The Journal of Neurophysiology Vol. 78 No. 4 October 1997, pp. 2217-2220
Copyright ©1997 by the American Physiological Society

RAPID COMMUNICATION

GABA_A-Mediated Local Synaptic Pathways Connect Neurons in the Rat Suprachiasmatic Nucleus

George J. Strecker, Jean-Pierre Wuarin, and F. Edward Dudek

Department of Anatomy and Neurobiology, Colorado State University, Fort Collins, Colorado 80523-1670

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Strecker, George J., Jean-Pierre Wuarin, and F. EdwardDudek. GABA_A-mediated local synaptic pathways connect neurons in the rat suprachiasmatic nucleus. J. Neurophysiol. 78: 2217-2220, 1997. The suprachiasmatic nucleus (SCN) in mammals functions as the biological clock controlling circadian rhythms, but the synaptic circuitry of the SCN is largely unexplored. Most SCN neurons use the neurotransmitter -aminobutyric acid (GABA), and anatomic studies indicate many GABAergic synapses and local axon collaterals; however, physiological evidence for synaptic communication among SCN neurons is indirect. We have used three approaches to investigate local circuitry in the SCN in acute hypothalamic slices from rat. First, tetrodotoxin was used to block action-potential-dependent synaptic release, which resulted in a decrease in the frequency of spontaneous synaptic currents in SCN neurons, suggesting that spontaneously active neurons in the slice connect synaptically to SCN neurons. Postsynaptic currents in SCN neurons were also evoked by the selective stimulation of other SCN neurons with glutamate, which avoids direct activation of axons that might originate outside the SCN. Two different methods of glutamate microapplication (i.e., pressure ejection and ultraviolet photolysis of caged glutamate) indicated that SCN neurons receive GABA_A-receptor-mediated synaptic input from other SCN neurons. In contrast, glutamate-receptor-mediated synaptic connections between SCN neurons were not detected. The GABAergic synapses that comprise the network described here could conceivably be a substrate for the synchronization and amplification of the circadian rhythm of SCN firing. Alternatively, this circuitry might mediate other aspects of clock function such as the integration of environmental and physiological information.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Numerous neuroactive substances have been detected in the suprachiasmatic nucleus (SCN), but only -aminobutyric acid (GABA)- and glutamate-receptor-mediated postsynaptic events have been reported (see Strecker et al. 1995, for review). It is unknown whether these events originate from local neurons or from neurons located outside the SCN. The SCN contains extensive GABAergic terminals andsomata (van den Pol 1986), and Golgi studies have revealed local axon collaterals of SCN neurons (van den Pol 1980). With anatomic evidence alone, however, it is difficult to know whether SCN neurons actually form functionalGABAergic synapses with each other. Physiological experiments to probe synaptic connectivity have typically utilized electrical stimulation, but this approach could also activate axons originating outside the SCN. In the present study, glutamate was used to stimulate neurons without activating axons. The postsynaptic currents (PSCs) evoked by glutamate microstimulation in the SCN provide physiological evidence for the presence of GABA_A-receptor-mediated synaptic pathways between SCN neurons.

	METHODS

Abstract Introduction Methods Results Discussion References

Sprague-Dawley rats were housed under a 12-h light:dark cycle and recordings were made predominantly during the light phase. Coronal slices (150-400 µm) of hypothalamus were cut and maintained in artificial cerebrospinal fluid (ACSF) containing (in mM) 125 NaCl, 2.5 KCl, 1 CaCl₂, 1 MgCl₂, 24 NaHCO₃, 1.25 NaH₂PO₄, and 10 glucose, bubbled with 5% CO₂-95% O₂, pH 7.4. Recordings were made at room temperature (20-22°C) from 2- to 4-week-old rats.

Patch pipettes (3-5 M) contained (in mM) 140 cesium gluconate (or 130 KCl in photostimulation experiments), 1 CaCl₂, 1 MgCl₂, 1 NaCl, 5 bis-(o-aminophenoxy)-N,N,N,N-tetraacetic acid, or 5-10 ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid, and 2-4 magnesium ATP, pH 7.2. Whole cell currents were amplified, filtered (corner frequency 5 kHz, Axopatch 1D), and stored digitally. Recordings were refiltered (1 or 2 kHz), redigitized (5 kHz), and analyzed off-line (pClamp software).

The frequencies and amplitudes of spontaneous PSCs were measured before and after the addition of tetrodotoxin (TTX) to block action-potential-dependent synaptic activity. Cumulative amplitude distributions of inhibitory PSCs (IPSCs) were compared with the use of the Kolmogorov-Smirnov test. The one-sample ² test was used to assess the effect of TTX on IPSC frequency. Data are generally expressed as means ± SE.

