Multiple NPY receptors coexist in pre- and postsynaptic sites: inhibition of GABA release in isolated self-innervating SCN neurons

Abstract

Although NPY has been shown to influence the action of many transmitters in the brain, modulation of GABA, the primary inhibitory transmitter, has not been detected with electrophysiology. Using whole-cell patch-clamp recording, we found that NPY has a large modulatory effect on GABAergic neurons of the suprachiasmatic nucleus (SCN) that act as the circadian clock in the mammalian brain. NPY, acting at both Y1- and Y2-like receptors, reduced the frequency of spontaneous miniature inhibitory postsynaptic currents while having little effect on the postsynaptic GABA receptors, suggesting a presynaptic mechanism of NPY action. In single self-innervating neurons, application of either Y1 or Y2 agonists to the same neuron significantly inhibited the evoked autaptic GABA release. The use of single-neuron microcultures has allowed the demonstration that a single peptide, NPY, has two different receptors coded for by different genes in the same axon terminal. The Y1 and Y2 agonists also inhibited whole-cell calcium currents when applied to the same neuron, indicating a coexistence of Y1- and Y2-like receptors in the postsynaptic cell body. The self-innervating cell model we use here may be applicable generally for discriminating presynaptic versus postsynaptic actions of other neurotransmitters and neuromodulators and locating their subtype receptors.

Fig. 2.

NPY reversibly blocks the evoked autaptic GABA release in single-neuron microcultures. A, A single SCN neuron in a microisland culture. The neuron was filled by biocytin during whole-cell recording and stained with avidin-HRP.B, C, Action potentials and the following autaptic IPSPs initiated by a brief current injection (2 msec, 20 pA) recorded in the single neuron illustrated in A.B, The evoked autaptic IPSP was reversibly blocked by bicuculline (BIC, 30 μm).C, The evoked autaptic IPSP also was reversibly blocked by NPY (200 nm).

Fig. 1.

NPY reduces mIPSP frequency but has little effect on postsynaptic GABA responses. A1, Consecutive traces showing that NPY (80 nm) reduced the miniature inhibitory postsynaptic potentials (mIPSPs) in the presence of TTX (1 μm) and glutamate receptor antagonists AP5 (100 μm) and CNQX (10 μm). The upward IPSPs are attributable to a KCl-based pipette solution. A2, Quantification indicating that NPY depressed the mIPSP frequency to approximately one-half of the control (p < 0.01). B, NPY has little effect on postsynaptic GABA responses. B1, Pressure-ejected (5 psi, 50 μm) GABA-induced postsynaptic responses before, during, and after the application of NPY (200 nm).B2, Quantitative illustration of the null effect of NPY on postsynaptic GABA responses (p > 0.1).

Fig. 4.

Both NPY Y1- and Y2-like receptors mediate the inhibition of miniature spontaneous GABA activity. A, Superimposed four consecutive traces illustrating control mIPSCs in the presence of TTX and AP5/CNQX. B, NPY Y1 receptor agonist, [Leu³¹, Pro³⁴]-NPY (80 nm) reduced the mIPSC frequency. C, The inhibition of [Leu³¹, Pro³⁴]-NPY was reversible. D, NPY Y2 receptor agonist NPY 13-36 (80 nm) also reduced the mIPSC frequency recorded from the same neuron. E, All of the mIPSCs were abolished by 30 μm bicuculline. F, Quantitative analysis showing that both [Leu³¹, Pro³⁴]-NPY and NPY 13-36 significantly reduced the mIPSC frequency to <60% of the control.

Fig. 3.

NPY inhibits presynaptic GABA release, but not the postsynaptic GABA response, in the same autaptic SCN neuron.A, Presynaptically released GABA (triggered by 2 msec, 60 mV voltage pulse) induced IPSC and pressure-ejected GABA-evoked response in the same recording. B, NPY (200 nm) eliminated the presynaptic release but did not affect the postsynaptic response. The box shows the sodium action current in the presence of NPY. C, The inhibition of NPY on presynaptic release was reversible. D, Bicuculline abolished both the pre- and the postsynaptic GABA responses. Capacitance currents and action currents were truncated.

Fig. 5.

NPY Y1 and Y2 agonists both inhibit the evoked autaptic GABA release in the same single SCN neuron. A, [Leu³¹, Pro³⁴]-NPY (80 nm) and NPY 13-36 (80 nm) both substantially inhibited the evoked autaptic IPSC. The recording traces on the top panel correspond to the four points (i, ii, iii, iv) in the graph below.B, Group data showing the reduction of the evoked IPSC by NPY, [Leu³¹, Pro³⁴]-NPY, NPY 13-36, and PYY.

Fig. 6.

NPY Y1- and Y2-like receptors mediate the inhibition of whole-cell barium currents in the same SCN neuron.A, Inhibition by Y2 agonist NPY 13-36 (80 nm). A1, Barium currents (I_Ba) evoked by test potential from −80 mV to 0 mV before, during, and after the application of NPY 13-36.A2, I–V curve of theI_Ba before and during NPY 13-36 application.B, Inhibition of I_Ba by Y1 agonist [Leu³¹, Pro³⁴]-NPY (80 nm) in the same neuron. B1,I_Ba before, during, and after the application of [Leu³¹, Pro³⁴]-NPY. B2, I–Vcurve I_Ba before and during [Leu³¹, Pro³⁴]-NPY application. Notice that NPY 13-36 produced greater inhibition than [Leu³¹, Pro³⁴]-NPY in this neuron. Neither NPY 13-36 nor [Leu³¹, Pro³⁴]-NPY changed the I–V relationship of the barium currents.

Fig. 7.

Time course of the action of Y1 and Y2 agonists on barium currents. A, Typical example showing the rapid inhibition by and the rapid recovery of I_Baafter the application of NPY 13-36 and [Leu³¹, Pro³⁴]-NPY. I_Ba was inhibited within 20–30 sec on application of NPY 13-36 or [Leu³¹, Pro³⁴]-NPY and returned to the control level within 2 min after washing out the agonists. In this neuron, [Leu³¹, Pro³⁴]-NPY produced greater inhibition than NPY 13-36.B, Summarized data showing the reduction ofI_Ba by NPY, [Leu³¹, Pro³⁴]-NPY, NPY 13-36, and PYY. The amount of reduction inI_Ba was comparable to the reduction in the evoked IPSC (see Fig. 5).

Fig. 8.

Inhibition of both barium currents and the evoked autaptic GABA release in the same SCN neuron. A, Recording traces showing the inhibition of the evoked IPSCs and the I_Ba by [Leu³¹, Pro³⁴]-NPY and NPY 13-36. Each tracecorresponds to a point in B.B, Comparison between the inhibition of the evoked IPSC and the I_Ba by [Leu³¹, Pro³⁴]-NPY and NPY 13-36 in the same neuron. Note that the relative amount of the inhibition of the evoked IPSC is proportional to the amount of the inhibition of the I_Ba. The time course of the inhibition and the recovery of the evoked IPSC is slower than and follows the I_Ba.

Fig. 9.

Schematic diagram showing the coexistence of NPY Y1- and Y2-like receptors in both the presynaptic nerve terminals and the postsynaptic cell body.