Splitting of the circadian rhythm of activity is abolished by unilateral lesions of the suprachiasmatic nuclei.

The circadian rhythm of activity in vertebrates often splits into two components after continuous exposure to constant light. This observation suggests that at least two circadian pacemakers underlie the activity rhythm. After unilateral ablation of the hypothalamic suprachiasmatic nuclei in hamsters, the splitting phenomenon was eliminated and a single rhythm of activity was established. The period of the new circadian activity rhythm different from the periods of the split rhythm and that preceding the split. These results suggest an interaction between the bilaterally paired suprachiasmatic nuclei in the generation of the circadian rhythm of activity.

Abundance and ultrastructural diversity of neuronal gap junctions in the OFF and ON sublaminae of the inner plexiform layer of rat and mouse retina.

Neuronal gap junctions are abundant in both outer and inner plexiform layers of the mammalian retina. In the inner plexiform layer (IPL), ultrastructurally-identified gap junctions were reported primarily in the functionally-defined and anatomically-distinct ON sublamina, with few reported in the OFF sublamina. We used freeze-fracture replica immunogold labeling and confocal microscopy to quantitatively analyze the morphologies and distributions of neuronal gap junctions in the IPL of adult rat and mouse retina. Under "baseline" conditions (photopic illumination/general anesthesia), 649 neuronal gap junctions immunogold-labeled for connexin36 were identified in rat IPL, of which 375 were photomapped to OFF vs. ON sublaminae. In contrast to previous reports, the volume-density of gap junctions was equally abundant in both sublaminae. Five distinctive morphologies of gap junctions were identified: conventional crystalline and non-crystalline "plaques" (71% and 3%), plus unusual "string" (14%), "ribbon" (7%) and "reticular" (2%) forms. Plaque and reticular gap junctions were distributed throughout the IPL. However, string and ribbon gap junctions were restricted to the OFF sublamina, where they represented 48% of gap junctions in that layer. In string and ribbon junctions, curvilinear strands of connexons were dispersed over 5 to 20 times the area of conventional plaques having equal numbers of connexons. To define morphologies of gap junctions under different light-adaptation conditions, we examined an additional 1150 gap junctions from rats and mice prepared after 30 min of photopic, mesopic and scotopic illumination, with and without general anesthesia. Under these conditions, string and ribbon gap junctions remained abundant in the OFF sublamina and absent in the ON sublamina. Abundant gap junctions in the OFF sublamina of these two rodents with rod-dominant retinas revealed previously-undescribed but extensive pathways for inter-neuronal communication; and the wide dispersion of connexons in string and ribbon gap junctions suggests unique structural features of gap junctional coupling in the OFF vs. ON sublamina.

5-HT1B receptor-mediated presynaptic inhibition of retinal input to the suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) receives glutamatergic afferents from the retina and serotonergic afferents from the midbrain, and serotonin (5-HT) can modify the response of the SCN circadian oscillator to light. 5-HT1B receptor-mediated presynaptic inhibition has been proposed as one mechanism by which 5-HT modifies retinal input to the SCN (Pickard et al., 1996). This hypothesis was tested by examining the subcellular localization of 5-HT1B receptors in the mouse SCN using electron microscopic immunocytochemical analysis with 5-HT1B receptor antibodies and whole-cell patch-clamp recordings from SCN neurons in hamster hypothalamic slices. 5-HT1B receptor immunostaining was observed associated with the plasma membrane of retinal terminals in the SCN. 1-[3-(Trifluoromethyl)phenyl]-piperazine HCl (TFMPP), a 5-HT1B receptor agonist, reduced in a dose-related manner the amplitude of glutamatergic EPSCs evoked by stimulating selectively the optic nerve. Selective 5-HT1A or 5-HT7 receptor antagonists did not block this effect. Moreover, in cells demonstrating an evoked EPSC in response to optic nerve stimulation, TFMPP had no effect on the amplitude of inward currents generated by local application of glutamate. The effect of TFMPP on light-induced phase shifts was also examined using 5-HT1B receptor knock-out mice. TFMPP inhibited behavioral responses to light in wild-type mice but was ineffective in inhibiting light-induced phase shifts in 5-HT1B receptor knock-out mice. The results indicate that 5-HT can reduce retinal input to the circadian system by acting at presynaptic 5-HT1B receptors located on retinal axons in the SCN.

