of 38
The neuropeptide Tac2 controls a distributed brain state induced
by chronic social isolation stress
Moriel Zelikowsky
1,*
,
May Hui
1
,
Tomomi Karigo
1
,
Andrea Choe
1
,
Bin Yang
1
,
Mario Blanco
1
,
Keith Beadle
1
,
Viviana Gradinaru
1
,
Benjamin E. Deverman
1
, and
David J. Anderson
1,2,3,4,*
1
Division of Biology and Biological Engineering 156-29, California Institute of Technology
Pasadena, CA 91125, USA
2
Howard Hughes Medical Institute, California Institute of Technology Pasadena, CA 91125, USA
3
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.).
4
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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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.
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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
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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
et
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
et
al.
, 1963
;
Valzelli, 1969
,
1973
;
Weiss
et al.
, 2004
;
Matsumoto
et al.
, 2005
;
Arrigo and
Bullock, 2008
;
An
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
In
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
or
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
2
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
in
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
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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 (
Lu
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
,
a
) 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
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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
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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
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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
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.
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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
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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
C
GTGGGACCAAAGGAGACATCACTTCCACAGAAACGTGACATGCACGACTTCTTTG
T
GGGACTTATGGGCAAGAGGAACAGCCAACCAGACACTCCCACCGACGTGGTTGA
A
GAGAACACCCCCAGCTTTGGCATCCTCAAAGGAAGCGGAGCTACTAACTTCAGCC
TGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTATGGTGAGCAAGG
GCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACAT
GGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCC
GCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCC
TGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTA
CGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGC
TTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACC
CAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGC
ACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAG
GCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAG
CAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACC
TACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAG
T
TGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGC
CGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAAGGCGCGCC
ATAACTTCGTATAATGTATGCTATACGAAGTTATTAAGAGGTTTCATATTGCTAATAG
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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
12
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
11
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
11
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
12
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
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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
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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
2
O
2
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
2
) to produce the number of
positively labeled cells/mm
2
.
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
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