Insulin signaling in the hippocampus and amygdalaregulates metabolism and neurobehaviorMarion Sotoa,b,1, Weikang Caia,b,1, Masahiro Konishia,b, and C. Ronald Kahna,b,2

aSection of Integrative Physiology and Metabolism, Joslin Diabetes Center, Boston, MA 02215; and bDepartment of Medicine, Harvard Medical School,Boston, MA 02215

Contributed by C. Ronald Kahn, January 2, 2019 (sent for review October 17, 2018; reviewed by Suzanne Craft and Sam Gandy)

Previous studies have shown that insulin and IGF-1 signaling in thebrain, especially the hypothalamus, is important for regulation ofsystemic metabolism. Here, we develop mice in which we havespecifically inactivated both insulin receptors (IRs) and IGF-1 receptors(IGF1Rs) in the hippocampus (Hippo-DKO) or central amygdala (CeA-DKO) by stereotaxic delivery of AAV-Cre into IRlox/lox/IGF1Rlox/lox mice.Consequently, both Hippo-DKO and CeA-DKO mice have decreasedlevels of the GluA1 subunit of glutamate AMPA receptor and displayincreased anxiety-like behavior, impaired cognition, and metabolicabnormalities, including glucose intolerance. Hippo-DKO mice alsodisplay abnormal spatial learning and memory whereas CeA-DKOmice have impaired cold-induced thermogenesis. Thus, insulin/IGF-1signaling has common roles in the hippocampus and central amyg-dala, affecting synaptic function, systemic glucose homeostasis, be-havior, and cognition. In addition, in the hippocampus, insulin/IGF-1signaling is important for spatial learning and memory whereasinsulin/IGF-1 signaling in the central amygdala controls thermo-genesis via regulation of neural circuits innervating interscapularbrown adipose tissue.

insulin | hippocampus | amygdala | metabolism | cognition

Insulin, insulin-like growth factor 1 (IGF-1), and IGF-2 actthrough two cognate receptors to control distinct physiological

processes throughout the body. Insulin binds with highest affinityto the insulin receptor (IR) and low affinity to the IGF-1 receptor(IGF1R) and thus regulates systemic metabolism (1, 2). In contrast,IGF-1 and IGF-2 bind with higher affinity to the IGF1R and act asgrowth factors important for tissue development and remodeling(3–5). Differences between insulin and IGF-1/2 are in part explainedby the different expression patterns of these receptors, and in partby differences in signals generated by the IR and IGF1R (6).In the central nervous system, both IR and IGF1R are widely

expressed, and their actions have been implicated in the brain incontrol of metabolism and energy homeostasis, as well as braindevelopment, injury repair, and higher neural processes, in-cluding cognition and mood (7, 8). At the disease level, alteredinsulin/IGF-1 signaling in the brain has been linked to increasedrisks for Alzheimer’s disease, premature cognitive decline, anddementia, as well as depression and anxiety (9–11). Patients withAlzheimer’s disease show decreased expression of both IR andIGF1R (12), as well as abnormal distribution and cellular lo-calization of these receptors (13). These CNS phenotypes havealso been observed in states in which there is insulin resistance inthe brain, such as obesity and diabetes (14, 15).Loss of IR in the whole brain causes hyperphagia, insulin re-

sistance, central hypogonadism, impaired response to hypoglyce-mia, and increased depressive-like behaviors (16–18) whereas lossof IGF1R (or one of its major substrates, IRS-2) has been shown toimpair brain development (19, 20). However, single IR or IGF1Rknockout models do not completely eliminate insulin or IGF-1signaling since both ligands can elicit signaling through the receptorthat remains intact. Furthermore, up to now, most studies havefocused on either the whole brain or hypothalamus (16, 19, 21, 22).Hence, the roles of IR/IGF1R in other nuclei controlling higherneural functions, including mood and cognition, are not known.

