Rip-Cre transgenic mice were crossed with cre-dependent green fluorescent protein (GFP) reporter mice () to generate Rip-Cre, lox-GFP mice, and immunohistochemistry for GFP was performed. GFP-positive neurons (i.e., RIP-Cre neurons) were primarily found in the hypothalamus and occasionally in the cortex and striatum ( Figure 1 A; see Table S1 available online). Within the hypothalamus, GFP-positive neurons were observed in the ARC, the ventromedial hypothalamus (VMH), the medial tuberal nucleus (MTu), the suprachiasmatic nucleus (SCN), and the dorsomedial hypothalamus (DMH). These observations are consistent with previous reports by, and

(D and E) Quantitative PCR results of Vgat mRNA in (D) mediobasal hypothalamus and (E) isolated pancreatic islets of 2-month-old littermates (mean ± SEM; n = 4). N.D., nondetectable. ∗ p < 0.05, unpaired t tests.

(B and C) In situ hybridization for Vgat mRNA in the brain of (B) control (Vgat flox/flox ) and (C) Rip-Cre, Vgat flox/flox littermates. Arrows indicate the regions with notable reduction of Vgat mRNA signal in Rip-Cre, Vgat flox/flox mice.

Vgat mRNA was undetectable in both control and Rip-Cre, Vgatpancreatic islets ( Figure 1 E), whereas, as noted above, using the identical assay, Vgat mRNA was readily detected in the hypothalamus ( Figure 1 D). Confirming the intact nature of pancreatic islet RNA, Ucp2, a gene expressed in islets (), was readily detected in the same samples ( Figure 1 E). Thus, mouse islets express little or no Vgat mRNA. Consequently, deletion of the Vgat gene in pancreatic β cells should produce no effects.

Rip-Cre transgenic mice were crossed with Vgatmice. The resulting Rip-Cre, Vgatmice were then crossed with Vgatmice to obtain Rip-Cre, Vgatstudy subjects and their control littermates (Vgatmice and Rip-Cre, Vgatmice). Rip-Cre, Vgatlittermates were included as controls to rule out nonspecific effects of the Rip-Cre transgene (). In situ hybridization studies for Vgat mRNA were then performed to assess for GABAergic neurons and also for sites where the Rip-Cre transgene disrupts Vgat expression. In Vgatcontrol mice, Vgat mRNA signal was detected in sites known to contain GABAergic neurons. With regard to sites shown in Figure 1 B, these include, within the hypothalamus, the ARC, DMH and MTu; and beyond the hypothalamus, the central amygdala (CeA) and the reticular nucleus of thalamus (RT). In Rip-Cre, Vgatmice ( Figure 1 C), Vgat signal was substantially reduced in sites containing RIP-Cre neurons, such as the ARC, DMH, and MTu. In sites where RIP-Cre neurons are not found, such as the CeA and RT, Vgat signal, as expected, was unchanged. Finally, we measured Vgat mRNA levels in the mediobasal hypothalamus, a region that includes the ARC, DMH, and MTu, which are three sites suggested by the aforementioned analysis to contain GABAergic RIP-Cre neurons. This quantitative analysis confirmed that Vgat mRNA, but not that of a control transcript (Ucp2 mRNA), was substantially reduced in Rip-Cre, Vgatmice ( Figure 1 D).

Given that Rip-Cre transgenic mice express cre in some SCN neurons ( Figure S1 B) and that virtually all SCN neurons are GABAergic ( Figure S1 C), Rip-Cre, Vgatmice could have altered circadian regulation, which could in turn affect energy balance (). To address this possibility, body temperature (Tb), a reliable indicator of circadian clock activity (), was measured using implanted biotelemetry. As shown in Figures S1 D and S1E, Rip-Cre, Vgatmice displayed diurnal and circadian Tb patterns (phasing, amplitude, or period) that were comparable to controls. Thus, the circadian clock does not appear to be altered or otherwise dysfunctional in Rip-Cre, Vgatmice and unlikely contributes to the metabolic phenotypes of the Rip-Cre, Vgatmice. Likewise, serum T4 and corticosterone levels, two other potential regulators of energy balance, were found to be unchanged in Rip-Cre, Vgatmice ( Figures S1 F and S1G).

