Interrogating synaptic transmission at circadian time scales

Daily circadian rhythms impact physiology of organisms throughout the evolutionary scale. Organisms, including humans, possess an internal biological clock that adapts to the rhythm of the daily solar cycle. It is well documented that disruptions in this daily rhythm precipitated by changes in the length of days, shift work or travel alter mood and result in cognitive and systemic function deficits. Although these disruptions impact sleep–wake cycles and multiple aspects of human physiology, the light cycle has been shown to regulate mood, learning and memory directly (LeGates et al., 2014). These earlier findings implicated a direct role for the light cycle in regulation of synaptic communication and neuronal physiology independent of its systemic effects. However, the mechanisms underlying this direct circadian regulation of synaptic circuitry remain poorly understood. Ragozzino et al. (2023) examine circadian regulation of synaptic transmission in the brainstem nucleus of the solitary tract (NTS). The NTS is the ideal system to interrogate circadian regulation, because it surveys changes in physiological states and coordinates autonomic responses to maintain bodily homeostasis. During this process, the NTS relays information regarding the status of peripheral organs to the CNS via primary vagal afferent neurons that synapse onto second-order NTS neurons. In addition to this strong glutamatergic input, NTS neurons are modulated directly by changes in metabolic status and circulating hormones. The NTS, in turn, impacts various autonomic reflex pathways, feeding, satiety and the resulting gastrointestinal physiological responses. Importantly, all these physiological functions that are influenced by the NTS show robust circadian rhythmicity (Ragozzino et al., 2023). In the initial set of experiments, Ragozzino et al. (2023) showed that the NTS and nodose ganglia have strongly rhythmic clock gene expression. For instance, canonical gene regulatory components, such as Per1, Per2 and NR1D1 (Rev-erbα), that underlie cellular circadian rhythmicity showed significantly diurnal expression patterns in both the NTS and the nodose ganglia (Takahashi, 2017). Next, using high-resolution whole-cell recordings to isolate synaptic responses, Ragozzino et al. (2023) observed strong circadian rhythmicity of spontaneous quantal glutamatergic neurotransmission onto NTS neurons. In these experiments, the highest rate of spontaneous glutamate release occurred during the light phase, and lowest levels prevailed in the dark. Interestingly, this circadian oscillation of spontaneous release was sufficient to regulate action potential firing. Ragozzino et al. (2023) also observed that the regulation of action potential-evoked release was completely the opposite, in that it was enhanced through the night and diminished during the day. The presynaptic changes that regulate evoked release were also coupled with alterations in neuronal conductance. Overall, these observations put forward a striking example, whereby the spontaneous and evoked forms of neurotransmitter release are regulated opposite directions during distinct phases of the daily cycle. In this way, spontaneous glutamate release and associated activity dominate the day, whereas action potential-driven afferent-evoked activity takes over in the night. In future experiments, it will be important to find out whether these fluctuations in levels of neurotransmission are attributable to circadian rhythmicity of the core clock machinery by testing them in mutant models. For instance, it will be particularly insightful to elucidate whether there is a causal link between the rhythmicity of clock genes and synaptic transmission and whether this putative link is cell autonomous. Taken together, these findings and the approach adopted by Ragozzino et al. (2023) have implications beyond their primary focus on the NTS and nodose ganglia. Synaptic transmission is typically monitored in short time frames, often in response to brief stimulation patterns, and responses are followed for up to a maximum of a few hours, as in the case of long-term synaptic plasticity. This study, therefore, opens up a new avenue focusing on long-term fluctuation and stability of neurotransmission patterns in an intact circuit over the course of daily cycles. This approach will be especially crucial to examine the function of low-probability spontaneous events. Spontaneous neurotransmission is slow and typically occurs with a frequency of 0.01–0.1 Hz per synapse, although its levels can be modulated substantially by neuromodulators and by disease-associated genetic variants (Kavalali, 2015). These release events, in turn, regulate resting neuronal calcium signalling, protein translation and gene transcription and can also elicit action potential firing in neurons in certain electrotonic conditions. However, given its low frequency at the level of individual synapses, information transfer via spontaneous release will probably occur over longer time periods than a traditional synaptic physiology experiment. Therefore, to uncover the full physiological significance of this poorly understood form of neuronal communication, long-term (days, weeks or months) continuous monitoring of neurotransmission in intact circuits is needed. Alterations in the level of spontaneous neurotransmission can elicit homeostatic synaptic plasticity within hours (Kavalali & Monteggia, 2020), thus long-term rhythmicity of spontaneous events will be likely to impact synaptic strength and modify circuit dynamics in unforeseen ways. The systematic study by Ragozzino et al. (2023) presents a great road map for future investigations into these poorly understood aspects of synaptic biology.