Changes in food availability alter the output of hypothalamic nuclei that underlie energy homeostasis. Here, we asked whether food deprivation impacts the ability of GABA synapses in the dorsomedial hypothalamus (DMH), an important integrator of satiety signals, to undergo activity-dependent changes. GABA synapses in DMH slices from satiated rats exhibit endocannabinoid-mediated long-term depression (LTD GABA ) in response to high-frequency stimulation of afferents. When CB1Rs are blocked, however, the same stimulation elicits long-term potentiation (LTP GABA ), which manifests presynaptically and requires heterosynaptic recruitment of NMDARs and nitric oxide (NO). Interestingly, NO signaling is required for eCB-mediated LTD GABA . Twenty-four hour food deprivation results in a CORT-mediated loss of CB1R signaling and, consequently, GABA synapses only exhibit LTP GABA . These observations indicate that CB1R signaling promotes LTD GABA and gates LTP GABA . Furthermore, the satiety state of an animal, through regulation of eCB signaling, determines the polarity of activity-dependent plasticity at GABA synapses in the DMH.

Because food deprivation increases circulating CORT, which, in other systems, downregulates CB1Rs (), we hypothesized that the absence of food, through associated changes in eCB signaling, would play a deterministic role in the ability of GABA synapses in the DMH to undergo activity-dependent plasticity. DMH neurons receive abundant GABAergic input from various hypothalamic nuclei, including the arcuate nucleus (), and primarily send glutamatergic projections to the paraventricular nucleus of the hypothalamus (PVN) (), where they play a role in the integration of satiety and stress signals. Our results indicate that in satiated animals, plasticity at GABA synapses relies on the combined effects of eCBs and NO and is biased, particularly during prolonged, repetitive recruitment of afferents, toward long-term depression (LTD). Following food deprivation, however, CORT-induced impairment of eCB signaling converts this system to one that only exhibits NO-dependent potentiation of GABA synapses (LTP).

Forebrain origins of glutamatergic innervation to the rat paraventricular nucleus of the hypothalamus: differential inputs to the anterior versus posterior subregions.

Organization of inputs to the dorsomedial nucleus of the hypothalamus: a reexamination with Fluorogold and PHAL in the rat.

Nitric oxide (NO), like the eCBs, is a retrograde signal that is produced in response to a rise in intracellular Ca. Unlike eCBs, however, NO has stimulatory effects on GABA release (). Although these retrograde transmitters have opposing actions at GABA synapses, accumulating evidence hints at a more nuanced interaction between eCBs and NO in mediating changes in synaptic strength. Specifically in some conditions, NO appears to be necessary for the induction of eCB-mediated plasticity (), although the exact mechanism is unclear. We therefore asked how the control of GABAergic transmission in feeding circuits is regulated by eCBs and NO under conditions of satiety and food deprivation.

The synapses connecting key nuclei in circuits regulating energy homeostasis are sensitive to a number of chemical mediators, but no signal is as ubiquitously expressed as endocannabinoids (eCBs). These lipophilic molecules are produced in response to increases in postsynaptic Caand act as retrograde signals to quench both glutamate and GABA release at nerve terminals (). Although there is widespread support for the hypothesis that eCBs are orexigenic signals and that targeting the eCB system is beneficial for the treatment of eating disorders (), emerging evidence suggests the relationship between eCBs and energy homeostasis is more complex. Using a genetic and pharmacological approach, recent work has revealed that eCBs have divergent actions on food intake. eCB-mediated hyperphagic actions appear to be the result of actions at CB1Rs located on glutamate terminals. By contrast, eCB actions at GABA terminals suppress food intake ().

Abrupt changes in an organism's environment precipitate requisite and rapid adaptive changes in neural circuits. In particular, synapses in hypothalamic nuclei that form the neural network underlying energy balance and food intake are remarkably susceptible to variations in the availability of food. The dearth of food is of such importance to an organism that it triggers both direct changes in food-related signals and the immediate activation of the stress response that increases circulating corticosteroids (CORT) (). The dorsomedial nucleus of the hypothalamus (DMH) regulates food intake and serves as a center for the integration of food and stress signals (). More recently, the DMH has also been implicated as being the key food entrainable oscillator in the brain that exhibits synchronous activity in response to food deprivation (). Although both of these roles are key to an organism's survival, surprisingly little is known about synaptic processing in the DMH and even less is known about the effects of food deprivation on synaptic function and plasticity in this nucleus.

