Drugs that prevent mouse sleep also block light-induced locomotor suppression, circadian rhythm phase shifts and the drop in core temperature.

Exposure of mice to a brief light stimulus during their nocturnal active phase induces several simultaneous behavioral or physiological responses, including circadian rhythm phase shifts, a drop in core body temperature (Tc), suppression of locomotor activity and sleep. Each response is triggered by light, endures for a relatively fixed interval and does not require additional light for expression. The present studies address the ability of the psychostimulant drugs, methamphetamine (MA), modafinil (MOD) or caffeine (CAF), to modify the light-induced responses. Drug or vehicle (VEH) was injected at CT11 into constant dark-housed mice then exposed to 5-min 100µW/cm(2) light or no light at CT13. Controls (VEH/Light) showed approximately 60-min phase delays. In contrast, response was substantially attenuated by each drug (only 12-15min delays). Under a 12-h light:12-h dark (LD12:12) photoperiod, VEH/light-treated mice experienced a Tc drop of about 1.3°C coincident with locomotor suppression and both effects were abolished by drug pre-treatment. Each drug elevated activity during the post-injection interval, but there was also evidence for CAF-induced hypoactivity in the dark prior to the photic test stimulus. CAF acutely elevated Tc; MA acutely lowered it, but both drugs reduced Tc during the early dark (ZT12.5-ZT13). The ability of the psychostimulant drugs to block the several effects of light exposure is not the result of drug-induced hyperactivity. The results raise questions concerning the manner in which drugs, activity, sleep and Tc influence behavioral and physiological responses to light.

Separation of function for classical and ganglion cell photoreceptors with respect to circadian rhythm entrainment and induction of photosomnolence.

Four studies were performed to further clarify the contribution of rod/cone and intrinsically photoreceptive retinal ganglion cells to measures of entrainment, dark preference, light-induced locomotor suppression and photosomnolence. Wild type (WT), retinally degenerate (rd/rd), and melanopsin-less (OPN4⁻/⁻) mouse strains were compared. In Experiment 1, mice were exposed to a graded photoperiod in which approximately 0.26 µW/cm² irradiance diminished to dark over a 6-h interval. This method enabled "phase angle titration," with individual animals assuming activity onsets according to their sensitivity to light. WT and OPN4⁻/⁻ animals entrained with identical phase angles (effective irradiance=0.078 µW/cm²), but rd/rd mice required a more intense irradiance (0.161 µW/cm²) and entrainment occurred about 2.5 h earlier. In Experiment 2, all three strains preferred the dark side of a divided light-dark chamber until the irradiance dropped to 0.5 µW/cm² at which point, rd/rd mice no longer showed a preference. Experiments 3 and 4 determined that WT and rd/rd mice showed equivalent light-induced locomotor suppression, but the response was greatly impaired in OPN4⁻/⁻ mice. Closer examination of open field locomotion using infrared video-based methods and Any-maze(tm) software revealed two opposing effects of light. Locomotor suppression was equivalent in WT and rd/rd mice. Responses by OPN4⁻/⁻ mice varied from being absent (n=17) to normal (similar to WT and rd/rd mice; n=8). Light onset was associated with a significant, but brief, locomotion increase in WT and OPN4⁻/⁻ mice, but not in rd/rd mice. Any-maze(tm) analysis supports the view that light-induced locomotor quiescence is followed by behavioral sleep (photosomnolence), a fact that was visually validated from the raw video files. The data show that (a) classical photoreceptors, most likely rods, allow mice to prefer and entrain to very dim light such as found in natural twilight; (b) the presence of melanopsin photopigment enables light-induced locomotor suppression and photosomnolence; (c) light-induced locomotor suppression/photosomnolence is rod/cone mediated in 36% of mice lacking melanopsin, but not in 64% of the same OPN4⁻/⁻ strain; and (d) light-induced locomotor suppression encompasses an interval of behavioral sleep.

Millisecond light pulses make mice stop running, then display prolonged sleep-like behavior in the absence of light.

