Circadian phase alteration by GABA and light differs in diurnal and nocturnal rodents during the day

These studies investigated the circadian effects of light and gamma aminobutyric acid-A (GABAA) receptor activation in the suprachiasmatic nucleus (SCN) of the diurnal unstriped Nile grass rat (Arvicanthis niloticus). Microinjection of the GABAA agonist muscimol into the SCN during the day produced phase shifts that were opposite in direction to those previously reported in nocturnal rodents. In addition, light had no significant effect on the magnitude of muscimol-induced phase delays during the daytime. Injection of muscimol during the night, however, significantly inhibited light-induced phase delays and advances in a manner similar to that previously reported in nocturnal rodents. Therefore, the circadian effects of GABAA receptor activation are similar in diurnal and nocturnal species during the night but differ significantly during the day.     

Enhanced circadian photoresponsiveness after prolonged dark adaptation in seven species of diurnal and nocturnal rodents

Previous studies in mice and Syrian hamsters have described an enhancement of circadian photoresponsiveness after exposure to darkness for several weeks. The present study investigated the generality of the phenomenon in 3 diurnal and 4 nocturnal rodent species. In four of the species tested, phase delays of the running-wheel activity rhythm evoked by 1-h light pulses were several-fold larger after 3 to 4 weeks of exposure to darkness than after a single day. This drastic change in photoresponsiveness has important implications for the understanding of the process of photic entrainment. Differences between species that showed a significant effect of dark adaptation and species that showed no effect were not accounted for by temporal niche (diurnal versus nocturnal) or photic sensitivity (albino versus pigmented). Further research is needed to elucidate the mechanisms responsible for inter-species differences in the occurrence of enhanced photoresponsiveness after dark adaptation and to identify the neural substrates of this phenomenon in species that exhibit it.     

Circadian profile and photic regulation of clock genes in the suprachiasmatic nucleus of a diurnal mammal Arvicanthis ansorgei

The molecular mechanisms of the mammalian circadian clock located in the suprachiasmatic nucleus have been essentially studied in nocturnal species. Currently, it is not clear if the clockwork and the synchronizing mechanisms are similar between diurnal and nocturnal species. Here we investigated in a day-active rodent Arvicanthis ansorgei, some of the molecular mechanisms that participate in the generation of circadian rhythmicity and processing of photic signals. In situ hybridization was used to characterize circadian profiles of expression of Per1, Per2, Cry2 and Bmal1 in the suprachiasmatic nucleus of A. ansorgei housed in constant dim red light. All the clock genes studied showed a circadian expression. Per1 and Per2 mRNA increased during the subjective day and decreased during the subjective night. Also, Bmal1 exhibited a circadian expression, but in anti-phase to that of Per1. The expression of Cry2 displayed a circadian pattern, increasing during the late subjective day and decreasing during the late subjective night. We also obtained the phase responses to light for wheel-running rhythm and clock gene expression. At a behavioral level, light was able to induce phase shifts only during the subjective night, like in other diurnal and nocturnal species. At a molecular level, light pulse exposure during the night led to an up-regulation of Per1 and Per2 concomitant with a down-regulation of Cry2 in the suprachiasmatic nucleus of A. ansorgei. In contrast, Bmal1 expression was not affected by light pulses at the circadian times investigated. This study demonstrates that light exposure during the subjective night has opposite effects on the expression of the clock genes Per1 and Per2 compared with that of Cry2. These differential effects can participate in photic resetting of the circadian clock. Our data also indicate that the molecular mechanisms underlying circadian rhythmicity and photic synchronization share clear similarities between diurnal and nocturnal mammals.     

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.     

Neurotransmission and electrophysiological mechanisms in the suprachiasmatic nucleus

Neurons in the mammalian suprachiasmatic nucleus (SCN) collectively show a circadian rhythm of firing rate, and the electrophysiological properties of SCN neurons support this periodicity. The SCN receives many afferents containing a wide variety of neuroactive substances. Nevertheless, physiological evidence indicates that GABA and glutamate are the primary neurotransmitters involved in fast synaptic transmission in the SCN. Other neurotransmitters such as serotonin are clearly important in the SCN, but their function is poorly understood, and most of these probably play a modulatory role. Nonsynaptic mechanisms in the SCN may also be important in communication among neurons and glia. Detailed examination of SCN function with traditional intracellular electrophysiological approaches, and recently, with patch-clamp recording, has begun to reveal the electrophysiological properties and functional circuitry of SCN neurons — an essential first step in understanding how this biological clock works.     