Finally, we asked whether these synaptic changes could be reversed by the re-introduction of food. Following food deprivation, animals were given unlimited access to food for 24 hr and then slices containing the DMH were prepared for electrophysiology. Refeeding following food deprivation restores circulating CORT to basal levels within 6 hr of food presentation (). Following refeeding, HFS did not elicit LTP(88% ± 12.7% of baseline, n = 5, p = 0.919; Figure 6 C), potentially suggesting partial functional recovery of CB1Rs. In agreement with this finding, WIN 55,212-2–induced depression of GABA synapses was restored after 24 hr of refeeding (63% ± 11.0% of baseline, n = 4, p = 0.021). Taken together, these results indicate that a food deprivation–induced rise in CORT leads to a downregulation of CB1Rs, thus creating a permissive state that favors the induction of LTP Figure 7 ).

Both eCBs and NO are produced in postsynaptic cells following a burst of activity in afferent inputs (high-frequency stimulation; HFS) but exhibit different thresholds for production. A brief synaptic burst (1 s) generates NO but not eCBs. Longer bursts (2 and 4 s) generate increasing amounts of eCBs in addition to NO. The different thresholds for NO or eCB production creates local environments that are exposed to only NO or both NO and eCBs. NO stimulates GABA release while eCBs through actions at presynaptic CB1Rs inhibit GABA release. In satiated animals, eCBs gate the ability of NO to induce LTP. Food deprivation inactivates CB1Rs via an increase in the circulating levels of CORT. In the absence of eCB signaling, HFS results in a uniform NO-mediated potentiation.

Schematic Diagram of the Effect of NO and eCBs on GABA Synapses following Different Durations of Stimulation and Food Deprivation

Figure 7 Schematic Diagram of the Effect of NO and eCBs on GABA Synapses following Different Durations of Stimulation and Food Deprivation

We have demonstrated thus far that CB1R signaling precludes NO-mediated LTPin the DMH. Here, we hypothesized that a physiological state in which CB1R signaling is compromised should favor the induction of LTP. There is compelling evidence that CORT reduces the expression of CB1Rs in the brain () and that acute food deprivation results in significant elevations in circulating CORT (). We first examined the impact of food deprivation on CB1R function in DMH neurons by testing the ability of WIN 55,212-2 to depress GABA synapses. Animals were food-deprived for 24 hr prior to slice preparation. Unlike naïve animals ( Figure 4 A), WIN 55,212-2 had no effect on the amplitude of evoked IPSCs (99% ± 6.6% of baseline, n = 6, p = 0.370, Figure 6 A), PPR (baseline: 0.938 ± 0.062; post-drug: 0.967 ± 0.114; p = 0.460), or CV (baseline: 0.103 ± 0.015; post-drug: 0.137 ± 0.052; p = 0.234) in food-deprived animals. To determine whether elevated levels of CORT were responsible for the loss of CB1R signaling, we administered the genomic glucocorticoid receptor antagonist, RU486 (25 mg/kg, subcutaneous) at 12 hr intervals during the 24 hr food deprivation period. In slices obtained from animals receiving RU486, CB1R agonist-mediated depression was recovered (64% ± 12.3% of baseline, n = 6, p = 0.037; Figure 6 A ). We next asked whether food deprivation unmasked LTP. Indeed, in neurons from food-deprived animals, HFS elicited a robust LTP(177% ± 26.9% of baseline, n = 7, p = 0.029; Figure 6 B). This was accompanied by a decrease in PPR (baseline: 1.276 ± 0.113; post-HFS: 0.833 ± 0.064; p = 0.006) and CV (baseline: 0.376 ± 0.061; post-HFS: 0.240 ± 0.026; p = 0.035), and an increase in the frequency of sIPSCs (269% ± 46.6% of baseline, p = 0.049), but a decrease in sIPSC amplitude (79% ± 4.4% of baseline, p = 0.006), suggesting an increase in the probability of GABA release from the presynaptic terminal. These observations indicate that acute food deprivation converts LTDto LTPin DMH neurons. RU486 treatment in food-deprived animals completely abolished LTPand unmasked an activity-dependent depression (68% ± 6.6% of baseline, n = 7, p = 0.018; Figure 6 B). In food-deprived animals receiving vehicle, HFS potentiated GABA synapses (148% ± 9.4% of baseline, n = 8, p = 0.0020; Figure 6 C), confirming the specificity of the effect of RU486. These experiments provide direct evidence that elevations in CORT accompanying food deprivation are necessary for these synapses to undergo LTP. Similar to LTPin slices from naïve animals following CB1R blockade or from CB1Ranimals, this synaptic potentiation was completely abolished in the presence of either L-NAME (102% ± 14.7% of baseline, n = 7, p = 0.921; Figure 6 D) or APV (117% ± 10.3% of baseline, n = 5, p = 0.157; Figure 6 D), indicating that it is mediated by NO produced by heterosynaptic activation of NMDARs. To determine whether these changes are specific to the prolonged stress of food deprivation, we conducted two additional experiments. First, animals were subjected to 30 min of immobilization, a stressor that transiently increases CORT () prior to experimentation. This manipulation failed to elicit LTPin response to HFS (119% ± 11.5% of baseline, n = 8, p = 0.284; Figure 6 C). These data suggest that prolonged activation of the HPA axis (∼24 hr) is required to shift synapses from a depressing to a potentiating state. One additional prolonged stressor may result from animals being housed individually during the food deprivation period. To rule out the possibility that social isolation alone (as a mild stressor) is sufficient to unmask LTP, we investigated whether HFS would elicit LTPin animals housed alone but given ad libitum access to food for 24 hr prior to slice preparation. Under these conditions, synapses did not exhibit LTP, but instead underwent an activity-dependent depression in GABA transmission (71% ± 12.2% of baseline, n = 5, p = 0.032; Figure 6 C), indicating that social isolation is not sufficient to shift the polarity of the plasticity.

