the hippocampal dentate gyrus. Psychoneuroendocrino. 2015;51:547556.
Hikosaka O. The habenula: from stress evasion to value-based decision-making. Nat Rev Neurosci.
2010;11(7):503-513.
Honig WK, Urcuioli PJ. The legacy of Guttman and Kalisch (1956): Twenty-five years of research on
stimulus generalization. J Exp Anal Behav. 1981;36(3):405-445.
Jacobs BL, Azmitia EC. Structure and function of the brain serotonin system. Physiol Rev.
1992;72(1):165-229.
Jennings JH, Ung RL, Resendez SL, Stamatakis AM, Taylor JG, Huang J, Veleta K, Kantak PA, Aita M,
Shilling-Scrivo K, Ramakrishnan C, Deisseroth K, Otte S, Stuber GD. Visualizing hypothalamic network
dynamics for appetitive and consummatory behavior. Cell. 2015;160(3):516-527.
Kempermann G, Kuhn HG, Gage FH. More hippocampal neurons in adult mice living in an enriched
environment. Nature. 1997;386(6624):493-495.
Kessler RC, Sonnega A, Bromet E, Hughes M, Nelson CB. Posttraumatic stress disorder in the National
Comorbidity Survey. Arch Gen Psychiat. 1995;52(12):1048-1060.
Kheirbek MA, Klemenhagen KC, Sahay A, Hen R. Neurogenesis and generalization: a new approach to
stratify and treat anxiety disorders. Nat Neurosci. 2012;15(12):1613-1620.
Konermann S, Brigham MD, Trevino AE, Hsu PD, Heidenreich M, Cong L, Platt RJ, Scott DA, Church
GM, Zhang F. Optical control of mammalian endogenous transcription and epigenetic states. Nature.
2013;500(7463):472-476.
LeDoux JE. Emotion circuits in the brain. Annu Rev Neurosci. 2000;23:155-184.
Lesch KP, Waider J. Serotonin in the modulation of neural plasticity and networks: implications for
neurodevelopmental disorders. Neuron. 2012;76(1):175-191.
Letzkus JJ, Wolff SB, Meyer EM, Tovote P, Courtin J, Herry C, Luthi A. A disinhibitory microcircuit for
associative fear learning in the auditory cortex. Nature.2011;480(7377): 331-335.
Leuner B, Mendolia-Loffredo S, Kozorovitskiy Y, Samburg D, Gould E, Shors TJ. Learning enhances the
survival of new neurons beyond the time when the hippocampus is required for memory. J Neurosci.
2004;24(34):7477-7481.
Liebe S, Hoerzer GM, Logothetis NK, Rainer G. Theta coupling between V4 and prefrontal cortex
predicts visual short-term memory performance. Nat Neurosci. 2012;15(3):456-462.
Lissek S, Bradford DE, Alvarez RP, Burton P, Espensen-Sturges T, Reynolds RC, Grillon C. Neural
substrates of classically conditioned fear-generalization in humans: a parametric fMRI study. Soc Cogn
Affect Neurosci. 2014;9(8):1134-1142.
Liu X, Ramirez S, Pang PT, Puryear CB, Govindarajan A, Deisseroth K, Tonegawa S. Optogenetic
stimulation of a hippocampal engram activates fear memory recall. Nature. 2012;484(7394):381-385.
Liu Z, Zhou J, Li Y, Hu F, Lu Y, Ma M, Feng Q, Zhang JE, Wang D, Zeng J, Bao J, Kim JY, Chen ZF, El
Mestikawy S, Luo M. Dorsal raphe neurons signal reward through 5-HT and glutamate. Neuron.
2014;81(6): 1360-1374.
Malberg JE, Eisch AJ, Nestler EJ, Duman RS. Chronic antidepressant treatment increases neurogenesis
in adult rat hippocampus. J Neurosci. 2000;20(24):9104-9110.
McDevitt RA, Tiran-Cappello A, Shen H, Balderas I, Britt JP, Marino RA, Chung SL, Richie CT, Harvey BK,
Bonci A. Serotonergic versus nonserotonergic dorsal raphe projection neurons: differential
participation in reward circuitry. Cell Rep. 2014;8(6):1857-1869.
McEwen BS. Plasticity of the hippocampus: adaptation to chronic stress and allostatic load. Ann NY
Acad Sci. 2001;933:265-277.
Mehta D, Klengel T, Conneely KN, Smith AK, Altmann A, Pace TW, Rex-Haffner M, Loeschner A, Gonik
M, Mercer KB, Bradley B, Moeller-Myhsok B, Ressler KJ, Binder EB. Childhood maltreatment is
associated with distinct genomic and epigenetic profiles in posttraumatic stress disorder. Proc Natl
Acad Sci. 2013;110(20):8302-8307.
