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The suprachiasmatic nucleus (SCN) of the hypothalamus is the site of a central circadian clock that orchestrates overt rhythms of physiology and behavior. Circadian timekeeping requires intercellular communication among SCN neurons, and multiple signaling pathways contribute to SCN network coupling. Gamma-aminobutyric acid (GABA) is produced by virtually all SCN neurons, and previous work demonstrates that this transmitter regulates coupling in the adult SCN but is not essential for the nucleus to sustain overt circadian rhythms. Here, we show that the deletion of the gene that codes for the GABA vesicular transporter Vgat from neuromedin-S (NMS) + neurons-a subset of neurons critical for SCN function-causes arrhythmia of locomotor activity and sleep. Further, NMS- Vgat deletion impairs intrinsic clock gene rhythms in SCN explants cultured ex vivo. Although vasoactive intestinal polypeptide (VIP) is critical for SCN function, Vgat deletion from VIP-expressing neurons did not lead to circadian arrhythmia in locomotor activity rhythms. Likewise, adult SCN-specific deletion of Vgat led to mild impairment of behavioral rhythms. Our results suggest that while the removal of GABA release from the adult SCN does not affect the pacemaker's ability to sustain overt circadian rhythms, its removal from a critical subset of neurons within the SCN throughout development removes the nucleus ability to sustain circadian rhythms. Our findings support a model in which SCN GABA release is critical for the developmental establishment of intercellular network properties that define the SCN as a central pacemaker., Competing Interests: Competing interests statement:The authors declare no competing interest.

Visual input to the hypothalamus from intrinsically photosensitive retinal ganglion cells (ipRGCs) influences several functions including circadian entrainment, body temperature, and sleep. ipRGCs also project to nuclei such as the supraoptic nucleus (SON), which is involved in systemic fluid homeostasis, maternal behavior, social behaviors, and appetite. However, little is known about the SON-projecting ipRGCs or their relationship to well-characterized ipRGC subtypes. Using a GlyT2 Cre mouse line, we show a subtype of ipRGCs restricted to the dorsal retina that selectively projects to the SON. These ipRGCs tile a dorsal region of the retina, forming a substrate for encoding ground luminance. Optogenetic activation of their axons demonstrates they release the neurotransmitter glutamate in multiple regions, including the suprachiasmatic nucleus (SCN) and SON. Our results challenge the idea that ipRGC dendrites overlap to optimize photon capture and suggests non-image forming vision operates to sample local regions of the visual field to influence diverse behaviors., (© 2023. The Author(s).)

Synaptic and extrasynaptic GABA A receptor (GABA A R)-mediated neurotransmission is a critical component of the suprachiasmatic nucleus (SCN) neuronal network. However, the properties of the GABA A tonic current ( I tonic ) and its origin remain unexplored. Spontaneous GABA A postsynaptic currents (sGPSCs) and I tonic were recorded from SCN neurons with the whole cell voltage-clamp technique at different times of the day. GABA A R antagonists (bicuculline, gabazine, and picrotoxin) inhibited sGPSC and induced an outward shift of the holding current, which defined the I tonic amplitude. The sGPSC frequency, synaptic charge transfer, and I tonic amplitude all demonstrated significant diurnal rhythms, with peaks in the middle of the day [zeitgeber time (ZT)7-8] and nadirs at night (ZT19-20). The I tonic amplitude increased proportionally with the sGPSC frequency and synaptic charge transfer during the day and required action potential-mediated GABA release, which was confirmed by TTX application. The activation of presynaptic GABA B receptors by baclofen did not significantly alter the I tonic of neurons with low-frequency sGPSC. The equilibrium potential (Eq) for I tonic was similar to the Eq for chloride and GABA A receptor-activated currents. I tonic showed outward rectification at membrane potentials over the range of -70 to -10 mV and then was linear at voltages greater than -10 mV. GABA A R containing α4-, α5-, and δ-subunits were expressed in SCN, and their contribution to I tonic was confirmed by application of the GABA A R agonist 4,5,6,7-tetrahydroisoxazolo[5,4- c ]pyridin-3-ol (THIP) and the GABA A R inverse agonist 11,12,13,13 a -tetrahydro-7-methoxy-9-oxo-9 H -imidazo[1,5- a ]pyrrolo[2,1- c ][1,4]benzodiazepine-1-carboxylic acid ethyl ester (L655,708). Thus, the I tonic was mediated by extrasynaptic GABA A Rs activated predominantly by GABA diffusing out of GABAergic synapses. NEW & NOTEWORTHY A tonic current ( I tonic ) mediated by GABA A receptors (GABA A Rs) containing α4-, α5- and δ-subunits was observed in the suprachiasmatic nucleus. The I tonic amplitude strongly depended on the action potential-mediated synaptic release of GABA. The equilibrium potential for I tonic corresponds to that for GABA A currents. The frequency of GABA A postsynaptic currents and I tonic amplitude increased during the day, with peak in the middle of the day, and then gradually declined with a nadir at night, thus showing a diurnal rhythm.

