Retinohypothalamic tract synapses in the rat suprachiasmatic nucleus demonstrate short-term synaptic plasticity

Abstract

The master circadian pacemaker located in the suprachiasmatic nucleus (SCN) is entrained by light intensity-dependent signals transmitted via the retinohypothalamic tract (RHT). Short-term plasticity at glutamatergic RHT-SCN synapses was studied using stimulus frequencies that simulated the firing of light sensitive retinal ganglion cells. The evoked excitatory postsynaptic current (eEPSC) was recorded from SCN neurons located in hypothalamic brain slices. The eEPSC amplitude was stable during 0.08 Hz stimulation and exhibited frequency-dependent short-term synaptic depression (SD) during 0.5 to 100 Hz stimulus trains in 95 of 99 (96%) recorded neurons. During SD the steady-state eEPSC amplitude decreased, whereas the cumulative charge transfer increased in a frequency-dependent manner and saturated at 20 Hz. SD was similar during subjective day and night and decreased with increasing temperature. Paired-pulse stimulation (PPS) and voltage-dependent Ca(2+) channel (VDCC) blockers were used to characterize a presynaptic release mechanism. Facilitation was present in 30% and depression in 70% of studied neurons during PPS. Synaptic transmission was reduced by blocking both N- and P/Q-type presynaptic VDCCs, but only the N-type channel blocker significantly relieved SD. Aniracetam inhibited AMPA receptor desensitization but did not alter SD. Thus we concluded that SD is the principal form of short-term plasticity at RHT synapses, which presynaptically and frequency-dependently attenuates light-induced glutamatergic RHT synaptic transmission protecting SCN neurons against excessive excitation.

Fig. 1.

Frequency dependence of synaptic depression during repetitive stimulation of the optic chiasm. A: evoked excitatory postsynaptic currents (eEPSCs) were recorded from the same neuron during trains of 25 stimuli at 0.08, 10, and 50 Hz (the onset latency of eEPSC: 4.72 ± 0.05 ms). Each eEPSC recording is an average of (n) trials: 0.08 Hz (n = 3), 10 Hz (n = 7), 50 Hz (n = 10). Note: these records are not shown on a timescale (the dots show the stimuli number). Dashed line is the steady-state eEPSC amplitude (mean of last 10 eEPSCs in the train). B: the frequency dependence of the steady-state eEPSC amplitude during the subjective day (Zeitgeber Time [ZT]: 6.5–10.0; n = 10) and night (ZT: 13.5–17.0; n = 7). C: frequency dependence of the cumulative charge transfer estimated during the first second of 1–25 Hz stimulation. Data were normalized to the charge transfer at 1 Hz and averaged across the recorded neurons: means ± SE (n = 5). D: temperature dependence of synaptic depression. eEPSCs were recorded from the same neurons at 28 and 36°C. *P < 0.001, n = 4 (paired t-test, 2-tailed). B and D: the amplitude of each eEPSC was normalized (%) to the amplitude of the first eEPSC in the stimulus train, after which the steady-state eEPSC amplitude was calculated. In some cases the error bars were sometimes smaller than the symbols.

Fig. 2.

Monosynaptic glutamatergic transmission in retinohypothalamic (RHT) synapses revealed depression during repetitive stimulation of the optic nerve. A: EPSC evoked by optic nerve stimulation in the same neuron. From left to right: superposition of 10 recordings during 0.08 Hz (control) and 10 Hz stimulation (the recording demonstrates the constant latency and persistence of the response during high-frequency stimulation), block of eEPSC during 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20 μM) and d-(−)-2-amino-5-phosphonopentanoic acid (d-AP5, 50 μM) coapplication, persistence of eEPSC in 40 mM extracellular Ca²⁺ (0.08 Hz stimulation). B: EPSCs evoked in the same neuron by 0.08, 10, and 50 Hz stimulation of the optic nerve with trains of 25 stimuli. Each eEPSC recording is an average of 3 trials. Note: these records are not shown on a timescale (the dots show the stimuli number). Dashed line: the mean eEPSC amplitude during steady state. C: frequency dependence of steady-state eEPSC amplitude during optic nerve stimulation. The amplitude of eEPSC was normalized (see Fig. 1). Open circles: neurons satisfying all the criteria for monosynaptic transmission (n = 9); black circles: other recorded neurons that were not tested for monosynaptic transmission (n = 12). P ≥ 0.28 for all applied stimulus frequencies (unpaired t-test, 2-tailed). D: the distribution of eEPSCs onset latency (top) and the conduction velocity of the optic nerve fibers (bottom) (n = 27 neurons).

Fig. 3.

Inhibition of presynaptic voltage-dependent Ca²⁺ channels decreased synaptic transmission and relieved synaptic depression. A, C, and E: frequency-dependent decrease of steady-state eEPSC amplitude (pA) during 0.08–25 Hz optic chiasm stimulation with trains of 25 stimuli in control and following application of N- and P/Q-type voltage-dependent Ca²⁺ channel blockers: ω-conotoxin GVIA (1 μM) and ω-agatoxin TK (500 nM), respectively. A: ω-agatoxin (Atx, n = 6). C: ω-conotoxin (Ctx, n = 7). E: coapplication of Atx and Ctx (n = 3). B, D, and F: the same data as in A, C, and E normalized (%) to the amplitude of the first eEPSC in the stimulus train at each condition (see Fig. 1). *P < 0.05, ***P < 0.001.

Fig. 4.

Contribution of AMPA receptor desensitization to synaptic depression. A: aniracetam (5 mM) inhibition of AMPA receptor desensitization increased (1.2–1.4 times) the steady-state eEPSC amplitude (pA) during repetitive stimulation (0.08–25 Hz). *P < 0.05, **P < 0.01, n = 4. Recordings in control and during aniracetam application were made in the same neurons. B: the frequency dependence of the steady-state eEPSC amplitude in control and after aniracetam application. eEPSC amplitude of each eEPSC was normalized to the first eEPSC in the stimulus train (see Fig. 1) and steady-state amplitudes before and after aniracetam application were compared. NMDA receptors were not activated during glutamate release from RHT axon terminals due to the configuration of our experiments (1.2 mM extracellular Mg²⁺, V_h −60 mV). AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; NMDA, N-methyl-d-aspartate; V_h, holding potential.

Fig. 5.

Synaptic depression and facilitation during paired-pulse stimulation (PPS) of optic nerve and optic chiasm. A: synaptic depression during optic nerve (n = 7) and optic chiasm (n = 8) PPS. B: facilitation during optic nerve (n = 2) and optic chiasm (n = 4) PPS. A and B: paired-pulse ratio (PPR) estimated as a ratio of the mean amplitude of the second EPSC to the mean amplitude of the first EPSC (mean EPSC₂/mean EPSC₁). For all applied stimulus frequencies P ≥ 0.11 for depression and P ≥ 0.33 for facilitation (unpaired t-test, 2-tailed). C and D: EPSC recordings in 2 neurons during 0.08 Hz (control) and 50 to 200 Hz PPS of the optic nerve. C: synaptic depression (mean of 5 sweeps). D: facilitation (mean of 35 sweeps). The stimulus artifact is shown attenuated. E: percentage of the neurons demonstrating depression or facilitation during PPS.