Milad MR, Pitman RK, Ellis CB, Gold AL, Shin LM, Lasko NB, Zeidan MA, Handwerger K, Orr SP, Rauch
SL. Neurobiological basis of failure to recall extinction memory in posttraumatic stress disorder. Biol
Psychiat. 2009;66(12):1075-1082;
Nihongaki Y, Yamamoto S, Kawano F, Suzuki H, Sato M. CRISPR-Cas9-based photoactivatable
transcription system. Chem Biol. 2015;22(2):169-174.
Pitman RK, Rasmusson AM, Koenen KC, Shin LM, Orr SP, Gilbertson MW, Milad MR, Liberzon I.
Biological studies of post-traumatic stress disorder. Nat Rev Neurosci. 2012;13(11):769-787.
Pollak Dorocic I, Furth D, Xuan Y, Johansson Y, Pozzi L, Silberberg G, Carlen M, Meletis K. A wholebrain atlas of inputs to serotonergic neurons of the dorsal and median raphe nuclei. Neuron.
2014;83(3):663-678.
Priebe S, Bogic M, Ashcroft R, Franciskovic T, Galeazzi GM, Kucukalic A, Lecic-Tosevski D, Morina N,
Popovski M, Roughon M, Schutzwohl M, Ajdukovic D. Soc Sci Med. 2010;71(12):2170-2177.
Raghanti MA, Stimpson CD, Marcinkiewicz JL, Erwin JM, Hof PR, Sherwood CC. Differences in cortical
serotonergic innervation among humans, chimpanzees, and macaque monkeys: a comparative study.
Cereb Cortex. 2008;18(3):584-597.
Redondo RL, Kim J, Arons AL, Ramirez S, Liu X, Tonegawa S. Bidirectional switch of the valence
associated with a hippocampal contextual memory engram. Nature. 2014;513(7518):426-430.
Sahay A, Scobie KN, Hill AS, O’Carroll CM, Kheirbek MA, Burghardt NS, Fenton AA, Dranovsky A, Hen
R. Increasing adult hippocampal neurogenesis is sufficient to improve pattern separation. Nature.
2011;472(7344):466-470.
Santarelli L, Saxe M, Gross C, Surget A, Battaglia F, Dulawa S, Weisstaub N, Lee J, Duman R, Arancia O,
Belzung C, Hen R. Requirement of hippocampal neurogenesis for the behavioral effects of
antidepressants. Science. 2003;301(5634):805-809.
Sherin JA, Nemeroff CB. Post-traumatic stress disorder: the neurobiological impact of psychological
trauma. Dial Clin Neurosci. 2011;13(3):263-278.
Squire LR, Wixted JT. The cognitive neuroscience of human memory since HM. Annu Rev Neurosci.
2011;34:259-288.
Tronel S, Belnoue L, Grosjean N, Revest JM, Piazza PV, Koehl M, Abrous DN. Adult-born neurons are
necessary for extended contextual discrimination. Hippocampus. 2012;22(2):292-298.
van Hagen BT, van Goethem NP, Lagatta DC, Prickaerts J. The object pattern separation (OPS) task: A
behavioral paradigm derived from the object recognition task. Behav Brain Res. 2014;S01664328(14)00706-2, doi:10.1016:j.bbr.2014.10.041.
Vangeneugden J, Mazo C, Lepousez G. Fleeting memories: transient character of adult-generated
olfactory granule cells committed to odour memories. Front Neurosci. 2015;
doi:10.3389/fnins.2015.00110.
van Praag H, Schinder AF, Christie BR, Toni N, Palmer TD, Gage FH. Functional neurogenesis in the
adult hippocampus. Nature. 2002;415(6875):1030-1034.
Vertes RP, Linley SB, Hoover WB. Pattern of distribution of serotonergic fibers to the thalamus of the
rat. Brain Struct Funct. 2008;215(1):1-28.
Wolff SB, Grundermann J, Tovote P, Krabbe S, Jacobson GA, Moeller C, Herry C, Ehrlich I, Friedrich
RW, Letzkus JJ, Luthi A. Amygdala interneuron subtypes control fear learning through disinhibition.
Nature. 2014;509(7501):453-458.
Yang CF, Chiang MC, Gray DC, Prabhakaran M, Alvarado M, Juntti SA, Unger EK, Wells JA, Shah NM.
Sexually dimorphic neurons in the ventromedial hypothalamus govern mating in both sexes and
aggression in males. Cell. 2013;153:896-909.
Yizhar O, Fenno LE, Davidson TJ, Mogri M, Deisseroth K. Optogenetics in neural systems. Neuron.
2011;71(1):9-34.
Zhang F, Cong L, Lodato S, Kosuri S, Church GM, Arlotta P. Efficient construction of sequence-specific
TAL effectors for modulating mammalian transcription. Nat Biotech. 2011;29(2):149-154.