Masking, measured as a decrease in nocturnal rodent wheel running, is a visual system response to rod/cone and retinal ganglion cell photoreception. Here, the authors show that a few milliseconds of light are sufficient to initiate masking, which continues for many minutes without additional photic stimulation. C57J/B6 mice were tested using flash stimuli previously shown to elicit large circadian rhythm phase shifts. Ten flashes, 2 msec each and equally distributed over 5 min, activate locomotor suppression that endures for an additional 25 to 35 min in the dark and does not differ in magnitude or duration from that elicited by 5-min saturating light pulse. Locomotor activity by mice without access to running wheels is also suppressed by light flashes. The effects of various light flash patterns on mouse locomotor suppression are similar to those previously described for hamster phase shifts. Video analysis of active mice indicates that light flashes initiated at ZT13 rapidly induce an interval of behavioral quiescence that lasts about 10 min at which time the animals assume a typical sleep posture that is maintained for an additional 25 min. Thus, the period coincident with light-induced wheel running suppression appears to consist of two distinct behavioral states, one interval during which locomotor quiescence is initiated and maintained, followed by a second interval characterized by behavioral sleep. Given this sequence effected by light stimulation, we suggest that it be referred to as "photosomnolence," the term reflecting upon both the nature of the stimulus and the associated behavioral change.

Intergeniculate leaflet: contributions to photic and non-photic responsiveness of the hamster circadian system.

The circadian visual system is able to integrate light energy over time, enabling phase response and Fos induction in the suprachiasmatic nucleus to increase in proportion to the total energy of the photic stimulus. In the present studies, the contribution of the intergeniculate leaflet to light energy integration by the hamster circadian rhythm system was evaluated. Fos protein is induced in intergeniculate leaflet neurons at much lower irradiance levels than seen in suprachiasmatic nucleus neurons. Bilateral N-methyl-d-aspartate lesions of the intergeniculate leaflet decreased phase response of the circadian locomotor rhythm to high irradiance and, in animals exposed to long duration light stimuli, reduced Fos induction in the suprachiasmatic nucleus. Normal photon integration, as indicated by attenuated rhythm phase shifts and Fos induction in suprachiasmatic nucleus cells in response to the energy in light stimuli, does not occur in the absence of the intergeniculate leaflet and is likely to be a property of the circadian rhythm system, rather than solely of the suprachiasmatic nucleus. Anatomical analysis showed that virtually no intergeniculate leaflet neurons projecting to the suprachiasmatic nucleus contain Fos induced by either light or locomotion in a novel wheel. However, cells projecting to the pretectum were found to contain novel-wheel induced Fos. The intergeniculate leaflet is implicated in the normal assessment of light by the circadian rhythm system, but the circuitry by which either photic or non-photic information gains access to the suprachiasmatic nucleus may be more complex than previously thought.

The circadian visual system, 2005.

The primary mammalian circadian clock resides in the suprachiasmatic nucleus (SCN), a recipient of dense retinohypothalamic innervation. In its most basic form, the circadian rhythm system is part of the greater visual system. A secondary component of the circadian visual system is the retinorecipient intergeniculate leaflet (IGL) which has connections to many parts of the brain, including efferents converging on targets of the SCN. The IGL also provides a major input to the SCN, with a third major SCN afferent projection arriving from the median raphe nucleus. The last decade has seen a blossoming of research into the anatomy and function of the visual, geniculohypothalamic and midbrain serotonergic systems modulating circadian rhythmicity in a variety of species. There has also been a substantial and simultaneous elaboration of knowledge about the intrinsic structure of the SCN. Many of the developments have been driven by molecular biological investigation of the circadian clock and the molecular tools are enabling novel understanding of regional function within the SCN. The present discussion is an extension of the material covered by the 1994 review, "The Circadian Visual 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.

Hypothalamic and zona incerta neurons expressing hypocretin, but not melanin concentrating hormone, project to the hamster intergeniculate leaflet.

