cAMP-dependent inward rectifier current in neurons of the rat suprachiasmatic nucleus

Electrophysiological properties of the inward rectification of neurons in the rat suprachiasmatic nucleus (SCN) were examined by using the single-electrode voltage-clamp method, in vitro. Inward rectifier current (I _H) was produced by hyperpolarizing step command potentials to membrane potentials negative to approximately −60 mV in nominally zero-Ca²⁺ Krebs solution containing tetrodotoxin (1 μM), tetraethylammonium (40 mM), Cd²⁺ (500 μM) and 4-aminopyridine (1 mM).I _H developed during the hyperpolarizing step command potential with a duration of up to 5 s showing no inactivation with time.I _H was selectively blocked by extracellular Cs⁺ (1 mM). The activation of the H-channel conductance (G _H) ranged between −55 and −120 mV. TheG _H was 80–150 pS (n=4) at the half-activation voltage of −84±7 mV (n=4). The reversal potential ofI _H obtained by instantaneous current voltage (I/V) relations was −41±6mV (n=4); it shifted to −51±8mV (n=3) in low-Na⁺ (20 mM) solution and to −24±4 mV (n=4) in high-K⁺ (20 mM) solution. Forskolin (1–10 μM) produced an inward current and increased the amplitude ofI _H. Forskolin did not change the half-activation voltage ofG _H. 8-Bromo-adenosine 3′,5′-cyclic monophosphate (8-Br-cAMP, 0.1–1 mM) and dibutyryl-cAMP (0.1–1 mM) enhancedI _H. 3-Isobutyl-1-methylxanthine (IBMX, 1 mM) also enhancedI _H. The results suggest that the inward rectifier cation current is regulated by the basal activity of adenylate cyclase in neurons of the rat SCN.     

Effects of restricted feeding on single neuron activity of suprachiasmatic neurons in rat hypothalamic slice preparation

Influence of restricted feeding on single neuron activity in the rat suprachiasmatic nucleus (SCN) was examined using hypothalamic slice preparations. Feeding restriction for one month did not change SCN neuron activity in either normal light-dark schedule (LD) or constant light schedule (LL). Locomotor activity in vivo was increased for a few hours before and during feeding time by food restriction of both LD and LL rats. The present experiment suggested that the memory of feeding time produced by food restriction is independent of SCN neuron activity.     

Circadian difference in firing rate of isolated rat suprachiasmatic nucleus neurons

The suprachiasmatic nucleus (SCN) of the hypothalamus contains the primary circadian clock in mammals. Dissociated SCN neurons in long-term culture exhibit a circadian modulation of spontaneous electrical activity. To evaluate the presence of circadian differences in spontaneous activity of isolated SCN neurons without synaptic connections, dissociated rat SCN neurons were studied with on-cell recording 3-4 days after preparation, before the formation of dendrites, axons and synapses. A day-night difference in spontaneous electrical firing rate was found in acutely dissociated SCN neurons. During the first subjective day, the average firing rate (0.87+/-0.12 Hz) was significantly higher than during the first subjective night (0.24+/-0.06 Hz), while the firing rate on the next day (0.68+/-0.11 Hz) was significantly higher than during the preceding night. These data suggest that populations of isolated SCN neurons with no synaptic interactions contain a functioning circadian clock, and are particularly amenable to biophysical experiments.     