In TTX and glutamate microapplication experiments, 150- to 200-µm slices were submerged in ACSF and SCN neurons were recorded while being viewed with Nomarski water-immersion optics (Edwards et al. 1989). Glutamate (1-2 mM; 5-10 mM in initial experiments) in ACSF was applied by pressure pipette (~0.2 s) onto the SCN during recording. Photostimulation experiments were performed in 400-µm-thick slices with the use of the blind-patch technique (Blanton et al. 1989). Slices were submerged in ACSF containing 250-500 µM caged glutamate [-(-carboxy-2-nitrobenzyl) ester, trifluoroacetate; Molecular Probes]. Flashes of ultraviolet light (xenon lamp; duration ~ 0.5 ms) were focused from beneath the slice into the SCN to uncage glutamate (Callaway and Katz 1993).

Data from neurons that showed clear synaptic responses evoked by glutamate microstimulation were analyzed for both magnitude and repeatability of the effect of glutamate on IPSC frequency. Cells that did not show clear responses were counted as unresponsive and not analyzed further. Magnitude was assessed by statistically comparing (1-sample ² test) the number of IPSCs occurring 10 s before and 10 s after the stimulus in the case of microapplication. In the photostimulation trials, the duration of the effect was shorter because stimulation was more focal, so a shorter poststimulus interval (2-8 s) was examined.

To assess repeatability, the binomial theorem was applied to evaluate whether any series of sequential trials displayed significantly more positive responses than were likely by chance (P < 0.05). The probability of positive or negative responses was assumed conservatively to be equal (i.e., 0.5). For evaluating a series of trials, a trial with a positive response was defined as having more (P < 0.2, ² test) IPSCs after stimulation. Positive responses so defined showed evoked increases in IPSC frequency by a factor of 2.4 ± 0.2 (mean ± SE; n = 81 positive responses) with microapplication, or a factor of 2.6 ± 0.2 (n = 30) with photostimulation. Some cells appeared clearly responsive, but not in enough consecutive trials to achieve significance of repeatability. In these cases, IPSC increases in individual trials were held to more stringent levels of significance (P < 0.05 rather than 0.2, ² test). Thus we defined responsive cells as having either 1) a binomially significant series of responses, with each response at the P < 0.2 level, or 2) at least three consecutive responses, each at the P < 0.05 level. Our approach is likely to underestimate the extent of local interactions in the SCN, but provides a reliable minimum value.

	RESULTS

Abstract Introduction Methods Results Discussion References

Spontaneous IPSCs were detected in 151 of 152 SCN neurons. Bath application of bicuculline (10 µM) blocked spontaneous IPSCs in 10 of 10 neurons, confirming that the IPSCs were mediated by GABA_A receptors. Spontaneous excitatory PSCs (EPSCs) were much less frequently detected (i.e., 15 of 47 SCN neurons).

A dependence of spontaneous IPSCs on action potentials would suggest that active presynaptic neurons were contained in the slice, possibly within the SCN itself. The mean frequency of spontaneous IPSCs in control solutions was 17.7 ± 14.4 Hz (n = 7 neurons). In each cell, the addition of TTX (1-3 µM) significantly reduced the frequency of IPSCs (P < 0.005, ² test, n = 7) to 44 ± 8% of the pre-TTX frequency (Fig. 1). Cumulative amplitude distributions of spontaneous IPSCs revealed a significant reduction by TTX in two of seven cells (Kolmogorov-Smirnov 2-sample test, 1-tailed). These results suggest that at least some spontaneously firing neurons in the slice formed inhibitory synapses on the recorded SCN neurons, but these presynaptic neurons were not necessarily in the SCN.

View larger version (30K): [in this window] [in a new window]   FIG. 1. Effects of tetrodotoxin (TTX) on spontaneous inhibitory postsynaptic currents (IPSCs). A: consecutive 16-s segments. B: amplitude distribution (normalized) of IPSCs in control solution and in TTX (1 µM). TTX reduced the frequency of IPSCs by a factor of 0.18 in this cell. C: cumulative amplitude distribution. Shown is the probability of observing IPSCs smaller than or equal to the given amplitude. This cell tended toward a slight decrease in the probability of the largest IPSCs with TTX; however, it did not reach significance. Data are from the same cell. Holding potential: 25 mV.

To test for the presence of local synaptic connections more directly, we examined whether microapplication of glutamate to the SCN could induce GABA-receptor-mediated PSCs in SCN neurons. Unlike electrical stimulation, glutamate is expected to stimulate only somata and dendrites and not axons (Christian and Dudek 1988). Of 32 cells, 8 (25%) responded significantly and reproducibly with increases in IPSC frequency to glutamate microapplied by pressure ejection (Fig. 2). Four additional neurons (i.e., 12 of 32 cells, 38%) showed at least one trial with a significant (P < 0.05, ² test) increase in IPSC frequency. Glutamate microapplication failed to evoke EPSCs in 17 of 17 cells.