The hypocretins (Hcrt; also known as orexins) and melanin-concentrating hormone comprise distinct families of neuropeptides synthesized in cells located in the lateral hypothalamus and adjacent areas. The Hcrts are thought to modulate food intake and sleep/wake patterns in mammals. Melanin-concentrating hormone has a well-documented role in energy metabolism. A moderate plexus of Hcrt immunoreactive terminals has been described in the hamster intergeniculate leaflet, part of the circadian rhythm system. This study investigated the origin of Hcrt-immunoreactive terminals in the intergeniculate leaflet and determined whether melanin-concentrating hormone neurons also project to the intergeniculate leaflet. The tracer, cholera toxin beta-subunit, was injected into the intergeniculate leaflet of the golden hamster. Double-label fluorescent immunohistochemistry for cholera toxin beta-subunit and Hcrt or melanin-concentrating hormone was then performed to identify retrogradely labeled cells also containing immunoreactive peptide. Most cholera toxin beta-subunit-labeled cells were detected in the medial zona incerta and sub-incertal zone, with few observed in the lateral hypothalamus. Hcrt-immunoreactive cells were abundant and found largely in the lateral hypothalamus and adjacent nuclei. Melanin-concentrating hormone cells were also abundant in the medial zona incerta, in close proximity to cholera toxin beta-subunit-labeled cells, but ventral to them. Cells containing both cholera toxin beta-subunit and Hcrt-immunoreactive, were present in the dorsal aspect of the lateral hypothalamus. The number observed was small, < or = 1% of the total number of Hcrt cells counted in the hamster. No cholera toxin beta-subunit-immunoreactive cells also contained melanin-concentrating hormone and no melanin-concentrating hormone-immunoreactive processes were evident in the intergeniculate leaflet. The results show that a small number of lateral hypothalamus cells containing Hcrt-immunoreactivity project to the intergeniculate leaflet, but they are scattered rather than collected into a discrete group. At the present time there is no information regarding the function of these cells, although they may contribute to the regulation of sleep/arousal, circadian rhythmicity, or vestibulo-oculomotor function.

Reciprocal serotonergic connections between the hamster median and dorsal raphe nuclei.

The median (MnR), but not the dorsal (DR) raphe, sends a serotonergic projection to the suprachiasmatic (SCN) nucleus. Stimulation of either nucleus by electrode or serotonin agonist yields equivalent effects on circadian rhythmicity. This and other evidence suggests the existence of a functional serotonergic pathway from the DR to the MnR that may participate in circadian rhythm regulation. The present investigation was designed to identify such a connection. Tract tracer studies revealed cells in the DR that project to the MnR, as well as cells in the MnR that project to the DR. Double label immunofluorescence methods demonstrated that some of the cells projecting from either nucleus to the other contain serotonin immunoreactivity. The results support the existence of a reciprocal pathway between the DR and MnR that is at least partially serotonergic.

The intergeniculate leaflet, but not the visual midbrain, mediates hamster circadian rhythm response to constant light.

Several studies have demonstrated a variety of effects of intergeniculate leaflet (IGL) lesions on circadian rhythm regulation. Recent studies have suggested the possibility that certain rhythm functions attributed to the IGL are actually controlled by retinorecipient midbrain nuclei or other brain areas connected to the IGL. The present investigations evaluated whether midbrain lesions previously shown to block the phasic actions of benzodiazepine would also reduce or eliminate the period-lengthening effect of constant light (LL), a function that has been attributed to the IGL. Experiment 1 established that the circadian period of controls lengthened by about 0.57 h when the animals were transferred from constant dark (DD) to LL, but the magnitude of change was attenuated by about 50% in animals with IGL lesions caused by the neurotoxin N-methyl-D-aspartate (NMDA). In experiment 2, controls were compared to groups receiving either NMDA lesions of the pretectum or tectum or knife cuts designed to sever connections between the IGL and more medial retinorecipient nuclei. As in experiment 1, there were no differences between groups with respect to circadian period in DD. However, unlike experiment 1, all groups lengthened period equally in LL (overall mean increase = 0.57 h). Thus, the effect of LL on circadian period appears to be a joint result of photic information arriving at the circadian clock directly from the retinohypothalamic tract and indirectly through the IGL via the geniculohypothalamic tract, without involvement of visual midbrain. The results may have implications for the anatomical basis of Aschoff's rule.