(C) Summary data showing percent change in IPSC amplitude following HFS under different conditions as shown in graph.

(B) Summary data showing LTP GABA in food-deprived animals, which is blocked with RU486. Sample traces of averaged IPSCs before and after HFS (arrow and dashed line) in food-deprived animals and in food-deprived animals receiving RU486 are shown to the left.

(A) Summary data showing the effect of WIN 55,212-2 (5 μM) in animals experiencing 24 hr of food deprivation prior to experimentation and food-deprived animals receiving the genomic glucocorticoid receptor antagonist RU486 (25 mg/kg; 2 s.c. injections, 12 hr apart during the food deprivation period). Sample traces of averaged IPSCs before and during incubation of slices in WIN 55,212-2 in food-deprived animals and food-deprived animals receiving RU486 are shown to the left.

Next, we conducted experiments to determine the consequences of NO production on eCB-mediated LTD. When NO synthesis was inhibited by L-NAME, HFS (100 Hz for 4 s × 2, 0.05 Hz interval) failed to elicit LTD(111% ± 11.3% of baseline, n = 8, p = 0.350; Figure 5 E), the changes in PPR (baseline: 0.860 ± 0.086; post-HFS: 0.826 ± 0.102; p = 0.369), or the changes in CV (baseline: 0.311 ± 0.028; post-HFS: 0.336 ± 0.07; p = 0.452). This suggests that NO signaling is required either for eCB production or CB1R signaling. Consistent with the latter idea, direct activation of CB1Rs by WIN 55,212-2 in the presence of L-NAME failed to significantly depress evoked IPSC amplitude (88% ± 10.8% of baseline, n = 6, p = 0.375; Figure 5 F), PPR (baseline: 0.903 ± 0.129; post-drug: 0.889 ± 0.092; p = 0.850), or CV (baseline: 0.362 ± 0.067; post-drug: 0.410 ± 0.094; p = 0.168). Overall, these data point to an inherent complexity in the signaling of the retrograde transmitters eCBs and NO in the DMH. Specifically, they argue that eCB signaling prevents NO-mediated potentiation of GABA synapses but that NO signaling is required for eCB-induced depression of GABA signaling.

Our data demonstrate that NO-mediated LTPis more prevalent in the absence of CB1R signaling. This raises the question as to how these signaling pathways interact at DMH synapses. If CB1R activation at the presynaptic terminal precludes the effects of NO to enhance GABA release, then the application of a CB1R agonist should block the potentiation of GABA transmission by the NO donor, SNAP. Consistent with this idea, SNAP failed to increase evoked IPSC amplitude when applied to slices that were continuously perfused with WIN 55,212-2 (104% ± 12.6% of WIN 55,212-2, n = 5, p = 0.646; Figures 5 A and 5C). Similarly, it did not affect PPR (baseline: 0.961 ± 0.119; post-drug: 0.883 ± 0.178; p = 0.544) or CV (baseline: 0.502 ± 0.071; post-drug: 0.500 ± 0.045; p = 0.962). Conversely, WIN 55,212-2 still effectively depressed IPSCs that were first potentiated by SNAP (36% ± 12.0% of SNAP, n = 7; Figures 5 B and 5D ). This change was accompanied by an increase in PPR (baseline: 0.663 ± 0.109; post-drug: 0.950 ± 0.099; p = 0.048) and CV (baseline: 0.332 ± 0.084; post-drug: 0.593 ± 0.117; p = 0.049), consistent with the effect of WIN 55,212-2 in the absence of SNAP. Interestingly, the onset of the WIN 55,212-2–induced depression was accelerated in the presence of SNAP when compared with WIN 55,212-2 alone, as evidenced by a decrease in the decay constant of the depression after drug application by approximately 80% (from 13.0 ± 2.8 min to 2.5 ± 0.7 min; Figure 5 D). These data suggest that activation of CB1Rs attenuates the NO-induced increase in GABA release, whereas NO itself enhances the effects of a CB1R ligand.

