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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]

The suprachiasmatic nucleus (SCN) of the hypothalamus is the brain structure that controls circadian rhythms in mammals. The SCN is formed by two neuroanatomical regions: the ventral and dorsal. Gamma-aminobutyric acid (GABA) neurotransmission is important for the regulation of circadian rhythms. Excitatory GABA effects have been described in both SCN regions displaying a circadian variation. Moreover, the GABAergic system transfers photic information from the ventral to the dorsal SCN. However, there is almost no knowledge about GABA neurotransmission during the prenatal or postnatal development of the SCN. Here, we used whole-cell patch-clamp recordings to study spontaneous inhibitory postsynaptic currents (IPSCs) in the two SCN regions, at two zeitgeber times (day or night), and at four postnatal (P) ages: P3-5, P7-9, P12-15, and P20-25. The results herein show that the three analyzed parameters of the IPSCs, frequency, amplitude, and decay time, were significantly affected by the postnatal age: mostly, the IPSC frequency increased with age, principally in the ventral SCN in both day and night recordings; similarly, the amplitude of IPSCs augmented with age, especially at night, whereas the IPSC decay time was reduced (it was faster) with postnatal age, mainly during the day. Our findings first reveal that parameters of GABA neurotransmission are modified by postnatal development, implying that synaptic adjustments are required for an appropriate maturation of the GABAergic system in the SCN. [ABSTRACT FROM AUTHOR]

The hypothalamic suprachiasmatic nucleus (SCN) is the leading circadian pacemaker in mammals, which synchronizes with environmental light through the retinohypothalamic tract (RHT). Although the SCN regulates circadian rhythms before birth, postnatal synaptic changes are needed for the RHT‐SCN pathway to achieve total functional development. However, it is unknown whether visual experience affects developmental maturation. Here, we studied the effects of constant darkness (DD) rearing on the physiology (at pre‐ and postsynaptic levels) of glutamatergic neurotransmission between RHT and SCN during postnatal development in rats. Upon recording spontaneous and evoked excitatory postsynaptic currents (EPSCs) by electrical stimulation of RHT fibers, we found that DD animals at early postnatal ages (P3–19) exhibited different frequencies of spontaneous EPSCs and lower synaptic performance (short‐term depression, release sites, and recruitment of RHT fibers) when compared with their normal light/dark (LD) counterparts. At the oldest age evaluated (P30–35), there was a synaptic response strengthening (probability of release, vesicular re‐filling rate, and reduced synaptic depression) in DD rats, which functionally equaled (or surmounted) that of LD animals. Control experiments evaluating EPSCs in ventral SCN neurons of LD rats during day and night revealed no significant differences in spontaneous or evoked EPSCs by high‐frequency trains in the RHT at any postnatal age. Our results suggest that DD conditions induce a compensatory mechanism in the glutamatergic signaling of the circadian system to increase the chances of synchronization to light at adult ages, and that the synaptic properties of RHT terminals during postnatal development are not critically influenced by environmental light. [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]