Heterozygosity mapping of partially congenic lines: mapping of a semidominant neurological mutation, Wheels (Whl), on mouse chromosome 4.

We identified a semidominant, chemically induced, mouse mutation with a complex array of abnormal behaviors including bidirectional circling and hyperactivity, abnormal circadian rhythmicity and abnormal responses to light. In this report, we genetically and phenotypically characterized the circling/waltzing component of the abnormal behavior. We mapped the locus controlling this trait by heterozygosity mapping of partially congenic lines carrying the mutagenized chromosome outcrossed to different inbred strains for three generations. Analysis of 68 PCR-based markers in 13 affected individuals indicated that the mutant locus, named Wheels (Whl), resides in the subcentromeric portion of mouse chromosome 4. The statistical evaluation of data obtained by heterozygosity mapping validates this efficient mapping approach. Further characterization of the Whl mutation demonstrated that Whl/Whl homozygotes die during embryonic life and that the penetrance of circling behavior depends on genetic background. Morphological analysis of the inner ears of Whl/+ mice revealed a variable number of abnormalities in the sensory and nonsensory portions of their semicircular canals. Abnormalities ranged from slight atrophy of one or more cristae to complete absence of the lateral crista and canal. The molecular characterization of the gene disrupted in the Whl mutation will provide insight into developmental mechanisms involved in inner ear formation.

The intergeniculate leaflet partially mediates effects of light on circadian rhythms.

Photic signals affect circadian activity rhythms by both phasic and tonic mechanisms that modulate pacemaker phase and period. In mammals, the effects of light on circadian activity are mediated by the retina, which communicates with the suprahiasmatic nucleus (SCN) by two different anatomical routes: the retino-hypothalamic tract (RHT), originating in the retina, and the geniculo-hypothalamic tract (GHT), arising from a retino-recipient nucleus, the intergeniculate leaflet (IGL). We assessed the roles of these two afferent systems in mediating phasic and tonic effects of light on circadian activity in IGL-lesioned animals. Destruction of the IGL significantly affected phase shifts produced by brief light pulses (phasic effect) and modified the change in period (tau) of the free-running activity rhythm produced by changing the level of constant light (LL) (tonic effect). Phase advances produced by brief light pulses were decreased in amplitude while phase delays were increased in IGL-lesioned animals as compared to controls. The free-running period in constant dark (tau DD) of IGL-lesioned animals was greater than tau DD of controls, and the lengthening of tau normally produced by LL was not observed or was greatly reduced in IGL-lesioned animals. Entrainment to light-dark cycles was unaffected by the lesions, as were other aspects of the circadian activity rhythm that normally change in response to LL (e.g., activity-rest ratio, total activity, splitting). Our data support the interpretation that the IGL plays a significant role in relaying information regarding illumination intensity to the SCN.

Serotonergic modulation of retinal input to the mouse suprachiasmatic nucleus mediated by 5-HT1B and 5-HT7 receptors.