(F) Summary data showing that WIN 55,212-2 fails to depress IPSCs in the presence of L-NAME. Sample traces of averaged IPSCs before and during incubation of slices in WIN 55,212-2 with continuous incubation in L-NAME are shown above. All values are mean ± SEM; ∗ p < 0.05.

(E) Summary data showing L-NAME (200 μM) prevents eCB-mediated LTD GABA . Sample traces of averaged IPSCs before and after HFS (arrow and dashed line) are shown above.

(D) Summary data showing the effect of WIN 55,212-2 alone and in the presence of SNAP (left) and summary bar graph showing percent of baseline in IPSC amplitude with WIN 55,212-2 alone and WIN 55,212-2 in the presence of SNAP (right). A single exponential fit of each time course is shown and is used to calculate the time constant for the depression.

(C) Summary data showing the effect of SNAP alone and in the presence of WIN 55,212-2 (left) and summary bar graph showing percent of baseline in IPSC amplitude with SNAP alone and SNAP in the presence of WIN 55,212-2 (right).

(B) Summary data showing WIN 55,212-2–induced depression in IPSCs in the presence of SNAP. Sample traces of averaged IPSCs before SNAP (d), during SNAP (e), and during SNAP + WIN 55,212-2 (f).

(A) Summary data showing that SNAP (200 μM) fails to potentiate IPSCs in the presence of WIN 55,212-2 (5 μM). Sample traces of averaged IPSCs before WIN 55,212-2 (a), during WIN 55,212-2 (b), and during WIN 55,212-2 + SNAP (c) are shown above.

We next examined whether these synapses were sensitive to pharmacological manipulations using exogenous ligands that either activate CB1Rs or liberate NO directly. Bath application of the CB1R agonist WIN 55,212-2 (5 μM) elicited a robust depression in evoked IPSC amplitude (51% ± 7.0% of baseline, n = 10, p = 0.0001; Figure 4 A). This was accompanied by an increase in the PPR (baseline: 0.887 ± 0.110; post-drug: 1.087 ± 0.112; p = 0.020; Figure 4 A) and CV (baseline: 0.394 ± 0.058; post-drug: 0.707 ± 0.174; p = 0.001; Figure 4 A), suggesting that these effects are localized at the presynaptic terminal. This is consistent with its action elsewhere in the hypothalamus () and throughout the brain (). To determine whether the NO donor SNAP (200 μM) modulates GABA release in the DMH, we assessed its effects on evoked IPSCs. SNAP caused a rapid increase in IPSC amplitude (145% ± 14.7% of baseline, n = 13, p = 0.0003, Figure 4 B) and a decrease in PPR (baseline: 0.757 ± 0.074; post-drug: 0.655 ± 0.056; p = 0.042; Figure 4 B) and CV (baseline: 0.343 ± 0.037; post-drug: 0.284 ± 0.031; p = 0.039; Figure 4 B). This is also consistent with previous reports that NO increases GABA release in the CNS (). Together, our findings confirm that GABA synapses in the DMH are sensitive to manipulations that directly activate CB1Rs or deliver NO to the tissue.

(B) Representative neuron depicts effects of SNAP (200 μM) on IPSCs. Sample traces of averaged IPSCs before and after SNAP are shown above. Data from multiple cells are summarized below. Panels on the right summarize SNAP-induced changes in PPR and CV.

(A) Representative neuron depicts effects of WIN 55,212-2 (5 μM) on IPSCs. Sample traces of averaged IPSCs before and after WIN 55,212-2 are shown above. Data from multiple cells are summarized below. Panels on the right summarize WIN 55,212-2-induced changes in PPR and CV.

The effects of NO on GABA release require the activation of presynaptic soluble guanylate cyclase (sGC), with a subsequent rise in cyclic GMP (cGMP) (). Consistent with these observations, we failed to elicit LTP(87% ± 6.7% of baseline, n = 6, p = 0.053; Figure 3 C) in the presence of both AM251 and the sGC inhibitor, ODQ (10 μM). When taken together, these findings are consistent with the hypothesis that GABA synapses are potentiated by NO recruited in a heterosynaptic fashion by the activation of NMDARs.

