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Prefrontal microcircuit underlies contextual learning after hippocampal loss

Prefrontal microcircuit underlies contextual learning after hippocampal loss

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Published by Dimitrije
Specific brain circuits have been classically linked to dedicated
functions. However, compensation following brain damage suggests
that these circuits are capable of dynamic adaptation. Such
compensation is exemplified by Pavlovian fear conditioning following
damage to the dorsal hippocampus (DH). Although the
DH normally underlies contextual fear and fear renewal after extinction,
both can be learned in the absence of the DH, although the
mechanisms and nature of this compensation are currently unknown.
Here, we report that recruitment of alternate structures, specifically
the infralimbic and prelimbic prefrontal cortices, is required for compensation
following damage to the hippocampus. Disconnection of
these cortices in DH-compromised animals and immediate early gene
induction profiles for amygdala-projecting prefrontal cells revealed
that communication and dynamic rebalancing within this prefrontal
microcircuit is critical. Additionally, the infralimbic cortex normally
plays a role in limiting generalization of contextual fear. These discoveries
reveal that plasticity through recruitment of alternate circuits
allows the brain to compensate following damage, offering promise
for targeted treatment of memory disorders.
Specific brain circuits have been classically linked to dedicated
functions. However, compensation following brain damage suggests
that these circuits are capable of dynamic adaptation. Such
compensation is exemplified by Pavlovian fear conditioning following
damage to the dorsal hippocampus (DH). Although the
DH normally underlies contextual fear and fear renewal after extinction,
both can be learned in the absence of the DH, although the
mechanisms and nature of this compensation are currently unknown.
Here, we report that recruitment of alternate structures, specifically
the infralimbic and prelimbic prefrontal cortices, is required for compensation
following damage to the hippocampus. Disconnection of
these cortices in DH-compromised animals and immediate early gene
induction profiles for amygdala-projecting prefrontal cells revealed
that communication and dynamic rebalancing within this prefrontal
microcircuit is critical. Additionally, the infralimbic cortex normally
plays a role in limiting generalization of contextual fear. These discoveries
reveal that plasticity through recruitment of alternate circuits
allows the brain to compensate following damage, offering promise
for targeted treatment of memory disorders.

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Prefrontal microcircuit underlies contextual learningafter hippocampal loss
Moriel Zelikowsky
a,b
, Stephanie Bissiere
c
, Timothy A. Hast
a
, Rebecca Z. Bennett
a
, Andrea Abdipranoto
d,e
,Bryce Vissel
d,e
, and Michael S. Fanselow
a,b,f,1
a
Department of Psychology,
b
Integrative Center for Learning and Memory, and
f
Department of Psychiatry and Biobehavioral Sciences, University ofCalifornia, Los Angeles, CA 90095;
c
European Molecular Biology Laboratory, Monash University, Melbourne, VIC 3800, Australia;
d
NeurodegenerationResearch Group, The Garvan Medical Research Institute, Sydney, NSW 2010, Australia; and
e
Faculty of Medicine, University of New South Wales, Sydney,NSW 2052, AustraliaEdited by Richard F. Thompson, University of Southern California, Los Angeles, CA, and approved April 17, 2013 (received for review January 25, 2013)
Speci
c brain circuits have been classically linked to dedicatedfunctions. However, compensation following brain damage sug-gests that these circuits are capable of dynamic adaptation. Suchcompensation is exempli
ed by Pavlovian fear conditioning fol-lowing damage to the dorsal hippocampus (DH). Although theDH normally underlies contextualfearandfearrenewalafterextinc-tion, both can be learned in the absence of the DH, although themechanismsandnatureofthiscompensationarecurrentlyunknown.Here, we report that recruitment of alternate structures, speci
callythe infralimbic and prelimbic prefrontal cortices, is required for com-pensation following damage to the hippocampus. Disconnection ofthese cortices in DH-compromised animals and immediate early geneinduction pro
les for amygdala-projecting prefrontal cells revealedthat communication and dynamic rebalancing within this prefrontalmicrocircuit is critical. Additionally, the infralimbic cortex normallyplays a role in limiting generalization of contextual fear. These dis-coveriesrevealthatplasticitythroughrecruitmentofalternatecircuitsallows the brain to compensate following damage, offering promisefor targeted treatment of memory disorders.
