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The suprachiasmatic nucleus (SCN) is the most important circadian clock in mammals. The SCN synchronizes to environmental light via the retinohypothalamic tract (RHT), which is an axon cluster derived from melanopsin‐expressing intrinsic photosensitive retinal ganglion cells. Investigations on the development of the nonimage‐forming pathway and the RHT are scarce. Previous studies imply that light stimulation during postnatal development is not needed to make the RHT functional at adult stages. Here, we examined the effects of light deprivation (i.e., constant darkness (DD) rearing) during postnatal development on the expression in the ventral SCN of two crucial proteins for the synchronization of circadian rhythms to light: the presynaptic vesicular glutamate transporter type 2 (vGluT2) and the GluN2B subunit of the postsynaptic NMDA receptor. We found that animals submitted to DD conditions exhibited a transitory reduction in the expression of vGluT2 (at P12–19) and of GluN2B (at P7–9) that was compensated at older stages. These findings support the hypothesis that visual stimulation during early ages is not decisive for normal development of the RHT‐SCN pathway. [ABSTRACT FROM AUTHOR]

Inhibitory regulation of the heart is determined by both cholinergic M2 receptors (M2R) and adenosine A1 receptors (A1R) that activate the same signaling pathway, the ACh-gated inward rectifier K+ (KACh) channels via Gi/o proteins. Previously, we have shown that the agonist-specific voltage sensitivity of M2R underlies several voltage-dependent features of IKACh, including the 'relaxation' property, which is characterized by a gradual increase or decrease of the current when cardiomyocytes are stepped to hyperpolarized or depolarized voltages, respectively. However, it is unknown whether membrane potential also affects A1R and how this could impact IKACh. Upon recording whole-cell currents of guinea-pig cardiomyocytes, we found that stimulation of the A1R-Gi/o-IKACh pathway with adenosine only caused a very slight voltage dependence in concentration-response relationships (~1.2-fold EC50 increase with depolarization) that was not manifested in the relative affinity, as estimated by the current deactivation kinetics (τ = 4074 ± 214 ms at -100 mV and τ = 4331 ± 341 ms at +30 mV; P = 0.31). Moreover, IKACh did not exhibit relaxation. Contrarily, activation of the M2R-Gi/o-IKACh pathway with acetylcholine induced the typical relaxation of the current, which correlated with the clear voltage-dependent effect observed in the concentration-response curves (~2.8-fold EC50 increase with depolarization) and in the IKACh deactivation kinetics (τ = 1762 ± 119 ms at -100 mV and τ = 1503 ± 160 ms at +30 mV; P = 0.01). Our findings further substantiate the hypothesis of the agonist-specific voltage dependence of GPCRs and that the IKACh relaxation is consequence of this property. [ABSTRACT FROM AUTHOR]

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairment in communication and social interaction, repetitive or stereotypical behaviors, altered sensory perception, and sleep disorders. In general, the causes of ASD remain unknown, but in Phelan–McDermid syndrome, it is known that the disorder is related to the haploinsufficiency of the Shank3 gene. We used an autism model with compromised glutamatergic signaling, the Shank3+/– mouse, to study the circadian rhythm architecture of locomotion behavior and its entrainment to light. We also analyzed the synapse between the retinohypothalamic tract (RHT) and the suprachiasmatic nucleus (SCN), employing tract tracing and immunohistochemical techniques. We found that Shank3+/– mice were not impaired in the SCN circadian clock, as indicated by a lack of differences between groups in the circadian architecture in entrained animals to either long or short photoperiods. Circadian rhythm periodicity (tau) was unaltered between genotypes in constant darkness (DD, dim red light). Similar results were obtained in the re-entrainment to shifts in the light–dark cycle and in the entrainment to a skeleton photoperiod from DD. However, Shank3+/– mice showed larger phase responses to light pulses, both delays and advances, and rhythm disorganization induced by constant bright light. Immunohistochemical analyses indicated no differences in the RHT projection to the SCN or the number of SCN neurons expressing the N -methyl-D-aspartate (NMDA) receptor subunit NR2A, whereas the Shank3+/– animals showed decreased c-Fos induction by brief light pulses at CT14, but increased number of vasoactive intestinal polypeptide (VIP)-positive neurons. These results indicate alterations in light sensitivity in Shank3+/– mice. Further studies are necessary to understand the mechanisms involved in such increased light sensitivity, probably involving VIP neurons. [ABSTRACT FROM AUTHOR]