Circadian rhythms are 24-h cycles in physiology regulated by the suprachiasmatic nucleus (SCN) in the brain, where daily cues act on SCN neurons to alter clock timing. Cannabinoid signaling modulates SCN neuronal activity, although the mechanism remains unclear. We propose that neuronal activity generates endocannabinoid release, activating astrocyte Ca 2+ signaling, which releases adenosine and activates adenosine-1 receptors (A1Rs) on the presynaptic axon terminals, decreasing GABA release. We demonstrated, in mice, that activation of cannabinoid-1 receptors (CB1R) with the agonist WIN 55,212-2 (WIN) reduced the miniature GABA receptor-mediated postsynaptic current (mGPSC) frequency by a mechanism that requires astrocytes and A1R. WIN activated an intracellular Ca 2+ signaling pathway in astrocytes. Activating this intracellular Ca 2+ pathway with designer receptors exclusively activated by designer drugs (DREADDs) also decreased the mGPSC frequency and required A1R activation. The frequency of spontaneous Ca 2+ events, including those induced by depolarization of a postsynaptic SCN neuron, was reduced by blocking CB1R activation with AM251, demonstrating neuronal endocannabinoid signaling modulates astrocytic Ca 2+ signaling in the SCN. Finally, daytime application of WIN or adenosine phase advanced the molecular circadian clock, indicating that this cannabinoid signaling pathway is vital for the timing of circadian rhythms., (Copyright © 2020 Hablitz et al.)

GABA is a principal neurotransmitter in the hypothalamic suprachiasmatic nucleus (SCN) that contributes to intercellular communication between individual circadian oscillators within the SCN network and the stability and precision of the circadian rhythms. GABA transporters (GAT) regulate the extracellular GABA concentration and modulate GABA A receptor (GABA A R)-mediated currents. GABA transport inhibitors were applied to study how GABA A R-mediated currents depend on the expression and function of GAT. Nipecotic acid inhibits GABA transport and induced an inward tonic current in concentration-dependent manner during whole cell patch-clamp recordings from SCN neurons. Application of either the selective GABA transporter 1 (GAT1) inhibitors NNC-711 or SKF-89976A, or the GABA transporter 3 (GAT3) inhibitor SNAP-5114, produced only small changes of the baseline current. Coapplication of GAT1 and GAT3 inhibitors induced a significant GABA A R-mediated tonic current that was blocked by gabazine. GAT inhibitors decreased the amplitude and decay time constant and increased the rise time of spontaneous GABA A R-mediated postsynaptic currents. However, inhibition of GAT did not alter the expression of either GAT1 or GAT3 in the hypothalamus. Thus GAT1 and GAT3 functionally complement each other to regulate the extracellular GABA concentration and GABA A R-mediated synaptic and tonic currents in the SCN. Coapplication of SKF-89976A and SNAP-5114 (50 µM each) significantly reduced the circadian period of Per1 expression in the SCN by 1.4 h. Our studies demonstrate that GAT are important regulators of GABA A R-mediated currents and the circadian clock in the SCN. NEW & NOTEWORTHY In the suprachiasmatic nucleus (SCN), the GABA transporters GAT1 and GAT3 are expressed in astrocytes. Inhibition of these GABA transporters increased a tonic GABA current and reduced the circadian period of Per1 expression in SCN neurons. GAT1 and GAT3 showed functional cooperativity: inhibition of one GAT increased the activity but not the expression of the other. Our data demonstrate that GABA transporters are important regulators of GABA A receptor-mediated currents and the circadian clock., (Copyright © 2017 the American Physiological Society.)