anxiety
|
recovery of function
|
amnesia
|
medial prefrontal cortex
A
 widely accepted view is that the brain is comprised of mul-tiple independent circuits dedicated to performing speci
cfunctions and encoding speci
c information. This view is exem-pli
ed in the
eld of learning and memory, where it is held thatdifferent circuits specialize in integrating and storing differentclasses of memories (1). However, studies looking at learningfollowing brain damage clearly demonstrate that the brain canalso behave dynamically. For example, in Pavlovian fear condi-tioning, contextual memories can be formed in the absence of circuits classically thought to underpin integration and storage of information about an animal
s environment or context (2).In fear conditioning, contexts play two key roles in controllingfearlearningandexpression.First,acontextcanactasaconditionalstimulus (CS), to which fear can be directly conditioned when anaversive experience, such as a foot shock [unconditional stimulus(US)] is signaled by the context. Second, contexts can modulateresponding to a discrete cue, such as a tone-CS, which has acquiredmultiple meanings. For example, in extinction, a tone previously conditioned to elicit fear is presented in the absence of the aversivefoot shock US, such that the conditional fear response begins toextinguish. However, because extinction is not an erasure of theoriginal fear but rather newlearning that interferes with retrieval of the original fear memory, an extinguished cue has multiple mean-ings(shock/noshock),whichcompeteforbehavioralexpression(3).This competition is mediated by context, as fear renewal occurs whenanextinguishedstimulusispresentedoutsideoftheextinctioncontext (4).These context-sensitive effects provide excellent models to testthe
exibility of learning and memory systems following damage,asbothhavebeenshowntorequire thedorsalhippocampus (DH).Speci
cally, the DH is thought to normally underlie contextualfear memories, as posttraining DH damage results in retrogradeamnesia for recently acquired contextual fear memories (5). Im-portantly,however,animalsthatsufferdamagetothehippocampusbefore conditioning show a striking ability to overcome hippo-campal loss provided they are given adequate training (2). Thisability to overcome anterograde amnesia suggests that plasticity intheabsenceofthehippocampusallowsthebraintocompensateforcontextual fearmemoryformation (6).Thispatternof 
ndingsis paralleled by fear renewal after extinction, as posttraininglesions of the DH cause a de
cit in fear renewal (7, 8), whereasimpairments due to pretraining lesions can be overcome (7, 9).One possibility is that rats recruit alternate circuitry to com-pensate following damage to primary memory structures, such asthe DH. Although compensation for DH damage has been welldocumented, the identity of compensatory structures is unknown.This question is of vital importance, as it has the potential togenerate a broader understanding of how different anatomicalstructures within the fear circuit normally interact and how suchdynamic interplay could allow for compensation following braindamage. Successful identi
cation of regions responsible for hip-pocampal compensation could provide sites to target for thetreatment of memory loss-related disorders.We hypothesized that the medial prefrontal cortex (mPFC)could potentially be a site of compensation in the absence of theDH for several reasons. First, the mPFC has already been iden-ti
edasakeystructureunderlyingthelong-termstorageofremotecontextual fear memories, suggesting that under normal con-ditions, contextual fear is comprised and controlled by a hippo-campal-prefrontal-amygdalacircuit (10, 11).Moreover, anmPFC-hippocampus-amygdala circuit has been implicated in controllingfearextinction(12
14), indicatingthat this regionhastheability tomodulate fear responses generated in the amygdala. Second, themPFC has been implicated in spatially sensitive, hippocampus-dependentworkingmemorytasks (15),and neuronal ensemblesinthe mPFC are thought to encode rich contextual representations(16, 17), suggesting that the mPFC is capable of processing spatialinformation that would otherwise be encoded by the hippocam-pus.These
ndings,combinedwithstudiesshowingthatthemPFCis phase-locked to hippocampal theta (18) and that the two aresynchronized during spatial tasks (19), suggest that the mPFC andhippocampus are intimately connected with regard to context-sensitive learning and memory.Importantly, the mPFC sits in an anatomical position that mightallow it to mediate context-sensitive fear following loss of the
Author contributions: M.Z., S.B., B.V., and M.S.F. designed research; M.Z., T.A.H., R.Z.B.,and A.A. performed research; A.A. and B.V. contributed new reagents/analytic tools; M.Z.analyzed data; and M.Z., S.B., B.V., and M.S.F. wrote the paper.The authors declare no con
ict of interest.This article is a PNAS Direct Submission.
