The neuropeptide Tac2 controls a distributed brain state induced by chronic social isolation stress

The neuropeptide Tac2 controls a distributed brain state induced

by chronic social isolation stress

Moriel Zelikowsky

1,*

May Hui

Tomomi Karigo

Andrea Choe

Bin Yang

Mario Blanco

Keith Beadle

Viviana Gradinaru

Benjamin E. Deverman

, and

David J. Anderson

1,2,3,4,*

Division of Biology and Biological Engineering 156-29, California Institute of Technology

Pasadena, CA 91125, USA

Howard Hughes Medical Institute, California Institute of Technology Pasadena, CA 91125, USA

Tianqiao and Chrissy Chen Institute for Neuroscience, California Institute of Technology

Pasadena, CA 91125, USA

Abstract

Chronic social isolation causes severe psychological effects in humans, but their neural bases

remains poorly understood. Two weeks (but not 24 hrs) of social isolation stress (SIS) caused

multiple behavioral changes in mice, and induced brain-wide up-regulation of the neuropeptide

tachykinin 2 (Tac2)/neurokinin B (NkB). Systemic administration of an Nk3R antagonist

prevented virtually all of the behavioral effects of chronic SIS. Conversely, enhancing NkB

expression and release phenocopied SIS in group-housed mice, promoting aggression and

converting stimulus-locked defensive behaviors to persistent responses. Multiplexed analysis of

Tac2/NkB function in multiple brain areas revealed dissociable, region-specific requirements for

both the peptide and its receptor in different SIS-induced behavioral changes. Thus, Tac2

coordinates a pleiotropic brain state caused by SIS, via a distributed mode of action. These data

reveal the profound effects of prolonged social isolation on brain chemistry and function, and

suggest potential new therapeutic applications for Nk3R antagonists.

Correspondence: moriel@caltech.edu (M.Z.), wuwei@caltech.edu (D.J.A.).

Lead Contact

DECLARATION OF INTERESTS

The authors declare no competing interests. B.E.D. and V.G. are listed as inventors on a patent related to AAV-PHP.B

(#US9585971B2).

SUPPLEMENTAL INFORMATION

Supplemental Information includes seven figures and two tables and can be found with this article online.

AUTHOR CONTRIBUTIONS

M.Z. and D.J.A. contributed to the study design. M.Z., M.H., A.C., M.B. and B.D. contributed to the data collection, scoring and

analysis. M.Z. and M.H. conducted the surgeries, behavior experiments, and histological analyses. K.B. performed PHP.B viral

packaging and B.E.D. helped design PHP.B experiments and performed retro-orbital viral injections. T.K. contributed the shRNA and

Tac2 cDNA constructs. B.Y. contributed to packaging of viral constructs. M.B. contributed to qRT-PCR analyses. V.G. provided

funding to support K.B. and B.E.D. and comments on manuscript. M.Z. and D.J.A. wrote the paper. All authors discussed and

commented on the manuscript.

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Author manuscript

Cell

. Author manuscript; available in PMC 2019 May 17.

Published in final edited form as:

Cell

. 2018 May 17; 173(5): 1265–1279.e19. doi:10.1016/j.cell.2018.03.037.

Author Manuscript

Graphical Abstract

The Tac2 neuropeptide system orchestrates the complex behavioral effects of chronic social

isolation stress by acting locally in multiple brain regions, suggesting the therapeutic potential of

Nk3R antagonists for managing behavioral changes upon prolonged social isolation.

Keywords

Tac2; neuropeptides; social isolation; stress; aggression; fear

INTRODUCTION

Internal states of arousal, motivation and emotion exert a major influence on how the brain

processes sensory information to control behavior (

Berridge, 2004

;

Bargmann, 2012

;

LeDoux, 2012

;

Anderson and Adolphs, 2014

;

Anderson, 2016

). An important class of

internal states is that produced by exposure to psychogenic stressors (

McEwen

et al.

, 2015

Chronic stress in particular has profound, long-lasting effects on both physical and mental

health (

Selye, 1936

;

House

et al.

, 1988

;

Sapolsky, 1996

;

Cacioppo and Hawkley, 2009

;

Kessler

et al.

, 2009

;

Holt-Lunstad

et al.

, 2010

;

Cacioppo

et al.

, 2014

;

Holt-Lunstad

et al.

2015

). However, most animal models of chronic stress entail repeated administration of

acute stressors, and hence contain within them a reprieve from the stressor (

Katz

et al.

1981

). Thus, although the stress is repeatedly administered, it is intermittent.

Chronic social isolation stress (SIS) provides one of the few paradigms in which a stressor

can be applied continuously for extended periods (days or weeks) (

Hilakivi

et al.

, 1989

;

Weiss

et al.

, 2004

). Social isolation stress is widespread in humans and has detrimental

effects on health (

House

et al.

, 1988

). However its neurobiological basis remains poorly

understood. For example, there is conflicting evidence on whether or not prolonged SIS

chronically activates the HPA axis (

Hawkley

et al.

, 2012

;

Cacioppo

et al.

, 2015

). A recent

study implicated dorsal raphe dopaminergic neurons in mediating effects of relatively brief

Zelikowsky et al.

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(24 hr) social isolation in mice (

Matthews

et al.

, 2016

), but a subsequent study described a

broader role for these neurons in promoting arousal (

Cho

et al.

, 2017

Neuropeptides, most notably CRH, have been implicated in mediating stress responses in a

variety of systems (reviewed in

Kormos and Gaszner, 2013

;

Witkin

et al.

, 2014

;

Kash

et al.

2015

;

Chen, 2016

), but the logic underlying their actions is not yet clear (Fig. 1A–D).

Guided by our previous studies of aggression in

Drosophila

(

Wang

et al.

, 2008

;

Asahina

al.

, 2014

), we have investigated a potential role for tachykinins in mediating social isolation

stress (SIS)-induced aggression in mice (

Maggio, 1988

). Studies of Tac2/NkB in the central

amygdala have implicated the peptide in fear memory consolidation (

Andero

et al.

, 2014

;

Andero

et al.

, 2016

), suggesting a role in fear learning and expression. Here we report a

broader and unanticipated role for Tac2/NkB as an important peptide mediator of the effects

of chronic SIS. Tac2/NkB is dramatically up-regulated by SIS throughout the brain, and

coordinates a pervasive change in brain state, affecting not only aggression but many other

behaviors, via distributed local actions in multiple brain regions.

RESULTS

Chronic social isolation stress produces widespread effects on multiple defensive

behaviors

Prolonged SIS is known to promote multiple behavioral effects, including increased

aggression and persistent responses to threats, in both humans and animal models (

Hatch

al.

, 1963

;

Valzelli, 1969

1973

;

Weiss

et al.

, 2004

;

Matsumoto

et al.

, 2005

;

Arrigo and

Bullock, 2008

;

et al.

, 2017

). As an initial step, therefore, we examined the effects of 2

weeks of SIS in wildtype C57Bl6/N mice using multiple behavioral assays: aggression in the

resident-intruder (RI) assay (

Thurmond, 1975

), innate freezing to an overhead looming disk

(LD) (

Yilmaz and Meister, 2013

), learned freezing to a conditioned tone (2.8 kHz)

(

Fanselow, 1980

), and reactivity to a footshock (0.7mA) (Fig. 1E–I and S1B–D). SIS

produced a robust increase in offensive aggression towards a submissive intruder, compared

to non-aggressive group housed controls (Fig. 1F), confirming previous studies (

Valzelli,

1969

;

Matsumoto

et al.