GABA is a principal neurotransmitter in the suprachiasmatic hypothalamic nucleus (SCN), the master circadian clock. Despite the importance of GABA and GABA uptake for functioning of the circadian pacemaker, the localization and expression of GABA transporters (GATs) in the SCN has not been investigated. The present studies used Western blot analysis, immunohistochemistry and electron microscopy to demonstrate the presence of GABA transporter 1 (GAT1) and GAT3 in the SCN. By using light microscopy, GAT1 and GAT3 were co-localized throughout the SCN, but were not expressed in the perikarya of arginine vasopressin- or vasoactive intestinal peptide-immunoreactive (-ir) neurons of adult rats, nor in the neuronal processes labelled with the neurofilament heavy chain. Using electron microscopy, GAT1- and GAT3-ir was found in glial processes surrounding unlabelled neuronal perikarya, axons, dendrites, and enveloped symmetric and asymmetric axo-dendritic synapses. Glial fibrillary acidic protein-ir astrocytes grown in cell culture were immunopositive for GAT1 and GAT3 and both GATs could be observed in the same glial cell. These data demonstrate that synapses in the SCN function as 'tripartite' synapses consisting of presynaptic axon terminals, postsynaptic membranes and astrocytes that contain GABA transporters. This model suggests that astrocytes expressing both GATs may regulate the extracellular GABA, and thereby modulate the activity of neuronal networks in the SCN., (© 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Environmental synchronization of the endogenous mammalian circadian rhythm involves glutamatergic and GABAergic neurotransmission within the hypothalamic suprachiasmatic nucleus (SCN). The neuropeptide nociceptin/orphanin FQ (N/OFQ) inhibits light-induced phase shifts, evokes K(+)-currents and reduces the intracellular Ca(2+) concentration in SCN neurons. Since these effects are consistent with a modulatory role for N/OFQ on synaptic transmission in the SCN, we examined the effects of N/OFQ on evoked and spontaneous excitatory and inhibitory currents in the SCN. N/OFQ produced a consistent concentration-dependent inhibition of glutamate-mediated excitatory postsynaptic currents (EPSC) evoked by optic nerve stimulation. N/OFQ did not alter the amplitude of currents induced by application of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-d-aspartate (NMDA) nor the amplitude of miniature EPSC (mEPSC) consistent with a lack of N/OFQ effect on postsynaptic AMPA or NMDA receptors. N/OFQ significantly reduced the mEPSC frequency. The inhibitory actions of N/OFQ were blocked by omega-conotoxin GVIA, an N-type Ca(2+)channel antagonist and partially blocked by omega-agatoxin TK, a P/Q type Ca(2+) channel blocker. These data indicate that N/OFQ reduces evoked EPSC, in part, by inhibiting the activity of N- and P/Q-type Ca(2+) channels. In addition, N/OFQ produced a consistent reduction in baseline Ca(2+) levels in presynaptic retinohypothalamic tract terminals. N/OFQ also inhibited evoked GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSC) in a concentration dependent manner. However, N/OFQ had no effect on currents activated by muscimol application or on the amplitude of miniature IPSC (mIPSC) and significantly reduced the mIPSC frequency consistent with an inhibition of GABA release downstream from Ca(2+) entry. Finally, N/OFQ inhibited the paired-pulse depression observed in SCN GABAergic synapses consistent with a presynaptic mechanism of action. Together these results suggest a widespread modulatory role for N/OFQ on the synaptic transmission in the SCN.

Myelin sheaths, wrapping axons, perform the following important functions: support, protection, feeding and isolation. Injury of myelin compact structure leads to an impairment and severe illness of the nerve system. Exact mechanisms underlying the myelination process and myelin sheaths damage have not established yet. Therefore search for substances, which provide regulatory and protective effects on the normal myelination as well as stimulating action on the remyelination after myelin damage, is of special interest. Recently it was shown that extract from mushroom Hericium erinaceus had activating action on the nerve tissue. So the aim of the present work was to study an influence of an extract from H. erinaceus on the cerebellar cells and the process of myelination in vitro. Obtained data revealed the normal growth of the nerve and glial cells with extract at cultivating. No pathologic or toxic action of the extract has been found. The cell ultrastructure was intact and similar to that observed in vivo. The process of myelination in the presence of the extract began earlier as compared to controls and was characterised by a higher rate. Thus, extract of H. erinaceus promoted normal development of cultivated cerebellar cells and demonstrated a regulatory effect on the process of myelin genesis process in vitro.