In the present study, we used stereotactic surgery and AAV-Cre toinduce IR and IGF1R double knockout (DKO) in the hippocampusand central amygdala. We found that IR/IGF1R deletion specificallydown-regulates the expression of an AMPA receptor subunit, glu-tamate receptor 1, in synaptosomes from both hippocampus andamygdala. This is accompanied by multiple metabolic and behavioralabnormalities, including glucose intolerance and increased anxiety-like behaviors. In addition, while deletion of both IR and IGF1R inthe hippocampus and central amygdala leads to impaired recogni-tion memory, IR/IGF1R loss in hippocampus also results in im-paired spatial memory. Finally, deletion of IR and IGF1R in thecentral amygdala impairs cold-induced thermogenesis.

ResultsDeletion of IR and IGF1R in the Hippocampus and Central Amygdala.We used bilateral stereotaxic injection to deliver AAV encodinga Cre-GFP fusion protein into the hippocampus and centralamygdala of IRlox/lox/IGF1Rlox/lox mice to delete both IR andIGF1R in these nuclei in the brain (Fig. 1 A and C). AAVencoding GFP alone was injected to generate control groups.The positions of injection and extent of coverage were confirmedby GFP expression (Fig. 1A). Western blotting of total tissuelysates taken 8 wk later revealed about 50% reduction in IR andIGF1R protein in the hippocampus from AAV-Cre-GFP–in-jected mice (Hippo-DKO) compared with AAV-GFP–injected

Significance

Loss of insulin receptors in the brain causes metabolic andbehavioral abnormalities whereas loss of IGF-1 receptors in thebrain leads to a developmental defect in the brain and pe-riphery. However, less is known about the impact of brain in-sulin and IGF-1 receptor (IR/IGF1R) loss in adult mice, especiallyin higher neural processing regions. Here, we show that loss ofIR/IGF1R in the hippocampus and central amygdala of adultmice results in decrease in glutamate receptors, accompaniedby glucose intolerance, anxiety-like behavior, and impairedcognition. In addition, we identify an insulin/IGF-1 signaling-dependent neural circuit originating from the central amyg-dala, which regulates interscapular brown fat activity andthermogenesis. Thus, brain insulin/IGF-1 signaling is importantfor higher neural processing and systemic metabolism.

Author contributions: M.S., W.C., and C.R.K. designed research; M.S., W.C., and M.K.performed research; M.S., W.C., and M.K. contributed new reagents/analytic tools; M.S.,W.C., and M.K. analyzed data; M.S., W.C., and C.R.K. wrote the paper; and C.R.K. super-vised the work.

Reviewers: S.C., Wake Forest University; and S.G., Mount Sinai School of Medicine.

Conflict of interest statement: C.R.K. and S.G. are coauthors on a 2016 review article.

Published under the PNAS license.

See Commentary on page 5852.1M.S. and W.C. contributed equally to this work.2To whom correspondence should be addressed. Email: c.ronald.kahn@joslin.harvard.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1817391116/-/DCSupplemental.

Published online February 14, 2019.

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control mice (Hippo-CTR) (Fig. 1B). A similar decrease wasobserved in synaptosomes isolated from the hippocampus (SIAppendix, Fig. S1A). Likewise, there was an ∼50% decrease inprotein levels of IR and IGF1R in the central amygdala of CeA-DKO mice compared with CeA-CTR mice (Fig. 1D) and syn-aptosomes (SI Appendix, Fig. S1B). Thus, using stereotaxicapproaches, we were able to significantly reduce IR and IGF1Rin the hippocampus and central amygdala of adult mice.

Decreased Expression of Glutamate Receptor 1 in Synaptosomes ofHippo-DKO and CeA-DKO Mice. Both insulin signaling and IGF-1signaling modulate synaptic plasticity, especially in excitatoryglutamatergic neurons (23, 24). Glutamate acts via AMPA andNMDA receptors, both of which are ionotropic transmembranecation channels that mediate fast synaptic transmission (25, 26).Both Hippo-DKO and CeA-DKO mice displayed 60 to 70%reductions of the GluA1 (also known as GluR1) subunit of theAMPA receptor in the isolated synaptosomes from these re-gions, compared with their controls, whereas the expression ofthe other major AMPA receptor, subunit GluA2, was not af-fected by IR/IGF1R loss (Fig. 2). These occurred with no changein the phosphorylation of either GluA1 or GluA2 (SI Appendix,Fig. S1 C and D), suggesting that insulin/IGF-1 signaling was notimportant for the phosphorylation of the AMPA receptor sub-units but did affect protein levels of GluA1. The NMDA re-ceptor subunits NR2A and NR2B, on the other hand, were notaffected by IR/IGF1R deletion (SI Appendix, Fig. S1 C and D).Expression of the synaptic marker PSD95 was similar in both thehippocampus and amygdala between control and DKO mice (SI