The suprachiasmatic nucleus (SCN) is the main brain clock in mammals. The SCN synchronizes to the light-dark cycle through the retinohypothalamic tract (RHT). RHT axons release glutamate to activate AMPA-kainate and N-methyl-D-aspartate (NMDA) postsynaptic receptors in ventral SCN neurons. Stimulation of SCN NMDA receptors is necessary for the activation of the signaling cascades that govern the advances and delays of phase. To our knowledge, no research has been performed to analyze the functional synaptic modifications occurring during postnatal development that prepare the circadian system for a proper synchronization to light at adult ages. Here, we studied the pre- and postsynaptic developmental changes between the unmyelinated RHT-SCN connections. Spontaneous NMDA excitatory postsynaptic currents (EPSCs) were greater in amplitude and frequency at postnatal day 34 (P34) than at P8. Similarly, both quantal EPSCs (miniature NMDA and evoked quantal AMPA-kainate) showed a development-dependent increase at analyzed stages, P3-5, P7-9, and P13-18. Moreover, the electrically evoked NMDA and AMPA-kainate components were augmented with age, although the increment was larger for the latter, and the membrane resting potential was more depolarized at early postnatal ages. Finally, the short-term synaptic plasticity was significantly modified during postnatal development as was the estimated number of quanta released and the initial release probability. All of these synaptic modifications in the unmyelinated RHT-SCN synapses suggest that synchronization to light at adult ages requires developmental changes similar to those that occur in myelinated fast communication systems. [ABSTRACT FROM AUTHOR]

The acetylcholine (ACh)-gated inwardly rectifying K+ current (IKACh) plays a vital role in cardiac excitability by regulating heart rate variability and vulnerability to atrial arrhythmias. These crucial physiological contributions are determined principally by the inwardly rectifying nature of IKACh. Here, we investigated the relative contribution of two distinct mechanisms of IKACh inward rectification measured in atrial myocytes: a rapid component due to KACh channel block by intracellular Mg2+ and polyamines; and a time- and concentration-dependent mechanism. The time- and ACh concentration-dependent inward rectification component was eliminated when IKACh was activated by GTPγS, a compound that bypasses the muscarinic-2 receptor (M2R) and directly stimulates trimeric G proteins to open KACh channels. Moreover, the time-dependent component of IKACh inward rectification was also eliminated at ACh concentrations that saturate the receptor. These observations indicate that the time- and concentration-dependent rectification mechanism is an intrinsic property of the receptor, M2R; consistent with our previous work demonstrating that voltage-dependent conformational changes in the M2R alter the receptor affinity for ACh. Our analysis of the initial and time-dependent components of IKACh indicate that rapid Mg2+-polyamine block accounts for 60-70% of inward rectification, with M2R voltage sensitivity contributing 30-40% at sub-saturating ACh concentrations. Thus, while both inward rectification mechanisms are extrinsic to the KACh channel, to our knowledge, this is the first description of extrinsic inward rectification of ionic current attributable to an intrinsic voltage-sensitive property of a G protein-coupled receptor. [ABSTRACT FROM AUTHOR]

The anterior paraventricular thalamus (aPVT) projects to the SCN, and a lesion of the aPVT leads to phase delays of circadian rhythms, instead of advances, produced by light pulses at CT23. As a first step to understanding the underlying mechanism, the authors characterized the monosynaptic responses of SCN neurons to aPVT in whole-cell recordings from brain slices in rats. Stimulation of aPVT evoked excitatory and inhibitory postsynaptic potentials in SCN neurons. Pharmacological isolation of such components indicated that the excitatory postsynaptic potential (EPSP) involves AMPA and NMDA glutamate receptors while the inhibitory postsynaptic potential (IPSP) involves GABAA receptors. Since the SCN comprises mostly GABA neurons, the persistence of IPSP after the blockade of glutamate receptors ruled out the possibility that GABA was released from SCN interneurons responsive to glutamate released from the paraventricular thalamus. Altogether, the present evidence demonstrates that glutamate and GABA are released in synapses between aPVT and the SCN. [ABSTRACT FROM AUTHOR]