Rationale: We have previously shown that in rats, capsaicin (Cap) has antidepressant-like properties when assessed using the forced swimming test (FST) and that a sub-threshold dose of amitriptyline potentiates the effects of Cap. However, synergistic antidepressant-like effects of the joint administration of Cap and the selective serotonin reuptake inhibitor citalopram (Cit) have not been reported. Objectives: To assess whether combined administration of Cap and Cit has synergistic effects in the FST and to determine whether this combination prevents the side effects of Cit. Methods: Cap, Cit, and the co-administration of both substances were evaluated in a modified version of the FST (30-cm water depth) conducted in rats, as well as in the open field test (OFT), elevated plus maze (EPM), and Morris water maze (MWM). Results: In line with previous studies, independent administration of Cap and Cit displayed antidepressant-like properties in the FST, while the combined injection had synergistic effects. In the OFT, neither treatment caused significant increments in locomotion. In the EPM, the time spent in the closed arms was lower in groups administered either only Cap or a combination of Cap and Cit than in groups treated with Cit alone. In the MWM, both Cap and the joint treatment (Cap and Cit) improved the working memory of rats in comparison with animals treated only with Cit. Conclusion: Combined administration of Cap and Cit produces a synergistic antidepressant-like effect in the FST and reduces the detrimental effects of Cit on anxiety and working memory. [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 suprachiasmatic nucleus (SCN) is the main brain clock that regulates circadian rhythms in mammals. The SCN synchronizes to the LD cycle through the retinohypothalamic tract (RHT), which projects to ventral SCN neurons via glutamatergic synapses. Released glutamate activates N-methyl-Daspartate (NMDA) receptors, which play a critical role in the activation of signaling cascades to enable phase shifts. Previous evidence indicates that presynaptic changes during postnatal development consist of an increase in RHT fibers impinging on SCN neurons between postnatal day (P) 1 to 4 and P15. The aim of this study was to evaluate postsynaptic developmental changes in the NR2 subunits that determine the pharmacological and biophysical properties of the neuronal NMDA receptors in the ventral SCN. To identify the expression of NR2 subtypes, we utilized RT-PCR, immunohistochemical fluorescence, and electrophysiological recordings of synaptic activity. We identified development-dependent changes in NR2A, C, and D subtypes in mRNA and protein expression, whereas NR2B protein was equally present at all analyzed postnatal ages. The NR2A antagonist PEAQX (100 nM) reduced the frequency of NMDA excitatory postsynaptic currents (EPSCs) at P8 significantly more than at P34, but the antagonists for NR2B (3 μM Ro 25-6981) and NR2C/D (150 nM PPDA) did not influence NMDA EPSCs differently at the 2 analyzed postnatal ages. Our results point to P8 as the earliest analyzed postnatal age that shows mRNA and protein expression similar to those found at the juvenile stage P34. Taken together, our findings indicate that postsynaptic development-dependent modifications in the NR2 subtypes of the NMDA receptor could be important for the synchronization of ventral SCN neurons to the LD cycle at adult stages. [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]

Recently, it has been shown that G protein-coupled receptors (GPCRs) display intrinsic voltage sensitivity. We reported that the voltage sensitivity of M2 muscarinic receptor (M2R) is also ligand specific. Here, we provide additional evidence to understand the mechanism underlying the ligand-specific voltage sensitivity of the M2R. Using ACh, pilocarpine (Pilo), and bethanechol (Beth), we evaluated the agonist-specific effects of voltage by measuring the ACh-activated K current ( I) in feline and rabbit atrial myocytes and in HEK-293 cells expressing M2R-Kir3.1/Kir3.4. The activation of I by the muscarinic agonist Beth was voltage insensitive, suggesting that the voltage-induced conformational changes in M2R do not modify its affinity for this agonist. Moreover, deactivation of the Beth-evoked I was voltage insensitive. By contrast, deactivation of the ACh-induced I was significantly slower at −100 mV than at +50 mV, while an opposite effect was observed when I was activated by Pilo. These findings are consistent with the voltage affinity pattern observed for these three agonists. Our findings suggest that independent of how voltage disturbs the receptor binding site, the voltage dependence of the signaling pathway is ultimately determined by the agonist. These observations emphasize the pharmacological potential to regulate the M2R-parasympathetic associated cardiac function and also other cellular signaling pathways by exploiting the voltage-dependent properties of GPCRs. [ABSTRACT FROM AUTHOR]

The suprachiasmatic nucleus (SCN) in mammals is the master clock which regulates circadian rhythms. Neural activity of SCN neurons is synchronized to external light through the retinohypothalamic tract (RHT). The paraventricular thalamic nucleus (PVT) is a neural structure that receives synaptic inputs from, and projects back to, the SCN. Lesioning the anterior PVT (aPVT) modifies the behavioral phase response curve induced by short pulses of bright light. In order to study the influence of the aPVT on SCN neural activity, we addressed whether the stimulation of the aPVT can modulate the electrical response of the SCN to either retinal or RHT stimulation. Using in vitro and in vivo recordings, we found a large population of SCN neurons responsive to the stimulation of either aPVT or RHT pathways. Furthermore, we found that simultaneous stimulation of the aPVT and the RHT increased neuronal responsiveness and spontaneous firing rate (SFR) in neurons with a low basal SFR (which also have more negative membrane potentials), such as quiescent and arrhythmic neurons, but no change was observed in neurons with rhythmic firing patterns and more depolarized membrane potentials. These results suggest that inputs from the aPVT could shift the membrane potential of an SCN neuron to values closer to its firing threshold and thus contribute to integration of the response of the circadian clock to light. [ABSTRACT FROM AUTHOR]