Appendix, Fig. S1 E and F), indicating that the defect of GluA1expression in the DKO mice was not likely due to the impair-ment of the structural integrity of the synapsis in these mice.Thus, IR signaling and IGF1R signaling modulate synapticplasticity specifically through regulating the expression of GluA1in both the hippocampus and central amygdala.

Glucose Homeostasis Is Regulated by IR/IGF1R Signaling in theHippocampus and Central Amygdala. Central insulin signaling hasbeen shown to affect systemic energy homeostasis (16, 21, 22).Following AAV injection, both controls and DKO mice showedsimilar body weight gain and food intake (SI Appendix, Fig. S2A–D). However, 3 wk after injection, Hippo-DKO mice displayedsignificantly higher random-fed blood glucose levels (Fig. 3A) anda strong trend toward decreased plasma insulin levels 9 wk afterinjection (SI Appendix, Fig. S2G). This was accompanied with animpaired oral glucose tolerance test (Fig. 3B). IR/IGF1R deletionin the hippocampus also resulted in moderate impairment of theglucose lowering effect of i.p. insulin injection (SI Appendix, Fig.S2E) and glucose-stimulated insulin secretion (SI Appendix, Fig.S2F), both of which might contribute to the glucose intolerance inHippo-DKO mice. AUC, area under the curve.CeA-DKO mice also displayed increased random-fed blood glu-

cose levels compared with control mice (Fig. 3C) and glucose in-tolerance (Fig. 3D), but this did not appear until 3 wk after AAVinjection. However, loss of IR/IGF1R in the central amygdala did nothave any significant impact on plasma insulin levels, insulin sensitivity,or glucose-stimulated insulin secretion (SI Appendix, Fig. S2 H–J).Both Hippo-DKO and CeA-DKO mice housed in the meta-

bolic cages under the fed and fasted conditions displayed similarspontaneous activity, energy expenditure measured by oxygenconsumption (VO2) and CO2 production (VCO2), and re-spiratory exchange ratio (RER) as their control mice (SI Ap-pendix, Fig. S3), indicating no major role for hippocampal andamygdala IR and IGF1R signaling on circadian rhythm, energyexpenditure, and substrate preference.

IR/IGF1R Signaling in the Amygdala Contributes to Cold-InducedThermogenesis. To investigate whether the IR/IGF1R in thehippocampus or amygdala is required for normal thermogenesis,mice were challenged to cold (∼6 °C), and their rectal temper-ature was measured every 30 min for 3 h. Core body temperature

Fig. 1. AAV-Cre mediates efficient gene recombination in the hippocampus(Hippo-DKO) and central amygdala (CeA-DKO) of IR/IGF1Rlox/lox mice. (A)Bilateral AAV injection sites of the anterior and posterior hippocampus.Representative images of immunohistochemical staining of GFP and DAPI inthe anterior and posterior hippocampus. (B) Immunoblotting of IR and IGF1Rin the hippocampus of adult IR/IGF1Rlox/lox mice injected with AAV-GFP orAAV-Cre-GFP. Bottom, densitometry analysis. (C) Bilateral AAV injection sitesof the central amygdala. Representative images of immunohistochemicalstaining of GFP and DAPI in the central amygdala. (D) Immunoblotting of IRand IGF1R in the central amygdala of adult IR/IGF1Rlox/lox mice injected withAAV-GFP or AAV-Cre-GFP. Bottom, densitometry analysis. *P < 0.05 by un-paired t test, n = 6. Data are presented as mean ± SEM.