1
To whom correspondence should be addressed. E-mail:mfanselow@gmail.com.This article contains supporting information online atwww.pnas.org/lookup/suppl/doi:10.1073/pnas.1301691110/-/DCSupplemental.
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hippocampus. Both the infralimbic (IL) and prelimbic (PL) sub-regions of the mPFC normally receive input from the ventral hip-pocampal formation (20, 21), as well as from extrahippocampalregions involved in processing information about the environment[e.g., entorhinal cortex (22); retrosplenial cortex; and perirhinalcortex (20)]. Finally, any region compensating for the DH in con-textual fear conditioning must be able to control fear responses. IL and PL both send projections to the amygdala complex (23
25).To test the role of the mPFC in context-sensitive fear formed inthe absence of the DH, we developed a unique behavioral para-digm that allowed us to assess both context fear and fear renewal within the same animal. We found that the compensatory contextfear and fear renewal found in DH-damaged animals was abol-ishedinanimalsthatsufferedadditionalbilateraldamage toeitherthe PL or IL. Blocking communication between the IL and PL cortices using a disconnection approach replicated the profoundde
cit in hippocampal compensation. Immediate early gene andtract tracing analyses in DH-lesioned animals revealed that com-pensatory context fear correlated with a dramatic rearrangementin the balance of activity between the PL and IL cortices inamygdala-projecting cells. Additionally, we discovered that IL lesions on their own enhanced generalization of contextual fear.Collectively, these data identify a microcircuit within mPFC that isrequired for context-sensitive fear in the absence of the DH.
Results
DH Lesions Produce Retrograde but Not Anterograde Amnesia.
Toinvestigate the structures underlying context-sensitive fearformed in the absence of the DH, we
rst sought to replicate thecompensation effect (2). Rats (
 n
=
8) were tone fear conditioned,given excitotoxic lesions of the DH or sham lesions, and tested forcontext fear (Fig. 1
 A
). Consistent with previous
ndings (2), wefound that rats showed a signi
cant retrograde amnesia for con-text fear when DH lesions where made after training (
t
=
4.050;
 P 
<
0.01;Fig. 1
 B
). Following this initial context test, ratswere fearconditioned again (retraining) and retested such that their prior
post
training lesions were now
pre
training lesions withrespectto retraining (Fig. 1
 A
). Differences in context fear between shamand DH rats were attenuated, indicating that DH-lesionedanimals were able to overcome anterograde amnesia for contextfear (
t
=
1.654;
>
0.05; Fig. 1
 B
).
Context Fear in the Absence of the DH Requires IL and PL.