The LTPobserved here is reminiscent of NO-dependent LTPdescribed in the ventral tegmental area (). To test the hypothesis that retrograde NO signaling mediates LTP, we first blocked NO production with the NO synthase inhibitor, Nω-nitro-L-arginine methyl ester (L-NAME; 200 μM) and repeated the HFS in the presence of AM251. This abolished LTP(77% ± 14.1% of baseline, n = 6, p = 0.175; Figure 3 A ) and prevented the change in PPR (baseline: 0.884 ± 0.131; post-HFS: 0.856 ± 0.103; p = 0.928) and CV (baseline: 0.133 ± 0.023; post-HFS: 0.150 ± 0.032; p = 0.691). Next, to determine whether activity-dependent production of NO in DMH neurons relies on an increase in intracellular Ca, which is often secondary to the activation of NMDARs (), we conducted two independent experiments. First, we delivered HFS in the presence of AM251 and the NMDAR antagonist APV (50 μM). Under these conditions, HFS failed to elicit LTP(94% ± 10.7% of baseline, n = 7, p = 0.436; Figure 3 B). In the second experiment, the postsynaptic cell was loaded with the Cachelator BAPTA (10 mM), and HFS was delivered. This manipulation also completely abolished LTPin the presence of AM251 (99% ± 14.8% of baseline, n = 5, p = 0.944; Figure 3 C), indicating that a rise in postsynaptic Cais necessary for NO-mediated potentiation of GABA synapses.

(C) Summary data showing percent change in IPSC amplitude after HFS under different conditions as shown in graph. “+” indicates the presence of the drug. All values are mean ± SEM; ∗ p < 0.05.

(B) Summary data showing no activity-dependent plasticity following HFS in the presence of AM251 and APV (50 μM). Sample traces of averaged IPSCs before and after HFS are shown above.

(A) Summary data showing no activity-dependent plasticity following HFS (arrow and dashed line) in the presence of AM251 (5 μM) and L-NAME (200 μM). Sample traces of averaged IPSCs before and after HFS are shown above.

We next asked what impact the duration of stimulation had on the ability of these synapses to undergo plasticity. We examined the effects of recruiting fibers at the same frequency but in shorter stimulation epochs (1 and 2 s) in control and AM251. Following stimulation with 1s epochs (100 Hz for 1 s × 2, 0.05 Hz interval), GABA synapses exhibited heterogeneous responses that were biased toward LTPin control conditions (142% ± 18.7% of baseline, n = 5, p = 0.085). Synaptic potentiation was more reliable in the presence of AM251 (161% ± 23.6% of baseline, n = 7, p = 0.041, Figures 2 E and 2F). When the duration of each stimulus epoch was increased to 2 s, we failed to observe any reliable changes in synaptic strength (100% ± 4.0% of baseline, n = 5, p = 0.960; Figures 2 E and 2F). Once again, in the presence of AM251, we observed a robust potentiation (160% ± 16.0% of baseline, n = 5, p = 0.024, Figures 2 E and 2F). At 4 s, there is clear evidence of LTDthat shifts to LTPin the presence of AM251. Overall, these data indicate that increasing the duration of the presynaptic burst shifts GABA synapses from those that are unreliable, but favor potentiation, to ones that exhibit reliable depression. In the absence of CB1R signaling, stable LTPis observed regardless of burst duration, suggesting that CB1Rs cause LTDand gate LTPat these synapses. To delve more deeply into the mechanisms responsible for LTDversus LTP, the remaining experiments were all conducted using 4 s stimulus epochs.

In other systems, eCBs, acting either at CB1Rs () or TRPV channels () have been implicated in similar activity-dependent LTD. We therefore tested whether eCBs were necessary for LTDin the DMH. Surprisingly, inclusion of the CB1R antagonist, AM251 (5 μM), rather than blocking LTD, unmasked a robust, long-term potentiation of GABA synapses (154% ± 16.9% of baseline, n = 12, p = 0.005; Figures 2 A and 2B). LTPwas accompanied by a decrease in PPR (baseline: 1.061 ± 0.045; post-HFS: 0.879 ± 0.065; p = 0.003; Figure 2 C ) and CV (baseline: 0.379 ± 0.032; post-HFS: 0.278 ± 0.028; p = 0.004; Figure 2 D), and an increase in the frequency of sIPSCs (177% ± 32.9% of baseline, p = 0.048), with no change in sIPSC amplitude (97% ± 12.0% of baseline, p = 0.793). In agreement with an essential role for CB1Rs in gating LTP, HFS also elicited LTPin CB1mice (138% ± 6.9% of baseline, n = 5, p = 0.039; Figure S2 ). These results suggest that eCBs produced during HFS act as a retrograde signal to induce LTDthrough their actions at presynaptic CB1Rs. In the absence of CB1Rs, LTD does not manifest and the same stimulus induces LTP.