Serotonin (5-HT) and 5-HT receptor agonists can modify the response of the mammalian suprachiasmatic nucleus (SCN) to light. It remains uncertain which 5-HT receptor subtypes mediate these effects. The effects of 5-HT receptor activation on optic nerve-mediated input to SCN neurons were examined using whole-cell patch-clamp recordings in horizontal slices of ventral hypothalamus from the male mouse. The hypothesis that 5-HT reduces the effect of retinohypothalamic tract (RHT) input to the SCN by acting at 5-HT1B receptors was tested first. As previously described in the hamster, a mixed 5-HT(1A/1B) receptor agonist, 1-[3-(trifluoromethyl)phenyl]-piperazine hydrochloride (TFMPP), reduced the amplitude of glutamatergic excitatory postsynaptic currents (EPSCs) evoked by selectively stimulating the optic nerve of wild-type mice. The agonist was negligibly effective in a 5-HT1B receptor knockout mouse, suggesting minimal contribution of 5-HT1A receptors to the TFMPP-induced reduction in the amplitude of the optic nerve-evoked EPSC. We next tested the hypothesis that 5-HT also reduces RHT input to the SCN via activation of 5-HT7 receptors. The mixed 5-HT(1A/7) receptor agonist, R(+)-8-hydroxy-2-(di-n-propylamino) tetralin hydrobromide (8-OH-DPAT), reduced the evoked EPSC amplitude in both wild-type and 5-HT1B receptor knockout mice. This effect of 8-OH-DPAT was minimally attenuated by the selective 5-HT1A receptor antagonist WAY 100635 but was reversibly and significantly reduced in the presence of ritanserin, a mixed 5-HT(2/7) receptor antagonist. Taken together with the authors' previous ultrastructural studies of 5-HT1B receptors in the mouse SCN, these results indicate that in the mouse, 5-HT reduces RHT input to the SCN by acting at 5-HT1B receptors located on RHT terminals. Moreover, activation of 5-HT7 receptors in the mouse SCN, but not 5-HT1A receptors, also results in a reduction in the amplitude of the optic nerve-evoked EPSC. The findings indicate that 5-HT may modulate RHT glutamatergic input to the SCN through 2 or more 5-HT receptors. The likely mechanism of altered RHT glutamatergic input to SCN neurons is an alteration of photic effects on the SCN circadian oscillator.

The injury resistant ability of melanopsin-expressing intrinsically photosensitive retinal ganglion cells.

Neurons in the mammalian retina expressing the photopigment melanopsin have been identified as a class of intrinsically photosensitive retinal ganglion cells (ipRGCs). This discovery more than a decade ago has opened up an exciting new field of retinal research, and following the initial identification of photosensitive ganglion cells, several subtypes have been described. A number of studies have shown that ipRGCs subserve photoentrainment of circadian rhythms. They also influence other non-image forming functions of the visual system, such as the pupillary light reflex, sleep, cognition, mood, light aversion and development of the retina. These novel photosensitive neurons also influence form vision by contributing to contrast detection. Furthermore, studies have shown that ipRGCs are more injury-resistant following optic nerve injury, in animal models of glaucoma, and in patients with mitochondrial optic neuropathies, i.e., Leber's hereditary optic neuropathy and dominant optic atrophy. There is also an indication that these cells may be resistant to glutamate-induced excitotoxicity. Herein we provide an overview of ipRGCs and discuss the injury-resistant character of these neurons under certain pathological and experimental conditions.

The afferent connections of the suprachiasmatic nucleus of the golden hamster with emphasis on the retinohypothalamic projection.

The afferent connections of the hypothalamic suprachiasmatic nucleus (SCN) of the golden hamster were examined using horseradish peroxidase (HRP) as the retrograde tracer molecule. Unilateral iontophoretic deposition of HRP into the SCN labeled ganglion cells bilaterally in the retinae. The labeled ganglion cells all had large somata and were randomly distributed across the retina. A similar number were labeled in each retina, which contrasted with the findings from injections into the optic chiasm and lateral geniculate body. Chiasm and geniculate injections both labeled three classes of ganglion cell (small, large, and giant) predominantly in the contralateral retinae. Telencephalic afferent projections to the SCN included the ventral subicular cortex and the septum. Notable diencephalic afferents included the dorsal lamina of the internal division of the ventral lateral geniculate nucleus (vLGN); the ipsilateral input was twice that of the contralateral projection. The same region of the vLGN was also noted to be reciprocally connected to the contralateral vLGN. The thalamic paraventricular nucleus was also heavily labeled but only ipsilaterally. Of functional significance, the SCN was discovered to innervate its contralateral homologue. Other less numerous afferents in the midbrain included the dorsal and median raphe nuclei and the dorsal nucleus of the lateral lemniscus. The afferent projections to the SCN determined in this study are discussed in regard to the known physiological role of the SCN as part of the circadian clock system.