To test whether the IL or PL could be potential sites of compensation,animals were given double lesions of either the IL 
+
DH (
 n
=
15)or PL 
+
DH (
 n
=
15) and compared to animals with lesions toonly the DH (
 n
=
16), IL (
 n
=
20), PL (
 n
=
17), or sham controls(
 n
=
24; lesions depicted inFig. S1). We chose to separately examine IL and PL contributions to DH compensation becausethese regions have been shown to play opposing roles in modu-lating fear expression (13, 26). We assessed compensation usinglesions of DH as opposed to ventral hippocampus (VH) becauseDH lesions produce the same compensatory effects as lesionsto the entire hippocampal formation (2), and the DH has beendirectly implicated in spatial and episodic memory (27). Addi-tionally, the VH, but not the DH, projects monosynaptically tomPFC, and we wanted to avoid changes that could be a result of direct deafferentation. We hypothesized that if either the IL orPL was recruited to compensate in the absence of the DH, thenpretraining damage to either would result in a failure to expresscontext fear in DH-compromised animals.Following surgery, rats underwent tone fear conditioning,context testing, tone extinction, and tone fear renewal testingusing a protocol we developed to test for direct context fear andcontext-modulated fearrenewalwithinthesameanimal (Fig.2
 A
). All animals acquired signi
cant tone fear (indexed by percentfreezing;
 F 
(3,303)
=
239.9;
 P 
<
0.0001;Fig. S2
), regardless of theirlesion condition (
 F 
(5,303)
=
1.294;
 P 
>
0.05). Importantly, baseline(BL) freezing to the context before the start of fear conditioning was
<
1%, demonstrating that the lesions alone did not result innonspeci
c freezing to novel environments (Fig. S2
).The following day, rats were tested for context fear in thetraining context or fear generalization in a novel context (Fig.2
 B
). Two-way ANOVA revealed a main effect of lesion (
 F 
(5,95)
=
8.669;
 P 
<
0.0001), context (
 F 
(1,95)
=
18.58;
 P 
<
0.0001), and theirinteraction (
 F 
(5,95)
=
4.179;
<
0.01). As previously demon-strated, context fear in DH-lesioned animals was not signi
cantly different then shams (
t
=
1.629;
>
0.05) (2, 28). DH-lesionedrats froze more in the training compared with novel context (
t
=
3.525;
<
0.01), which deviates from studies showing that theDH is required for context discrimination (29). However, in thosestudies, the contextual discrimination was more dif 
cult than thatused here. Rodents with hippocampal impairments easily dis-criminate very different contexts even though they have troublediscriminating very similar ones (5, 29, 30). Crucially, the com-pensatory context fear seen in DH animals was lost in double-le-sioned animals (DHIL vs. DH:
t
=
3.229;
<
0.01; DHPL vs. DH:
t
=
3.604;
<
0.01), suggesting that both regions are required forcontext fear in the absence of the DH. This reduction in freezingcould not be explained by a failure to freeze, as animals withdouble lesions showed normal acquisition (Fig. S2
).Interestingly, rats with IL lesions were unable to differentiatebetween a context they were shocked in and a novel context, suchthat they froze no differently between the two (
t
<
1). This effect was due to a combination of reduced freezing in the trainingcontext (IL vs. sham:
t
=
2.665;
<
0.05) and enhanced freezingin the novel context. This failure to distinguish between contextsin IL-lesioned rats is surprising given that these animals have anintact hippocampus and thus might be expected to discriminatebetween two contexts (29). Low baseline freezing to the contextbefore fear conditioning (Fig. S2
) suggests that an IL lesion onits own does not cause freezing to a novel context. Additionally,the failure for IL-lesioned rats to exhibit an anxiety-like pheno-type, as indexed by elevated plus maze (EPM) testing (Fig. S3),suggests that their inability to discriminate was not caused by a general increase in anxiety. This failure to appropriately andselectively freeze in a shocked context rather than a novel
0255075100
Sham
DH
**
n.s.
   F  r  e  e  z   i  n  g   (   %   )
Retrograde Amnesia Probe
Lesion with respect to training
PostPre
 Anterograde Amnesia Probe
A
   L   E   S   I   O   N
10d 
Fear Cond.
4X Tone+Shk(ITI: 60s)
24h
ContextFear Test
8 min
24h
Fear Cond.
4X Tone+Shk(ITI: 60s)
24h
ContextFear Test
8 min
B
Retrograde Amnesia Probe Anterograde Amnesia Probe
Fig. 1.
Retrograde but not anterograde amnesia for the same contextual in-formation. (
 A
) Experimental design. (
B
) Lesions made to the DH posttrainingresultedinasigni
cantreductionofcontextfearcomparedwithshamcontrols(retrogradeamnesia). When ratswereretrained in the absenceoftheDH,theydemonstrated contextual fear expression at test that was equivalent to theirshamcounterparts(attenuatedanterogradeamnesia).Resultsarepresentedasmean
±
SEM percent freezing.**
<
0.01; n.s.
 ,
not signi
cant; shk, shock.