View larger version (28K): [in this window] [in a new window]   FIG. 2. Effect of glutamate microapplication. A: glutamate applications by pressure ejection onto the suprachiasmatic nucleus (SCN) evoked barrages of IPSCs. Traces represent separate, consecutive stimulation trials occurring at the arrow. More IPSCs occurred in the 10-s period after stimulation than before. Shown are 5 of a series of 10 significant (P < 0.05) increases in IPSC frequency (holding potential: 10 mV; cesium gluconate intracellular solution). B: mean frequency of IPSCs for these 5 trials relative to the time of microstimulation.

To improve the temporal and spatial characteristics of glutamate application, a second method was usedphotolysis of caged glutamate. Of 12 SCN cells tested, 3 (25%) responded to glutamate uncaging inside the SCN with significant and reproducible increases in IPSC frequency (Fig. 3), and a 4th cell responded (P < 0.05 level) repeatedly but not consecutively enough to satisfy statistical reproducibility. Because of the enhanced temporal and spatial resolution with this method, we were able to observe one other neuron that repeatedly displayed a single IPSC 25 ms after each glutamate photolysis stimulus, rather than the burst of PSCs more commonly seen. As expected, bicuculline blocked all evoked IPSCs (n = 2 neurons). No EPSCs could be evoked in 11 of 11 neurons tested with photostimulation.

View larger version (30K): [in this window] [in a new window]   FIG. 3. Photolysis of caged glutamate within the SCN evoked barrages of IPSCs. Five consecutive 20-s traces are shown. This SCN neuron displayed a series of 9 consecutive trials with significant (P < 0.05) increases in IPSC frequency (holding potential: 60 mV; KCl intracellular solution). Bottom trace is shown in expanded time. Large inward current of ~2 s in duration underlying the barrage of evoked IPSCs reflects the additional effect of applied glutamate contacting the recorded cell. In another cell, IPSCs could also be evoked in the absence of a direct effect.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Experiments showing that TTX reduced the frequency and occasionally the amplitude of IPSCs indicate that at least some of the neurons responsible for GABAergic synaptic input onto SCN neurons were present in the slices, possibly within the SCN (see also Jiang et al. 1995). In these experiments it is assumed that cut axons lacking a soma or axon hillock are less likely to fire spontaneously compared with intact axons connected to SCN somata, which fire spontaneously at ~10 Hz during the day.

Our experiments with glutamate microstimulation provide more specific evidence that local synaptic pathways exist in the SCN and are predominantly GABAergic. Only GABA_A-receptor-mediated IPSCs, either spontaneous or locally evoked, were detected in SCN neurons. Spontaneous EPSCs were also detected, but no EPSCs could be locally evoked. In both the microapplication and photolysis experiments, 25% of neurons displayed local GABAergic input. This is likely to be a lower bound, given the stringency of our criteria for the identification of responsive cells and the partial deafferentation that is characteristic of slice preparations. It is unlikely that glutamate microapplication caused synaptic release by direct action through metabotropic receptors on presynaptic terminals. Such receptors in mammals are typically found to inhibit IPSCs (Desai et al. 1994; Jouvenceau et al. 1995; Llano and Marty 1995; Schrader and Tasker 1997).

The GABA-mediated synaptic network demonstrated here has important implications for SCN function. This network might be involved in the synchronization of the circadian rhythm of neuronal firing. SCN neurons fire at a higher rate during the day than at night (Inouye and Kawamura 1979). Although GABA is typically inhibitory, action potentials can be initiated in other regions by synaptically mediated postinhibitory rebound (see Llinás 1988 for review). GABA-mediated synaptic currents in the SCN also might lead to action potentials and synchronize neuronal activity. Recent evidence suggests that GABA excites SCN neurons during the day and inhibits them during the night, because of shifts in the chloride equilibrium potential (Wagner et al. 1997). Thus this network might be used to synchronize SCN neurons or to increase the amplitude of the circadian change in firing frequency. Alternatively, it might be involved in phase shifting or in the integration of environmental information rather than in the rhythm of firing per se.

	ACKNOWLEDGEMENTS

  We are grateful to Drs. Gary Pickard and Patricia Sollars for comments on this manuscript.

  This work was supported by grants from the Air Force Office of Scientific Research to F. E. Dudek and the National Institute of Neurological Disorders and Stroke (R01 NS-32662 to J.-P. Wuarin).

	FOOTNOTES

  Address reprint requests to F. E. Dudek.

  Received 12 May 1997; accepted in final form 30 June 1997.

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