Direct retinal projections to the hypothalamus, piriform cortex, and accessory optic nuclei in the golden hamster as demonstrated by a sensitive anterograde horseradish peroxidase technique.

The central projections of the retinal ganglion cells of the golden hamster were examined using horseradish peroxidase (HRP) as the anterograde tracer molecule. Following monocular injections of HRP into the vitreous, retinofugal fibers were histochemically demonstrated using the chromagen tetramethylbenzidine. This procedure, being more sensitive than the 3H-amino acid radioautographic technique, provided a clear demonstration of previously controversial retinal projections, clearer definition of established projections, and the discovery of new retinal pathways. An inferior accessory optic system was shown to be unequivocally present in this species and to consist of both crossed and uncrossed components. A direct retinal projection to the suprachiasmatic nucleus (SCN) of the hypothalamus was confirmed in this study. But the distribution of terminals as seen by this procedure was substantially different than previously reported; both rostrocaudal and mediolateral asymmetries in the distribution of label between the ipsilateral and contralateral SCN were observed. Substantial differences in the retinal projection to the SCN in the hamster and the rat were also noted. It is suggested that these differences may reflect the different effects photic input has on the neuro-endocrine-gonadal axis in these two species. Finally, labeled retinal axons were followed leaving the optic tract and coursing anteriorly through the plexiform layer of the piriform cortex; other labeled fibers were seen to enter the septal region. The physiological significance of these previously undescribed retinal projections is not known.

Subcellular distribution of 5-HT(1B) and 5-HT(7) receptors in the mouse suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN), a circadian oscillator, receives glutamatergic afferents from the retina and serotonergic (5-HT) afferents from the median raphe. 5-HT(1B) and 5-HT(7) receptor agonists inhibit the effects of light on SCN circadian activity. Electron microscopic (EM) immunocytochemical procedures were used to determine the subcellular localization of 5-HT(1B) and 5-HT(7) receptors in the SCN. 5-HT(1B) receptor immunostaining was associated with the plasma membrane of thin unmyelinated axons, preterminal axons, and terminals of optic and nonoptic origin. 5-HT(1B) receptor immunostaining in terminals was almost never observed at the synaptic active zone. To a much lesser extent, 5-HT(1B) immunoreaction product was noted in dendrites and somata of SCN neurons. 5-HT(7) receptor immunoreactivity in gamma-aminobutyric acid (GABA), vasoactive intestinal polypeptide (VIP), and vasopressin (VP) neuronal elements in the SCN was examined by using double-label procedures. 5-HT(7) receptor immunoreaction product was often observed in GABA-, VIP-, and VP-immunoreactive dendrites as postsynaptic receptors and in axonal terminals as presynaptic receptors. 5-HT(7) receptor immunoreactivity in terminals and dendrites was often associated with the plasma membrane but very seldom at the active zone. In GABA-, VIP-, and VP-immunoreactive perikarya, 5-HT(7) receptor immunoreaction product was distributed throughout the cytoplasm often in association with the endoplasmic reticulum and the Golgi complex. The distribution of 5-HT(1B) receptors in presynaptic afferent terminals and postsynaptic SCN processes, as well as the distribution of 5-HT(7) receptors in both pre- and postsynaptic GABA, VIP, and VP SCN processes, suggests that serotonin plays a significant role in the regulation of circadian rhythms by modulating SCN synaptic activity.

Effects of photoperiod on hypothalamic luteinizing hormone releasing hormone in the male hamster.