, 2005

;

Toth

et al.

, 2011

). It also caused persistent freezing to both

the LD and CS (Fig. 1G, H,

post

, red bars), in contrast to GH controls where freezing

terminated with stimulus offset. However the magnitude of freezing to both the overhead LD

and the conditioned tone was unaffected by SIS (Fig. 1G, H,

during

), as was the rate and

asymptotic value of conditioned fear acquisition (Fig. S1A). SIS mice also showed

significantly enhanced reactivity to a footshock (Fig. 1I), increased freezing to a threatening

ultrasonic stimulus (USS) (

Mongeau

et al.

, 2003

) (Fig. 1J–K), increased tail rattling to the

LD (Fig. S1E), increased sensitivity to sub-threshold acoustic startle stimuli (Fig. S1I), and a

decreased latency to flinch to a mild footshock (Fig. S1H).

SIS mice were also tested for anxiety-like behavior in the open field test (OFT) and the

elevated plus maze (EPM) (Figs. 1L–N, S1F). They showed a modest but significant

reduction in time spent in the center of the OFT arena, without a change in velocity (Fig.

1M), but were no different from GH mice on the EPM test (Fig. 1N). However, SIS mice

showed an increased propensity to jump off the EPM platform (Fig. S1F). Lastly, SIS mice

spent less time interacting with a novel mouse in a social interaction assay (although their

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latency to initially approach the mouse was reduced; Fig. S1J) but more time closer to a

predator (rat) (Fig. S1G). Collectively, these findings demonstrate that SIS alters behavioral

responses to a variety of stimuli (summarized in Fig. 1O). This profile appears different

from anxiety (

Blanchard

et al.

, 2003

;

Bourin

et al.

, 2007

), consistent with earlier studies in

mice (

Hilakivi

et al.

, 1989

). Importantly, when we isolated mice for just 24 hr, we failed to

detect any of the behavioral alterations we observed following 2 weeks of SIS (Fig. S3A–E),

distinguishing this paradigm from the effects of short-term SI studied previously (

Matthews

et al.

, 2016

Chronic SIS causes widespread up-regulation of

Tac2

transcription

Drosophila,

an unbiased screen of peptidergic neurons identified DTK (

Drosophila

tachykinin)-expressing neurons, and the DTK peptide, as required for social isolation-

induced aggression (

Asahina

et al.

, 2014

). To determine whether this function might be

conserved, we investigated the role of tachykinins in SIS. In rodents, the tachykinin gene

family comprises

Tac1

and

Tac2

(

Maggio, 1988

Tac1

encodes the peptides substance P

(SP), as well as neurokinin A (NkA);

Tac2

encodes neurokinin B (NkB). These peptides

bind with the highest affinities to the G-protein coupled Nk1, Nk2, and Nk3 receptors,

respectively (Fig. 2A) (

Ebner

et al.

, 2009

Tac1

and

Tac2

are expressed in a variety of brain

regions implicated in emotion and social behavior (Fig. 2B) (

Culman and Unger, 1995

To determine whether

Tac

gene expression is influenced by SIS, we crossed

Tac2-IRES-Cre

Tac1-IRES-Cre

knock-in mice (

Tasic

et al.

, 2016

) to a Cre-reporter mouse (line Ai6)

expressing zsGreen (

Madisen

et al.

, 2010

). Double-heterozygous mice were socially isolated

for two weeks or group housed prior to sacrifice. Strikingly, freshly dissected brains from

isolated

Tac2-Cre; Ai6

(but not

Tac1-Cre; Ai6

) mice exhibited enhanced cortical reporter

expression that could be detected by the naked eye under ambient lighting (Fig. S2A).

Histology confirmed a widespread increase in zsGreen expression, in both males (Fig. 2C;

S2B) and females (Fig. S2C). Up-regulation was evident in the anterior dorsal bed nucleus

of the stria terminalis (dBNSTa), central nucleus of the amygdala (CeA), dorsomedial

hypothalamus (DMH), as well as the cortex and striatum (Fig. S2A, B). Cell-specific

markers indicated that most zsGreen expression occurred in neuronal cells (Fig. S2D).

Increased zsGreen expression was also detected in peripheral endocrine tissues, such as the

pancreas, testes and submandibular gland (not shown).

Similar results were obtained using a different Cre reporter mouse, Ai14 (

Madisen

et al.

2010

) expressing tdTomato (Fig. S2E), indicating that the induction was not a peculiarity of

the Ai6 line. Importantly, no such change was observed in SIS

Tac1-Cre; Ai6

mice (Figs.

2D; S2A). These data suggest that the induction of zsGreen observed in SIS mice is specific

to the

Tac2

Cre

allele, and is not a non-specific effect of SIS to increase Cre-mediated

recombination at the

Rosa-26

locus, or a peculiarity of the zsGreen reporter.

To confirm that SIS up-regulated endogenous

Tac2

expression, we quantified Tac2 mRNA in

selected brain regions using qRT-PCR and RNA fluorescent in situ hybridization (FISH).

QRT-PCR indicated that SIS caused a large (~3–8 –fold) and significant increase in Tac2

mRNA levels in the dBNSTa, DMH and CeA, with trends to an increase in the ACC and

dHPC (Fig. 2E). An increase in NkB protein expression was also observed in these regions

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by immunostaining (Fig. S2I). A time-course revealed a gradual increase in Tac2 mRNA

from 30 min to 2 weeks of SIS (Fig. S2F). No increase in Tac1 mRNA was observed

following SIS (Fig. 2F, S2G).

Endogenous Tac2 mRNA up-regulation was also observed by FISH in in dBNSTa, CeA and

DMH (Figs. 2G–P). Up-regulation in CeA was significant in both its medial and lateral

subdivisions (

Kim

et al.

, 2017

). The fold-increase in fluorescence intensity per mm

was

much greater than the fold-increase in the number of Tac2 mRNA

cells (Fig. 2L–P). In

contrast, the Cre reporter transgene, which integrates and amplifies changes in expression,

yielded a larger fold-increase in the number of positive cells (Fig. S2B). This difference was

particularly evident in the ACC or dHPC (Fig. 2J–K, O–P and Fig. S2B), suggesting

amplification of induction by the Cre reporter. Despite these quantitative differences, the

mRNA data confirm that SIS up-regulates endogenous Tac2 expression in multiple brain

regions.

Acute systemic antagonism of Nk3Rs attenuates the effects of SIS

To investigate a causal role for NkB in mediating behavioral effects of SIS, we systemically

administered osanetant (Fig. 3A) (

Emonds-Alt

et al.

, 1995

), a specific Nk3R antagonist that

crosses the blood-brain barrier (

Spooren

et al.

, 2005

). Osanetant delivered after SIS, but 20

min prior to each test, strongly reduced aggression enhanced by SIS (Fig. 3B), but not by

sexual experience (

Remedios

et al.

, 2017

) (Fig. S3J–K). It also attenuated persistent freezing

to both the LD and the fear-conditioned tone (Fig. 3C, D,

post,

green bars), but not acute

freezing during stimulus presentation. Osanetant also attenuated other SIS-induced

behaviors including increased shock reactivity (Fig. 3E), increased tail-rattling (Fig. S3G),

decreased social interaction (Fig. S3H) and enhanced responding in the acoustic startle assay

(Fig. S3I). Thus, systemic antagonism of Nk3Rs blocked virtually all of the measured

behavioral effects of chronic SIS, while leaving non-SIS altered behaviors intact (Fig. S3K).

