Henk Albus

A GABAergic Mechanism Is Necessary for Coupling Dissociable Ventral and Dorsal Regional Oscillators within the Circadian Clock

BACKGROUND Circadian rhythms in mammalian behavior, physiology, and biochemistry are controlled by the central clock of the suprachiasmatic nucleus (SCN). The clock is synchronized to environmental light-dark cycles via the retino-hypothalamic tract, which terminates predominantly in the ventral SCN of the rat. In order to understand synchronization of the clock to the external light-dark cycle, we performed ex vivo recordings of spontaneous impulse activity in SCN slices of the rat. RESULTS We observed bimodal patterns of spontaneous impulse activity in the dorsal and ventral SCN after a 6 hr delay of the light schedule. Bisection of the SCN slice revealed a separate fast-resetting oscillator in the ventral SCN and a distinct slow-resetting oscillator in the dorsal SCN. Continuous application of the GABA(A) antagonist bicuculline yielded similar results as cut slices. Short application of bicuculline at different phases of the circadian cycle increased the electrical discharge rate in the ventral SCN but, unexpectedly, decreased activity in the dorsal SCN. CONCLUSIONS GABA transmits phase information between the ventral and dorsal SCN oscillators. GABA can act excitatory in the dorsal SCN and inhibits neurons in the ventral SCN. We hypothesize that this difference results in asymmetrical interregional coupling within the SCN, with a stronger phase-shifting effect of the ventral on the dorsal SCN than vice versa. A model is proposed that focuses on this asymmetry and on the role of GABA in phase regulation.

Seasonal Encoding by the Circadian Pacemaker of the SCN

The circadian pacemaker of the suprachiasmatic nucleus (SCN) functions as a seasonal clock through its ability to encode day length [1-6]. To investigate the mechanism by which SCN neurons code for day length, we housed mice under long (LD 16:8) and short (LD 8:16) photoperiods. Electrophysiological recordings of multiunit activity (MUA) in the SCN of freely moving mice revealed broad activity profiles in long days and compressed activity profiles in short days. The patterns remained consistent after release of the mice in constant darkness. Recordings of MUA in acutely prepared hypothalamic slices showed similar differences between the SCN electrical activity patterns in vitro in long and short days. In vitro recordings of neuronal subpopulations revealed that the width of the MUA activity profiles was determined by the distribution of phases of contributing units within the SCN. The subpopulation patterns displayed a significantly broader distribution in long days than in short days. Long-term recordings of single-unit activity revealed short durations of elevated activity in both short and long days (3.48 and 3.85 hr, respectively). The data indicate that coding for day length involves plasticity within SCN neuronal networks in which the phase distribution of oscillating neurons carries information on the photoperiods duration.

Spontaneous epileptiform discharges in hippocampal slices induced by 4-aminopyridine

4-Aminopyridine (4-AP) induced 2 types of spontaneous field potentials (SFPs) in the hippocampal slice. Type I resembled spontaneous activity induced by other convulsants. They occurred at a rate of approximately 1 Hz, started in the CA2/CA3 region and spread at a velocity of 0.3 m/s to area CA1. Transsection experiments and laminar profiles indicated that they spread synaptically along the Schaffer collateral pathway. Synaptic blockade by low Ca2+/high Mg2+ or kynurenic acid reversibly abolished type I SFPs. Increasing [Ca2+]o lowered the rate and slightly increased the amplitude. Possibly, increased spontaneous transmitter release, and not disinhibition, is responsible for the generation of type I SFPs. Type II occurred at a rate of about 0.15 Hz and travelled in the same direction, but a factor 10 slower. They could not be blocked by separation of the CA1 and CA3 region; coupling remained until stratum moleculare was severed. Type II could not be suppressed by blockade of synaptic transmission. The laminar profile is similar in shape to that of type I but not identical. Increasing [Ca2+]o had the same but stronger effect as on type I. Type II SFPs depressed evoked population spikes up to a second and delayed the next type I SFP. The mechanisms involved remain largely speculative; further analysis is needed to help understand the epileptogenic action of 4-AP.

Multiunit activity recordings in the suprachiasmatic nuclei : in vivo versus in vitro models

The suprachiasmatic nuclei (SCN) of the hypothalamus continue to oscillate when they are isolated in a brain slice preparation. We recorded multiunit activity in the SCN of the rat both in vivo and in vitro to determine the circadian discharge pattern. The variability of the discharge pattern is larger and the amplitude of the rhythm is smaller in vivo than in vitro. Moreover we found evidence for a direct effect of the animals behavioural activity on electrical activity of the SCN in vivo. These findings may provide an electrophysiological basis for the known effects of behavioural stimuli on the circadian pacemaker. This study underscores the importance of recordings in intact preparations in addition to in vitro work when generalisations to physiological conditions are to be made.