We next analyzed brown adipose tissue (BAT), a well-established mediator of thermogenesis (). Interscapular BAT (iBAT) of Rip-Cre, Vgatmice was markedly enlarged and pale in comparison with that from control littermates. As shown in Figure 2 G, BAT from Rip-Cre, Vgatmice contained larger cells with unilocular triglyceride deposits, similar to that observed in animals with defective sympathetic activation of BAT (). As assessed by biotelemetry probes implanted subcutaneously in the interscapula fossa beneath iBAT (), iBAT temperature, an index of BAT thermogenesis, was reduced in Rip-Cre, Vgatmice ( Figure 2 H). In contrast, subcutaneous temperature of a flank site devoid of BAT was similar in the two groups ( Figure S1 A). Finally, expression of Ucp1 mRNA, which encodes the BAT-specific thermogenic molecule, uncoupling protein 1, was significantly lower in Rip-Cre, Vgatmice ( Figure 2 I). These results indicate that GABA release from RIP-Cre neurons regulates BAT thermogenic function and suggest that decreased BAT activity is, at least in part, responsible for reduced energy expenditure in Rip-Cre, Vgatmice.

(D and E) Circadian rhythms of body temperature (Tb) over 30 days recorded in 2-month old control (Vgat flox/flox , n = 3) and Rip-Cre, Vgat flox/flox (n = 5) male littermates using i.p. biotelemetry implants. The animals were housed under 12:12 light-dark cycles (LD) for 15 days, followed by exposure to constant darkness (DD); Two representative actograms from each genotype are shown. Under LD conditions, the diurnal rhythm of Tb in Rip-Cre, Vgat flox/flox mice exhibited stable entrainment with a phase angle that was comparable to that of the wild-types. Similarly, under DD conditions, Rip-Cre, Vgat flox/flox mice showed a persisting consolidated “free-running” circadian Tb rhythm with a period close to or slightly shorter than 24 hr, indicating that deletion of GABA release from SCN RIP-Cre neurons does not disturb circadian rhythms.

(C) In situ hybridization for Vgat mRNA ( 35 S-labbeld cRNA probe, silver grains) in control (Vgat flox/flox ) and Rip-Cre, Vgat flox/flox littermates. Arrows indicate the SCN with notable reduction of Vgat mRNA signal in Rip-Cre, Vgat flox/flox mice.

When fed a standard chow diet, Rip-Cre, Vgatmice have modestly increased body weight ( Figure 2 A) and, at 3 months of age, markedly increased fat stores ( Figure 2 B). Lean body mass, on the other hand, was unchanged (data not shown). The possible causes of positive energy balance were then assessed in 2-month-old animals. Food intake ( Figure 2 C) and locomotor activity ( Figure 2 D) were found to be unchanged. Obesity, in the face of normal food intake, strongly implicates reduced energy expenditure in Rip-Cre, Vgatmice. This was confirmed by direct assessments of energy expenditure. Oxygen consumption was markedly reduced in Rip-Cre, Vgatmice, and this was apparent when data were expressed per body weight ( Figure 2 E) or per animal ( Figure 2 F). Thus, obesity in Rip-Cre, Vgatmice is due entirely to a selective reduction in energy expenditure.

Data are presented as mean ± SEM.p < 0.05 andp < 0.001, unpaired t tests compared with Vgatgroup. See also Figures S1 and S2

(A–D) (A) Body weight, (B) body fat mass (3 months old), (C) daily food intake (2 months old), and (D) locomotor activity (2 months old) of ad libitum chow-fed male littermates (n = 8–10). Black bars in (D) indicate dark cycles.

Some RIP-Cre neurons are glutamatergic, such as those in the VMH. To assess if glutamatergic RIP-Cre neurons regulate energy homeostasis, mice lacking VGLUT2 in RIP-Cre neurons (Rip-Cre, Vglut2mice) were generated as was done for Rip-Cre, Vgatmice. In situ hybridization analyses revealed that, as expected, Vglut2 mRNA was dramatically reduced in the VMH of Rip-Cre, Vglut2mice ( Figures S2 A and S2B). Of note, Rip-Cre, Vglut2mice had normal body weight, oxygen consumption, and food intake ( Figures S2 C–S2E), indicating that glutamate release from RIP-Cre neurons is not required for regulation of energy balance.

(C) Body weight, (D) oxygen consumption, and (E) daily food intake of ad libitum chow-fed male mice at 3 months of age (mean ± SEM; n = 8–10).