Notably, osanetant also blocked persistent freezing to the LD caused by prior footshock (Fig.

S3L–M), suggesting involvement of NkB in responses to other stressors.

Chronic systemic antagonism of Nk3Rs during SIS has a protective effect

To investigate whether Tac2 signaling is required during SIS, mice were administered

osanetant daily in their home cage during the two-week isolation period, but were then

tested off-drug. To control for carry-over of the drug from the last home-cage administration

into the testing period (24 hrs later), an additional group of SIS mice was given a single

home-cage administration of osanetant 24 hours prior to testing (Fig. 3F).

Treatment with daily osanetant prevented SIS-enhanced aggression (Fig. 3G), persistent

freezing to the LD (Fig. 3H), and persistent freezing to the fear conditioned tone (Fig. 3I).

The SIS-induced increase in shock reactivity was reduced, but not significantly (Fig. 3J, K).

Strikingly, mice that had been treated with osanetant during SIS could be returned to

housing with their pre-isolation cagemates without any subsequent fighting observed, in

contrast to control SIS mice which vigorously attacked their cagemates when reintroduced

to the group (data not shown).

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Nk3Rs act in different brain regions to mediate effects of SIS on different behaviors

We next asked where in the brain NkB signaling is required to mediate the behavioral effects

of SIS. The dBNSTa, DMH, and CeA exhibited strong induction of Tac2 by SIS (Fig. 2),

and also contain cells expressing Nk3Rs (Fig. S4A). Since Tac2

neurons in CeA and

dBNSTa project to multiple distal targets (Fig. S4B–D; Table S2), as a first step, we

pharmacologically inhibited Nk3Rs locally in these Tac2-expressing regions. SIS mice

received bilateral microinfusions of osanetant into a given region 20 minutes prior to each

behavioral test (Fig. 4A). We selected four assays – the RI assay, LD, fear conditioning, and

shock reactivity – because they exhibited robust SIS-induced changes and could be

performed sequentially within the same animals without affecting each other (as indicated

by initial experiments in which the assays performed following aggression testing were

performed independently, Fig. S1C–D).

Local infusion of osanetant in dBNSTa selectively inhibited persistent, but not acute,

freezing to both the LD and the conditioned tone (Figs. 4C–D), but had no effect on

aggression (Fig. 4B) or shock reactivity (Fig. S4F). By contrast, osanetant microinfused into

the DMH abolished aggression (Fig. 4E), but had no effect on persistent responses to the LD

(Fig. 4F) or the conditioned tone (Fig. 4G), or on footshock reactivity (Fig. S4G). (However,

DMH-infused mice showed an increase in the latency to first orient and freeze to the LD;

Fig. S4E). Lastly, osanetant infusion into the CeA reduced acute (and thereby persistent)

freezing to the innate and conditioned threatening stimuli, as well as reactivity to the

footshock (Figs. 4H–K, S4H), but not aggression. Infusion of osanetant into the ACC or

striatum failed to yield significant effects on SIS-induced persistent freezing to the LD (Figs.

S4I–J).

Region-specific chemogenetic silencing of Tac2

neurons blocks distinct behavioral

responses to SIS

To determine whether Tac2 up-regulation in dBNSTa, DMH and CeA reflected a

requirement for NkB release in these structures, we first asked whether the activity of Tac2

neurons in these regions was required for the effects of SIS. Tac2/c-fos dFISH experiments

revealed a significant induction of c-fos in Tac2

cells in dBNSTa and CeA following

exposure to the LD or conditioned tone, but not during aggression. Conversely, Tac2

cells

in DMH were activated during aggression but not during threat exposure (Fig S5A–D).

To determine whether silencing Tac2

cells could prevent the effects of SIS, Tac2-IRES-Cre

mice were bilaterally injected in dBNSTa, CeA or DMH with a Cre-dependent AAV

encoding hM4DREADD (AAV2-DIO-hM4D-mCherry) (

Conklin

et al.

, 2008

). Following 3

weeks to allow viral expression (Fig. S5E) and 2 weeks of SIS, mice were tested for SIS-

induced behavioral changes 20 minutes after injection of clozapine-N-oxide (CNO) or

vehicle (Fig. 5A).

Chemogenetic silencing of Tac2

cells in dBNSTa, DMH, and CeA essentially phenocopied

the effect of local osanetant infusions. In dBNSTa, persistent freezing responses were

selectively attenuated (Figs. 5B–D,

post

), while in DMH aggression was inhibited (Figs. 5E–

G), and in CeA acute freezing and shock reactivity were suppressed (Figs. 5H–K, S5H). No

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effects of CNO were observed in control mice subjected to the same series of behavioral

assays (Fig. 5SI–N), excluding off-target effects (

Gomez

et al.

, 2017

). Thus the activity of

Tac2

neurons, like Nk3R function, is differentially required in different brain regions for

different behavioral effects of SIS.

Tac2 synthesis is differentially required in dBNSTa, CeA, and DMH

We asked next whether Tac2 synthesis was required in each of the three brain regions

studied, via targeted shRNAi-mediated knockdown of Tac2. Mice were injected

stereotaxically in dBNSTa, DMH, or CeA with adeno-associated viruses (AAVs) expressing

small hairpin RNAs (shRNAs), together with a CMV promoter-driven zsGreen fluorescent

reporter (AAV5-H1-shRNA-CMV-zsGreen). Two shRNAs (shRNA-1 and shRNA-2) proved

effective as determined by FISH and qRT-PCR, with shRNA-2 yielding the strongest

reductions in Tac2 mRNA (Fig. S6E–G). Control mice were injected with an AAV encoding

an shRNA targeted to the

luciferase

gene. Injections were histologically verified by zsGreen

fluorescence. The number of zsGreen

neurons was not significantly different between

animals injected with control vs. experimental shRNA’s, suggesting that the reduction in the

number of Tac2 mRNA

cells was not due to cell death (Fig. S6D).

In DMH both shRNAs strongly attenuated SIS-induced aggression, but had no significant

effect on freezing (Fig. 6E–G), similar to the effect of Tac2

neuron silencing or local

infusion of osanetant in this region (Fig. 4E–G and Fig. 5E–G). Conversely, in the dBNSTa

shRNA-1 strongly reduced persistent freezing to both the LD and the conditioned tone (Fig.

6C–D, red bars,

post

), but had no effect on acute freezing to the threatening stimuli (Fig. 6C,

D, red bars,

during

), or on SIS-induced aggression (Fig. 6B). Notably, unlike the case with

Tac2

neuron silencing and local osanetant infusion, the stronger shRNA-2 expressed in

dBNSTa significantly reduced acute as well as persistent freezing to both the LD and

conditioned tone (Fig. 6C–D, I–J,

during

, orange bars). In CeA, Tac2 shRNA2 reduced acute

freezing during and after stimulus presentation (Fig. 6I), but had no effect on aggression

(Fig. 6H). The fact that local inhibition of Tac2 synthesis or of Tac2

neuronal activity

yielded similar behavioral effects (Fig. 5K, 6K) supports a requirement for Tac2 release in

the effects of SIS.

Enhancement of Tac2 expression and Tac2

neuronal activity mimics the effects of SIS

The foregoing findings indicate that Tac2 is required for the collective behavioral effects of

SIS. However, because there is baseline Tac2 expression in these regions in GH mice (Fig.

