Hunger states switch a flip-flop memory circuit via a synaptic AMPK-dependent positive feedback loop

Abstract

Synaptic plasticity in response to changes in physiologic state is coordinated by hormonal signals across multiple neuronal cell types. Here, we combine cell-type-specific electrophysiological, pharmacological, and optogenetic techniques to dissect neural circuits and molecular pathways controlling synaptic plasticity onto AGRP neurons, a population that regulates feeding. We find that food deprivation elevates excitatory synaptic input, which is mediated by a presynaptic positive feedback loop involving AMP-activated protein kinase. Potentiation of glutamate release was triggered by the orexigenic hormone ghrelin and exhibited hysteresis, persisting for hours after ghrelin removal. Persistent activity was reversed by the anorexigenic hormone leptin, and optogenetic photostimulation demonstrated involvement of opioid release from POMC neurons. Based on these experiments, we propose a memory storage device for physiological state constructed from bistable synapses that are flipped between two sustained activity states by transient exposure to hormones signaling energy levels.

Figure 1. Deprivation-induced synaptic plasticity in AGRP and POMC neurons

(A) mEPSCs from fed and food-deprived (dep) mice. (B) f_mEPSC in AGRP neurons from deprived mice in the light period or from fed mice at transition to the dark period (DP) were both significantly increased over f_mEPSC from fed mice in the light period. (C) Fluorescence micrograph of arcuate nucleus from pomc-topazFP, npy-sapphireFP double transgenic mouse. POMC neurons (green) and AGRP neurons (blue) are intermingled. (D) In POMC neurons, f_mEPSC is decreased by food-deprivation. (E) Elevated spontaneous firing rate in AGRP neurons from deprived mice (n = 11) is reduced to the level of fed mice (n = 12) by CNQX. ***P < 0.001. Data are represented as mean ± s.e.m. See also Figure S1.

Figure 2. Role of calcium and ghrelin in synaptic plasticity at AGRP neurons

(A) Comparison of f_mEPSC in AGRP neurons from fed and food-deprived (dep) mice with no treatment (nt, data from Figure 1B) or treated with BAPTA-AM, ryanodine, or ghrelin (left panel). The right panel shows treatments in the presence of CdCl₂, which blocks VGCCs. All pairwise interactions were tested and P values were corrected with Holm’s method. Significant differences are denoted by any interaction across the red dashed line; interactions on the same side of the line are not significant (P > 0.05). Left and right panels were analyzed separately. (B) Caffeine increases f_mEPSC (n = 8), which is blocked by ryanodine pretreatment (red, n = 3). (C) Time course of ghrelin-mediated f_mEPSC increase in an AGRP neuron. (D) Ghrelin increases f_mEPSC in AGRP neurons (n = 6), which is blocked by d-Lys³-GHRP6 (blue, n = 5) or ryanodine (red, n = 7). (E) For fed mice, ghrelin injection increased f_mEPSC relative to saline treatment. (F) I.c.v. d-Lys³-GHRP6 (blue) during deprivation blocked the f_mEPSC increase observed with i.c.v. saline (black). ***P < 0.001. Data are represented as mean ± s.e.m.

Figure 3. AMPK signaling mediates deprivation- and ghrelin-induced synaptic activity

(A) AICAR increases f_mEPSC in AGRP neurons from fed (n = 8) but not food deprived (n = 8) mice. (B) Cpd C reduces f_mEPSC in AGRP neurons from deprived (n = 8) but not fed (n = 6) mice and blocks the ghrelin-mediated increase in f_mEPSC. (C) AGRP neuron dialysis with the AMPK activator ZMP (3 mM) in the patch pipette internal solution (int.) does not increase f_mEPSC relative to AGRP neurons recorded with standard internal solution (nt, data from Fig. 1B). (D) Targeted AMPK inhibition with Cpd C (int.) in AGRP neurons does not significantly (P > 0.05) change f_mEPSC after neuron dialysis (15 min). (E) AGRP neuron firing rate is increased by bath-applied AICAR after intracellular blockade of AMPK with Cpd C (int.). (F) AGRP neuron firing is decreased by bath-applied Cpd C after dialysis with Cpd C (int.). (G) Targeted AMPK inhibition with Cpd C (int.) does not block ghrelin-mediated f_mEPSC increase. (H) Ghrelin activates firing in AGRP neurons from fed (n = 8) but not deprived (n = 7) mice with postsynaptic AMPK blockade by Cpd C (int.). (I) Glutamate receptor blockade prevents ghrelin activation of AGRP neuron firing. (J) Paired pulse ratio (PPR) in AGRP neurons from fed and food deprived mice in 2 mM Ca²⁺. (K) PPR in AGRP neurons from fed mice (0.5 mM external Ca²⁺) is reduced by ghrelin and this is reversed by Cpd C. Average EPSC responses from two cells are also shown (inset). (L,M) PPR in AGRP neurons from deprived mice (0.5 mM external Ca²⁺) is unaffected by ghrelin and increased by Cpd C. (M, inset) Average EPSC response from one cell is shown. (N) Inhibition of CAMKK with STO-609 in AGRP neurons from fed mice blocks ghrelin-mediated but not AICAR-mediated increase of f_mEPSC. (O) AICAR does not increase f_mEPSC in the presence of ryanodine. (P) 8-Br-cADP ribose blocks the ghrelin-mediated increase of f_mEPSC. (Q) Diagram of the signaling pathway supported by these experiments. Pointed and “T” arrows represent activation and inhibition, respectively. n.s. P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. Data are represented as mean ± s.e.m. See also Figure S2.