(F) Summary data showing the effect of different HFS durations on the ability of GABA synapses to undergo plasticity in control and AM251.

(E) Post-HFS traces in control and AM251 are scaled to the peak of the average baseline trace. Baseline traces for each HFS duration (1, 2, and 4 s) are normalized. Only one representative baseline trace is shown for clarity.

(C and D) HFS-induced changes in PPR and CV in the presence of AM251. HFS duration impacts plasticity differently in control versus AM251.

(A and B) Representative neuron depicts effects of HFS (arrow and dashed line) on IPSCs in the presence of the CB1R antagonist AM251 (5 μM). Sample traces of averaged IPSCs are shown above. Data from multiple cells are summarized.

In an effort to examine this more closely, we conducted a more systematic analysis of individual cells following HFS. There appear to be two types of neurons in the DMH with distinct electrophysiological fingerprints. Some neurons display a low-threshold spike in response to depolarizing pulses when held at hyperpolarized potentials (6 of 16 cells; Figure S1 A), whereas others show continuous firing (10 of 16 cells; see Figure S1 A available online). We examined the magnitude of depression in these two groups and observed no difference in the ability of HFS to induce plasticity ( Figure S1 B). Therefore, both cell types were pooled for the remainder of the experimental analyses. The variability in the PPR and CV did not correlate with the postsynaptic cell types and were evenly distributed in the cells with a low-threshold spike (40% and 80% for ≥10% PPR and CV increase, respectively) and continuously firing cells (38% and 63% for PPR and CV, respectively).

To investigate the locus of LTDwe examined the paired pulse ratio (PPR), the coefficient of variation (CV), and the frequency and amplitude of spontaneous IPSCs (sIPSCs). HFS did not significantly affect the PPR (baseline: 0.763 ± 0.067, post-HFS: 0.872 ± 0.054; p = 0.221, Figure 1 C) or the coefficient of variation (baseline: 0.366 ± 0.033, post-HFS: 0.533 ± 0.08; p = 0.127, Figure 1 D). Analysis of sIPSCs also indicated that HFS had no effect on either the frequency (88% ± 13.5% of baseline, p = 0.404) or the amplitude (99% ± 5.3% of baseline, p = 0.853). We did note, however, that changes in these parameters, regardless of the magnitude of LTD, were highly variable across different cells ( Figures 1 E and 1F).

Although eCBs and NO have opposing effects on GABA release, they are both produced following bursts of afferent activity that release glutamate on to postsynaptic glutamate receptors and increase intracellular Ca. HFS (100 Hz for 4 s × 2, 0.05 Hz interval) elicited LTD, as assessed by examining the amplitude of evoked inhibitory postsynaptic currents (IPSCs; 65% ± 7.7% of baseline, n = 16, p = 0.0004, Figures 1 A and 1B ).

Representative neuron depicts effects of HFS (arrow and dashed line) on IPSCs (A). Sample traces of averaged IPSCs before and after HFS are shown above. Data from multiple cells are summarized in (B). HFS does not alter PPR (C) or CV (D). Changes in PPR (E) and CV (F) were examined before and after HFS-induced LTD GABA in individual cells. Cells that displayed ≥10% LTD GABA and ≥10% PPR/CV increase are shown in red. Cells that displayed ≥10% LTD GABA but <10% PPR/CV increase are shown in blue. Cells with <10% LTD GABA are shown in black regardless of PPR/CV increase. PPR and CV changes were variable and were not observed in all cells displaying LTD GABA. Traces in all figures depict IPSCs averaged from the 5 min period immediately before (baseline) and 10–15 min after (post-HFS) HFS. Scale bars in all figures represent 25 pA and 10 ms.

In addition to being innervated by axons immunoreactive for CB1Rs, neurons in the DMH contain the machinery required for retrograde NO signaling including the Ca-dependent enzyme NO synthase (), soluble guanylate cyclase (sGC; the NO receptor), and the cGMP-dependent protein kinase 1α (). We first examined the capacity of GABA synapses onto DMH neurons in slices obtained from satiated male Sprague-Dawley rats (postnatal days 21–30) to undergo activity-dependent plasticity in response to high-frequency stimulation (HFS) of synaptic inputs.

Discussion

The data presented here demonstrate that the feeding state of an animal determines the polarity of plasticity exhibited by GABA synapses in the DMH in response to repetitive synaptic stimuli. In satiated animals, GABA synapses undergo eCB-mediated LTD that requires NO. Following acute food deprivation, however, only LTP is evident. LTP GABA , which requires the heterosynaptic activation of NMDARs, is constrained in satiated animals by eCBs. Blockade of CB1Rs or their down-regulation during food deprivation by circulating CORT biases the synapses toward LTP. These findings provide, to the best of our knowledge, the first demonstration of state-dependent plasticity of a feeding circuit in response to acute food deprivation, and highlight a complex interaction between retrograde signals in which NO is necessary for LTD, and eCBs gate LTP GABA .