Fig. 2. Decreased expression of glutamate receptor 1 in the synaptosome ofHippo-DKO and CeA-DKO mice. (A and B) Representative Western blots andquantification of glutamate receptor 1 and 2 (GluA1 and GluA2) in synap-tosomes extracted from the hippocampus in Hippo-DKO and control mice(A) and the central amygdala in CeA-DKO and control mice (B). GAPDH serveas loading controls. *P < 0.05, **P < 0.01 by unpaired t test, n = 6. Quan-titative data are presented as mean ± SEM.

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of Hippo-DKO mice dropped minimally from 38 °C to 37.6 °Cfollowing 3-h cold exposure, similar to the drop observed incontrol mice (Fig. 4A). In contrast, the core body temperature ofthe CeA-DKO mice decreased by ∼1.0 °C during the 3-h coldexposure, significantly more than control mice, whose tempera-ture was decreased by ∼0.5 °C (Fig. 4 B and C). Since brownadipose tissue (BAT) plays an important role for thermoregu-lation in rodents by producing heat through uncoupling protein 1(UCP-1) (27), we assessed the expression of UCP-1 in BAT andfound that both messenger and protein levels of UCP-1 in CeA-DKO mice were slightly, but not significantly, reduced comparedwith control mice (SI Appendix, Fig. S4 A and B).Sympathetic outflow is a key regulator of thermogenic acti-

vation of brown fat upon cold exposure (28). The defective cold-induced thermogenesis of CeA-DKO mice led us to hypothesizethat some neuronal population expressing IR and IGF1R in thecentral amygdala are connected to the neural circuitry thatcontrols sympathetic nerves innervating the interscapular brownadipose tissue (iBAT). To test this, we injected pseudorabiesvirus PRV-765 encoding red fluorescent protein (RFP) intoiBAT to perform retrograde tracing of neuronal connectionsfrom iBAT to the brain. Seven days after viral injection, RFP-expressing neurons were detected in the spinal cord, nucleus ofthe solitary tract (NTS) of the medulla, and parabrachial nucleus(PBN) of the pons, as well as several areas in the hypothalamus,including the paraventricular nucleus (PVN), dorsomedial hy-pothalamus (DMH), and lateral hypothalamic area (LHA) (SIAppendix, Fig. S4C and Table S1), highlighting the neuronalcircuit from the hypothalamus to the iBAT. Interestingly, theamygdala was also highly RFP-labeled (Fig. 4D), demonstratingpreviously unrecognized input of the neural circuits in theamygdala for the regulation of iBAT.

Hippo-DKO and CeA-DKO Mice Display Increased Anxiety-LikeBehaviors. Behavior of the mice was assessed 8 wk after the de-letion of IR/IGF1R in the hippocampus and central amygdala.During an open field test, Hippo-DKO mice exhibited a 40% re-duction in the number of center zone entries and spent ∼75% lesstime in the center zone compared with controls (Fig. 5A), indicatingincreased anxiety in these mice. These mice also showed signifi-cantly greater marble-burying activity (Fig. 5B), another indicator

for anxiety-like behavior in rodents. In the dark–light box test,however, both control and DKO mice showed similar time spent inthe light zone (Fig. 5C). Loss of IR and IGF1R in the centralamygdala also resulted in ∼40% reduction in entries and time in thecenter zone in the open field test (Fig. 5D) and an ∼25% increase inmarble-burying activity (Fig. 5E). In these mice, there was also an∼33% decrease in time spent in the light compartment of a dark–light box compared with control (Fig. 5F). Thus, IR/IGF1R sig-naling in both the hippocampus and amygdala is important forcontrolling anxiety-like behavior in mice.