(A and B) In situ hybridization for Vglut2 mRNA ( 35 S-labbeld cRNA probe, silver grains) in the brain of (A) control (Vglut2 flox/flox ) and (B) Rip-Cre, Vglut2 flox/flox littermates.

Increased energy expenditure following the ingestion of highly palatable, calorically dense diets, a phenomenon often referred to as diet-induced thermogenesis, plays an important role in resisting diet-induced obesity (). To determine if GABAergic RIP-Cre neuron-driven energy expenditure is involved in this adaptive response, Rip-Cre, Vgatmice and control animals were fed a high-fat, high-sucrose diet (HFD) from between 6 and 26 weeks of age. As shown in Figure 3 A, compared to control mice, Rip-Cre, Vgatmice developed massive obesity. Of interest, this diet-induced obesity was not caused by increased food intake ( Figure 3 B), strongly implicating impaired diet-induced thermogenesis. To directly assess this, oxygen consumption was measured during the transition from chow to HFD (3 days on chow followed by 3 days on HFD) in 7-week-old mice (note, body weight is normal at this early age). As shown in Figure 3 C, oxygen consumption of Rip-Cre,Vgatmice, on both chow and HFD, was markedly lower than that observed in control animals. Importantly, whereas HFD increased energy expenditure by 7%–12% in control mice, this response was markedly blunted in Rip-Cre, Vgatmice (increased by only 3%–5% in response to HFD). In addition, HFD-treated Rip-Cre,Vgatmice (6 months old and 20 weeks on HFD) exhibited reduced Ucp1 mRNA expression in BAT ( Figure 3 D). In summary, Rip-Cre, Vgatmice are extremely sensitive to diet-induced obesity, and this is due entirely to a defect in diet-induced thermogenesis.

(C) Oxygen consumption expressed per body weight during the transition from chow to HFD (n = 8). CD, averaged oxygen consumption over 3 days on chow; HD1, HD2 and HD3, oxygen consumption during day 1, 2, and 3, respectively, on HFD. The percent increase in oxygen consumption on HFD above that on chow diet is indicated above each bar.

(A and B) Body weight on HFD (A) and daily food intake (B) averaged over the first 2 weeks on HFD (n = 8–10).

To determine which subset of RIP-Cre neurons in the hypothalamus expresses LEPRs, leptin-induced phosphorylation of STAT3 (Tyr705, pSTAT3), a marker for LEPR activity (), was assessed in Rip-Cre, lox-GFP mice. The neurons double positive for pSTAT3 and RIP-Cre activity were mainly observed in the ARC and the VMH ( Figures 4 H, 4I, and S3 C–S3N). The DMH, which contained both RIP-Cre neurons and pSTAT3-positive neurons, exhibited negligible colocalization ( Figures 4 I and S3 I–S3K). Because VMH neurons are glutamatergic and not GABAergic (), all leptin-responsive, GABAergic RIP-Cre neurons appear to be located in the ARC. Collectively, given the aforementioned observations, it is likely that GABAergic RIP-Cre neurons in the ARC mediate leptin’s stimulatory effect on energy expenditure.

Genetic deletion of leptin receptors (LEPRs) from RIP-Cre neurons causes marked obesity without affecting food intake (), suggesting a defect in energy expenditure. Given that Rip-Cre,Vgatmice also have defective energy expenditure, this raises the possibility that GABA release from RIP-Cre neurons mediates leptin’s effects on energy expenditure. To test this, changes in body weight and food intake were monitored following injection of saline or leptin. In control mice, treatment with leptin reduced body weight ( Figures 4 A and 4B) and food intake ( Figures 4 C and 4D). Of note, whereas the ability of leptin to reduce food intake in Rip-Cre,Vgatmice was completely intact ( Figure 4 D), its ability to reduce body weight was markedly attenuated ( Figure 4 B). Attenuation of body weight loss in the face of intact inhibition of food intake indicates that leptin’s ability to increase energy expenditure is impaired in Rip-Cre, Vgatmice. To directly assess leptin action on energy expenditure, in particular, in stimulating BAT activity, iBAT temperature and Ucp1 mRNA were measured. In control mice, leptin but not saline dramatically and rapidly increased the temperature of iBAT (as previously described by) ( Figures 4 E and 4F), but not the temperature of a subcutaneous flank site devoid of BAT ( Figures S3 A and S3B). Leptin also markedly increased Ucp1 mRNA levels ( Figure 4 G). Of note, these stimulatory effects of leptin on iBAT temperature and Ucp1 mRNA levels were attenuated in Rip-Cre,Vgatanimals ( Figures 4 E–4G). Thus, GABA release from RIP-Cre neurons is required for leptin to fully stimulate energy expenditure, but not for leptin to inhibit feeding.

