Neuropeptide Y depresses GABA-mediated calcium transients in developing suprachiasmatic nucleus neurons: a novel form of calcium long-term depression

Abstract

In contrast to its inhibitory role in mature neurons, GABA can exert excitatory actions in developing neurons, including mediation of increases in cytosolic Ca2+. Modulation of this excitatory activity has not been studied previously. We used Ca2+ digital imaging with Fura-2 to test the hypothesis that neuropeptide Y (NPY) would depress GABA-mediated Ca2+ rises in neurons cultured from the developing suprachiasmatic nucleus (SCN). SCN neurons were chosen as a model system for this study because SCN neurons are primarily GABAergic, they express high levels of NPY and GABA receptors, and functionally, NPY causes profound phase-shifts in SCN-generated circadian rhythms. Vigorous GABA-mediated Ca2+ activity was found in young SCN neurons that were maintained in vitro for 4-14 d. NPY showed a dose-dependent rapid depression of the amplitude of Ca2+ rises generated by GABA released from presynaptic SCN axons. NPY exerted a long-term depression of cytosolic CA2+ in the majority of neurons tested, which lasted more than 1 hr after NPY washout. The magnitude of the NPY depression was dose-dependent. NPY did not affect Ca2+ levels when GABAA receptor activity was blocked by bicuculline; however, when bicuculline and NPY were withdrawn from the perfusion solution, the subsequent CA2+ rise was either significantly reduced or completely absent, suggesting that the NPY receptor was activated in the absence of elevated intracellular Ca2+ and GABAA receptor activity, and that the latent effect of NPY was revealed only after depolarizing GABA stimulation was renewed. Pretreating neurons with pertussis toxin greatly reduced the ability of NPY to depress GABAergic Ca2+ rises, suggesting that the NPY modulation of the GABA activity was based largely on a mechanism involving pertussis toxin-sensitive Gi/Go proteins. NPY receptor stimulation depressed (< 30%) postsynaptic Ca2+ rises evoked by GABA (20 microM) application in the presence of tetrodotoxin (TTX). The effects of NPY were mimicked by the NPY Y1 receptor agonist [Pro34,Leu31] NPY and the Y2 receptor agonist NPY 13-36 and by peptide YY (PYY). Together, our data suggest that the Y1 and Y2 type NPY receptors act both presynaptically and postsynaptically to depress GABA-mediated Ca2+ rises. If related mechanisms exist in peptide modulation of inhibitory GABA activity in mature neurons, this could underlie long-term changes in the behavior of neurons of the SCN necessary for phase-shifting the circadian clock by NPY, NPY also modulated GABA responses in neuroendocrine neurons from the hypothalamic arcuate nucleus. NPY thus can play an important role in evoking long-term depression of GABA-mediated Ca2+ activity in these developing neurons, allowing NPY-secreting cells to modulate the effects of GABA on neurite outgrowth, gene expression, and physiological stimulation. This is the first example of such a cellular memory: that is, long-term Ca2+ depression based on modulation of depolarizing GABA activity.

Fig. 1.

A, The removal of the GABA_A receptor antagonist bicuculline (BIC) (20 μm) from the perfusion solution caused an immediate Ca²⁺ rise in neurons from the SCN cultured for 4 d in vitro (DIV). The addition of NPY (100 nm) caused a rapid depression in the GABAergic Ca²⁺ rise. The ability of neurons to recover to pre-NPY Ca²⁺levels differed significantly, with some showing a modest recovery (A2, A3) and others not recovering (A1). B, The effects of NPY on endogenous, GABA-mediated, Ca²⁺ rises persisted with time in culture (11 DIV). C, The effect of NPY on GABA-mediated Ca²⁺ rises was concentration-dependent. The Ca²⁺ rise from a bicuculline-defined baseline was determined 15 sec before and 100 sec after the application of NPY. These values were normalized such that the Ca²⁺ rise just before the application of NPY (white bar) was set equal to 100%. Error barsrepresent the SEM. N refers to the total number of neurons assayed. The Ca²⁺ rise defined from a bicuculline (20 μm) baseline was determined 15 sec before and 100 sec after the application of NPY receptor agonists (see Materials and Methods for a full description). D, Control experiments show that in the absence of NPY, the Ca²⁺ rise was maintained for an extended time. Ca²⁺ levels in the presence of bicuculline were usually between 45 and 80 nm. Vertical bars to the right of each neuron represent the calibrated Ca²⁺ level. Horizontal bars represent the time scale for a group of neurons. The ionotropic glutamate receptor antagonists AP5 and CNQX were present in all perfusion solutions to eliminate the potentially complicating interactions between NPY and glutamatergic neurons.