Hippo-DKO and CeA-DKO Mice Display Impaired Cognition. The ef-fect of IR/IGF1R loss in the hippocampus and central amygdalashowed differential effects on learning and memory. In the ha-bituation stage of the novel object recognition test, both controland Hippo-DKO mice showed equal exploration time with eachof the two identical objects (SI Appendix, Fig. S5A). When one ofthe familiar objects was replaced with a novel object in the teststage 6 h later, control mice spent ∼65% of the time exploringthe novel object (Fig. 6A). By contrast, Hippo-DKO miceshowed equal interacting time between the familiar and novelobjects (Fig. 6A), suggesting impaired recognition memory. Inthe novel object location test, one of the familiar objects wasmoved to a new location in the testing stage 6 h after the ha-bituation phase. Control mice explored the object in the newlocation ∼75% of the total exploration time whereas Hippo-DKO mice explored both objects equally regardless of the lo-cation of the objects (Fig. 6B and SI Appendix, Fig. S5B), which isa sign of a spatial memory deficit.

Fig. 4. IR/IGF1R signaling in the amygdala contributes to cold-inducedthermogenesis. (A) Rectal temperature in Hippo-CTR (n = 4) and Hippo-DKO mice (n = 5) during a 3-h exposure to a 6 °C environment. (B) Rectaltemperature in CeA-CTR (n = 9) and CeA-DKO mice (n = 10) during a 3-hexposure to a 6 °C environment. *P < 0.05 by unpaired t test. (C) Thermalimages using a FLIR T300 Infrared Camera showing surface temperatureafter 3 h at 6 °C between CeA-CTR and CeA-DKO mice. (D) Fluorescent im-ages of brain sections of mice 7 d after injection of the PRV-765 virus in theBAT. BLA, basolateral amygdalar area; BMA, basomedial amygdalar nucleus;COA, cortical amygdalar area; ENTl, entorhinal area lateral part; PA,piriform-amygdalar area; PIA, piriform area; TR, postpiriform transition area.Data are presented as mean ± SEM.

Fig. 3. Glucose homeostasis is regulated by IR/IGF1R signaling in the hip-pocampus and central amygdala. (A) Blood glucose levels in the random-fedstate of Hippo-DKO (n = 31) or Hippo-CTR mice (n = 27). (B) Oral glucosetolerance test (OGTT) performed in Hippo-DKO or Hippo-CTR mice (n =13).(C) Blood glucose levels in the random-fed state of CeA-DKO (n = 29) or CeA-CTR mice (n = 28). (D) OGTT performed in CeA-DKO or CeA-CTR mice (n = 6).*P < 0.05, **P < 0.01, ***P < 0.001 by unpaired t test. Data are presented asmean ± SEM. AUC, area under the curve.

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We further analyzed spatial learning and memory using aStone T maze. In this test, a mouse needs to learn the correctsequence of 13 left/right turns to successfully escape a water-filled maze and reach the goal box (SI Appendix, Fig. S5E).During the first 10 trials of learning conducted over 3 d, controlmice displayed continuous improvement of learning, with fewererrors and shorter latency to reach the goal box (Fig. 6C and SIAppendix, Fig. S5F). In contrast, Hippo-DKO mice displayedsignificantly slower learning (Fig. 6C and SI Appendix, Fig.S5F). One week after the last learning trial, mice were exposedto the same maze to assess their memory. Control mice wereable to finish the maze with low numbers of errors and shortlatency, similar to how they performed during the last trial oflearning. By contrast, after a 1-wk hiatus, Hippo-DKO micemade significantly more errors and took a longer time tocomplete the maze (Fig. 6C and SI Appendix, Fig. S5F), andthese mice performed even worse on this task after a 1-mohiatus (Fig. 6C and SI Appendix, Fig. S5F). In addition, whenthe maze was rotated 180° from its original setting, only con-trols, but not Hippo-DKO mice, showed worsened performancein the T maze, indicating that control mice used spatial cues tofinish the maze while Hippo-DKO mice did not (Fig. 6C and SIAppendix, Fig. S5F).Effects on memory and learning were selective and dependent

more on the specific task in CeA-DKO mice. Thus, CeA-DKOmice failed to recognize the novel object compared with controlsin the novel object recognition test (Fig. 6D and SI Appendix, Fig.S5C). However, CeA-DKO mice were able to remember thelocation of the object as well as control mice (Fig. 6E and SIAppendix, Fig. S5D). In the Stone T maze test, CeA-DKO miceshowed normal learning and only slightly impaired ability toremember the maze (Fig. 6F and SI Appendix, Fig. S5G). Thesedata suggest that loss of IR and IGF1R in the central amygdala

significantly impairs the recognition memory of the mice but hasonly minor effects on spatial memory.