Dallman et al., 1999 Dallman M.F.

Akana S.F.

Bhatnagar S.

Bell M.E.

Choi S.

Chu A.

Horsley C.

Levin N.

Meijer O.

Soriano L.R.

et al. Starvation: early signals, sensors, and sequelae. Di Marzo et al., 2001 Di Marzo V.

Goparaju S.K.

Wang L.

Liu J.

Bátkai S.

Járai Z.

Fezza F.

Miura G.I.

Palmiter R.D.

Sugiura T.

Kunos G. Leptin-regulated endocannabinoids are involved in maintaining food intake. Kirkham et al., 2002 Kirkham T.C.

Williams C.M.

Fezza F.

Di Marzo V. Endocannabinoid levels in rat limbic forebrain and hypothalamus in relation to fasting, feeding and satiation: stimulation of eating by 2-arachidonoyl glycerol. Squadrito et al., 1994 Squadrito F.

Calapai G.

Altavilla D.

Cucinotta D.

Zingarelli B.

Campo G.M.

Arcoraci V.

Sautebin L.

Mazzaglia G.

Caputi A.P. Food deprivation increases brain nitric oxide synthase and depresses brain serotonin levels in rats. Johnstone et al., 2006 Johnstone L.E.

Fong T.M.

Leng G. Neuronal activation in the hypothalamus and brainstem during feeding in rats. Renner et al., 2010 Renner E.

Szabó-Meltzer K.I.

Puskás N.

Tóth Z.E.

Dobolyi A.

Palkovits M. Activation of neurons in the hypothalamic dorsomedial nucleus via hypothalamic projections of the nucleus of the solitary tract following refeeding of fasted rats. GABA of evoked synaptic responses following HFS in food-deprived animals, this is accompanied by a small decrease in the amplitude of spontaneous IPSCs. Under these conditions, in which CB1Rs are compromised, we surmise that HFS will still produce eCBs, but they have no presynaptic binding partner available. Thus, the eCBs may preferentially bind to postsynaptic TRPV channels and promote a postsynaptic LTD GABA. This putative postsynaptic LTD may rely on a mechanism similar to that reported at glutamate synapses in other brain regions ( Chávez et al., 2010 Chávez A.E.

Chiu C.Q.

Castillo P.E. TRPV1 activation by endogenous anandamide triggers postsynaptic long-term depression in dentate gyrus. Grueter et al., 2010 Grueter B.A.

Brasnjo G.

Malenka R.C. Postsynaptic TRPV1 triggers cell type-specific long-term depression in the nucleus accumbens. In young rats, acute food deprivation results in a plethora of metabolic changes including a considerable reduction in body weight, an increase in circulating CORT levels () and robust changes in eCB and NO levels in the hypothalamus (). While we investigate GABA signaling in the DMH following food deprivation, other studies have reported an increase in neuronal activity in the DMH, as assessed by changes in Fos expression, in response to refeeding following food deprivation (). Our finding that food deprivation produces an increase in GABA drive to DMH neurons is in agreement with these studies and suggests that enhanced inhibition of these neurons is a mechanism to cope with the lack of food. These neurons are then activated upon refeeding following a period of food deprivation. Although we report LTPof evoked synaptic responses following HFS in food-deprived animals, this is accompanied by a small decrease in the amplitude of spontaneous IPSCs. Under these conditions, in which CB1Rs are compromised, we surmise that HFS will still produce eCBs, but they have no presynaptic binding partner available. Thus, the eCBs may preferentially bind to postsynaptic TRPV channels and promote a postsynaptic LTDThis putative postsynaptic LTD may rely on a mechanism similar to that reported at glutamate synapses in other brain regions () and may explain why we observe a slight depression following HFS when both the CB1R and NO signaling pathways are blocked.

GABA is favored over NO-mediated potentiation. With shorter durations of stimulation, however, we observed a shift from LTD GABA to LTP GABA . It is likely that shorter bursts of afferent activity favor the production of NO over eCBs. Although both retrograde signals are produced following a rise in intracellular Ca2+, it is possible that NO may be synthesized at a faster rate because of coupling of NO synthase to the NMDA receptor ( Bredt and Snyder, 1989 Bredt D.S.

Snyder S.H. Nitric oxide mediates glutamate-linked enhancement of cGMP levels in the cerebellum. Garthwaite et al., 1989 Garthwaite J.

Garthwaite G.

Palmer R.M.