(C–N) Double immunohistochemistry for GFP (green), leptin-induced phosphorylated STAT3 (Tyr105, pSTAT3) (purple) in the ARC (C–E), VMH (F–H), DMH (I–K), and SCN (L–N) of Rip-Cre, lox-GFP mice. Neurons with co-expressed GFP and pSTAT3 (indicated by arrows) were mainly observed in the ARC and VMH, with few in the DMH and none in the SCN. See also Figure 4 I for quantification.

(A and B) Subcutaneous flank temperature of control (Vgat flox/flox ) and Rip-Cre, Vgat flox/flox mice, upon (A) saline or (B) leptin (6 mg/Kg, i.p.) stimulation (mean ± SEM, n = 8–10).

Data are presented as mean ± SEM. See also Figures S3 and S5

(I) Quantification of the neurons that expressed GFP, pSTAT3, or both in the hypothalamic nuclei (n = 3 mice).

(H) Double immunohistochemistry for GFP (green) and leptin-induced phosphorylation of STAT3 (Tyr105, pSTAT3, magenta) in the ARC of Rip-Cre, lox-GFP mice. Arrows indicate the neurons with coexpression of GFP and pSTAT3.

(G) Ucp1 mRNA level in iBAT 4 hr after saline (Sal) or leptin (Lep) injection (n = 6). ∗ p < 0.05 and ∗∗ p < 0.01, unpaired t tests compared with saline-injected animals of the same genotype; # p < 0.05, unpaired t tests compared with Vgat flox/flox animals of the same treatment.

(A–F) The effects of saline or leptin on (A and B) body weight, (C and D) daily food intake, and (E and F) iBAT temperature in 2-month-old male littermates (n = 8–12). ∗ p < 0.05, paired t tests compared with animals of the same genotype before leptin injection (i.e., time point 0); # p < 0.05, unpaired t tests compared with control animals at given time point.

The pancreatic beta cell is a key site for mediating the effects of leptin on glucose homeostasis.

To assess effects on energy expenditure, virus-injected animals were housed individually in metabolic cages, and oxygen consumption was monitored following injection with saline or CNO. After an acclimation period, each mouse was injected with saline on the first day followed by CNO on the second day. Selective activation of ARC RIP-Cre neurons with CNO rapidly increased oxygen consumption ( Figure 5 F), and this effect lasted for approximately 9 hr. Importantly, CNO had no effect on oxygen consumption in Rip-Cre, Vgatmice ( Figure 5 G), which are notable for their inability to release GABA. Similarly, CNO had no effect on oxygen consumption in control mice (i.e., Vgatmice), which do not express hM3Dq ( Figure 5 E). Remarkably, and consistent with our earlier findings suggesting that GABAergic RIP-Cre neurons selectively control energy expenditure, food intake was unaltered by CNO treatment ( Figure S4 B). We next assessed BAT activity during stimulation of ARC RIP-Cre neurons. In Rip-Cre mice, CNO significantly increased iBAT temperature ( Figure 5 I), but not subcutaneous flank temperature ( Figure S4 C), and also markedly increased Ucp1 mRNA ( Figure 5 L). CNO did not stimulate iBAT temperature or Ucp1 mRNA in Vgatmice (which do not express hM3Dq) or in Rip-Cre, Vgatmice (which are unable to release GABA) ( Figures 5 H, 5J, 5K, and 5M). These results demonstrate that synaptic GABA release from ARC RIP-Cre neurons selectively stimulates BAT activity and energy expenditure.

