Benjamin Rusak

Neuropeptide Y immunoreactivity in the hamster geniculo-suprachiasmatic tract

The distributions of neuropeptide Y (NPY) and avian pancreatic polypeptide (APP) immunoreactivity were examined in the suprachiasmatic nucleus and the geniculate area of male golden hamster brains. In some cases, colchicine was injected intraventricularly to aid in visualization of immunoreactive cell bodies. A group of hamsters were given bilateral or unilateral radiofrequency lesions of the geniculate area and neuropeptide Y immunoreactivity was examined in the suprachiasmatic nucleus after survival times varying between 8 and 300 days. Another group of hamsters received unilateral intraocular injections of anterograde tracers and the overlap of NPY-immunoreactive cells in the geniculate area and labeled retinal afferents was assessed. It was found that NPY- and APP-immunoreactive fibers formed a dense plexus in the ventro-lateral suprachiasmatic nucleus. NPY-immunoreactive cell bodies were observed in the intergeniculate leaflet as well as in the external lamina of the anterior portion of the ventral lateral geniculate nucleus. Unilateral lesions of the geniculate produced a relative reduction in neuropeptide Y immunoreactivity in the ipsilateral suprachiasmatic nucleus whereas bilateral lesions produced a reduction of neuropeptide Y immunoreactivity in both suprachiasmatic nuclei. All NPY-immunoreactive cells in the intergeniculate leaflet were overlapped by bilateral retinal afferents. In the ventral lateral geniculate nucleus, all NPY-immunoreactive cells were overlapped by contralateral retinal afferents; however, not all such cells were in areas receiving ipsilateral retinal afferents. These results indicate that the hamster geniculo-suprachiasmatic tract originates in part from NPY-immunoreactive cell bodies and that these cells lie in areas receiving direct retinal afferents.

Lesions of the Thalamic Intergeniculate Leaflet Alter Hamster Circadian Rhythms

We have investigated the effects of destruction of the geniculo-hypothalamic tract (GHT) on the circadian system of golden hamsters. In the first experiment, intact hamsters were housed in constant darkness, and phase shifts in running-wheel activity rhythms were assessed following 15-min light pulses administered at circadian time (CT) 12 (defined as the beginning of activity), CT 14, CT 18, and CT 20. Responses to light pulses at the same CTs were then reassessed after GHT lesions. Hamsters with complete lesions showed decreases in phase advances caused by light pulses at CT 18 and CT 20. Phase delays elicited by light at CT 12 and CT 14 were not altered. In a second study, intact and GHT-ablated hamsters housed in constant light received 6-hr dark pulses at various CTs. Hamsters with complete GHT ablation showed smaller advances than controls to dark pulses centered on CT 8-10. After 110 days in constant light, 7 of 10 intact hamsters showed splitting of their activity rhythms into two components, while only 1 of the 8 similarly treated ablated hamsters displayed dissociated activity components. Ablated hamsters had significantly shorter free-running periods during the first 35 days of exposure to constant light than did the intact hamsters. These results demonstrate that destruction of the GHT in the hamster alters phase shifting in response to periods of light or dark, and they indicate a role for the GHT in mediating several photic effects on the circadian system.

Luminance coding in a circadian pacemaker: the suprachiasmatic nucleus of the rat and the hamster.

The hypothalamic suprachiasmatic nuclei (SCN) of mammals function as a pacemaker driving circadian rhythms. This pacemaker is entrained to the daily light-dark cycle in the environment via the retina and central retinal projections to the anterior hypothalamus. We carried out a comparative study of the visual properties of rat and hamster SCN neurons. Extracellular single cell activity was recorded in the SCN of urethane-anaesthetized animals. In both species, visual SCN neurons responded to retinal illumination with a sustained increase or a sustained decrease in electrical discharge. The majority (75%) of these cells were activated by light. In both the rat and the hamster SCN, visually responsive cells altered their discharge rate as a monotonic function of luminance. The intensity-response curve could be described by a Michaelis function with a small working range between threshold and saturation (2-3 log units) and a relatively high threshold. Intensity-response curves in both species were occasionally different for increasing as opposed to decreasing luminance. Thus, hysteresis effects of illumination may occur in the SCN. The spontaneous firing rates as well as the responsiveness of visual SCN cells were subject to marked variations between and within cells. The overall photic responsiveness of SCN neurons, however, indicated that they are specialized for luminance coding in the range of light intensities naturally occurring at dawn and dusk. This property makes these cells suitable to mediate photic entrainment of circadian rhythms as well as the measurement of photoperiod.