Cryptochrome-Deficient Mice Lack Circadian Electrical Activity in the Suprachiasmatic Nuclei

The mammalian master clock driving circadian rhythmicity in physiology and behavior resides within the suprachiasmatic nuclei (SCN) of the anterior hypothalamus. Circadian rhythms are generated by a set of clock genes via intertwined negative and positive autoregulatory transcription-translation feedback loops. The Cryptochrome 1 and 2 genes are indispensable for molecular core oscillator function, as evident from the arrhythmic wheel-running behavior and lack of rhythmic clock gene expression in mCry1/mCry2 double-mutant mice in constant darkness. In the present study, using real-time multiunit electrode activity recordings in hypothalamic slices, we show that SCN neurons from mCry-deficient mice kept in constant darkness lack circadian oscillations in firing patterns. This proves that cryptochromes, and thus an intact circadian clockwork, are prerequisites for circadian electrical activity in SCN neurons. Interestingly, when mCry-deficient mice were kept in normal light-dark conditions and SCN slices were prepared 2 hr after the beginning of the day, a single noncircadian peak in neuronal activity was detected. This light-induced rise in electrical activity of the SCN may explain why mCry-deficient mice lack the arrhythmic short bouts of wheel-running activity and instead show apparently normal behavior in normal day-night cycles.

The effects of glutamate on membrane potential and discharge rate of suprachiasmatic neurons

The suprachiasmatic nucleus (SCN) is a major pacemaker for circadian rhythms in mammals. Photic entrainment of the circadian pacemaker is mediated by the retinohypothalamic tract (RHT). Most likely, excitatory amino acids function as neurotransmitters in this pathway. We have now investigated the effect of glutamate on the membrane potential of cultured SCN cells of the rat with the aid of the patch clamp technique. It was found that 1 mM glutamate depolarizes the cells by about +44 mV. In spontaneously active neurons, the glutamate induced depolarization caused either an increase in discharge or a depolarization block. We then investigated the effect of 1 mM glutamate on SCN discharge in the acutely prepared hypothalamic slice of the hamster. In most cells glutamate induced an increase in discharge whilst in a few cells discharge was suppressed. Both series of experiments indicate that glutamate in the used dosage was effective and its effect reversible. The data are discussed with respect to the failure of 1 mM glutamate injections to mimic the effect of light on the circadian activity rhythm of the hamster.

Enhanced circadian phase resetting in R192Q Cav2.1 calcium channel migraine mice

Mammalian circadian rhythms are driven by the circadian pacemaker of the suprachiasmatic nucleus (SCN) and are synchronized to the external 24‐hour light/dark cycle. After advance time zone transitions (eastbound jet lag), overt circadian rhythms require several days to adjust. The retarded adaptation may protect against acute imbalance of different brain systems. Abrupt circadian rhythm changes may trigger migraine attacks, possibly because migraineurs have an inadequate adaptation mechanism. The novel R192Q knock‐in migraine mouse model carries mutated Cav2.1 calcium channels, causing increased presynaptic calcium influx and neurotransmitter release. We investigated whether these mice have an abnormal adjustment to phase advance shifts.

The opioid fentanyl affects light input, electrical activity and Per gene expression in the hamster suprachiasmatic nuclei.

The suprachiasmatic nuclei (SCN) contain a major circadian pacemaker, which is regulated by photic and nonphotic stimuli. Although enkephalins are present in the SCN, their role in phase regulation of the pacemaker is largely unknown. The opioid agonist fentanyl, a homologue of morphine, is an addictive drug that induces phase shifts of circadian rhythms in hamsters. We observed that these phase shifts are blocked by naloxone, which is a critical test for true opioid receptor involvement, and conclude that opioid receptors are the sole mediators of the actions of fentanyl on the circadian timing system. A strong interaction between opioids and light input was shown by the ability of fentanyl and light to completely block each others phase shifts of behavioural activity rhythms. Neuronal ensemble recordings in vitro provide first evidence that SCN cells show direct responses to fentanyl and react with a suppression of firing rate. Moreover, we show that fentanyl induces a strong attenuation of light‐induced Syrian hamster Period 1 (shPer1) gene expression during the night. During the subjective day, we found no evidence for a role of shPer1 in mediation of fentanyl‐induced phase shifts. Based on the present results, however, we cannot exclude the involvement of shPer2. Our data indicate that opioids can strongly modify the photic responsiveness of the circadian pacemaker and may do so via direct effects on SCN electrical activity and regulation of Per genes. This suggests that the pathways regulating addictive behaviour and the circadian clock intersect.

Functional absence of extraocular photoreception in hamster circadian rhythm entrainment

The mammalian circadian pacemaker is entrainable by light via the retina. The putative role of extraocular light perception was investigated in blinded hamsters. These animals were shaved and exposed to a light-emitting pad for either 30 min or 3 h. The absence of any phase-shifting effects on wheel running activity rhythms indicates that extraocular light perception plays no functional role in photic entrainment of the circadian pacemaker in the hamster.

Light entrainment of the mammalian biological clock.

Publisher Summary Mammalian circadian rhythms are controlled by a pacemaker that is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN produces rhythms of roughly 24 hours. In the external world, circadian rhythms are entrained to the environmental light/dark cycle and adopt the external period of 24 hours. As a consequence, animals time their behaviour properly in view of the light/dark cycle that enhances their chance of survival. In this chapter, the current knowledge on photo entrainment is reviewed. The photic entrainment of the biological pacemaker is based on adjustments to the light/dark cycle made possible by a phase-dependent responsiveness of the pacemaker to light. Additional mechanisms increase the animals ability to entrain. The magnitude of a phase shift that can be obtained at a given phase of the circadian cycle depends on (a) light intensity, (b) duration of a light pulse, and (c) wavelength of light. The role of any of these three parameters is investigated by manipulating only one of them while keeping the other two constant.