To directly test the ability of ARC RIP-Cre neurons to drive energy expenditure, we used the pharmacogenetic approach referred to as designer receptors exclusively activated by designer drugs (DREADD). The stimulatory DREADD, hM3Dq, is activated by the otherwise inert, brain-penetrable compound, clozapine-N-oxide (CNO) (). The cre-dependent adeno-associated virus, AAV-Flex-hM3Dq-mCherry (), was stereotaxically injected into the ARC of 3- to 4-week-old Rip-Cre transgenic mice ( Figure 5 A), and studies were performed 2–3 weeks after injection. Brain slice electrophysiology studies confirmed that CNO depolarizes and increases the firing rate of hM3Dq-expressing RIP-Cre neurons ( Figure 5 B), but not control non-hM3Dq-expressing RIP-Cre neurons ( Figure S4 A). hM3Dq virus was then bilaterally injected into the ARC of 5- to 6-week-old Vgatmice, Rip-Cre mice, or Rip-Cre, Vgatmice. Studies were performed 3 weeks after injection. The mCherry fusion tag was exclusively detected in the ARC of Rip-Cre mice and Rip-Cre, Vgatmice and was absent in the ARC of Vgatmice (because these mice lack cre activity that enables hM3Dq expression) ( Figure 5 C). When CNO was injected in vivo, c-fos immunoreactivity was markedly increased in the ARC of Rip-Cre mice and Rip-Cre, Vgatmice, but not in the ARC of Vgatmice ( Figure 5 D). Thus, in vivo treatment with CNO activates hM3Dq-expressing RIP-Cre neurons in the ARC.

(B) Food intake over the first 4 hr following saline or CNO injection in AAV-Flex-hM3Dq-mCherry virus-injected Vgat flox/flox ,(left), Rip-Cre (middle), and Rip-Cre, Vgat flox/flox (right) mice (mean ± SEM, n = 8).

(A) A representative voltage tracing recorded in an ARC RIP-Cre neuron that was negative for mCherry fluorescence. The brain slice was prepared from a Rip-Cre, lox-GFP mouse that was injected with AAV-Flex-hM3Dq-mCherry virus into the ARC. 5 μM CNO was applied to the bath as indicated.

(K–M) Ucp1 mRNA in iBAT 6 hr after saline or CNO injection (n = 4–6). ∗ p < 0.05, unpaired t tests compared with saline-injected animals of the same genotype.

(H–J) iBAT temperature (Temp) over 4 hr following saline or CNO injection (n = 6–8). ∗ p < 0.01, paired t test compared to animals of the same genotype before CNO injection; # p < 0.01, paired t tests compared to saline-injected animals at given time point.

(E–G) Oxygen consumption over a 24 hr period (left) and during the first 4 hr (right) following i.p. injection of saline or CNO (n = 8). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, paired t tests compared to saline groups.

(C and D) Immunohistochemistry for (C) mCherry and (D) CNO-induced c-Fos (DAB, black stain) in the ARC of virus-injected Vgat flox/flox (top), Rip-Cre (middle), and Rip-Cre, Vgat flox/flox (bottom) male mice.

(A) Diagram of AAV-Flex-hM3Dq-mCherry (left) and schematic indication of the stereotaxic injection into the ARC of Rip-Cre transgenic mice (right).

Whole-cell current-clamp recordings were performed as previously described byon arcuate RIP-Cre neurons visualized by expression of GFP in Rip-Cre, lox-GFP mice. In data not shown, ARC RIP-Cre neurons exhibited heterogeneous responses to leptin: 30% of neurons (6 of 20) were excited (depolarized membrane potential and increased firing rate), 35% (7 of 20) were inhibited (hyperpolarized membrane potential and decreased firing rate), and 35% (7 of 20) were not affected by leptin. As discussed in the following section, the paraventricular hypothalamus (PVH) is the likely downstream site that mediates the thermogenic effects of GABAergic ARC RIP-Cre neurons. To assess the effects of leptin on PVH-projecting ARC RIP-Cre neurons, retrograde red fluorescent beads were stereotaxically injected into the PVH of Rip-Cre, lox-GFP mice ( Figures S5 A and S5B). Retrogradely transported beads were observed in sites known to innervate the PVH, including the ARC, DMH, SCN, nucleus of the solitary tract (NTS), and posterodorsal medial amygdala (MEpd) (data not shown). In the ARC, 62% (194 of 312) of RIP-Cre neurons contained red beads ( Figure S5 C). Recordings were then performed on PVH-projecting (GFP/beads) and in non-PVH-projecting (GFP/beads) ARC RIP-Cre neurons. Ionotropic glutamate (kynurenate) and GABA (PTX) receptor blockers were added to minimize indirect effects of leptin. As shown in Figure S5 D, 9 of 15 PVH-projecting ARC RIP-Cre neurons were directly excited by leptin, 6 of 15 were unaffected by leptin, and 0 of 15 were inhibited by leptin. In contrast, 0 of 12 non-PVH-projecting ARC RIP-Cre neurons were excited by leptin, 9 of 12 were unaffected by leptin, and 3 of 12 were directly inhibited by leptin ( Figure S5 E). Thus, leptin excites the majority of PVH-projecting ARC RIP-Cre neurons.