Male hamsters were maintained on long (14 h light : 10 h darkness; 14L : 10D) or short (6L : 18D) photoperiods. Animals on short-days had reduced levels of LH in the serum and anterior pituitary gland, decreased androgen in the circulation and regressed testes and accessory sex organs. These same hamsters had significantly raised concentrations of hypothalamic luteinizing hormone releasing hormone (LH-RH). There was no significant difference in the response to exogenous LH-RH between groups maintained on long- and short-days. Castration significantly reduced levels of LH-RH in the hypothalamus in the long-day animals but had little effect on this parameter in short-day animals which had already undergone testicular regression. The increased levels of LH-RH in the hyothalami of both intact and castrated hamsters on non-stimulatory photoperiods is interpreted as a decreased release of the neurohormone which subsequently results in a decreased release of LH.

Intergeniculate leaflet ablation alters circadian rhythms in the mouse.

The intergeniculate leaflet (IGL) receives retinal input and sends afferents to the hypothalamic suprachiasmatic nucleus (SCN), a circadian oscillator. The effect of IGL ablation on the circadian rhythm of wheel-running activity was examined in mice maintained in constant dark (DD) and in two intensities of constant light (LL). IGL-lesioned animals demonstrated significantly longer free-running rhythms in DD conditions than IGL-intact mice similarly housed; there was no effect of IGL ablation on the period of the free-running activity rhythm in LL of either 10 or 100 lux intensity. A change in the period of the free-running activity rhythm in DD following IGL destruction is evidence that the IGL exerts an endogenous influence on the SCN circadian system.

Altered circadian rhythmicity in the Wocko mouse, a hyperactive transgenic mutant.

Induced changes in the level of daily activity can alter the period of the mammalian circadian clock. In this report, we examined the period of the circadian rhythm of wheel-running activity in a transgenic neurological mouse mutant, Wocko. Wocko mice display a dominant behavioral phenotype that consists of hyperactivity, circling and head tossing. The period of the circadian rhythm of wheel-running activity in constant dark conditions was significantly shorter in mice expressing the Wocko mutation than in their normal littermates. Total activity, monitored by the interruption of an array of infrared beams, was significantly elevated in Wocko mice. These findings support the view that spontaneous exercise can modulate the circadian timekeeping system.

Thyrotropin-releasing hormone phase shifts circadian rhythms in hamsters.

The role of thyrotropin-releasing hormone (TRH) in regulating circadian rhythms was investigated by assessing the ability of TRH microinjections into the suprachiasmatic nucleus (SCN) to induce phase shifts in hamster wheel-running behavior. TRH injected into the SCN at 10 and 100 nM doses produced phase advances in wheel-running activity of 18.3 +/- 1.9 and 34.8 +/- 2.9 minutes, respectively, when administered at circadian time (CT) 6. Injections at CT 18 produced no effects. The temporal sensitivity of the SCN to TRH administration was examined by administering TRH at specific circadian times. TRH produced significant phase advances at CT 4, 6, and 8, while no significant changes in wheel-running onset were observed at other CT times. These studies represent the first evidence of TRH's ability to affect circadian function.

Circadian rhythm of nociception in the golden hamster.

The response latency of golden hamsters to nociceptive stimuli was measured under cyclic lighting conditions and during constant illumination. A day-night rhythm of nociception was demonstrated; response latencies were significantly longer during the day. A circadian rhythm of nociception was displayed by hamsters maintained for 30 days in constant dim light. Short response latencies noted under these conditions were associated with the inactive period of the animals circadian cycle (subjective day). The experiments provide data which indicate the phase relationship between the circadian rhythms of nociception and locomotor activity differs under entrained and free-running conditions.

Entrainment of the circadian rhythm of wheel-running activity is phase shifted by ablation of the intergeniculate leaflet.