Moncada S. NMDA receptor activation induces nitric oxide synthesis from arginine in rat brain slices. GABA . We provide evidence that eCBs, through actions at CB1Rs, gate LTP at GABA synapses. In addition, our study also reveals two interesting interactions between the NO and eCB systems in regulating GABA transmission in the DMH. First, eCB signaling impairs NO-mediated potentiation of GABA synapses. This is evident following a prolonged burst of afferent activity where eCB-mediated LTDis favored over NO-mediated potentiation. With shorter durations of stimulation, however, we observed a shift from LTDto LTP. It is likely that shorter bursts of afferent activity favor the production of NO over eCBs. Although both retrograde signals are produced following a rise in intracellular Ca, it is possible that NO may be synthesized at a faster rate because of coupling of NO synthase to the NMDA receptor (). With longer stimulation, both NO and eCBs are present and eCB signaling impairs NO-mediated LTP Figure 7 summarizes our current hypothesis regarding the activity-dependent production and action of NO and eCBs in regulating GABA transmission in satiated and food-deprived conditions.

Barman et al., 2003 Barman S.A.

Zhu S.

Han G.

White R.E. cAMP activates BKCa channels in pulmonary arterial smooth muscle via cGMP-dependent protein kinase. Nugent et al., 2009 Nugent F.S.

Niehaus J.L.

Kauer J.A. PKG and PKA signaling in LTP at GABAergic synapses. The mechanism of the eCB-mediated blockade of NO action is not known, but our observation that the NO donor SNAP fails to potentiate GABA synapses in the presence of WIN 55,212-2 suggests that CB1R activation impedes NO signaling in the DMH. eCB-mediated LTD requires inhibition of protein kinase A (PKA). Thus, one possibility is that there may be an interaction between the cAMP-PKA and cGMP-PKG signaling pathways () such that inhibition of PKA interferes with PKG.

2+ in the postsynaptic cell, these retrograde signals have opposing actions on GABA release (reviewed in Feil and Kleppisch, 2008 Feil R.

Kleppisch T. NO/cGMP-dependent modulation of synaptic transmission. Wilson and Nicoll, 2002 Wilson R.I.

Nicoll R.A. Endocannabinoid signaling in the brain. GABA following acute food deprivation. This finding, that abolition of CB1R signaling is associated with a drive to eat, appears to be at odds with the overwhelming support for an orexigenic role of the eCB system. For example, following acute food deprivation, most evidence points to an increase in hypothalamic eCB levels ( Di Marzo et al., 2001 Di Marzo V.

Goparaju S.K.

Wang L.

Liu J.

Bátkai S.

Járai Z.

Fezza F.

Miura G.I.

Palmiter R.D.

Sugiura T.

Kunos G. Leptin-regulated endocannabinoids are involved in maintaining food intake. Kirkham et al., 2002 Kirkham T.C.

Williams C.M.

Fezza F.

Di Marzo V. Endocannabinoid levels in rat limbic forebrain and hypothalamus in relation to fasting, feeding and satiation: stimulation of eating by 2-arachidonoyl glycerol. −/− mice exhibit hypophagia compared with their wild-type littermates ( Bellocchio et al., 2010 Bellocchio L.

Lafenêtre P.

Cannich A.

Cota D.

Puente N.

Grandes P.

Chaouloff F.

Piazza P.V.

Marsicano G. Bimodal control of stimulated food intake by the endocannabinoid system. Bellocchio et al., 2010 Bellocchio L.

Lafenêtre P.

Cannich A.

Cota D.

Puente N.

Grandes P.

Chaouloff F.

Piazza P.V.

Marsicano G. Bimodal control of stimulated food intake by the endocannabinoid system. Although the production of eCBs and NO is similarly triggered by a rise in intracellular Cain the postsynaptic cell, these retrograde signals have opposing actions on GABA release (reviewed in). In this study, we demonstrate that a loss in CB1R signaling is a necessary prerequisite for NO-mediated LTPfollowing acute food deprivation. This finding, that abolition of CB1R signaling is associated with a drive to eat, appears to be at odds with the overwhelming support for an orexigenic role of the eCB system. For example, following acute food deprivation, most evidence points to an increase in hypothalamic eCB levels (), and CB1mice exhibit hypophagia compared with their wild-type littermates (). Recent work, however, provides clear evidence that activation of CB1Rs on GABA terminals is associated with a reduction in feeding. In the ventral striatum, CB1R-mediated inhibition of GABAergic transmission is associated with a hypophagic action of eCBs (). Thus, it is possible that a loss of CB1Rs at GABAergic terminals in the DMH in response to food deprivation is akin to removing the brake from the brain's feeding circuitry.