2B, E), these data do not distinguish whether Tac2 up-regulation

per se

mediates the effects

of SIS, or whether Tac2 is simply permissive. Therefore, we asked whether increasing the

level and/or release of Tac2 was sufficient to mimic any of the behavioral effects of SIS, in

group housed animals.

To do this, we injected intravenously into GH Tac2-IRES-Cre (gene-conserving) driver mice

(Fig. S7) Cre-dependent vectors encoding the DREADD neuronal activator hM3D; a Tac2

cDNA or control mCherry using the AAV serotype PHP.B, which crosses the blood-brain

barrier (

Deverman

et al.

, 2016

). Following three weeks to allow for viral expression, all mice

(including mCherry-expressing controls) were given CNO in their drinking water for 2

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weeks. Mice were then behaviorally tested 20 min following an i.p. CNO injection (Fig.

7A). This procedure was designed to achieve brain-wide Tac2 over-expression and/or

neuronal activation in Tac2

cells, during both a two-week mock SIS period, as well as

during behavioral testing.

Remarkably, combined over-expression of Tac2 and chemogenetic activation of Tac2

neurons recapitulated key behavioral effects of SIS in GH mice, including increased

aggression and persistent freezing to threats (Fig. 7B–D; summarized in Fig. 7F). In

contrast, increasing Tac2 expression, or activating Tac2

neurons, on its own was insufficient

to yield SIS-like effects in any of our assays (Figs. 7B–E, lavender and cyan bars), as was

injection of mCherry-only virus. Histological analysis confirmed expression of mCherry-

tagged AAV cargo genes in the dBNSTa, CeA and DMH (Fig. S7A–C), as well as in several

additional regions (Fig. S7D–E). The absence of any effects in CNO-treated mCherry virus-

injected mice rules out off-target effects of the drug (

Gomez

et al.

, 2017

DISCUSSION

Identification of Tac2/NkB as a key mediator of brain responses to chronic SIS

A large number of neuropeptides have been implicated in stress responses, most prominently

CRH (reviewed in (

Kormos and Gaszner, 2013

;

Kash

et al.

, 2015

;

Chen, 2016

)). Prior work

on the tachykinins in stress has focused primarily on Tac1/Substance P/NkA (

Bilkei-Gorzo

et al.

, 2002

;

Beaujouan

et al.

, 2004

;

Ebner

et al.

, 2004

;

Ebner

et al.

, 2008

). Previous

pharmacological and genetic studies have yielded conflicting results regarding the direction

of NkB influences on stress responses (

Ebner

et al.

, 2009

). Motivated by our previous results

Drosophila

(

Asahina

et al.

, 2014

)

we identified Tac2/NkB as an important and previously

unrecognized mediator of chronic SIS influences on the brain. The finding that tachykinins

play a role in the control of social isolation-induced aggression in both flies and mice is

consistent with evidence supporting an evolutionary conservation of neuropeptide function

in behavior across phylogeny (reviewed in (

Bargmann, 2012

;

Katz and Lillvis, 2014

)). It is

conceivable that Tac2/NkB may play a role in the well-known effect of solitary confinement

to increase violence in humans (

Arrigo and Bullock, 2008

CRH is considered the prototypic stress peptide (

Chen, 2016

). Approximately 50% of Tac2

cells in dBNSTa and CeA co-express CRH (Fig. S7F), raising the possibility that co-release

of CRH may play some role in effects of SIS exerted via these structures. However, Tac2

shRNAi and osanetant injections yielded similar effects as Tac2

neuronal silencing, while

activation of Tac2

neurons had no effect unless a Tac2 cDNA was co-expressed. Therefore

co-release of CRH is unlikely to explain the results of our chemogenetic manipulations of

Tac2

neuronal activity. Nevertheless, we cannot exclude that CRH may act genetically

upstream or downstream of Tac2 in these structures, to mediate the influences of SIS.

Interestingly, there was virtually no expression of CRH among Tac2

neurons in DMH,

where NkB controls aggression (Fig. S7F).

Our SIS paradigm differs from acute and repeated intermittent stressors (e.g., footshock,

restraint, forced swim) not only in its quality, but also in its extended duration and

continuous nature. The engagement of the Tac2/NkB system in chronic SIS, therefore, could

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reflect any of these differences. However, the fact that systemic delivery of osanetant

blocked acute footshock-induced persistent freezing in the LD assay suggests a more general

role for the peptide in responses to stressors. A role for Tac2/NkB in consolidation of a

conditioned fear memory in CeA has been reported (

Andero

et al.

, 2014

), but this effect was

interpreted to reflect a role in memory consolidation, rather than in stress. Understanding the

role of Tac2 in other forms of chronic and acute stress will be an interesting topic for future

studies.

Tac2/NkB acts in a distributed manner to control multiple components of the SIS response

With few exceptions (

Regev

et al.

, 2011

;

Regev

et al.

, 2012

), most previous studies of

neuropeptides in stress have focused on a single brain region, stressor and/or behavior (e.g.,

the BNST and anxiety assays; reviewed in

Kash

et al.

, 2015

), and have used a single type of

functional perturbation (but see (

McCall

et al.

, 2015

)). This, together with the variations in

stress and behavioral paradigms used in different laboratories, makes it difficult to

synthesize studies of the same peptide in different regions to understand how a peptide acts

more globally in the brain (

Kormos and Gaszner, 2013

;

Chen, 2016

). The multiplexed

approach used here permitted comparison of the same perturbation in different brain regions,

and of different perturbations in the same brain region, using a battery of behavioral assays.

This approach revealed a distributed mode of action in which up-regulation of Tac2 by stress

regulated different behavioral effects of SIS in different areas. Such a distributed mechanism

is reminiscent of that played by Pigment-Dispersing Factor (PDF) in controlling circadian

circuits in

Drosophila

(

Taghert and Nitabach, 2012

;

Dubowy and Sehgal, 2017

)

or roaming

vs. dwelling states in

C. elegans

(

Flavell

et al.

, 2013

), and may also explain some of the

diverse functions of CRH (

Regev

et al.

, 2011

;

Flandreau

et al.

, 2012

;

McCall

et al.

, 2015

The fact that local inhibition of Tac2/NkB synthesis, Tac2

neuronal activity and NkB

receptors yielded qualitatively similar results in each brain region is suggestive of local

actions of Tac2. In other systems, NkB acts in an autocrine or paracrine manner on Nk3R-

expressing neurons to increase their activity (

Navarro, 2013

); whether this occurs in the

regions studied here is not yet clear. Importantly, our results do not rule out requirements for

NkB signaling at distal targets of Tac2

neurons as well. The biochemistry of Nk3R action

suggests that Tac2/NkB should increase intracellular free calcium via an IP3/DAG pathway

(

Ebner et al., 2009

). In this way, NkB could potentiate the activation of target neurons by

glutamate or other excitatory transmitters, and/or promote release of additional peptides.

Activation and peptide overexpression in Tac2

neurons mimics the effects of SIS

Injection of stress peptides or receptor agonists can elicit behavioral responses (reviewed in

(

Koob, 1999

;

Bruchas

et al.