The phase angle of entrainment of golden hamster wheel-running activity was determined before and after bilateral destruction of the intergeniculate leaflet (IGL). Animals were maintained under continuous dim light which rhythmically varied in intensity (10-5 lux) in the form of a sine wave with a 24 h period. The phase angle of entrainment changed significantly following IGL ablation suggesting a role for the IGL in entrainment.

Individual pineal cells exhibit a circadian rhythm in melatonin secretion.

Pineal glands and dissociated pineal cells exhibit a circadian rhythm of melatonin secretion in vitro which persists for several cycles under constant conditions. It is not known whether individual, physically isolated pineal cells are capable of generating a circadian oscillation in melatonin release. This question was addressed by utilizing a reverse hemolytic plaque assay for the detection of melatonin secretion from individual pineal cells. Dissociated pineal cells from the anole lizard, maintained in short term culture, displayed a marked variation in melatonin secretion for up to 72 h under both cyclic lighting conditions and in constant dark. The persistence of daily fluctuations of melatonin secretion from individual cells strongly suggests that individual pineal cells can function as circadian oscillators.

Influence of deuterium oxide on circadian activity rhythms of hamsters: role of the suprachiasmatic nuclei.

The period of the free-running circadian activity rhythm of Syrian hamsters was measured before and during treatment with 10% deuterium oxide (D2O). Deuteration increased period length by approximately 0.5 h per cycle both pre- and postoperatively in hamsters sustaining complete, incomplete or no unilateral lesions of the suprachiasmatic nuclei (SCN). Neither coupling between the bilaterally paired SCN, nor elimination of 50% of SCN tissue affected period length during D2O treatment. However, variability of the response to D2O was much greater in lesioned than in intact hamsters. We propose that a small percentage of the normal complement of SCN neurons is sufficient to permit full responsiveness of the circadian system to D2O and that there is substantial redundancy in the neural system that responds to deuterium. Stability of the circadian system appears to be increased by the full complement of SCN neurons.

Time course of fiber outgrowth from fetal anterior hypothalamic heterografts.

Fetal anterior hypothalamic (AH) heterografts can restore circadian rhythmicity to animals rendered arrhythmic following ablation of the suprachiasmatic nucleus (SCN). Behavioral restoration of circadian activity typically begins between two and six weeks post-implantation. The time course of fiber outgrowth from fetal AH heterografts was examined to determine whether neuronal outgrowth from the implants precedes the typically observed effects of such implants upon circadian behavior. Fetal mouse or rat AH tissue containing the SCN was implanted into the third ventricle of SCN-lesioned hamsters. Using species-specific monoclonal antibodies generated against mouse or rat neuronal elements, fiber outgrowth into the host hypothalamus was examined at 2, 4, 7, 14, 30 and 45 days after implantation. Fibers were observed to have emerged from the implant at the earliest time point examined. Four days after surgery, individual fibers had extended up to 0.6 mm into the host neuropil. By 14 days post-implantation, outgrowth from the implant had formed a dense fiber plexus in the host hypothalamus. This observation demonstrates that neuronal integration of the implant with the host brain begins within 48 hours of implantation, and is extensively established well before a restoration of rhythmicity is typically observed. Thus, on the basis of the time course of fiber outgrowth, it is clear that neuronal contact between graft and host may mediate the observed restoration of circadian rhythmicity.

Ventral lateral geniculate nucleus afferents to the suprachiasmatic nucleus in the cat.

The lateral geniculate complex innervates the hypothalamic suprachiasmatic nucleus (SCN). The location of neurons in the cat ventral lateral geniculate nucleus (vLGN) that give rise to the geniculohypothalamic tract has not been described. In this study, retrogradely labeled neurons were noted throughout the rostrocaudal extent of the medial vLGN following tracer injection into the SCN region. In addition, neuropeptide Y immunoreactive processes were also observed in the vLGN in this same medial zone and in the SCN. The data suggest that the medial zone of the cat vLGN may be homologous to the rodent intergeniculate leaflet (IGL).