Riluzole-sensitive slowly inactivating sodium current in rat suprachiasmatic nucleus neurons

The persistent (i.e., slowly inactivating) fraction of the Na current (I(Na,P)) regulates excitability of CNS neurons. In isolated rat suprachiasmatic nucleus (SCN) neurons with a ramp-type voltage-clamp protocol, we have studied the properties of a robust current that has the general properties of I(Na,P) but exhibits a slow inactivation (I(Na,S)). The time dependence of the development of the inactivation was also studied by clamping of the membrane potential at different levels: time constants ranging from approximately 50 to approximately 700 ms, depending on the voltage level, were revealed. The I(Na,S) (50-150 pA) was present in both spontaneously active and silent neurons. The neurons exhibited I(Na,S) without visible rundown during approximately 1-h recordings. I(Na,S) had a threshold between -65 and -60 mV and was maximal at about -45 mV. Tetrodotoxin (TTX; 1 microM) completely and reversibly blocked I(Na,S). Riluzole, an effective blocker of I(Na,P), inhibited reversibly I(Na,S) with an EC(50) of 1-2 microM. Microapplication of 10 microM riluzole during either extracellular or intracellular recording suppressed spontaneous activity in isolated SCN neurons. In the slice preparation, bath application of 20 microM riluzole resulted in decreased firing rate or complete suppression of spontaneous activity in some neurons (9/14) but had no effect on other neurons (5/14). In riluzole-resistant neurons in cell-attached experiments, low-amplitude current spikes were present in 1 microM TTX. We concluded that I(Na,S) is ubiquitously expressed by all SCN neurons and that this current is a necessary but not sufficient depolarizing component of the mechanism for spontaneous firing.     

Changes in and dorsal to the rat suprachiasmatic nucleus during early pregnancy

Circadian rhythms in behavior and physiology change as female mammals transition from one reproductive state to another. The mechanisms responsible for this plasticity are poorly understood. The suprachiasmatic nucleus (SCN) of the hypothalamus contains the primary circadian pacemaker in mammals, and a large portion of its efferent projections terminate in the ventral subparaventricular zone (vSPZ), which also plays important roles in rhythm regulation. To determine whether these regions might mediate changes in overt rhythms during early pregnancy, we first compared rhythms in Fos and Per2 protein expression in the SCN and vSPZ of diestrous and early pregnant rats maintained in a 12:12-h light/dark (LD) cycle. No differences in the Fos rhythm were seen in the SCN core, but in the SCN shell, elevated Fos expression was maintained throughout the light phase in pregnant, but not diestrous, rats. In the vSPZ, the Fos rhythm was bimodal in diestrous rats, but this rhythm was lost in pregnant rats. Peak Per2 expression was phase-advanced by 4 h in the SCN of pregnant rats, and some differences in Per2 expression were found in the vSPZ as well. To determine whether differences in Fos expression were due to altered responsivity to light, we next characterized light-induced Fos expression in the SCN and vSPZ of pregnant and diestrous rats in the mid-subjective day and night. We found that the SCN core of the two groups responded in the same way at each time of day, whereas the rhythm of Fos responsivity in the SCN shell and vSPZ differed between diestrous and pregnant rats. These results indicate that the SCN and vSPZ are functionally re-organized during early pregnancy, particularly in how they respond to the photic environment. These changes may contribute to changes in overt behavioral and physiological rhythms that occur at this time.     

Persistent subthreshold voltage-dependent cation single channels in suprachiasmatic nucleus neurons

The hypothalamic suprachiasmatic nucleus (SCN) contains the primary circadian pacemaker in mammals, and transmits circadian signals by diurnal modulation of neuronal firing frequency. The ionic mechanisms underlying the circadian regulation of firing frequency are unknown, but may involve changes in membrane potential and voltage-gated ion channels. Here we describe novel tetrodotoxin- and nifedipine-resistant subthreshold, voltage-dependent cation (SVC) channels that are active at resting potential of SCN neurons and increase their open probability (P(o)) with membrane depolarization. The increased P(o) reflects changes in the kinetics of the slow component of the channel closed-time, but not the channel open-time or fast closed-time. This study provides a background for investigation of the possible role of SVC channels in regulation of circadian oscillations of membrane excitability in SCN neurons.     