, 2010

;

Kormos and Gaszner, 2013

)). However, in most studies,

injection or transgenic overexpression of a stress peptide does not fully mimic the behavioral

effects of stressors. For example, even CRH when exogenously administered to unstressed

animals in low arousal conditions does not produce stress-like responses (

Koob, 1999

Using a novel experimental design, we found that overexpression of Tac2 combined with

neuronal activation in Tac2

cells, but neither manipulation on its own, sufficed to mimic

several of the behavioral effects of SIS, in GH mice. This suggests that neuronal activity

Zelikowsky et al.

Page 9

Cell

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Author Manuscript

may be limiting for observing behavioral effects of neuropeptide overexpression in other

systems. This may explain why overexpression of CRH using genetic methods produced

different responses, depending on the mode and site of expression (

Regev

et al.

, 2011

;

Flandreau

et al.

, 2012

;

Regev

et al.

, 2012

;

Sink

et al.

, 2012

;

Kash

et al.

, 2015

Our experiments were enabled by a strategy that allows independent manipulation of Tac2

expression and Tac2

neuronal activity, in a brain-wide, noninvasive manner in adult mice

(

Deverman

et al.

, 2016

;

Chan

et al.

, 2017

), without the need to employ complex transgenic

strategies (

et al.

, 2008

). More spatially and temporally resolved applications of this

approach should reveal precisely where and when enhancing NkB signaling exerts its

effects. We anticipate that this approach will prove useful for studying other neuropeptides

as well.

Increased Tac2/NkB signaling converts stimulus-locked to persistent threat responses

It was striking to observe that acute freezing responses to threats in GH animals could be

converted to persistent ones, simply by artificially increasing Tac2 expression and release.

Preliminary data indicate that Tac2 is required for acute freezing in GH animals as well.

Together, these data suggest that the up-regulation of Tac2 expression caused by SIS may

serve to convert defensive reactions to threats from transient to more enduring responses

(Fig. 7H, lower). In this way, the scalable property of neuropeptides – their concentration

can vary continuously – may be used to promote persistence, a key component of emotion

and related internal states (

Anderson and Adolphs, 2014

With one exception (see below), manipulations of NkB signaling in dBNSTa reduced

persistent but not acute (during stimulus) freezing, while manipulations of CeA reduced

freezing during as well as following threat stimulus presentation This dissociation appears

consistent with the prevailing view that CeA controls phasic, stimulus-locked defensive

responses to threats (“fear”), while dBNSTa controls more persistent responses (“anxiety”)

(

Walker

et al.

, 2009

;

Kash

et al.

, 2015

). However, in dBNSTa the more potent Tac2

shRNA-2 inhibited acute as well as persistent freezing, while the less potent shRNA-1 only

reduced post-stimulus freezing. These data suggest that the effects of Tac2/NkB signaling on

acute vs. enduring responses to threats are not determined simply by the region(s) in which

the neuropeptide acts, but also by the level of peptide expression and by potentially different

thresholds for neuropeptide effects in each area (Fig. 7H). However we cannot exclude the

possibility that reciprocal connections between CeA and dBNST (

Dong

et al.

, 2001

;

Dong

and Swanson, 2006b

) may also contribute to the partially overlapping shRNAi phenotypes

we observed (Fig. 7G).

Nk3R antagonists as potential treatments for isolation-related stress

Social isolation is well known to promote poor health, clinical psychiatric symptoms and

increased mortality in humans (

Cacioppo and Hawkley, 2009

;

Umberson and Montez, 2010

;

Cacioppo

et al.

, 2015

;

Holt-Lunstad

et al.

, 2015

). Osanetant and several other Nk3R

antagonists have been tested in clinical trials as therapies for schizophrenia, bipolar and

panic disorder (

Spooren

et al.

, 2005

). These drugs were well tolerated but abandoned for

lack of efficacy (

Griebel and Holsboer, 2012

). The profound effect of osanetant to prevent

Zelikowsky et al.

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Author Manuscript

and reverse an SIS-induced deleterious brain state suggests that Nk3R antagonists may merit

re-examination as potential treatments for mood disorders caused by extended periods of

social isolation (or other stressors) in humans, and in domesticated animals as well.

STAR METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Antibodies

Rabbit polyclonal anti-proNKB

Invitrogen

PA1-16745

Rabbit polyclonal anti-NeuN

Millipore

ABN78

Chicken polyclonal anti-PLP

Millipore

AB15454

Rabbit polyclonal anti-NFIA

Deneen B., Baylor

N/A

Goat anti-rabbit, Alexa Fluor 594

Invitrogen

R37117

Goat anti-chicken, Alexa Fluor 594

Invitrogen

A-11042

Bacterial and Virus Strains

AAV2-EF1a-DIO-hM4D(Gi)-mCherry

UNC Vector Core

N/A

AAV2-EF1a-DIO-mCherry

UNC Vector Core

N/A

AAV1-CAG-FLEX-eGFP

UNC Vector Core

N/A

AAV5.H1.Tac2-shRNA1.CMV.ZsGreen.SV40

This paper

N/A

AAV5.H1.Tac2-shRNA2.CMV.ZsGreen.SV40

This paper

N/A

AAV5.H1.shRLuc.CMV.ZsGreen.SV40

This paper

N/A

AAVPHP.b-hSyn-Tac2-P2A-mCherry

This paper

N/A

AAVPHP.b-hSyn-Tac2-P2A-GFP

This paper

N/A

AAVPHP.b-hSyn-DIO-hM3D(Gq)-mCherry

This paper

N/A

AAVPHP.b -hSyn-DIO-mCherry

This paper

N/A

Chemicals, Peptides, and Recombinant Proteins

DAPI

Sigma

D9542

Vectashield

Vector Labs

H-1000

Fluoro-Gel with Tris Buffer

Electron Microscopy Sciences

17985-10

Clozapine N-oxide

Enzo

NS105-0005

Osanetant

Axon

1533; SR 142801

Senktide

Tocris

1068

Digoxigenin-labeled Tac2 RNA probe

This paper

N/A

Digoxigenin-labeled Cfos RNA probe

This paper

N/A

Digoxigenin-labeled CRH RNA probe

This paper

N/A

DNP-labeled Tac2 RNA probe

This paper

N/A

DIG RNA Labeling Mix

Roche

11277073910

DNP-11-UTP

PerkinElmer

NEL555

T7 RNA Polymerase

Roche

10881767001

Anti-digoxigenin-POD

Roche

11207733910

Zelikowsky et al.

Page 11

Cell

. Author manuscript; available in PMC 2019 May 17.

Author Manuscript

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Anti-DNP antibody, HRP conjugate

