Regulation of GABA and glutamate release from proopiomelanocortin neuron terminals in intact hypothalamic networks

Abstract

Hypothalamic proopiomelanocortin (POMC) neurons and their peptide products mediate important aspects of energy balance, analgesia, and reward. In addition to peptide products, there is evidence that POMC neurons can also express the amino acid transmitters GABA and glutamate, suggesting these neurons may acutely inhibit or activate downstream neurons. However, the release of amino acid transmitters from POMC neurons has not been thoroughly investigated in an intact system. In the present study, the light-activated cation channel channelrhodopsin-2 (ChR2) was used to selectively evoke transmitter release from POMC neurons. Whole-cell electrophysiologic recordings were made in brain slices taken from POMC-Cre transgenic mice that had been injected with a viral vector containing a floxed ChR2 sequence. Brief pulses of blue light depolarized POMC-ChR2 neurons and induced the release of GABA and glutamate onto unidentified neurons within the arcuate nucleus, as well as onto other POMC neurons. To determine whether the release of GABA and glutamate from POMC terminals can be readily modulated, opioid and GABA(B) receptor agonists were applied. Agonists for μ- and κ-, but not δ-opioid receptors inhibited transmitter release from POMC neurons, as did the GABA(B) receptor agonist baclofen. This regulation indicates that opioids and GABA released from POMC neurons may act at presynaptic receptors on POMC terminals in an autoregulatory manner to limit continued transmission. The results show that, in addition to the relatively slow and long-lasting actions of peptides, POMC neurons can rapidly affect the activity of downstream neurons via GABA and glutamate release.

Figure 1.

POMC cell-specific expression of functional ChR2. A, Confocal z-stack image of a brain slice containing cells expressing ChR2-mCherry 18 d after AAV injection (left panel) and cells immunoreactive for ACTH (center panel). A high degree of colocalization can been seen in the merged image (right panel). B, Images of the hypothalamus in a live brain slice from an AAV-injected mouse shown in DIC (top panel) and fluorescence (bottom panel). The arrow indicates a neuron with a high level of ChR2 expression within the focal plane. C, Whole-cell currents from a ChR2-expressing neuron elicited by pulses of blue light at increasing intensities (0 at top trace; 28 mW/mm² elicited the largest current, bottom-most trace). D, Light-induced depolarization of a ChR2-expressing neuron caused action potential firing in current-clamp recordings.

Figure 2.

POMC neurons release GABA and glutamate onto cells within the arcuate nucleus. Light-evoked IPSCs observed in the presence of DNQX (10 μm) (Ai) were abolished with the addition of BMI (10 μm) (Aii). Light-evoked EPSCs observed in the presence of BMI (Bi) were abolished with the addition of DNQX (Bii). C, An example of a cell with light-evoked IPSCs and EPSCs. Upon washout of DNQX and/or BMI evoked currents returned (Aiii, Biii, Civ). The images in A and B consist of three overlaid sweeps, whereas those in C are each an average of three sweeps. The transmitter(s) mediating evoked currents was determined in all cases in which a PSC was observed and is presented in D. E, A light-evoked IPSC in a POMC cell that also displays a direct ChR2 current (top trace). The evoked IPSC is ablated by BMI (bottom trace). F, Currents evoked onto an unidentified cell are completely inhibited by treatment with TTX (1 μm).

Figure 3.

Light-evoked IPSCs and EPSCs are inhibited by opioid receptor activation. A, Plot of light-evoked IPSC amplitudes over time shows an inhibition of light-evoked IPSCs by the nonspecific opioid agonist ME (10 μm). The IPSC inhibition is reversed by application of the MOR-selective antagonist CTAP (500 nm). B, Representative traces of IPSCs in control conditions and in the presence of ME. C, ME inhibits light-evoked EPSC amplitude and EPSC amplitude returns to baseline upon washout of ME. D, Representative traces of EPSCs in control conditions and in the presence of ME. The arrowheads in B and D indicate the timing of the light pulse.

Figure 4.

μ- and κ-opioid receptors mediate inhibition of evoked release from POMC terminals. A, Plot of light-evoked IPSC amplitudes over time shows no effect of the DOR-selective agonist DPDPE (100 nm) and inhibition of IPSC amplitude by the MOR-selective agonist DAMGO (10 μm). The DAMGO-induced inhibition is reversed by application of the MOR-specific antagonist CTAP (500 nm). B, Representative traces taken during control and agonist treatment. C, The specific KOR agonist U69593 (500 nm) inhibited evoked IPSC amplitude and was reversed by the addition of nor-BNI (100 nm). D, Representative traces taken during control conditions and during U69593 treatment.

Figure 5.

GABA_B receptor activation inhibits GABA release from POMC terminals. A, Light-evoked IPSC amplitudes are inhibited by baclofen (30 μm). The inhibition is reversed by washout of drug. B, Representative traces taken during control conditions and during baclofen treatment.