Electrophysiological and calcium mobilization experiments have suggested that the intracellular calcium release channel ryanodine receptors (RyRs) are involved in the circadian rhythmicity of the suprachiasmatic nucleus (SCN). In the present report the authors provide behavioral evidence that RyRs play a specific and major role in the output of the molecular circadian clock in SCN neurons. They measured the circadian rhythm of drinking and locomotor behaviors in dim red light before, during, and after administration of an activator (ryanodine 0.1 μM) or an inhibitor (ryanodine 100 μM) of the RyRs. Drugs were delivered directly into the SCN by cannulas connected to osmotic minipumps. Control treatments included administration of artificial cerebrospinal fluid, KCl (20 mM), tetrodotoxin (1 μM), and anysomicin (5 μg/μl). Activation of RyRs induced a significant shortening of the endogenous period, whereas inhibition of these Ca2+ release channels disrupted the circadian rhythmicity. After the pharmacological treatments the period of rhythmicity returned to basal values and the phase of activity onset was predicted from a line projected from the activity onset of basal recordings. These results indicate that changes in overt rhythms induced by both doses of ryanodine did not involve an alteration in the clock mechanism. The authors conclude that circadian modulation of RyRs is a key element of the output pathway from the molecular circadian clock in SCN neurons in rats. [ABSTRACT FROM AUTHOR]

Ryanodine-sensitive intracellular Ca2+ channels (RyRs) are present in suprachiasmatic nuclei (SCN) neurons, but the functions served by these channels are not known. Here we addressed whether mobilization of intracellular Ca2+ stores through the RyRs may be a link between the molecular clock and the firing rate in SCN neurons. Activation of the RyRs by administration of either 1 mm caffeine or 100 nm ryanodine increased the firing frequency, whereas inhibition of RyRs by 10 µm dantrolene or 80 µm ryanodine decreased firing rate. Similar results were obtained in experiments conducted at either midday or midnight. Furthermore, these effects were not mediated by synaptic transmission as blockade of GABA A, AMPA and NMDA receptors did not prevent the excitatory or inhibitory effects induced by either dose of ryanodine on SCN firing. We conclude that gating of RyRs is a key element of the intricate output pathway from the circadian clock within SCN neurons in rats. [ABSTRACT FROM AUTHOR]

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairment in communication and social interaction, repetitive or stereotypical behaviors, altered sensory perception, and sleep disorders. In general, the causes of ASD remain unknown, but in Phelan-McDermid syndrome, it is known that the disorder is related to the haploinsufficiency of the Shank3 gene. We used an autism model with compromised glutamatergic signaling, the Shank3 +/- mouse, to study the circadian rhythm architecture of locomotion behavior and its entrainment to light. We also analyzed the synapse between the retinohypothalamic tract (RHT) and the suprachiasmatic nucleus (SCN), employing tract tracing and immunohistochemical techniques. We found that Shank3 +/- mice were not impaired in the SCN circadian clock, as indicated by a lack of differences between groups in the circadian architecture in entrained animals to either long or short photoperiods. Circadian rhythm periodicity (tau) was unaltered between genotypes in constant darkness (DD, dim red light). Similar results were obtained in the re-entrainment to shifts in the light-dark cycle and in the entrainment to a skeleton photoperiod from DD. However, Shank3 +/- mice showed larger phase responses to light pulses, both delays and advances, and rhythm disorganization induced by constant bright light. Immunohistochemical analyses indicated no differences in the RHT projection to the SCN or the number of SCN neurons expressing the N -methyl-D-aspartate (NMDA) receptor subunit NR2A, whereas the Shank3 +/- animals showed decreased c-Fos induction by brief light pulses at CT14, but increased number of vasoactive intestinal polypeptide (VIP)-positive neurons. These results indicate alterations in light sensitivity in Shank3 +/- mice. Further studies are necessary to understand the mechanisms involved in such increased light sensitivity, probably involving VIP neurons., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Alamilla, Ramiro-Cortés, Mejía-López, Chavez, Rivera, Felipe and Aguilar-Roblero.)