2 of 6
|
 
context was seen in IL- but not PL-lesioned rats. PL animals, likesham controls, expressed appropriate levels of high contextualfear in the training context coupled with signi
cantly lower fearin the novel context (
t
=
3.344;
<
0.01).
Context-Modulated Fear Renewal in the Absence of the DH RequiresIL and PL.
Following the probe test for contextual fear, animalsunderwent extinction training (Fig. S2
C
). DH-lesioned rats dis-played enhanced rates of within-session extinction (
t
=
6.759;
 P 
<
0.001), and IL-lesioned rats showed a retarded rate of extinction(
t
=
4.026;
<
0.01). However, all groups reached similar lowlevels of fear by the end of extinction training (Fig. S2
). Fol-lowing extinction, rats were tested for context-modulated fearrenewal. Renewal was assessed by comparing animals tested inthe same vs. shifted context (with respect to the extinction con-text), thereby controlling for any differences due to surgicalcondition (e.g., baseline fear, spontaneous recovery). A two-way ANOVA found a main effect of context (
 F 
(1,95)
=
35.20;
<
0.0001), lesion (
 F 
(5,95)
=
12.05;
<
0.0001), and theirinteraction (
 F 
(5,95)
=
5.530;
<
0.001; Fig. 2
C
). Signi
cant fearrenewal was displayed by sham controls (
t
=
4.911;
<
0.001), as well as rats with DH damage (
t
=
2.710;
<
0.05), replicating ourprevious results (7). However, compensatory fear renewal in theabsence of the DH was lost for animals with additional lesions tothe IL or PL (DHIL:
t
=
0.1904;
>
0.05; DHPL:
t
=
2.059;
>
0.05), mimicking the pattern of 
ndings observed for context fear.It should be noted, however, that the loss of compensation forDHIL animals was more pronounced then that observed in DHPL rats. Importantly, baseline was
<
5% and did not differ betweengroups (Fig. S4), showing that alternate context exposure reducesbaseline context fear before test. Rats with IL-only lesions dem-onstrated signi
cant fear renewal (
t
=
5.955;
<
0.001). However,ILratsalsoshowedimpairedextinctionmemoryrecall,astheyfrozesigni
cantly more to the tone presented in the extinction contextcompared with shams (i.e., the contrast between Same-Sham andSame-IL is reliable,
t
=
2.339;
 P 
<
0.05; Fig. 2
C
). These results areconsistent with
ndings by Quirk and colleagues (31, 32).Last, animals with PL-only damage failed to mount a renewalresponse (
t
=
4265;
>
0.05), which can be partially explained by reduced freezing in the shifted condition compared with shamcontrols (
t
=
3.095;
<
0.01), consistent with studies implicatingthe PL in fear expression (33). A summary of the various be-havioral effects obtained with each lesion condition relative tosham controls is provided inTable S1.
Compensatory Context Fear Is Characterized by Changes in mPFC cfosInduction.
Our behavioral results revealed that in the absence of theDH, both the IL and PL are required for rats to express compen-satory context fear and fear renewal. That is, loss of only the IL oronly the PL prevented DH-lesioned rats from expressing context-sensitive fear. To investigate whether this role for the IL and PL corresponds to alterations in neuronal activity within these regions, we examined cfos expression as an indirect marker of neuronalactivation.Rodents with pretraining DH lesions (
 n
=
5) or sham controls(
 n
=
5)were fear conditionedandtested forcontext fear (Fig.3
 A
). Animals with DH lesions and shams froze similarly during testing(
t
<
1; Fig. 3
 B
). Brains were extracted and sections were processedfor cfos. The total number of cells positive for cfos (cfos
+
) in theIL and PL cortex was quanti
ed using rigorous unbiased stereo-logical methods (34). Additional home cage (HC
+
) control rats were added to the cfos analyses to provide a baseline level of cfosexpressionintheILandPLforeachsurgicalcondition(
 n
=
8).HC
+
rats were trained identically to experimental animals except the
****
###
***
###
n.s.n.s.n.s.