Fig. 2.

A, The ability of NPY to depress endogenous GABA-mediated Ca²⁺ rises was assayed for >60 min. The Ca²⁺ rise initiated by the removal of bicuculline (BIC) (20 μm) was depressed by a 2 min application of NPY (100 nm) (arrow). Representative SCN neurons were divided into two groups: those that showed a MINIMAL RECOVERY (A1–A2) and those that showed a rapidRECOVERY (A3–A4). Note that the reintroduction of bicuculline at the end of the experiment reduced the Ca²⁺ level in all neurons. B, The addition of the truncated NPY analog C2-NPY (100 nm) caused a rapid and sustained Ca²⁺ depression. Unlike NPY, C2-NPY has very low nonspecific binding characteristics and therefore tends not to remain associated with cells. C, The NPY receptor antagonist benextramine (10 μm) was added to the perfusion solution 3 min after NPY (100 nm) washout in an attempt to displace NPY that may have remained bound to the NPY receptor. Little effect was detected. D, NPY exhibited a latent regulation of GABAergic Ca²⁺ rises. The Ca²⁺ levels of the neurons shown in D1–D3 were elevated reversibly by the removal of bicuculline (20 μm). During the third bicuculline application, neurons were pulsed with NPY (100 nm) (vertical gray lines). Subsequent Ca²⁺rises induced by the removal of bicuculline were reduced drastically. The arrow points to the 30 sec application of the glutamate receptor agonist NMDA (30 μm).D4 is a graphical representation of the effect of NPY;1st and 2nd refer to the two bicuculline-sensitive Ca²⁺ rises before the addition of NPY; 3rd and 4th refer to the two bicuculline-sensitive Ca²⁺ rises after the pulse of NPY. The glutamate receptor antagonists AP5 (100 μm) and CNQX (10 μm) were in all solutions except during NMDA application. Error bars represent SEM.

Fig. 3.

NPY receptor subtypes. A, The application of NPY (NPY 13–36) (100 nm) reduced GABA-mediated Ca²⁺ rises. Similar results were seen for [Pro³⁴,Leu³¹] NPY (NPY PRO-34) (100 nm) (B), and PYY (100 nm) (C). For each pair shown, the top neuron is an example of a long-term effect, and the bottom shows neurons that showed either a smaller effect (A) or a more substantial recovery (B, C). D, Pretreatment of SCN neurons with pertussis toxin (200 ng/ml) for 20 hr before the start of the experiment blocked the NPY-mediated depression of GABAergic Ca²⁺ rises. E, GABAergic Ca²⁺ rises are in large part dependent on L-type Ca²⁺ channel activity. The response of a representative neuron shows that the addition of the L-type Ca²⁺ channel blocker nimodipine (1 μm) depressed the endogenous Ca²⁺ rise. NPY (100 nm) had no effect in the presence of nimodipine. F, Bar graph representation of the efficacy of NPY receptor agonists. The Ca²⁺ rise defined from a bicuculline (20 μm) baseline was determined 15 sec before and 100 sec after the application of NPY receptor agonists. Values were normalized such that the Ca²⁺ rise just before the application of NPY (white bar) was set equal to 100%. The agonists used are shown along the x-axis. The efficacy of NPY is shown for 11 DIV and 4 DIV. NPY PRO-34 refers to [Pro³⁴,Leu³¹] NPY. NPY+PTX refers to the efficacy of NPY when the neurons were pretreated with pertussis toxin. The breakdown by group for the total number of neurons assayed (n = 440) was 4 DIV NPY = 111, 11 DIV NPY = 10, NPY 13–36 = 81, NPY PRO-34 = 98, PYY = 75, and NPY+PTX = 65. Only data from neurons with a Ca²⁺ rise >20 nm are included. Glutamate receptor antagonists AP5 (100 μm) and CNQX (10 μm) were in all solutions. NPY receptor agonist-mediated Ca²⁺ depression was statistically different from pre-NPY Ca²⁺ levels for each group (p < 0.001). BIC, Bicuculline. Error bars represent SEM.