Transplantation of the neonatal suprachiasmatic nuclei into rats with complete bilateral suprachiasmatic lesions

The suprachiasmatic nuclei (SCN) obtained from neonatal day 1 rats were transplanted into the third ventricle of SCN lesioned rats which had shown circadian arrhythmicity in wheel-running activity for more than 2 months. In 8 out of 16 rats that received SCN transplantation, appearance of circadian rhythms in wheel-running activity was observed between 2 and 9 weeks after transplantation. Histological examination revealed ingrowth of the grafts into the periventricular zone, caudal from the lesioned SCN. These findings suggest that the recovery of circadian rhythmicity was the result of functional reinnervation of the periventricular zone by efferent fibers from the SCN.     

Suprachiasmatic nucleus communicates with anterior thalamic paraventricular nucleus neurons via rapid glutamatergic and gabaergic neurotransmission: state-dependent response patterns observed in vitro

The hypothalamic suprachiasmatic nucleus uniquely projects to the midline thalamic paraventricular nucleus. To characterize this projection, patch clamp techniques applied in acute rat brain slice preparations examined responses of anterior thalamic paraventricular nucleus neurons to focal suprachiasmatic nucleus stimulation. Whole cell recordings from slices obtained during daytime (n=40) revealed neurons with a mean membrane potential of -66+/-1.2 mV, input conductance of 1.5+/-0.1 nS and state-dependent tonic or burst firing patterns. Electrical stimulation (one or four pulses) in suprachiasmatic nucleus elicited monosynaptic excitatory postsynaptic potentials (mean latency of 12.6+/-0.6 ms; n=12), featuring both AMPA and N-methyl-D-aspartate-glutamate receptor-mediated components, and monosynaptic bicuculline-sensitive inhibitory postsynaptic potentials (mean latency of 16.6+/-0.6 ms; n=7) reversing polarity at -72+/-2.6 mV, close to the chloride equilibrium potential. Glutamate microstimulation of suprachiasmatic nucleus also elicited transient increases in spontaneous excitatory or inhibitory postsynaptic currents in anterior thalamic paraventricular neurons. Recordings from rats under reverse light/dark conditions (n=22) yielded essentially similar responses to electrical stimulation. At depolarized membrane potentials, suprachiasmatic nucleus-evoked excitatory postsynaptic potentials triggered single action potentials, while evoked inhibitory postsynaptic potentials elicited a silent period in ongoing tonic firing. By contrast, after manual adjustment of membrane potentials to hyperpolarized levels, neuronal response to the same "excitatory" stimulus was a low threshold spike and superimposed burst firing, while responses to "inhibitory" stimuli paradoxically elicited excitatory rebound low threshold spikes and burst firing. These data support the existence of glutamatergic and GABAergic efferents from the suprachiasmatic nucleus to its target neurons. Additionally, in thalamic paraventricular nucleus neurons, responses to activation of their suprachiasmatic afferents may vary in accordance with their membrane potential-dependent intrinsic properties, a characteristic typical of thalamocortical neurons.     

Characterization of an apamin-sensitive potassium current in suprachiasmatic nucleus neurons