Neuromodulator content of hamster intergeniculate leaflet neurons and their projection to the suprachiasmatic nucleus or visual midbrain.

The intergeniculate leaflet (IGL) of the lateral geniculate complex has widespread, bilateral, and reciprocal connections with nuclei in the subcortical visual shell. Its function is poorly understood with respect to its role in visual processing. The most well-known IGL projection, and the only one with a clear function, is the geniculohypothalamic tract (GHT) that terminates in the suprachiasmatic nucleus (SCN), site of the primary circadian clock. The hamster GHT is derived, in part, from IGL neurons containing neuropeptide Y and enkephalin. IGL neurons containing these peptides also project to the pretectal region. The present studies used a combination of immunohistochemical, lesion, and retrograde tracing techniques to study neuron types in the IGL and their projections to hamster SCN and pretectum. Two additional neuromodulators, gamma-aminobutyric acid (GABA) and neurotensin, are shown to be present in IGL neurons. The GABA- and neurotensin-immunoreactive neurons project to the SCN with terminal field patterns very similar to those for neuropeptide Y and enkephalin. IGL neurons of all four types also send projections to the pretectum, but rarely do individual cells project to both the SCN and the pretectum. Nearly all neurotensin is colocalized with neuropeptide Y in IGL neurons, although about half of the neuropeptide Y cells do not contain neurotensin. Otherwise, the extent to which the four neuromodulators are colocalized varies from 6% to 54%. Nearly every SCN neuron appears to contain GABA. In the IGL, the majority of cells studied are not identifiable by GABA immunoreactivity. Putative functions of the various neuromodulator projections from the IGL to pretectum or SCN are discussed.

Light augments FOS protein induction in brain of short-term enucleated hamsters.

FOS protein is synthesized in neuronal nuclei in response to a variety of environmental stimuli and has been used as a marker of stimulus-specific brain function. The present studies were initiated to examine the effects of ultraviolet light on the induction of FOS protein immunoreactivity (FOS-IR) in several brain regions of adult male hamsters. Experiment 1 confirmed previous observations of FOS-IR induced in visual cortex in response to ultraviolet light. However, protein was also induced by ultraviolet or white light in a variety of other areas and induction occurred in both sighted and enucleated animals. Therefore, experiments were conducted to evaluate the effects of a 514 nm light on FOS-IR induction in blind or sighted animals. Experiments 2 and 3 were performed during the early subjective night and mid-subjective day, respectively, using animals about 4 days after bilateral enucleation or sham surgery. In Experiment 2, light and enucleation independently and interactively resulted in increased FOS-IR neuronal nuclei counts. In Experiment 3, there was a main effect of enucleation and an interaction between enucleation and light condition, but no main effect of light. In Experiment 4, conducted during the early subjective night using animals enucleated 60 days earlier, there were neither effects of light or enucleation. The results support the view that, under certain conditions related to subjective time of day and time since enucleation, light can act through unknown extraocular mechanisms to modify brain activity. Further, short term enucleation itself induces widespread alteration in brain function as indicated by increased FOS-IR expression. The results specifically do not support a role for extraretinal photoreception with respect to direct circadian rhythm regulation.

Forebrain connections of the hamster intergeniculate leaflet: comparison with those of ventral lateral geniculate nucleus and retina.