Sham DH DHIL DHPL IL PL
0255075100
SameShifted
C
Fear Renewal Test
Sham DH DHIL DHPL IL PL
0255075100
DangerousNovel
B
*******
#######
Context Fear Probe
 
SameShifted
 
TrainingNovel
   F  r  e  e  z   i  n  g   (   %   )
A
      L      E      S      I      O      N
~12d 
Fear Cond.
4X Tone+Shk Alternate ContextExpos.1 & 2(48 min)
24h
+
 Alternate ContextExpos.1 & 2(48 min)
+
Fear RenewalTest
4X Tone-
24h
Context Fear Probe+ Extinction 1 & 230X Tone-Context Fear Probe+ Extinction 1 & 230X Tone-
   N  o  v  e   l   T  r  a   i  n   i  n  g
3h3h
Fig. 2.
Context fear and renewal in lesioned rats. (
 A
) Experimental designaimed at probing both the direct (Context Test) and indirect (Fear RenewalTest) contribution of context to fear learning and memory. Black and un
lledrectangles refer to different contexts; physical contexts were counter-balanced. Same and shifted refer to whether extinction occurred in the sameor novel context with respect to the training/test context. Acquisition andextinctiondataaredisplayed inFig.S2. (
B
) Test forcontextfearprobed duringthe 180-s baseline (BL) period before extinction 1. Context fear for DH ratswas not reliably different then sham rats indicating hippocampal compen-sation. Context fear was abolished in animals with double DHIL and DHPLlesions.Sham, DH,andPL lesion groupsshowed signi
cantlyreduced freezingwhen shifted to a novel context, whereas IL groups froze similarly whentested in thesame context as acquisition or shifted to a novel context.(
) Testfor fear renewal after extinction. Fear renewal was observed in the sham, DH,and IL groups but lost in the DHIL and DHPL groups. IL lesions produced poorextinction retention in the same and shifted condition compared with shams,and PL lesions resulted in no renewal and a de
cit in fear responding in theshifted condition. ***
<
0.001, **
<
0.01, *
<
0.05,
###
<
0.001,
##
<
0.01,
#
<
0.05; n.s.
 ,
not signi
cant. Results are presented as mean
±
SEM percentfreezing. ITI, intertrial interval; trial, tone-shock pairing.
0100200300400500600700
PLIL
BCA
Fear Cond.
4X Tone+ShkITI: 60s
24h82min
LESION
 
~12d 
ContextFear Test
8 min
Immediate EarlyGene Analyses
 
Sham DH
0255075100
   F  r  e  e  z   i  n  g   (   %   )
***
###
   T  o   t  a   l   #  c   f  o  s   +  n  e  u  r  o  n  s   (
       k
   )   P  r  e   l   i  m   b   i  c   I  n   f  r  a   l   i  m   b   i  c
Sham + Tested
 
cfos+cv
DH + Tested
 
   P  r  e   l   i  m   b   i  c   I  n   f  r  a   l   i  m   b   i  c
cfos+cv
DE
HC+ Fear Tested
Sham ShamDH DH
###
n.s.n.s.
**
Fig. 3.
Cfos expression in the IL and PL regions following compensatorycontext fear expression. (
 A
) Experimental design. (
B
) Context fear test. DH-lesioned rats showed similar context fear as their sham counterparts (com-pensation).(
)Totalnumberofcellsimmunoreactiveforcfos(estimatedusingunbiased stereology) in the PL (red bars) and IL (gray) of Sham (
Left 
) and DH(
Right 
) rats that were either homecaged controls (HC
+
) or tested for contextfear (Fear Tested). Fear testing disrupted the balance between PL and IL ac-tivity, with reduced IL activation for both shams and DH animals. Compensa-tionandrebalancinginDHratswasadditionallydrivenbyincreasedactivationof PL compared with shams and HC
+
rats. (
D
) Representative micrograph andmagni
ed insets showing cfos expression in both the IL and PL cortices insham (
D
) and DH lesioned animals (
). (Scale bars, 50
μ
m for the mPFCoverviews and 15
μ
m for inset images.) Results are presented as mean
±
SEM, ***
<
0.001, **
<
0.01,
###
<
0.001; n.s., not signi
cant.
Zelikowsky et al. PNAS Early Edition
|
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