The action of P. cubensis mushroom extract, containing psilocybin (PCB) and psilocin, on spike activity of hippocampal CA1 pyramidal neurons was studied in in vitro rat brain slices. In 38 (76%) out of 50 investigated neurons spike activity was decreased, in 2 (4%) cells it increased. There was no response 10 (20%) neurons. Application of the extract caused short burst firing in 12 (24%) neurons. All neurons showing inhibition during PCB-containing extract application, were also inhibited by serotonin (5-HT). Usually inhibitory reaction did not last over 4-5 min upon 3 min extract application and could be prolonged up to 10-43 min up on serotonin application. Part of neurons were inhibited by serotonin and did not react to extract application. Inhibitory reactions induced by extract application were blocked by ritanserin in half of the tested units and were induced due to activation of 5-HT2 serotonin receptors. The extract suppressed excitative spike reactions caused by application of L-glutamic acid. It is concluded, that application of PCB-containing extract in most cases reduced spike activity in hippocampal CA1 pyramidal neurons and suppressed glutamate transmission.

Application of heteromodal extrastimuli (ES) promotes selected excitation of most of the responding neurons in the thalamic reticular nucleus (R). Twelve of 32 tested neurons responded to visual ES and 4 from 21 tested neurons responded to auditory ES. Initial responses of neurons and quantity of cells with initial responses decreased during habituation to ES. Grouped discharges disappeared after external stimulation. Initial responses to ES and to the following conditional stimulus appeared during external inhibition (EI), while late responses usually accompanying conditioning were inhibited almost in half of the studied neurons. It was suggested that R neurons participated in EI and in habituation to ES.

Activity of 66 neurons of the reticular nucleus (R), 31 neurons of the ventroposterolateral nucleus and 14 neurons of the posterolateral nucleus-pulvinar complex of the thalamus was investigated during extinction of the conditioned instrumental alimentary reflex. The quantity of R neurons that show initial excitation in response to the conditional stimulus in the first 300 ms decreased during extinction. Conditioned placing reactions and late excitatory and inhibitory neuronal responses in the R and dorsal thalamic nuclei with latency above 300 ms disappeared during extinction simultaneously. The background unit activity decreased during extinction in the 2/3 of investigated neurons of R and dorsal thalamic nuclei. It is suggested that the efferent influence from the R decreased during extinction.

Spike reactions of 156 neurons of the thalamic reticular nucleus (R) have been studied during performance of instrumental alimentary conditioned reflex. These reactions consist of initial and late phases. The latencies of initial reaction phase are 10-100 ms and duration--50-250 ms. The minimum data of late phase latencies are 100-300 ms. Initial responses to the conditional sound click are found in 27 neurons: 26 of them show excitation and one neuron--inhibitory-excitatory responses. The late reaction phase has been observed in 134 neurons only during performance of conditioned placing reactions: 115 of them show excitation, 19 neurons--inhibition and 22 neurons--were not reactive. Responses of 30 neurons forestalled the performance of conditioned forelimb movement. 118 neurons responded during the ballistic phase of the movement. It is concluded that the thalamic reticular nucleus takes part in the conditional signal perception and in the preparation and control of conditioned movement performance.

Spike activity of neurons (area 7) was studied during conditioned placing reflex in the rest, during appearance and extinction of the orienting reaction in trained cats. Spike activity was studied also during unconditioned electro-cutaneous stimulation of the contralateral forelimb in untrained cats. Conditioned movements and electrocutaneous stimulation occurred without specific spike reactions of neurons due to the effect of the external stimulation on attention of animals. It is suggested that appearance of spike responses of neurons to the stimulation only reflects the processes in structures that take part in the triggering and realization of the reflex.