DiscussionInsulin signaling and IGF-1 signaling produce a range of effectson cellular metabolism, proteostasis, growth, and many other

Fig. 5. Hippo-DKO and CeA-DKO mice display increased anxiety-like be-haviors. (A) Time spent in the center zone and entries in center zone in openfield test in Hippo-CTR (n = 12) and Hippo-DKO mice (n = 15). (B) Assessmentof anxiety as number of buried marbles over 30 min during a marble-buryingtask in Hippo-CTR (n = 15) and Hippo-DKO mice (n = 17). (C) Time spent inlight compartment during light/dark box test in Hippo-CTR (n = 11) andHippo-DKO mice (n = 12). (D) Time spent in the center zone and entries incenter zone in open field test in CeA-CTR (n = 13) and CeA-DKO mice (n =14). (E) Assessment of anxiety as number of buried marbles over 30 minduring a marble-burying task test in CeA-CTR (n = 9) and CeA-DKO mice (n =9). (F) Time spent in light compartment during light/dark box test in CeA-CTR(n = 18) and CeA-DKO mice (n = 19). *P < 0.05, **P < 0.01 by unpaired t test.Data are presented as mean ± SEM.

Fig. 6. Hippo-DKO and CeA-DKO mice have impaired cognition. (A) Timespent exploring the novel object during the test session of the object rec-ognition task in Hippo-CTR (n = 8) and Hippo-DKO mice (n = 9). (B) Timespent exploring the object that was moved during the test session of theobject location task in Hippo-CTR (n = 4) and Hippo-DKO mice (n = 5). (C)Errors recorded during the 10 acquisition trials, 1-wk, and 1-mo memorytesting of the Stone T maze in Hippo-CTR (n = 7) and Hipp-DKO mice (n = 8).(D) Time spent exploring the novel object during the test session of theobject recognition task in CeA-CTR (n = 9) and CeA-DKO mice (n = 10). (E)Time spent exploring the object that was moved during the test session ofthe object location task in CeA-CTR (n = 6) and CeA-DKO mice (n = 6). (F)Errors recorded during the 10 acquisition trials, 1-wk, and 1-mo memorytesting of the Stone T maze in CeA-CTR (n = 7) and CeA-DKO mice (n = 9).#P < 0.05, ##P < 0.01, ###P < 0.001 by unpaired t test. *P < 0.05, **P < 0.01between DKO and control mice by unpaired t test. Data are presented asmean ± SEM.

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functions (6, 29–32). Recent studies have shown that the brain isa major target of insulin/IGF-1 signaling. Insulin and IGF-1 areable to cross the blood–brain barrier (BBB), in part throughreceptor-mediated transcytosis, and act on brain tissues (33, 34).In addition, a significant amount of IGF-1 is produced in thebrain. IR and IGF1R are widely distributed throughout the brainand are present on both neurons and glial cells (7). Thus, thebrain is able to respond to locally and systemically producedinsulin and IGF-1 and to modulate its activities accordingly. Inthe present study, we found that loss of IR and IGF1R in thehippocampus or central amygdala results in metabolic and be-havioral abnormalities, including impaired glucose homeostasisand cognition and increased anxiety-like behavior. We also showthat insulin/IGF-1 signaling in the central amygdala plays aspecific role in thermogenesis while insulin/IGF-1 signaling inthe hippocampus is important for spatial memory.At a molecular level, loss of IR and IGF1R results in a sig-