Compartmentalized expression of light-induced clock genes in the suprachiasmatic nucleus of the diurnal grass rat (Arvicanthis niloticus)

Photic responses of the circadian system are mediated through light-induced clock gene expression in the suprachiasmatic nucleus (SCN). In nocturnal rodents, depending on the timing of light exposure, Per1 and Per2 gene expression shows distinct compartmentalized patterns that correspond to the behavioral responses. Whether the gene- and region-specific induction patterns are unique to nocturnal animals, or are also present in diurnal species is unknown. We explored this question by examining the light-induced Per1 and Per2 gene expression in functionally distinct SCN subregions, using diurnal grass rats Arvicanthis niloticus. Light exposure during nighttime induced Per1 and Per2 expression in the SCN, showing unique spatiotemporal profiles depending on the phase of the light exposure. After a phase delaying light pulse (LP) in the early night, strong Per1 induction was observed in the retinorecipient core region of the SCN, while strong Per2 induction was observed throughout the entire SCN. After a phase advancing LP in the late night, Per1 was first induced in the core and then extended into the whole SCN, accompanied by a weak Per2 induction. This compartmentalized expression pattern is very similar to that observed in nocturnal rodents, suggesting that the same molecular and intercellular pathways underlying acute photic responses are present in both diurnal and nocturnal species. However, after an LP in early subjective day, which induces phase advances in diurnal grass rats, but not in nocturnal rodents, we did not observe any Per1 or Per2 induction in the SCN. This result suggests that in spite of remarkable similarities in the SCN of diurnal and nocturnal rodents, unique mechanisms are involved in mediating the phase shifts of diurnal animals during the subjective day.     

GABA release from suprachiasmatic nucleus terminals is necessary for the light-induced inhibition of nocturnal melatonin release in the rat.

The daily rhythm of melatonin production in the mammalian pineal is driven by the endogenous circadian pacemaker in the suprachiasmatic nuclei. The major release period of melatonin is closely linked to the dark phase of the 24-h day/night cycle. Environmental light will affect melatonin release in two ways: (i) it entrains the rhythm of the circadian oscillator; and (ii) it causes an acute suppression of nocturnal melatonin release. These two effects of light are both mediated by the suprachiasmatic nucleus and enable the pineal gland to convey information about day length to the reproductive system through changes in melatonin levels. Glutamate is currently believed to be the major transmitter in the retinal ganglion cell fibers reaching the suprachiasmatic nucleus. At present no information is available, however, about the transmitter(s) implicated in the further propagation, i.e. from the suprachiasmatic nucleus onwards, of the light information. In the present study we provide evidence that the endogenous release of GABA from suprachiasmatic nucleus terminals is implicated in the further transmission of light information to the pineal gland. Bilateral administration of the GABA-antagonist bicuculline to hypothalamic target areas of the suprachiasmatic nucleus completely prevents the inhibitory effect of nocturnal light on melatonin secretion and the present study thus documents that retina-mediated photic activation of suprachiasmatic nucleus neurons induces the release of GABA from efferent suprachiasmatic nucleus nerve terminals, resulting in an inhibition of melatonin release by the pineal gland. Together with our previous (electro)physiological data these results identify GABA as an important mediator of rapid synaptic transmission of suprachiasmatic nucleus output to its target areas.     

Self-innervation of dendrites in the rat suprachiasmatic nucleus

Summary Golgi-Cox impregnations of the rat suprachiasmatic nucleus show small dendritic side branches which appear to contact their neurons of origin. Electronmicroscopically a dendrite has been found forming a Gray-type-II synapse with one of its own branches. The arrangement is discussed as a general phenomenon of a feedback connection for temporal limitation of local excitation.     

Electrical synapses coordinate activity in the suprachiasmatic nucleus

In the suprachiasmatic nucleus (SCN), the master circadian pacemaker, neurons show circadian variations in firing frequency. There is also considerable synchrony of spiking across SCN neurons on a scale of milliseconds, but the mechanisms are poorly understood. Using paired whole-cell recordings, we have found that many neurons in the rat SCN communicate via electrical synapses. Spontaneous spiking was often synchronized in pairs of electrically coupled neurons, and the degree of this synchrony could be predicted from the magnitude of coupling. In wild-type mice, as in rats, the SCN contained electrical synapses, but electrical synapses were absent in connexin36-knockout mice. The knockout mice also showed dampened circadian activity rhythms and a delayed onset of activity during transition to constant darkness. We suggest that electrical synapses in the SCN help to synchronize its spiking activity, and that such synchrony is necessary for normal circadian behavior.     

Immunohistochemical characterization of the sheep suprachiasmatic nucleus

Using 19 antisera raised against neuropeptides, amines or enzymes of amine biosynthesis, an immunohistochemical characterization of the sheep suprachiasmatic nucleus was performed. The most distinguishing characteristic of the sheep suprachiasmatic nucleus was the low density of serotonin- and neuropeptide Y-immunoreactive fibres; their concentration was similar to that in surrounding areas. This is different from observations in rodents but similar to those in primates. Moreover, the sheep suprachiasmatic nucleus is also characterized by a dense plexus of methionine-enkephalin-immunoreactive fibres. This has not been observed in other species. As in other species, such as rodents, the sheep suprachiasmatic nucleus contains numerous neurophysin-immunoreactive neurons and a few tyrosine hydroxylase-immunoreactive neurons. After colchicine pretreatment, many intensely stained vasoactive intestinal peptide-, vasopressin- and somatostatin-immunoreactive perikarya appeared, and more neurophysin-immunoreactive cell bodies were observed. Thus, although similarities exist among species, there are distinct differences in the neuro-chemical organization of the suprachiasmatic nucleus in the sheep and other species.     