In neurons of the suprachiasmatic nucleus, spike frequency adaptation and membrane afterhyperpolarization occur during a train of action potentials. Extracellular Ca2+ may regulate neuronal excitability by several mechanisms, including activation of small conductance and large conductance Ca(2+)-activated K+ channels. The overall goal of this study was to examine the role of Ca(2+)-activated K+ currents in individual suprachiasmatic nucleus neurons. To this end, we used the nystatin-perforated patch technique to record currents from suprachiasmatic nucleus neurons. Iberiotoxin and tetraethylammonium, antagonists of large conductance Ca(2+)-activated K+ channels, had no effect on the membrane afterhyperpolarization. However, antagonists of small conductance Ca(2+)-activated K+ channels, apamin and d-tubocurarine, reduced the amplitude of the membrane afterhyperpolarization and inhibited the spike frequency adaptation that occurred during a train of action potentials. Although there was no significant difference in membrane AHP between different portions of the circadian day, apamin and d-tubocurarine increased the spontaneous firing frequency of suprachiasmatic nucleus neurons during the daytime. In voltage-clamp mode, membrane depolarization-activated currents were followed by an outward tail current reversing near the K+ equilibrium potential. The tail current decayed with a time constant of 220 ms at +20 mV and 149 ms at -40 mV. Apamin irreversibly and d-tubocurarine reversibly inhibited the tail current. The tail current amplitude was also reduced by the GABAA receptor antagonist, bicuculline methiodide, while picrotoxin (another GABAA receptor antagonist) was without effect. Removal of extracellular Ca2+ or the addition of Cd2+ reversibly inhibited the tail current. These results indicate that apamin- and d-tubocurarine-sensitive small conductance Ca(2+)-activated K+ channels have a modulatory function on the action potential firing frequency as well as the membrane afterhyperpolarization that follows a train of action potentials in suprachiasmatic nucleus neurons. Importantly, our data also indicate that a portion of the effects of bicuculline methiodide on suprachiasmatic nucleus neurons may be mediated by inhibition of small conductance Ca(2+)-activated K+ channels.     

Circadian periods of single suprachiasmatic neurons in rats

Neuronal activity of a single neuron was monitored continuously for more than 5 days by means of a multi-electrode dish in dispersed cell culture of the rat suprachiasmatic nucleus (SCN). Sixty-seven out of 88 neurons showed a robust circadian rhythm in firing rate. The mean circadian period was 24.2 h, which was almost identical to that of the locomotor activity rhythm in 114 weanling rats blinded on the day of birth. However, the circadian period in individual SCN neurons was scattered from 20.0 to 28.3 h (SD, 1.4 h), while the period of activity rhythm clustered from 24.0 to 24.8 h (SD, 0.2 h). It is concluded that a large number of SCN neurons contain the circadian oscillator, the period of which is more variable than the circadian period of the SCN as a whole. It is suggested that the circadian rhythms in individual SCN neurons are capable of synchronizing to each other and are integrated to constitute a multiple oscillator system(s) within the SCN.     

The role of GABAergic neuron on NMDA- and SP-induced phase delays in the suprachiasmatic nucleus neuronal activity rhythm in vitro

Gamma-aminobutyric acid (GABA), and its biosynthetic enzyme, glutamic decarboxylase, are widely distributed in the suprachiasmatic nucleus (SCN). In the present study, we examined the role of the GABA_A receptor on in vitro SCN responses to photic-like signals. We found that 100 μM GABA_A receptor antagonist bicuculline partially blocked field potentials evoked by optic nerve stimulation. NMDA- and SP-induced phase shifts of SCN neuronal activity rhythms, were blocked with 10 μM bicuculline. Application of 100 μM bicuculline alone induced phase advance of SCN neuronal activity rhythm. These results show that NMDA- and SP-induced phase shifts are blocked by bicuculline and suggest GABA has an important role as neurotransmitter in the neuronal network regulating phase shifts of the circadian clock.     

The suprachiasmatic nucleus participates in food entrainment: a lesion study

Daily feeding schedules entrain temporal patterns of behavior, metabolism, neuronal activity and clock gene expression in several brain areas and periphery while the suprachiasmatic nucleus (SCN), the biological clock, remains coupled to the light/dark cycle. Because bilateral lesions of the SCN do not abolish food entrained behavioral and hormonal rhythms it is suggested that food entrained and light entrained systems are independent of each other. Special circumstances indicate a possible interaction between the light and the food entrained systems and indicate modulation of SCN activity by restricted feeding. This study explores the influence of the SCN on food entrained rhythms. Food entrained temporal profiles of behavior, core temperature, corticosterone and glucose, as well as Fos and PER1 immunoreactivity in the hypothalamus and corticolimbic structures were explored in rats bearing bilateral SCN lesions (SCNX). In SCNX rats food anticipatory activity and the food entrained temperature and corticosterone increase were expressed with earlier onset and higher values than in intact controls. Glucose levels were lower in SCNX rats in all time points and SCNX rats anticipation to a meal induced higher c-Fos positive neurons in the hypothalamus, while a decreased c-Fos response was observed in corticolimbic structures. SCNX rats also exhibited an upregulation of the PER1 peak in hypothalamic structures, especially in the dorsomedial hypothalamic nucleus (DMH), while in some limbic structures PER1 rhythmicity was dampened. The present results indicate that the SCN participates actively during food entrainment modulating the response of hypothalamic and corticolimbic structures, resulting in an increased anticipatory response.     