(D and E) Whole-cell current-clamp recordings were performed in ARC neurons labeled with (D) both GFP and red beads or with (E) GFP only. Synaptic blockers (1 mM Kynurenate and 100 μM PTX) were included in aCSF to minimize leptin’s indirect effects. (D) Representative tracing of PVH-projecting ARC RIP-Cre neurons: (D i) that were activated by leptin (membrane potential: from −60.7 ± 2.1 mV to −54.3 ± 1.7 mV (n = 9, p < 0.001), firing rate: from 0.12 ± 0.09 Hz to 0.54 ± 0.23Hz (n = 9, p < 0.05); (D ii) that did not respond to leptin (membrane potential: from −59.4 ± 2.8 mV to −59.9 ± 2.8 mV [n = 6], firing rate: from 0.40 ± 0.28 Hz to 0.42 ± 0.30 Hz [n = 6]). (E) Representative tracing of non-PVH-projecting ARC RIP-Cre neurons: (E i) that did not respond to leptin (membrane potential: from −59.5 ± 2.0 mV to −60.1 ± 1.8 mV (n = 9), firing rate: from 0.9 ± 0.3 Hz to 1.0 ± 0.4 Hz (n = 9); (E ii) that were inhibited by leptin (membrane potential: from −60.2 ± 1.1 mV to −66.7 ± 0.9 mV (n = 3, p < 0.001), firing rate: from of 0.72 ± 0.58Hz to 0.06 ± 0.03 Hz [n = 3]). (mean ± SEM, unpaired t test).

(C) Immunohistochemistry against GFP (green) and native fluorescence of retrograde red beads (red) in the ARC of beads-injected Rip-Cre, lox-GFP animals. Arrows indicate RIP-Cre neurons that were labeled with both GFP and red beads (i.e., PVH-projecting RIP-Cre neurons). An adjacent RIP-Cre neuron that was labeled with GFP only but not with red beads is also shown (i.e., a RIP-Cre neuron that does not project to the PVH). There were also many ARC neurons labeled with red beads only but not with GFP (i.e., PVH-projecting, non-RIP-Cre neurons) (data not shown).

Leptin directly activates SF1 neurons in the VMH, and this action by leptin is required for normal body-weight homeostasis.

Downstream Neurocircuitry Engaged by ARC RIP-Cre Neurons

A receptor antagonist ( Biag et al., 2012 Biag J.

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Hahn J.D.

Toga A.W.

Dong H.W. Cyto- and chemoarchitecture of the hypothalamic paraventricular nucleus in the C57BL/6J male mouse: a study of immunostaining and multiple fluorescent tract tracing. Simmons and Swanson, 2009 Simmons D.M.

Swanson L.W. Comparison of the spatial distribution of seven types of neuroendocrine neurons in the rat paraventricular nucleus: toward a global 3D model. To determine if GABAergic RIP-Cre neurons are functionally, synaptically connected to PVH neurons, inhibitory postsynaptic currents (IPSCs) were assessed in PVH neurons following illumination of ChR2-expressing RIP-Cre terminals. Light-driven IPSCs were reliably evoked in a small subset of randomly selected PVH neurons that were surrounded by mCherry fluorescent terminals (2 out of 14), and these were completely blocked by bicuculline, a GABAreceptor antagonist ( Figure 6 F). The latency between onset of light and onset of IPSC in these two neurons was 1.2 and 2.3 ms. The low frequency of responders (i.e., 2 out of 14) likely relates to complexity within the PVH, which is composed of numerous subsets of functionally distinct neurons (). As explained below, we have used site of projection to enrich for PVH neurons likely to control energy expenditure and, therefore, likely to receive monosynaptic input from GABAergic RIP-Cre neurons.