Double-Labeling of Neuropeptide Y-Immunoreactive Neurons which Project from the Geniculate to the Suprachiasmatic Nuclei

A projection from the ventral geniculate area to the suprachiasmatic nuclei (SCN) has been demonstrated in rats and hamsters. Large lesions in this area of the geniculate cause a dramatic decrease in neuropeptide Y-immunoreactivity in the SCN. Since numerous neuropeptide Y-immunoreactive neurons are found in the lateral geniculate area, we and others proposed that these immunoreactive neurons project to the SCN. In the present study, neurons in the lateral geniculate area of golden hamster brains were examined for both neuropeptide Y-immunoreactivity and a retrograde tracer transported from the SCN. Two days after a pressure injection of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the SCN of hamsters, labeled neurons were found in the intergeniculate leaflet and in the external lamina of the anterior ventral lateral geniculate nucleus (VLGN). These neurons were compared with similarly located neurons which showed immunoreactivity for neuropeptide Y. Morphometric comparisons of neuropeptide Y- and WGA-HRP-labeled neurons indicated that they were comparable in terms of soma size, number of dendrites, orientation and location. In additional hamsters, neurons double-labeled with a retrograde tracer and neuropeptide Y-immunoreactivity were localized in the intergeniculate leaflet and in the external lamina of the anterior VLGN. These results demonstrate that many neuropeptide Y-immunoreactive neurons located in both the intergeniculate leaflet and in the external lamina of the anterior VLGN project to the SCN in hamsters.

Hamster circadian rhythms are phase-shifted by electrical stimulation of the geniculo-hypothalamic tract

The suprachiasmatic nuclei (SCN) contain the major pacemaker for mammalian circadian rhythms. The SCN receive photic input both directly, via the retinohypothalamic tract (RHT), and indirectly, via the geniculohypothalamic tract (GHT), which originates in cells in the intergeniculate leaflet (IGL) and anterior portions of the ventral lateral geniculate nucleus (vLGN). We tested whether electrical stimulation of the GHT would induce phase shifts in wheel-running activity rhythms of Syrian hamsters housed in continuous darkness or continuous illumination. In both lighting conditions, electrical stimulation of the GHT induced mainly phase advances when given during the late subjective day and small phase delays when given during the late subjective night and early subjective day. Stimulation in the thalamus outside the GHT failed to produce similar phase shifts. Repeated daily stimulation had only a weak entraining effect on the activity rhythm. Activation of GHT neurons appears to influence the pacemaker for activity rhythms in a phase-dependent manner.

Photic induction of Fos protein in the suprachiasmatic nucleus is inhibited by the NMDA receptor antagonist MK-801.

Exposure of rodents to light can induce expression of a number of immediate-early genes, including c-fos, in cells of the suprachiasmatic nucleus (SCN), a dominant pacemaker in the mammalian circadian system. We examined the effects of pre-treatment with the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801, on the induction of Fos-like immunoreactivity (Fos-lir) in cells in the hamster SCN. At doses of 3 and 5 mg/kg, MK-801 treatment blocked photic induction of Fos-lir in the rostral SCN and ventrolateral portions of the caudal SCN but failed to block induction of Fos-lir in a discrete region of the dorsolateral SCN. These results suggest that photic induction of Fos-lir in most of the SCN depends on activation of an NMDA-type receptor which is sensitive to MK-801, but that Fos-lir in one portion of the SCN is induced by a mechanism which is not antagonized by MK-801.