The hamster intergeniculate leaflet (IGL), part of the circadian rhythm regulatory system, has very extensive interconnections with subcortical visual nuclei. The present investigation describes IGL connections with the hamster diencephalon and telencephalon and compares them with ventral lateral geniculate nucleus (VLG) connections and retinal projections. Connections of the geniculate nuclei were evaluated using anterograde transport of iontophoretically injected Phaseolus vulgaris leucoagglutinin and by retrograde transport of cholera toxin beta fragment. The cholera fragment was also injected intraocularly to trace retinal efferents. The IGL has ipsilateral and contralateral projections to the anterior and posterior hypothalamic nuclei, the ventral preoptic, lateral and dorsal hypothalamic areas, but not to the core ventromedial nucleus and very sparsely to the paraventricular nucleus. There are also IGL projections to the medial and lateral zona incerta, anteroventral, anterodorsal, reuniens, parataenial, paraventricular, centrolateral, central medial, and laterodorsal thalamic nuclei. IGL projections to the telencephalon are found in the horizontal limb of the diagonal band, olfactory tubercle, nucleus of the lateral olfactory tract, posterior bed nucleus of the stria terminalis, ventral pallidum, and in nuclei of the medial amygdala. The only substantial VLG projections are to bed nucleus of the stria terminalis, IGL, medial zona incerta, central medial and laterodorsal thalamic nuclei. Several of the IGL targets, the bed nucleus of the stria terminalis and zona incerta in particular, send projections back to the IGL and VLG. In addition, cells are present in the caudal cingulate cortex that project to both nuclei. Retinal projections are found in many of the regions receiving IGL innervation, including nuclei of the medial basal telencephalon, the posteromedial bed nucleus of the stria terminalis, and nuclei of the hypothalamus. A retinal projection is also visible in the lateral olfactory tract from which it extends rostrally, then medially along the base of the rhinal fissure. Fibers also extend caudally, in a superficial location, to perirhinal cortex. The results further demonstrate the widespread connections of the IGL and support the idea that the IGL modulates olfactory, photic, and circadian rhythm regulation of regulatory physiology and behavior.

The hamster circadian rhythm system includes nuclei of the subcortical visual shell.

The clock regulating mammalian circadian rhythmicity resides in the suprachiasmatic nucleus. The intergeniculate leaflet, a major component of the subcortical visual system, has been shown to be essential for certain aspects of circadian rhythm regulation. We now report that midbrain visual nuclei afferent to the intergeniculate leaflet are also components of the hamster circadian rhythm system. Loss of connections between the intergeniculate leaflet and visual midbrain or neurotoxic lesions of pretectum or deep superior colliculus (but not of the superficial superior colliculus) blocked phase shifts of the circadian activity rhythm in response to a benzodiazepine injection during the subjective day. Such damage did not disturb phase response to a novel wheel stimulus. The amount of wheel running or open field locomotion were equivalent in lesioned and control groups after benzodiazepine treatment. Electrical stimulation of the deep superior colliculus, without its own effect on circadian rhythm phase, greatly attenuated light-induced phase shifts. Such stimulation was associated with increased FOS protein immunoreactivity in the suprachiasmatic nucleus. The results show that the circadian rhythm system includes the visual midbrain and distinguishes between mechanisms necessary for phase response to benzodiazepine and those for phase response to locomotion in a novel wheel. The results also refute the idea that benzodiazepine-induced phase shifts are the consequence of induced locomotion. Finally, the data provide the first indication that the visual midbrain can modulate circadian rhythm response to light. A variety of environmental stimuli may gain access to the circadian clock mechanism through subcortical nuclei projecting to the intergeniculate leaflet and, via the final common path of the geniculohypothalamic tract, from the leaflet to the suprachiasmatic nucleus.

Electrical stimulation of the median or dorsal raphe nuclei reduces light-induced FOS protein in the suprachiasmatic nucleus and causes circadian activity rhythm phase shifts.