Spike reactions of the same somatic cortex neurons (areas 3 and 4) were analyzed during unconditioned stimulation. They were registered in the untrained hungry cats under conditions of rest and with food motivation. A decrease of the intensity of initial spike reactions, disappearance of the late neuronal responses following electro-cutaneous stimulation as well as changes in the level of the background activity were observed during emergence of food motivation. Resemblance and correlation between time of appearance of neuronal reactions and respected movements during conditioned stimulation under various levels of food motivational excitation were shown. This correlation depended on the level of the animal satiation. The results show the possibility of considerable influence of food motivation on the patterns of spike reactions in the somatic cortex neurons.

In experiments on untrained and trained cats the impulse activity of cortical neurons (area 4) was studied in response to long (1s) acoustic stimulus (conditioned stimulus for trained animals) and to a fall of the platform under the studied limb (unconditioned stimulus). Only those neurons were studied which responded to the appearance of passive movement after the fall of the platform. In trained animals the number of neurons responding to conditioned stimuli in case of realization of the reflex was 100% and in the absence of conditioned movements--70%. This largely exceeded the number of neurons responding to the same sound in untrained animals (45%). In peristimulus histograms of neuronal reactions of the studied population in untrained and trained animals (in the absence of conditioned movements) only the initial impulse response was clearly seen with the latency less than 50 ms and duration up to 100 ms. In the presence of conditioned movements the impulse response consisted of many components: initial response, early and late after-responses. The early after-responses with latency of 100-150 ms and duration of 100-200 ms were associated with the start of the conditioned stimulus, and the pattern and duration of the late after-responses was determined by the appearance of the conditioned movements. It is established that the value of neuronal response to reinforcement in trained animals does not depend on the appearance of the conditioned movement.

Spike responses of 104 neurons of the nucleus reticularis thalami (R) and neighbouring thalamic nuclei to the acoustic stimuli, tactile and visual stimulation were studied in the chronic experiment. 29% of neurons responded to the acoustic stimulation and 11% of them were not specific to different acoustic stimuli. The minimal latency of the excitation to the acoustic stimulation was 12-37 ms and that of the inhibition--18-27 ms. The duration of the excitation in response to the acoustic stimulation was 50-250 ms and that of inhibition--27-190 ms. 16% of R neurons responded to the stimulation of different sensory systems. The majority of R neurons showed excitation in response to different stimuli and only 4-10% of neurons showed inhibition.

Spike reactions were recorded from motor cortex neurons in cats in the forelimb projection area during external and internal inhibition of the conditioned postural adjustment (transfer of the body weight) of the same limb. Spike responses evoked by external stimulation were of the same character as the responses during conditioned stimulation. As a rule, they were determined by the state of the animal and its habituation to the applied stimulus. Duration of the responses during external stimulation was shorter in trained cats than in untrained ones. Late after-discharges of units and associated conditioned movements disappeared simultaneously during external and internal inhibition. Sometimes the external stimuli were able to depress after-discharges even when the conditioned movements appeared. External stimuli of various modalities inhibited the reflex in different manners. The changes of spike reactions during conditioned posture adjustment resembled those during well learned local conditioned events.

Spike activity of unitary motor cortex (area 4) neurons was recorded during performance of conditioned reflex postural adjustment in cats. Changes in activity of most neurons (increase of discharge frequency) correlated with conditioned-reflex supporting movement of the forelimb. These neuronal reactions had specific polycomponent patterns and preceded the conditional movements by 50-600 ms. Neuronal reactions connected with erroneous positive movement responses to differential stimuli and spontaneous "interstimular" movements had the same direction but were less intensive and occurred in most cases synchronously with these movements. Neuronal reactions and postural adjustments simultaneously disappeared during reflex extinction. The proposed method of elaboration of conditioned reflex of postural adjustments permits obtaining standard natural coordinated forelimb movements which form the necessary initial component of more complex behavioral reactions.

Spike activity of neurons (areas 3, 4) was studied in cats during conditioned placing reaction before and after food satiation. After feeding the background activity increased in 2/3 of recorded neurons, tonic component of neuronal reaction decreased considerably and repeated contraction of m. biceps disappeared. Difference in the latencies of spike reactions in the same neuron to conditioned stimulation in the hungry and satisfied state of the animal during experiment was 50-300 ms. The latencies of conditioned placing reaction did not change. It was suggested that fluctuations of the food motivation level of animals lead to changes in the cortico-subcortical interrelations responsible for control of the start and realization of conditioned movements.