nificant reduction of the GluA1 subunit of the AMPA receptorpresent in the synaptosomal fraction. Unlike NMDA receptors,which are ligand-gated channels permeable to both calcium andsodium, AMPA receptors are ligand-gated sodium channels re-sponsible for the rapid depolarization of neuron membrane po-tential (25, 26). The AMPA receptor is a tetrameric channel,composed mainly of GluA1/GluA2 or GluA2/GluA3 hetero-dimers (25). A large proportion of GluA1 subunit-containingAMPA receptors localize in the endosomes close to the post-synaptic membrane (35), which can undergo rapid recruitment tothe synaptic membrane upon stimulation by insulin or by NMDAreceptor-mediated calcium influx (36). This is a key molecularmechanism of long-term potentiation (LTP) (37) and is impor-tant for learning and memory. Consistent with this, mice withboth IR and IGF1R deleted in the hippocampus display im-paired recognition and spatial memory, possibly due to thelimited GluA1 subunit-containing AMPA receptor pools in thesynapses of these neurons. In agreement with this, lentiviral-mediated deletion of IR in the hippocampus results in im-paired spatial memory due to impaired LTP (38). Also, knockoutof SCAP, a cholesterol sensing protein downstream of insulinaction, also results in impaired LTP (39). Intriguingly, IR/IGF1Rdeletion has no major effect on the phosphorylation of theGluA1 subunit of the AMPA receptor. Consistent with this,calmodulin kinase and cAMP/PKA pathways, which are re-sponsible for GluA1 phosphorylation (40, 41), are not generallyconsidered downstream signaling pathways regulated by IR/IGF1R. The mechanism by which IR/IGF1R regulates GluA1protein levels will require further investigation but might includetranscriptional or posttranscriptional alterations.Insulin is the key hormone regulating glucose handling and

energy homeostasis. Thus, it is not surprising that the majority ofresearch on brain insulin action has focused on the hypothala-mus, which controls many metabolic responses (21, 22, 42). Ourstudy clearly shows that metabolic control by central insulinsignaling is not limited to the hypothalamus since IR/IGF1Rdeletion in both hippocampus and amygdala leads to impairedglucose tolerance. In the hippocampus, this appears to be due toa combination of systemic insulin resistance and a decrease ininsulin secretion. The causal factor for impaired glucose toler-ance in mice lacking IR and IGF1R in the amygdala, on theother hand, is less clear. These mice show normal insulin sensi-tivity and glucose-stimulated glucose secretion, suggesting a de-fect in some peripheral insulin-independent pathway involved inglucose disposal. The sympathetic nervous system (SNS) inner-vates the liver and controls hepatic glucose production. Our dataindicate that the amygdala may play a role in the regulation ofhepatic glucose metabolism and, eventually, systemic glucoselevels, directly or through other brain regions with which it hasprojections, such as the hypothalamus (43). Indeed, it has beenshown that insulin injected in the central amygdala activates

neuronal populations in regions of the hypothalamus (44) knownto affect the autonomic output to the liver. Interestingly, it hasbeen noted that mice with diet-induced obesity develop insulinresistance in the central amygdala (44) similar to that reported inthe hypothalamus (45), and thus it is possible that insulin sig-naling in the central amygdala participates in abnormal glucosehomeostasis in the development of obesity.Interscapular brown adipose tissue (iBAT) is a major ther-

mogenic tissue in rodents and is important in the regulation ofcore body temperature, as well as systemic glucose and lipidmetabolism (27). Brown adipose tissue is also present in humanswhere it has both thermogenic and metabolic functions (46).Regulation of BAT is coordinated by the brain. Thus, whentemperature changes, warm- and cold-sensitive neurons signal tothe preoptic area of the hypothalamus (POA), which, through acircuit involving the dorsal medial hypothalamus (DMH) and therostral raphe pallidus nucleus (rRPa), controls sympatheticnervous system (SNS) outflow to BAT and thus its thermogenicactivity (47). Our retrograde transsynaptic tracing demonstratesthat the amygdala is directly involved in the neural circuit in-nervating BAT. This neural pathway is important for cold-induced thermogenesis, and, more importantly, it is regulatedby insulin/IGF-1 signaling since IR/IGF1R deletion in the cen-tral amygdala specifically leads to cold intolerance in mice.Several groups, including us, have previously observed thethermogenic effects of central insulin/IGF-1 signaling usingwhole brain IR knockout mice (7, 48, 49). The current studypinpoints the amygdala as a critical nucleus where insulin/IGF-1signaling exerts a major role in thermogenesis. How this con-verges with the POA → DMH → rRPa pathway requiresfurther studies.We have also identified a crucial role for IR and IGF1R in