Several pharmacological studies have suggested that the large median raphe serotonergic projection to the circadian clock in the suprachiasmatic nucleus may modulate circadian rhythm phase. The present experiments studied the role of dorsal and median raphe nuclei as regulators of circadian rhythmicity by evaluating the ability of electrical stimulation to shift rhythm phase or to alter photic induction of FOS protein synthesis. Male hamsters implanted with bipolar electrodes in either the median or dorsal raphe nucleus were stimulated during the early subjective night coincident with exposure to a saturating light pulse. About 90 min later, animals were anesthetized, perfused and the brains processed for FOS protein immunoreactivity. As previously demonstrated, light alone induces FOS immunoreactivity in nuclei of suprachiasmatic nucleus neurons. This was significantly attenuated by stimulation of either the median or dorsal raphe nucleus, with the extent of attenuation proportional to the intensity of stimulation. Electrical stimulation without light exposure had no effect on FOS expression. The effect of light on FOS expression in the suprachiasmatic nucleus was not modified by pre-treatment with the 5-HT1/2 serotonin receptor antagonist, metergoline, although it greatly reduced electrical stimulation-induced FOS expression in the hippocampus. In a second experiment, hamsters housed with running wheels in constant light were electrically stimulated in the median or dorsal raphe nucleus 6 h prior to (CT6) or 2 h after (CT14) expected activity onset. Regardless of which raphe nucleus was electrically stimulated, approximately 22 min phase advances were elicited at CT6 and 36 min phase delays were elicited at CT14. Despite the fact that the sole direct projection from the raphe complex to the suprachiasmatic nucleus is from the median nucleus, the present data do not distinguish between the median and dorsal raphe with respect to their impact on circadian rhythm regulation. Instead, two possible roles for each raphe nucleus are demonstrated. One main effect is that both raphe nuclei modulate rhythm phase. The second is an interaction between raphe efferent activity and light which, in the present studies, is demonstrated by the ability of raphe stimulation to modulate the action of light on the circadian system. While serotonin is a likely neurotransmitter mediating one or both effects, alternatives such as GABA, must be considered.

The ascending serotonergic system in the hamster: comparison with projections of the dorsal and median raphe nuclei.

The ascending serotonergic projections are derived largely from the midbrain median and dorsal raphe nuclei, and contribute to the regulation of many behavioral and physiological systems. Serotonergic innervation of the hamster circadian system has been shown to be substantially different from earlier results obtained with other methods and species. The present study was conducted to determine whether similar differences are observed in other brain regions. Ascending projections from the hamster dorsal or median raphe were identified using an anterograde tracer, Phaseolus vulgans leucoagglutinin, injected by iontophoresis into each nucleus. Brains were processed for tracer immunoreactivity, and drawings were made of the median raphe and dorsal raphe efferent projection patterns. The efferents were also compared to the distribution of normal serotonergic innervation of the hamster midbrain and forebrain. The results show widespread, overlapping projection patterns from both the median and dorsal raphe, with innervation generally greater from the dorsal raphe. In several brain regions, including parts of the pretectum, lateral geniculate and basal forebrain, nuclei are innervated by the dorsal, but not the median, raphe. The hypothalamic suprachiasmatic nucleus is the only site innervated exclusively by the median and not by the dorsal raphe. The pattern of normal serotonin fiber and terminal distribution is generally more robust than would be inferred from the anterograde tracer material. However, there is good qualitative similarity between the two sets of data. The oculomotor nucleus and the medial habenula are unusual to the extent that each has a moderately dense serotonin terminal plexus, although neither receives innervation from the median or dorsal raphe. In contrast, the centrolateral thalamic nucleus and lateral habenula have little serotonergic innervation, but receive substantial other neural input from the raphe nuclei. The normal serotonergic innervation of the hamster brain is similar to that in the rat, although there are exceptions. The anterograde tracing of ascending median or dorsal raphe projections reveals a high, but imperfect, degree of correspondence with the serotonin innervation data, and with data from rats derived from immunohistochemical and autoradiographic tract-tracing techniques.

Serotonin and the regulation of mammalian circadian rhythmicity.