Fig. 4.

To determine whether NPY exerted a postsynaptic effect, synaptic communication was blocked with TTX (1 μm), and GABA-induced Ca²⁺ rises were evoked repeatedly in combination with NPY receptor stimulation. A, The application ofGABA (20 μm) (arrows) to the perfusion solution elicited reproducible Ca²⁺rises. The administration of NPY (100 nm) starting 45 sec before GABA application caused a large reduction in the level of GABA-induced Ca²⁺ rises. NPY-dependent depression of GABA-induced Ca²⁺ increases either persisted (top neuron) or diminished (bottom neuron) after NPY was washed out. Cells were pulsed with NMDA (30 μm) to demonstrate that neurons were healthy and would respond to Ca²⁺-mobilizing transmitters at the end of an experimental series (wide arrow). Similar results were seen for NPY 13–36 (B), [Pro³⁴,Leu³¹] NPY (NPY PRO-34) (C), and PYY(D). E, The effect of NPY on high K⁺-induced Ca²⁺ rises was small. F, The GABA_A receptor-specific agonist muscimol (MUSCIMOL) (10 μm) elicited a Ca²⁺ rise, whereas the GABA_B receptor-specific agonist baclofen (BACLOFEN) (10 μm) had no effect.G, GABA-evoked Ca²⁺ rises could be inhibited by the coadministration of bicuculline (BICUCULLINE) (20 μm).

Fig. 5.

NPY receptor agonists suppressed GABA-evoked Ca²⁺ rises. Data compare sequential GABA-evoked Ca²⁺ rises. The graph shows that the peak response to the first control GABA-evoked Ca²⁺rise was not different from the second control GABA-evoked Ca²⁺ rise (white bars). The second control GABA-evoked Ca²⁺ rise was normalized and set equal to 100% and then compared with the first GABA-evoked Ca²⁺ rise in the presence of NPY receptor agonist. All NPY receptor agonists caused a statistically significant depression in the peak Ca²⁺ rise (p < 0.01). Data from high K⁺-induced Ca²⁺ increases (25 mm K⁺NPY) were analyzed in an identical manner to GABA-evoked responses. NPY PRO-34 refers to [Pro³⁴,Leu³¹] NPY.N refers to the total number of neurons assayed. Error bars represent SEM. Only data from neurons with a Ca²⁺ rise >20 nm were analyzed.

Fig. 6.

NPY (100 nm) depressed GABA-mediated Ca²⁺ rises in neurons from SCN cultured punches (A, B) and in neurons cultured from the arcuate nucleus (C, D). A,C, Removal of bicuculline (BIC) (20 μm) elicited a Ca²⁺ rise that was depressed by the addition of NPY. Glutamate receptor antagonists AP5 (100 μm) and CNQX (10 μm) were maintained in the perfusion solution during endogenous activity experiments. B, D, NPY depressed Ca²⁺ rises evoked by the application of GABA (20 μm) to the perfusion solution. TTX (1 μm) was included in all evoked-response perfusion solutions in B and D.