Effect of suprachiasmatic lesions on diurnal heart rate rhythm in the rat

Heart rate and locomotor activity of rats kept under 12L/12D illumination regimen was recorded every six minutes for ten days using implantable radio transmitters. Some of the rats then received bilateral radio frequency lesions into the suprachiasmatic nucleus (SCN). Control sham operations were performed on the rest of the animals. After recovery from surgery, recording of heart rate and locomotor activity was continued for ten days. SCN lesioned rats showed no significant diurnal fluctuation in heart rate, while normal and sham operated rats showed the normal diurnal rhythm in that function. The arrhythmic diurnal heart rate pattern of SCN rats appeared to be correlated to their sparadic activity pattern. The integrity of the suprachiasmatic nucleus therefore is necessary for the generation and/or the expression of diurnal rhythmicity in heart rate in the rat.     

Vasopressin-deficient suprachiasmatic nucleus grafts re-instate circadian rhythmicity in suprachiasmatic nucleus-lesioned arrhythmic rats

It was investigated whether grafts of the suprachiasmatic nucleus could re-instate circadian rhythmicity in the absence of its endogenous vasopressin production and whether the restored rhythm would have the long period length of the donor. Grafts of 17-days-old vasopressin-deficient homozygous Brattleboro rat fetuses, homotopically placed in arrhythmic suprachiasmatic nucleus-lesioned Wistar rats, re-instated circadian drinking rhythm within 20-50 days similar as seen for grafts of heterozygous control fetuses. Period length of the recovered rhythm revealed a similar difference (average 24.3 vs. 23.8 h) as reported for the rhythm between the adult Brattleboro genotypes. In all transplants, also those of the two-third non-recovery rats, a surviving suprachiasmatic nucleus was visible as a vasoactive intestinal polypeptide-positive neuronal cell cluster, whereas heterozygous transplants also revealed the complementary vasopressinergic cell part. Explanation of the absence of recovery failed since no undisputable correlation emerged between recovery of rhythm and vasoactive intestinal polypeptide, vasopressin and/or somatostatin immunocytochemical characteristics of the suprachiasmatic nucleus of the transplant. Special focus on the somatostatinergic neurons revealed their presence only occasionally near or in between the vasoactive intestinal polypeptidergic and (in the case of heterozygous grafts) vasopressinergic cell cluster. However their aberrant cytoarchitectural position appeared not to have affected the possibility to restore drinking rhythm of the suprachiasmatic nucleus-lesioned arrhythmic rat. It was concluded that grafted Brattleboro fetal suprachiasmatic nucleus develop their intrinsic rhythm conform their genotype and that vasopressin is not a crucial component in the maintenance nor in the transfer of circadian activity of the biological clock for drinking activity. Vasopressin of the suprachiasmatic nucleus may instead serve modulation within the circadian system.     

Complex organization of mouse and rat suprachiasmatic nucleus

The suprachiasmatic nucleus, site of the dominant mammalian circadian clock, contains a variety of different neurons that tend to form groups within the nucleus. The present investigation used single and multiple label tract tracing and immunofluorescence methods to evaluate the relative locations of the neuron groups and to compare them with the distributions of the three major afferent projections, the retinohypothalamic tract, geniculohypothalamic tract and the serotonergic pathway from the median raphe nucleus. The suprachiasmatic nucleus has a complex order characterized by peptidergic cell groups (vasopressin, gastrin releasing peptide, vasoactive intestinal polypeptide, calbindin, calretinin, corticotrophin releasing factor and enkephalin) that, in most cases, substantially overlap. The retinohypothalamic tract projects bilaterally to virtually all the suprachiasmatic nucleus in both rat (predominantly contralateral) and mouse (symmetric) and its terminal field overlaps that for the geniculohypothalamic tract, but with distinctions visible according to density criteria; neither provides more than sparse innervation of the dorsomedial suprachiasmatic nucleus. In the mouse, the serotonergic terminal field is densest medially and ventrally, but is also distributed elsewhere with varying density. The serotonergic terminal plexus in the rat is densest centromedially and largely, but not completely, overlaps the complete distribution of retinal terminals with density much reduced in the lateral suprachiasmatic nucleus. The locations of vasopressin neurons, retinohypothalamic tract terminals and serotonergic (mouse, rat) or geniculohypothalamic tract (rat) provide evidence for three clear, but not exclusionary, sectors of the suprachiasmatic nucleus. The data, in conjunction with emerging knowledge concerning rhythmically dynamic changes in the size of regions of neuropeptide gene expression in suprachiasmatic nucleus cells, support the view that suprachiasmatic nucleus organization is more complex than a simple "core" and "shell" arrangement. While generalizations about suprachiasmatic nucleus organization can be made with respect to location of cell phenotypes or terminal fields, oversimplification may hinder, rather than facilitate, understanding of suprachiasmatic nucleus structure-function relationships.