PerkinElmer

FP1129

Anti-DNP antibody, Alexa Fluor 488 conjugate

Invitrogen

A11097

Sheep serum

Sigma

S3772

TSA Blocking Reagent

PerkinElmer

FP1020

TSA Plus Biotin Kit

PerkinElmer

NEL749A001KT

TSA Plus DNP (HRP) kit

PerkinElmer

NEL747B001KT

Avidin/biotin blocking kit

Vector Labs

SP-2001

TSA Plus DNP (HRP) System

PerkinElmer

NEL747A001KT

Avidin/Biotin Blocking Kit

Vector Labs

SP-20001

Streptavidin Alexa Fluor 488 conjugate

Invitrogen

S11223

Streptavidin Alexa Fluor 594

Jackson ImmunoResearch

016-580-084

Proteinase K

NEB

P8107S

Yeast tRNA

Sigma

R8759

Calf Thymus DNA

Invitrogen

15633019

Dextran sulfate

Sigma

D8906

Denhardt’s Solution 50x

Sigma

D2532

PrimeSTAR Max DNA Polymerase

Takara

R045A

GeneArt Seamless Cloning and Assembly Kit

Invitrogen

A13288

RNAlater

Qiagen

76106

RNAeasy Plus Mini Kit

Qiagen

74134

TURBO DNase

Thermo Fisher

AM2238

Murine RNase Inhibitor

NEB

M0314L

Dynabeads MyOne Silane

Thermo Fisher

37002D

LightCycler 480 SYBR Green

Roche

4887352001

Superscript III Reverse Transcriptase

Life Technologies

18080093

Tac1 Primer

Integrated DNA Technologies

www.idtdna.com

Tac2 Primer

Integrated DNA Technologies

www.idtdna.com

GAPDH Primer

Integrated DNA Technologies

www.idtdna.com

18s Primer

Integrated DNA Technologies

www.idtdna.com

Experimental Models: Organisms/Strains

C57BL/6N

Charles River

N/A

Balb/c

Charles River

N/A

Tac2-Cre

Harris et al., 2014

N/A

Tac1-Cre

Harris et al., 2014

N/A

Ai6-zsGreen reporter

Madisen et al., 2010

N/A

Ai14-tdTomato reporter

Madisen et al., 2010

N/A

Recombinant DNA

pAAV.H1.Tac2-shRNA1.CMV.ZsGreen.SV40

This paper

N/A

pAAV.H1.Tac2-shRNA2.CMV.ZsGreen.SV40

This paper

N/A

Zelikowsky et al.

Page 12

Cell

. Author manuscript; available in PMC 2019 May 17.

Author Manuscript

REAGENT or RESOURCE

SOURCE

IDENTIFIER

pAAV.H1.shRLuc.CMV.ZsGreen.SV40

U Penn Vector Core

PL-C-PV1781

pAAV-GFP

Cell Biolabs Inc

AAV-400

pAAV-hSyn-Tac2-P2A-mCherry

This paper

N/A

pAAV-hSyn-Tac2-P2A-GFP

This paper

N/A

pAAV-hSyn-DIO-hM3D(Gq)-mCherry

Addgene

44361

pAAV-hSyn-DIO-mCherry

Addgene

50459

Software and Algorithms

Image J

NIH

https://imagej.nih.gov/ij

Prism 6

GraphPad Software

www.graphpad.com

MATLAB

MathWorks

www.mathworks.com

EthoVision XT

Noldus

www.noldus.com

Looming Code, MATLAB

Meister M., Caltech

N/A

Behavior Annotator, MATLAB

Perona P., Caltech

N/A

Behavioral Analysis Code, MATLAB

This paper

N/A

Metamorph

Technical Instrument

www.techinst.com

SiDirect 2.0

http://sidirect2.rnai.jp/

Other

Cannulae (guide, dummy, internal)

Plastics One

N/A

Microinfusion Pump

Harvard Apparatus

www.harvardapparatus.com

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for reagents may be directed and will be fulfilled by the

Lead Contact David J. Anderson (wuwei@caltech.edu).

EXPERIMENTAL MODEL AND SUBJECT DEATILS

Animals—

Wildtype (WT) C57BL/6N male mice (experimental), C57BL/6N female mice

(for sexual experience), and BALB/c male mice (intruders) were obtained from Charles

River (at 6–10 weeks of age). For visualization of Tac2 and Tac1 expression, we used

previously described Cre-dependent Ai6-zsGreen and Ai14-tdTomato fluorescent reporter

mice (

Madisen et al., 2010

), Tac2-IRES2-Cre (

Cai et al., 2014

), and Tac1-IRES2-Cre

knockin mice (obtained from the Allen Institute for Brain Science), which were backcrossed

to the C57BL/6N background in the Caltech animal facility. Tac2-IRES-Cre mice were used

for Cre-dependent LOF/GOF experiments (Figs. 5, 7). Animals were housed and maintained

on a reverse 12-hr light-dark cycle with food and water

ad libitum

. Behavior was tested

during the dark cycle. Care and experimental manipulation of animals were in accordance

with the National Institute of Health Guide for Care and Use of Laboratory Animals and

approved by the Caltech Institutional Animal Care and Use Committee.

Social isolation stress

WT males (Charles River) were housed in isolation (1 animal per cage), or in groups of 3.

Tac2-IRES-Cre males (bred in-house) were housed in isolation, or in groups of 2–5.

Zelikowsky et al.

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Author Manuscript

Animals were isolated post-weaning, at 8–16 weeks of age. All cage conditions remained

otherwise identical for group housed mice compared to isolated animals, and mice were

housed on the same rack in the same vivarium. Except where otherwise indicated, social

isolation was maintained for at least 2 weeks (this period was extended in the case of

surgical experiments, i.e. when adequate time for recovery and viral expression levels were

required). All mice were between 12–20 weeks of age at the time of behavioral testing.

METHOD DETAILS

Viral constructs—

The AAV2-EF1a-DIO-hM4D(Gq)-mCherry and AAV2-EF1a-DIO-

mCherry were acquired from the University of North Carolina (UNC) viral vector core. The

pAAV-Tac2-shRNA1-CMV-zsGreen, pAAV-Tac2-shRNA2-CMV-zsGreen, and pAAV-

shRLuc-CMV-zsGreen plasmids were constructed (see construction below) and serotyped

with AAV5 coat proteins and packaged in-house (see viral packaging below). The pAAV-

hSyn-Tac2-P2A-mCherry and pAAV-hSyn-Tac2-P2A-GFP plasmids were constructed (see

below) and packaged into AAV-PHP.B (see PHP.B section below). The pAAV-hSyn-DIO-

hM3D( Gq)-mCherry and pAAV-hSyn-DIO-mCherry were acquired from Addgene and

packaged into AAV-PHP.B (see below).

Construction of small hairpin RNA expressing AAV vector—

Small hairpin RNA

(shRNA) for mouse Tac2 gene (NM_009312.2) were designed using online designing tool

siDirect 2.0 (

http://sidirect2.rnai.jp/

) (

Naito et al., 2009

Oligonucleotides encoding Tac2 shRNAs were purchased from IDT. Oligonucleotides used

were as follows: shRNA1, 5

′

CCGACGTGGTTGAAGAGAACACCGCTTCCTGTCACGGTGTTCTCTTCAACCACGT

C TTTTTT -3

′

and 5

′

AAAAAAGACGTGGTTGAAGAGAACACCGTGACAGGAAGCGGTGTTCTCTTCAAC

C ACGTCGG -3

′

; shRNA2, 5

′

CCGCCTCAACCCCATAGCAATTAGCTTCCTGTCACTAATTGCTATGGGGTTGAGGC

TTTTTT -3

′

and 5

′

AAAAAAGCCTCAACCCCATAGCAATTAGTGACAGGAAGCTAATTGCTATGGGGTT

G AGGCGG -3

′

pAAV.H1.shRLuc.CMV.ZsGreen.SV40 (Luc shRNA) plasmid (PL-C-

PV1781, Penn Vector Core) was used as shRNA AAV vector backbone and control shRNA

construct.