Cell-type specific distribution of chloride transporters in the rat suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN) is a circadian oscillator and biological clock. Cell-to-cell communication is important for synchronization among SCN neuronal oscillators and the great majority of SCN neurons use GABA as a neurotransmitter, the principal inhibitory neurotransmitter in the adult CNS. Acting via the ionotropic GABA_A receptor, a chloride ion channel, GABA typically evokes inhibitory responses in neurons via Cl⁻ influx. Within the SCN GABA evokes both inhibitory and excitatory responses although the mechanism underlying GABA-evoked excitation in the SCN is unknown. GABA-evoked depolarization in immature neurons in several regions of the brain is a function of intracellular chloride concentration, regulated largely by the cation-chloride cotransporters NKCC1 (sodium/potassium/chloride cotransporter for chloride entry) and KCC1-4 (potassium/chloride cotransporters for chloride egress). It is well established that changes in the expression of the cation-chloride cotransporters through development determines the polarity of the response to GABA. To understand the mechanisms underlying GABA-evoked excitation in the SCN, we examined the SCN expression of cation-chloride cotransporters. Previously we reported that the K⁺/Cl⁻ cotransporter KCC2, a neuron-specific chloride extruder conferring GABA's more typical inhibitory effects, is expressed exclusively in vasoactive intestinal peptide (VIP) and gastrin-releasing peptide (GRP) neurons in the SCN. Here we report that the K⁺/Cl⁻ cotransporter isoforms KCC4 and KCC3 are expressed solely in vasopressin (VP) neurons in the rat SCN whereas KCC1 is expressed in VIP neurons, similar to KCC2. NKCC1 is expressed in VIP, GRP and VP neurons in the SCN as is WNK3, a chloride-sensitive neuron-specific with no serine–threonine kinase which modulates intracellular chloride concentration via opposing actions on NKCC and KCC cotransporters. The heterogeneous distribution of cation-chloride cotransporters in the SCN suggests that Cl⁻ levels are differentially regulated within VIP/GRP and VP neurons. We suggest that GABA's excitatory action is more likely to be evoked in VP neurons that express KCC4.     

Influence of aging on the seasonal rhythm of the vasopressin-expressing neurons in the human suprachiasmatic nucleus

The mammalian suprachiasmatic nucleus (SCN) is considered to be a major component of the biological clock implicated in the temporal organization of a variety of physiological endocrine and behavioral processes. There is now a great deal of evidence indicating that many of these rhythms are progressively disturbed during senescence. The present study was aimed at investigating the influence of aging on the seasonal rhythm of the vasopressin (VP)-expressing neurons in the human SCN. To that end brains obtained at autopsy of 48 human subjects ranging in age from 6 to 91 years were studied. Subjects were divided into two age groups viz. “young subjects” (up to 50 years) and “elderly subjects” (over 50 years). It is shown that the number of VP-immunoreactive neurons in the human SCN exhibits a marked annual oscillation in young but not in elderly people. Whereas in young subjects low VP-immunoreactive neuron numbers were found during the summer (May-July) and peak values in autumn (September-November) the SCN of elderly people showed a disrupted annual cycle with a reduced amplitude. These data suggest that the biosynthesis of vasopressin in the human SCN exhibits a seasonal rhythm that becomes disturbed later in life.     