The vesicular glutamate transporter 3 (VGLUT3) is expressed at several locations not normally associated with glutamate release. Although the function of this protein has been generally elusive, when expressed in non-glutamatergic synaptic terminals, VGLUT3 can not only allow glutamate co-transmission but also synergize the action of non-glutamate vesicular transporters. Interestingly, in the immature glycinergic projection between the medial nucleus of the trapezoid body (MNTB) and the lateral superior olive (LSO) of auditory brainstem, the transient early expression of VGLUT3 is required for normal developmental refinement. It has however been unknown whether the primary function of VGLUT3 in development of these inhibitory synapses is to enable glutamate release or to promote loading of inhibitory neurotransmitter through vesicular synergy. Using tissue from young mice in which Vglut3 had been genetically deleted, we evaluated inhibitory neurotransmission in the MNTB-LSO pathway. Our results show, in contrast to what has been seen at adult synapses, that VGLUT3 expression has little or no effect on vesicular synergy at the immature glycinergic synapse of brainstem. This finding supports the model that the primary function of increased VGLUT3 expression in the immature auditory brainstem is to enable glutamate release in a developing inhibitory circuit. [ABSTRACT FROM AUTHOR]

The medial nucleus of the trapezoid body (MNTB) is a key nucleus in high-fidelity temporal processing that underlies sound localization in the auditory brainstem. While the glycinergic principal cells of the MNTB project to all primary nuclei of the superior olive, during development the projection from MNTB to the lateral superior olive (LSO) is of interest because this immature inhibitory projection is known to undergo tonotopic refinement during an early postnatal period, and because during this period individual MNTB terminals in the LSO transiently release glycine GABA and glutamate. Developmental changes in calcium-dependent release are understood to be required to allow various auditory nuclei to follow high frequency activity; however, little is known about maturation of calcium-dependent release in the MNTB-LSO pathway, which has been presumed to have less stringent requirements for high-fidelity temporal following. In acute brainstem slices of rats age postnatal day 1 to 15 we recorded whole-cell responses in LSO principal neurons to electrical stimulation in the MNTB in order to measure sensitivity to external calcium, the contribution of different voltage-gated calcium channel subtypes to vesicular release, and the maturation of these measures for both GABA/glycine and glutamate transmission. Our results establish that release of glutamate at MNTB-LSO synapses is calcium-dependent. Whereas no significant developmental changes were evident for glutamate release, GABA/glycine release underwent substantial changes over the first two postnatal weeks: soon after birth L-type, N-type, and P/Q-type voltage-gated calcium channels (VGCCs) together mediated release, but after hearing onset P/Q-type VGCCs predominated. Blockade of P/Q-type VGCCs reduced the estimated quantal number for GABA/gly and glutamate transmission at P5–8 and the frequency of evoked miniature glycinergic events at P12–15, without apparent effects on spontaneous release of neurotransmitter, supporting a model in which P/Q-type VGCCs are required for mature synchronous synaptic transmission, but not for spontaneous vesicle release. [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]

ZnO nanoparticles (ZnONPs) have been shown to have therapeutic potential in some diseases such as diabetes and cancer. However, concentration-dependent adverse effects have also been reported. Studies which evaluate the effects of ZnONPs on the cardiovascular system are scarce. This study aimed to evaluate the cardiovascular effects of a low dose of ZnONPs administered chronically in healthy rats. Changes in dyslipidemia biomarkers, blood pressure, aortic wall structure, vascular contractility, and expression of cannabinoid receptors in the aorta wall were evaluated. Healthy rats were divided into two groups: control or treated (one, two, and three months). The treated rats received an oral dose of 10 mg/kg/day. The results showed that treatment with ZnONPs induced dyslipidemia from the first month, increasing atherosclerosis risk, which was confirmed by presence of atherosclerotic alterations revealed by aorta histological analysis. In in vitro assays, ZnONPs modified the aorta contractile activity in response to the activation of cannabinoid receptors (CB1 and CB2). The expression of CB1 and CB2 was modified as well. Moreover, ZnONPs elicited an increase in blood pressure. In conclusion, long-time oral administration of ZnONPs induce dyslipidemia and atherosclerosis eliciting alterations in aorta contractility, CB1 and CB2 receptors expression, and an increase in blood pressure in healthy rats. [ABSTRACT FROM AUTHOR]