NMDA and non-NMDA receptor antagonists inhibit photic induction of fos protein in the hamster suprachiasmatic nucleus

We previously reported that systemic treatment with a noncompetitive antagonist affecting the NMDA subtype of excitatory amino acid (EAA) receptor, MK-801, inhibits photic induction of Fos-like immunoreactivity (Fos-lir) in the hamster suprachiasmatic nucleus (SCN). Because MK-801 blocks the Ca2+ channel associated with the NMDA receptor, it may also influence the activity of other transmitters acting through Ca2+ channels. To assess the specificity of these effects, we compared the effects on photic induction of Fos-lir of several treatments: central injection of a competitive NMDA antagonist, CPP; central injection of a non-NMDA antagonist, DNQX; and systemic injection of the non-competitive NMDA antagonist, ketamine. Fos-lir was induced in SCN cells of vehicle-injected hamsters exposed to a light pulse 4-5 h after dark onset. Pretreatment with CPP (greater than 2 nmoles) or ketamine (greater than 100 mg/kg) caused a dose-related inhibition of photic induction of Fos-lir in portions of the SCN. These treatments reduced Fos-lir mainly in the rostral SCN and ventrolateral, but not dorsolateral, portions of the caudal SCN. Pretreatment with DNQX (greater than 20 nmoles) also inhibited photic induction of Fos-lir in the same regions of the SCN. These results indicate that photic induction of Fos protein in a portion of the hamster SCN is regulated by both NMDA and non-NMDA types of EAA receptor.

Circadian phase response curves for dark pulses in the hamster

SummaryHamsters maintained under constant illumination were exposed to 2- or 6-h pulses of darkness at various phases of their circadian activity rhythms. When presented around the time of activity onset, the pulses resulted in phase advances, and when presented toward the end of daily activity, they resulted in phase delays. Since others have shown that light pulses presented at the same phases in constant darkness cause phase shifts in the opposite directions, these results indicate that phase response curves for light and dark pulses are mirror images.Dark pulses also caused phase-dependent changes, both transient and long-lasting, in the period of the free-running rhythms, and a few pulses were immediately followed by splitting of the activity rhythms into two components. Such effects may reflect a differential responsiveness of two coupled oscillators to dark pulses.

Circadian variation in photic regulation of immediate-early gene mRNAs in rat suprachiasmatic nucleus cells

Exposure of rodents to light at daily times at which it can phase-shift circadian rhythms (subjective night) induces an increase in immunoreactivity for the immediate-early gene product Fos in cells of the circadian pacemaker, the suprachiasmatic nuclei (SCN). Light exposure at other phases (subjective day) does not increase Fos immunoreactivity in SCN cells, but it is not known whether this failure reflects the inability of light to induce transcription of appropriate mRNAs, or a post-transcriptional block. We used in situ hybridization studies to examine levels of mRNA in the SCN of rats exposed to light during the subjective day and subjective night. We studied levels of mRNAs for several immediate-early genes: c-fos, NGFI-A, NGFI-B, c-jun, junB and junD, before and after light exposure at these phases. Levels of mRNAs for all of the genes tested were unaffected by light exposure during the subjective day, and all were increased in response to light during the subjective night. With the exception of a weak constitutive label for junD, none of the genes were expressed in the SCN in darkness at either phase. Light-induced increases in the levels of several mRNAs in the SCN occur only during the subjective night; the mechanisms which prevent such responses during the subjective day remain unknown.

Palatable daily meals entrain anticipatory activity rhythms in free-feeding rats: Dependence on meal size and nutrient content

Circadian wheel-running rhythms were monitored continuously in 3 groups of female Sprague-Dawley rats under different palatable food availability schedules. All rats had free access to standard rat chow and water throughout the study. In addition, Group 1 rats received a palatable nutrient-rich mash for 2 hr each day for 28 days, beginning 3 hr after light onset of a 12:12 LD cycle. Group 2 rats received the same mash but were limited to 4 g daily. Group 3 rats received a palatable non-nutritive mash. Ten of 13 Group 1 rats, 2 of 13 Group 2 rats, and 0 of 13 Group 3 rats showed anticipatory running prior to the daily palatable meal. Palatable mash intake was generally lower among Group 3 rats than among Group 1 rats. However, several Group 3 rats consumed non-nutritive mash in amounts which equalled or exceeded the nutritive mash intake of Group 1 rats showing anticipatory running. The results indicate that temporally limited daily access to a palatable food can entrain anticipatory wheel-running in rats that are not food-deprived. They also indicate that entrainment to periodic food availability depends on stimuli associated with the concentrated intake of nutrients rather than on the absolute size or palatability of a meal.