the hippocampus and amygdala on mood and cognition. BothHippo-DKO and CeA-DKO mice displayed significantly in-creased anxiety-like behavior and impaired cognition, indicatinga beneficial role of insulin signaling on mood and cognition.Intranasal insulin delivery has been shown to improve mood andcognition in humans (50, 51). Our studies suggest that the ion-otropic glutamate AMPA receptor and glutamate signaling maybe a molecular link between brain insulin/IGF-1 signaling de-ficiency and altered neurobehavior. Other studies have shownthat inhibition of metabotropic glutamate receptors mGluR2/3can improve mood behavior, and this is accompanied by in-creased adult hippocampal neurogenesis (52). Both insulin/IGF-1 and BDNF have been shown to contribute to adult hippo-campal neurogenesis and have potential beneficial effects inAlzheimer’s disease and psychotic disorders (53, 54). Whetherinsulin/IGF-1 signaling and BDNF signaling share a commonmechanism for their antidepressive and antidementia effectsawaits further investigation. In addition, future studies areneeded to explore the electrophysiological mechanism of theamygdala GluA1-associated defects in cognition and mood inCeA-DKO mice.Interestingly, the phenotypes of the region-specific IR/IGF1R

double knockout mice are more severe than those of the micewith single IR-only knockout throughout the whole brain, sincemice with Nestin-Cre-dependent IR deletion (NIRKO) displayno apparent spatial memory deficit, and the anxiety-like behav-iors develop only with aging (18, 55). On the other hand, IRknockout in astrocytes does have significant effects on mood andbehavior in young mice (56). To what extent the phenotype ofthese region-specific IR/IGF1R knockouts involves astrocytes vs.neurons is uncertain. Combined IR and IGF1R deletion in pe-ripheral tissues, like muscle and adipose tissue, leads to moresevere phenotypes than IR-only knockout (29, 32), suggestingthat IGF1R might partially compensate for IR in peripheraltissues, and this could be also true in the brain. Previous reportshave shown higher expression of IGF1R than IR in the brain (7),

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indicating a potential important role for this insulin-IGF1Rcross-activation in the brain. Further studies will be necessaryto dissect the specific contributions of IR and IGF1R or thepotential role of IR/IGF1R hybrids in these phenotypes.In summary, we have shown important roles for hippocampal

and amygdala IR and IGF1R signaling in systemic glucose ho-meostasis and thermogenesis. At least a part of this effect may bethrough modulation of an amygdala-dependent neural circuit tocontrol sympathetic outflow to liver and brown adipose tissue,thus regulating peripheral glucose handling and cold-inducedthermogenesis. In addition, we demonstrate that IR and IGF1Rin the hippocampus and central amygdala are important formood and cognition. These defects in IR/IGF1R-deficient micemay be the result of a reduction of AMPA receptor subunitGluA1 in the synapse, which impairs synaptic plasticity in DKO

mice. Thus, both the hippocampus and amygdala are importantbrain regions for central insulin action and normal energy ho-meostasis and neural functions.

MethodsAll animal studies were conducted in compliance with the regulations andethics guidelines of the NIH and were approved by the Institutional AnimalCare and Use Committee (IACUC) of the Joslin Diabetes Center. Detailedmaterials and methods are available in SI Appendix.

ACKNOWLEDGMENTS. We thank Dr. Lynn W. Enquist (Princeton University)for sharing the PRV-765 virus for the retrograde tracing studies. This workwas supported by NIH Grants R01 DK031036 and R01 DK033201 (to C.R.K.).The Advanced Microscopy Core and Animal Physiology Core in the JoslinDiabetes Research Center (DRC) (P30 DK036836) also provided importanthelp.

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