Circadian rhythms in multiple unit activity inside and outside the suprachiasmatic nucleus in the diurnal chipmunk (Eutamias sibiricus)

In the siberian chipmunk, a diurnal rodent, a pair of bipolar electrodes were implanted bilaterally near the suprachiasmatic nucleus (SCN). Long-term simultaneous recordings of multiple unit activity (MUA) inside and outside the SCN revealed diurnal rhythms inside the SCN that showed a daytime peak in activity during a 24 h light-dark cycle (LD 12:12). These rhythms persisted during constant darkness. MUA outside the SCN and overt behavior showed circadian rhythms that paralleled MUA inside the SCN. No phase reversal similar to that found in the nocturnal rat was observed.     

A novel neuronal cell line derived from the ventrolateral region of the suprachiasmatic nucleus

The suprachiasmatic nucleus of the anterior hypothalamus is the center of an internal biological clock in mammals. Glutamate is the neurotransmitter of retino-hypothalamic tract responsible for mediating the circadian actions of light in rodents. N-methyl-d-aspartate receptors, particularly NR2B subunit are reported to be principally involved in photic resetting of the biological clock in vivo and in slice culture. But, the precise cellular mechanisms of the resetting are not elucidated, because no adequate neuronal cell lines derived from the suprachiasmatic nucleus have been established. We established a neuronal cell line, N14.5, derived from the suprachiasmatic nucleus of a transgenic rat harboring the temperature-sensitive simian virus 40 large T-antigen gene. When the cells were cultured at 39 degrees C, the morphological features were turned fibroblastic into neuronal round cell body with neurite extensions. These cells showed immunoreactivities for neuronal markers (betaIII-tubulin, microtubule-associated protein 2 and TAU2) and as well as for vasoactive intestinal peptide which is expressed in the ventrolateral region of the suprachiasmatic nucleus. The cells expressed N-methyl-d-aspartate receptors, particularly NR1 and NR2B subunits as revealed by quantitative PCR. N-methyl-d-aspartate activated phosphorylation of p44/42 mitogen-activated protein kinase and increased expression level of Per1 and Per2 mRNA. These results suggest that the N14.5 is a novel neuronal cell line derived from the ventrolateral region of the suprachiasmatic nucleus, and that N-methyl-d-aspartate receptors expressed in the cells are a functional receptor. The N14.5 cells may be a useful tool to elucidate numerous chronobiological processes, especially resetting mechanism induced by an external light signal.     

Number of Arginine-Vasopressin Neurons in the Suprachiasmatic Nuclei Is Not Related to Level or Circadian Characteristics of Wheel-Running Activity in House Mice

House mouse lines bidirectionally selected for nest-building behavior show a correlation between number of AVP cells in the suprachiasmatic nuclei (SCN), the master circadian clock in mammals, and level of nest-building behavior as well as a correlation between wheel-running activity and SCN AVP content. Similar genetic correlations between wheel-running activity and nest-building behavior have been reported in house mouse lines selected for increased voluntary wheel-running behavior. These similarities in genetic correlation structure in independently selected mouse lines allowed us to test whether AVP in the SCN and wheel running activity are truly correlated traits under identical testing procedures. In the mouse lines selected for voluntary wheel-running, no difference was found between the lines selected for high levels of voluntary wheel-running and randomly-bred control lines in the number of AVP immunoreactive neurons in the SCN (F _1,6 = 0.09, NS; replicate line effect: F _1,22 = 0.05, NS). This finding was confirmed at the level of individual variation, which revealed no relationship between number of AVP neurons in the SCN and total daily activity (R = –0.086, NS, n = 24), or circadian organization (i.e., the chi-squared periodogram waveform amplitude; R = –0.071, NS). Therefore our data do not support the hypothesis that differences in activity level and the circadian expression of activity in young adult mice are related to differences in the number of AVP-immunoreactive cells in the SCN.