Entire Luc shRNA plasmid except luciferase shRNA sequence was amplified by PCR with

the following primers: shRNA1, Forward -

AACCACGTCTTTTTTAATTCTAGTTATTAATAGTAATCAA; Reverse -

CTTCAACCACGTCGGCTGGGAAAGAGTGGTCTC; shRNA2, Forward -

GGTTGAGGCTTTTTTAATTCTAGTTATTAATAGTAATCAA ; Reverse -

ATGGGGTTGAGGCGGCTGGGAAAGAGTGGTCTC. All PCR reactions were performed

using PrimeSTAR Max DNA Polymerase (Takara Bio, Kusatsu, Japan). After PCR

amplification, template plasmid was digested by DpnI (NEB, Ipswich, MA) and PCR

amplicons were ligated with annealed shRNA oligoes using GeneArt Seamless Cloning and

Zelikowsky et al.

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Assembly Kit (Thermo Fisher Scientific, Waltham, MA) following the manufacturer’s

instruction.

Construction of Tac2-overexpression AAV vectors—

Tac2-P2A-mCherry gene

fragment was synthesized in the form of IDT gBlocks (see below*). pAAV-hSyn-Tac2-P2A-

mCherry was generated via ligation to AccI/NheI site of pAAV-hSyn-DIO-hM3D(Gq)-

mCherry plasmid (Addgene #44361) using DNA Ligation Kit Mighty Mix (Takara Bio,

Kusatsu, Japan). To generate pAAV-hSyn-Tac2-P2A-GFP plasmid, entire pAAV-hSyn-Tac2-

P2A-mCherry plasmid except mCherry sequence was amplified by PCR with the following

primers: Forward - CTCCTCGCCCTTGCTCAC; Reverse -

GGCGCGCCATAACTTCGTATAATG and GFP sequence was amplified from pAAV-GFP

plasmid (AAV-400, Cell Biolabs Inc, San Diego, CA) with the following primers: Forward -

CCTGGACCTATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTG; Reverse-

AGCATACATTATACGAAGTTATGGCGCGCCCTACTTGAGCTCGAGATCTGAGTAC.

Both PCR amplicons were treated with DpnI (NEB) and ligated together using GeneArt

Seamless Cloning and Assembly Kit (Thermo Fisher scientific) following the manufacture’s

instruction.

*Synthesized Tac2-P2A-mCherry gene fragment:

GCTAGCGCCACCATGAGGAGCGCCATGCTGTTTGCGGCTGTCCTCGCCCTCAGCT

TGGCTTGGACCTTCGGGGCTGTGTGTGAGGAGCCACAGGGGCAGGGAGGGAGG

CTCAGTAAGGACTCTGATCTCTATCAGCTGCCTCCGTCCCTGCTTCGGAGACTCTA

CGACAGCCGCCCTGTCTCTCTGGAAGGATTGCTGAAAGTGCTGAGCAAGGCTTG

GTGGGACCAAAGGAGACATCACTTCCACAGAAACGTGACATGCACGACTTCTTTG

GGGACTTATGGGCAAGAGGAACAGCCAACCAGACACTCCCACCGACGTGGTTGA

GAGAACACCCCCAGCTTTGGCATCCTCAAAGGAAGCGGAGCTACTAACTTCAGCC

TGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTATGGTGAGCAAGG

GCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACAT

GGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCC

GCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCC

TGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTA

CGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGC

TTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACC

CAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGC

ACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAG

GCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAG

CAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACC

TACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAG

TGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGC

CGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAAGGCGCGCC

ATAACTTCGTATAATGTATGCTATACGAAGTTATTAAGAGGTTTCATATTGCTAATAG

Zelikowsky et al.

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CAGCTACAATCCAGCTACCATTCTGCATAACTTCGTATAAAGTATCCTATACGAAGT

TATTCCGGAGTCGAC

Viral packaging—

rAAVs were produced by polyethylenimine (PEI) triple transfection of

HEK293T cells. Briefly, 40μg of equi-molar pHelper, pXR5 and pAAV-trans DNA plasmids

were mixed with 120μl of 1mg/ml Polyethylenimine HCl MAX (Polysciences) in PBS and

incubated at RT for 5 minutes. 90% confluent HEK293 cells grown on 15cm tissue culture

plates were transfected with the plasmid/PEI mixture. Cells were collected 72 hours post

transfection, freeze-thawed 3 timed and incubated with Benzonase (Millipore) at 5 units/mL

for 1 hour. The solution was then centrifuged at 5000xg for 20 minutes. The supernatant was

layered on top of a discontinuous gradient of iodixanol and centrifuged at 200,000xg for 2

hours at 18°C. The 40% iodixanol fraction was collected, concentrated, and buffer

exchanged with PBS using a Millipore 100kD centrifugal filter. AAV genomic titers were

determined by real-time PCR using primers against the ITR and normalized by dilution with

PBS to 1×10

genome copies per mL virus.

AAV-PHP.B production and intravenous administration—

The AAV-hSyn-DIO-

Tac2-P2A-mCherry, AAV-hSyn-DIO-Tac2-P2A-GFP, AAV-hSyn-DIO-hM3D-mCherry, and

AAV-hSyn-DIO-mCherry recombinant AAV genomes were separately packaged into the

AAV-PHP.B capsid by triple transfection of HEK293T cells and purified with iodixanol step

gradients as previously described (

Deverman et al., 2016

). 5×10

vector genomes (vg) of

each virus was administered intravenously (via the retro-orbital sinus) to Tac2-Cre animals

individually, or in combination. To equalize the amount of virus given to each mouse,

5×10

vg of AAV-PHP.B-hSyn-DIO-mCherry was administered to each animal to bring

them up to the amount injected in the double Tac2+hM3DREAAD group. Each animal

received a total vector dose of 1×10

vg.

Surgery and cannula implants—

Mice 8–16 weeks old were anesthetized with

isoflurane and mounted in a stereotaxic apparatus (Kopf Instruments). Anesthesia was

maintained throughout surgery at 1–1.5% isoflurane. The skull was exposed and small burr

holes produced dorsal to each injection site using a stereotaxic mounted drill. Virus was

backfilled into pulled fine glass capillaries (~50μm diameter at tip) and pressure injections of

300nl were made bilaterally into either the dBNSTa (AP +0.25, ML ±0.85, DV −4.1), DMH

(AP −1.3, ML ±0.35, DV −5.6), or CeA (AP −1.4, ML ±2.6, DV −4.73) at a rate of 30nl per

minute using a nanoliter injector (Nanoliter 2000, World Precision Instruments) controlled

by an ultra microsyringe pump (Micro4, World Precision Instruments). Capillaries remained

in place for 5 minutes following injections to allow for full diffusion of virus and to reduce

backflow up the injection tract. Skin above the skull was then drawn together and sealed

with GLUture (Zoetis). For bilateral cannula implantations, single or double guide cannula

(custom, Plastics One) aimed 0.5mm above each region were implanted and held in place

with dental cement (Parkell). Compatible dummy cannulas with a 0.5mm protrusion at the

tip were inserted to prevent cannula clogging. Directly following surgery, mice were given a

subcutaneous injection of ketoprofen (2mg/kg) and supplied with drinking water containing

400mg/L sulphamethoxazole and 200mg/L ibuprofen and monitored for 7 days. Dummies

Zelikowsky et al.

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were replaced every 2–3 days to keep cannula tracts clean. All injections were subsequently

verified histologically.