Figure 4. Synaptic hysteresis resulting from an AMPK-dependent positive feedback loop

(A) Ghrelin upregulation of f_mEPSC shows hysteresis; f_mEPSC is sustained after ghrelin washout and Ghsr1 blockade with SP*. Duration of each transition is in parentheses (minutes). (B) Caffeine increases f_mEPSC which remains elevated after washout. Synaptic activity returned to baseline after treatment with Cpd C, consistent with a positive feedback loop. (C-F) Procedure for testing duration of persistent activity by transient exposure of brain slices to ghrelin or caffeine (5 min), transfer to a wash solution (10 min), transfer again to a solution containing either aCSF alone or with d-Lys³-GHRP6 (3-5 h). (D,E) After transient ghrelin exposure and prolonged d-Lys³-GHRP6 incubation, f_mEPSC remained elevated but was rapidly (10 min) reduced by (D) Cpd C or (E) STO-609. Control brain slices that were not treated (nt) with ghrelin but otherwise incubated as above (D) had low f_mEPSC. (F) After transient exposure to caffeine and prolonged incubation in caffeine-free aCSF (3-5 h), f_mEPSC was still elevated but was rapidly (10 min) reduced by Cpd C, consistent with the operation of a positive feedback loop. n.s. P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. Data are represented as mean ± s.e.m. See also Figure S3.

Figure 5. Persistent synaptic upregulation is reversed by leptin-mediated opioid release

(A) Synaptic activity before, during, and after food-deprivation and re-feeding (fed and deprived data from Figure 1C, 1h: n = 21, 24h: n = 27, 48h: n = 23). P values for multiple pair-wise comparisons were adjusted with Holm’s correction. The significant differences are denoted by any interaction across the red dashed line. (B) AGRP neuron firing rate 24 h after re-feeding was still elevated, which was dependent on glutamatergic synaptic input. (C) Injection of leptin in deprived mice reduced f_mEPSC in AGRP neurons relative to saline injection. (D) DAMGO reduced f_mEPSC in AGRP neurons under VGCC block (CdCl₂). The f_mEPSC remained at this level during a 15 min wash, but was increased by treatment with AICAR. (E) f_mEPSC in AGRP neurons from deprived mice treated with DAMGO are insensitive to Cpd C. (F) NTX pretreatment of deprived mice blocks leptin-mediated reduction in f_mEPSC observed with saline pretreatment. (G) Co-injection of ghrelin and NTX, but not ghrelin alone, leads to elevated f_mEPSC in brain slices prepared after 3 h. *P < 0.05, **P < 0.01, ***P < 0.001. Data are represented as mean ± s.e.m.

Figure 6. POMC neurons release an opioid that resets persistent synaptic activity

(A) Epifluorescence micrograph of brain slice with POMC neurons expressing ChR2-tdtomato. Blue circles: photostimulation sites; ARC: arcuate nucleus; 3V: third ventricle; D: dorsal; V: ventral. (B) ChR2-mediated photostimulation of POMC neurons in brain slices from deprived mice in the presence or absence of NTX. Photostimulation of POMC neurons reduces f_mEPSC unless performed in the presence of NTX. (C) Subset of neurons in (B) subjected to AICAR after photostimulation. POMC neuron photostimulation in the absence of NTX (n = 5) rendered f_mEPSC in AGRP neurons sensitive to AICAR, while those photostimulated in the presence of NTX (n = 5) were insensitive to AICAR, indicating that a POMC neuron-derived opioid, inactivates AMPK. **P < 0.01. Data are represented as mean ± s.e.m.

Figure 7. SR Flip-flop model of a neural circuit with synaptic memory of physiological state

(A) A core circuit in which AGRP neurons synaptically inhibit POMC neurons and are regulated by circulating hormones is controlled by ghrelin-responsive excitatory synapses. These synapses give this circuit a memory property based on an AMPK-dependent positive feedback loop (inset), which can be reversed by POMC neuron output, likely β-endorphin. (B) A heuristic for the logic of this circuit is the SR flip-flop memory storage circuit. In the analogy with the neural circuit reported here, the set signal is ghrelin, which activates the green NOR gate, representing the conglomeration of AGRP neurons and their ghrelin-sensitive excitatory presynaptic terminals. The reset signal is leptin which interacts with POMC neurons represented as the blue NOR gate. Notably, when R and S are both high, the circuit does not support memory, and this condition is consistent with the case of ghrelin treatment of fed mice where opioid signaling is sufficiently high to prevent persistent synaptic upregulation (Figure 5G).