Immunohistochemistry—

Immunofluorescence staining proceeded as previously

described (

Anthony et al., 2014

;

Cai et al., 2014

;

Hong et al., 2014

;

Kunwar et al., 2015

Briefly, mice were perfused transcardially with 0.9% saline followed by 4%

paraformaldehyde (PFA) in 1XPBS. Brains were extracted and post-fixed in 4% PFA

overnight at 4°C followed by 48 hours in 15% sucrose. Brains were embedded in OCT

mounting medium, frozen on dry ice, and stored at −80°C for subsequent sectioning. Sect

ions 40–50 μm thick were cut on a cryostat (Leica Biosystems). Sections were either directly

mounted onto Superfrost slides for histological verification of injections/cannula placements

or were cut free floating for antibody staining. For antibody staining, brain sections were

washed 3× in 1XPBS and blocked in PBS-T (0.3% Triton X-100 in 1XPBS) with 10%

normal goat or donkey serum for 1hr at room temperature (RT). Sections were then

incubated in primary antibody diluted in blocking solution at 4°C for 48–72 hours. We

stained for neurokinin B (rabbit anti-proNKB; 1:1000; Invitrogen); the glial marker nuclear

factor I-A (rabbit anti-mouse NFIA; 1:1000; Deneen lab) (

Deneen et al., 2006

); the

oligodendrocyte marker proteolipid protein (chicken anti-PLP; 1:1,000; Millipore) or the

nuclear marker NeuN (rabbit anti-NeuN; 1:1000; Millipore). Sections were then washed 3×

and incubated in secondary antibodies diluted in blocking buffer (goat anti-rabbit, goat anti-

chicken, Alexa Fluor 594, 1:500) overnight at 4°C. Sections were then washed 3X,

incubated for 20 minutes at RT in DAPI diluted in 1XPBS (1:2000) for counterstaining,

washed again, mounted on Superfrost slides, and coverslipped for imaging on a confocal

microscope (Olympus FluoView FV1000).

Fluorescent in situ hybridization—

Digoxigenin (DIG)-labeled

Tac2, Cfos, Crh

RNA

probes and dinitrophenyl (DNP)-labeled

Tac2

probe were generated following a previously

described protocol (

http://help.brain-map.org/display/mousebrain/Documentation

) (

Lein et

al., 2007

) with the following primer sets: Tac2; Forward - AGCCAGCTCCCTGATCCT;

Reverse -TTGCTATGGGGTTGAGGC (NM_009312.2, 36–608bp

Cfos

; Forward -

agaatccgaagggaacgg and Reverse -ggaggccagatgtggatg (NM_010234.2, 560–1464bp)

Crh

;

Forward - agggaggagaagagagc and Reverse agccacccctcaagaatg (NM_205769.3, 219–

1185bp). Fluorescent in situ hybridization (FISH) or double fluorescent in situ hybridization

(dFISH) was carried out according to the protocol used in (

Thompson et al., 2008

) with

modifications. Briefly, mice were transcardially perfused with 1 × PBS followed by 4%

paraformaldehyde/PBS (PFA) in 1 × PBS. Brains were fixed in 4% PFA 3–4 hours at 4°C

and cryoprotected for overnight in 15% sucrose at 4°C. Brains were embedd ed in OCT

Compound (Fisher Scientific) and cryosectioned in 30 μm thickness and mounted on

Superfrost Plus slides (Fisher Scientific). Sections were fixed in 4% PFA for 30 min,

acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine for 10 min, dehydrated

with increasing concentrations of EtOH (50, 70, 95 and 100%), gently treated with

proteinase K (6.3 μg/mL in 0.01M Tris-HCl pH7.4 and 0.001M EDTA) for 10 min, and

fixed in 4% PFA for 20 min. All procedures were performed at room temperature (RT). The

hybridization buffer contained 50% deionized formamide, 3 × standard saline citrate (SSC),

0.12 M PB (pH 7.4), 10% dextran sulfate, 0.12 mg/ml yeast tRNA, 0.1 mg/mL calf thymus

Zelikowsky et al.

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DNA, and 1× Dehardt solution. The sections were prehybridized at 63°C in hybridization

buffer for 30 min and then hybridized withRNA probes (300 ng/ml for each probe) in

hybridization buffer at 63 °C for 16 hours. After hybridization, the sections were washed

with 5 × SSC for 10 min, 4× SSC / 50% formamide for 20 min, 2× SSC / 50% formamide

for 30min, and 0.1× SSC for 20min twice each at 61 °C. The sections were blocked with 4%

sheep serum in TNT buffer (Tris-HCl pH7.5, 0.15 M NaCl and 0.00075% tween 20) for 30

min and TNB Blocking buffer (TSA blocking reagent, PerkinElmer, Waltham, MA) for 30

min at RT. The sections were incubated with anti-digoxigenin-POD antibody (1:600, Roche

Diagnostics) in TNB buffer for overnight at RT. The sections were washed with TNT buffer

and tyramide-biotin signal amplification was performed using the TSA Plus Biotin Kit

(PerkinElmer) and signals were visualized after 1 hr incubation with Alexa Fluor 594

Streptavidin (Jackson ImmunoResearch) or Alexa Fluor 488 Streptavidin (Invitrogen) at RT.

The sections were washed with TNT buffer and fixed with 4% PFA for 20 min at RT,

washed with PBS, blocked with avidin/biotin blocking kit (Vector), and then treated with

0.3% H

for 15 min at RT. Subsequently, the sections were washed with PBS, blocked

with TNB blocking buffer for 20 min at RT, and incubated with anti-DNP HRP conjugated

antibody (1:250, PerkinElmer) in TNB blocking buffer for overnight at RT. On the following

day, the sections were washed with TNT buffer and tyramide-DNP signal amplification was

performed using the TSA Plus DNP (HRP) Kit (PerkinElmer) and signals were visualized

with Anti-DNP Alexa Fluor 488 conjugated antibody (1:125, Invitrogen) at RT. The Sections

were counterstained with DAPI (0.5ug/mL in PBS), washed with 1 × PBS, and coverslipped

using Fluoro-Gel with Tris Buffer (Electron Microscopy Sciences). Tissue images of entire

coronal brain sections were taken using a slide-scanner (VS120-S6-W, Olympus) or a

confocal microscope (FluoView FV1000, Olympus) and cells positive for the probes were

counted as described below.

Cell counting—

Following confocal or slide-scanner imaging, quantification of labeled

cells was performed using ImageJ and Metamorph. Cells were counted by an observer blind

to experimental conditions. Brain images were converted to greyscale (16-bit) in ImageJ and

adjusted using automatic thresholding and watershed separation. Cells were either counted

automatically using ImageJ’s particle analysis algorithm (random sections were counted

manually to cross-check that automated scoring was consistent with manual human scoring);

otherwise, cells were counted manually using MetaMorph. Cells that were not entirely

contained within a given region of interest (ROI) were excluded from analyses. Relative

fluorescent intensities (for cell body or projection terminal labeling with a fluorescent

protein) were measured automatically using MetaMorph for a given ROI. Raw cell counts

within an ROI were divided by the size of the ROI (mm

) to produce the number of

positively labeled cells/mm

Quantitative real-time reverse transcription PCR—

Group housed or isolated (30

minutes, 24 hours, 2 weeks) mice were decapitated and brains were quickly removed and

placed in RNA Later (Qiagen) at 4°C. Tissue from dBNSTa, DMH, CeA, ACC, and dHPC

was micro-dissected and placed in RNA Later. Tissue was then homogenized and RNA

purified using an RNAeasy Plus Mini Kit (Qiagen). 150ng of total RNA/region/condition

was then incubated with 3μl of Turbo DNase, 1μl of Murine RNase Inhibitor, in 1× Turbo

Zelikowsky et al.

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