The suprachiasmatic nucleus (SCN), the site of the primary mammalian circadian clock, contains one of the densest serotonergic terminal plexes in the brain. Although this fact has been appreciated for some time, only in the last decade has there been substantial approach toward the understanding of the function of serotonin in the circadian rhythm system. The intergeniculate leaflet, which projects to the SCN via the geniculohypothalamic tract, receives serotonergic innervation from the dorsal raphe nucleus, and the SCN receives its serotonergic input from the median raphe nucleus. This separation of serotonergic origins provides the opportunity to investigate the function of the two projections. Loss of serotonergic neurones of the median raphe yields earlier onset and later offset of the nocturnal activity phase, longer duration of the activity phase, and increased sensitivity of circadian rhythm response to light. Despite the simplicity of the origins of serotonergic anatomy with respect to the circadian rhythm system, the actual involvement of serotonin in rhythm modulation is not so obvious. A variety of pharmacological studies have clearly implicated serotonin as a direct regulator of circadian rhythm phase, but others employing different methods suggest that simple elevation of SCN serotonin concentrations does not modify rhythm phase. The most convincing role of serotonin is its apparent ability to modulate sensitivity of the circadian rhythm to light. The putative method for such modulation is via a presynaptic 5-HT1B receptor on the retinohypothalamic tract, the activation of which attenuates photic input to the SCN thereby reducing phase response to light. Serotonin may modulate phase response to benzodiazepines, but does not appear to modify such response to environmentally induced locomotor activity. Current interest in serotonergic modulation of circadian rhythmicity is strong and the research is vigorous. There is an abundance of information about serotonin and circadian rhythm function that lacks a satisfactory framework for its interpretation. The next decade is likely to see the gradual evolution of this framework as the role of serotonin in circadian rhythm regulation is further elucidated.

Destruction of serotonergic neurons in the median raphe nucleus blocks circadian rhythm phase shifts to triazolam but not to novel wheel access.

Systematic treatment of hamsters with triazolam (TRZ) or novel wheel (NW) access will yield PRCs similar to those for neuropeptide Y. Both TRZ and NW access require an intact intergeniculate leaflet (IGL) to modulate circadian rhythm phase. It is commonly suggested that both stimulus types influence rhythm phase response via a mechanism associated with drug-induced or wheel access-associated locomotion. Furthermore, there have been suggestions that one or both of these stimulus conditions require an intact serotonergic system for modulation of rhythm phase. The present study investigated these issues by making serotonin neuron-specific neurotoxic lesions of the median or dorsal raphe nuclei and evaluating phase response of the hamster circadian locomotor rhythm to TRZ treatment or NW access. The expected effect of TRZ injected at CT 6 h on the average phase advance was virtually eliminated by destruction of serotonin neurons in the median, but not the dorsal, raphe nucleus. No control or lesioned animal engaged in substantial wheel running in response to TRZ. By contrast, all median raphe-lesioned hamsters that engaged in substantial amounts of running when given access to a NW had phase shifts comparable to control or dorsal raphe-lesioned animals. The results demonstrate that serotonergic neurons in the median raphe nucleus contribute to the regulation of rhythm phase response to TRZ and that it is unlikely that these neurons are necessary for phase response to NW access. The data further suggest the presence of separate pathways mediating phase response to the two stimulus conditions. These pathways converge on the IGL, a nucleus afferent to the circadian clock, that is necessary for the expression of phase response to each stimulus type.

Interconnections among nuclei of the subcortical visual shell: the intergeniculate leaflet is a major constituent of the hamster subcortical visual system.

The intergeniculate leaflet (IGL), a major constituent of the circadian visual system, is one of 12 retinorecipient nuclei forming a "subcortical visual shell" overlying the diencephalic-mesencephalic border. The present investigation evaluated IGL connections with nuclei of the subcortical visual shell and determined the extent of interconnectivity between these nuclei. Male hamsters received stereotaxic, iontophoretic injections of the retrograde tracer, cholera toxin beta fragment, or the anterograde tracer, Phaseolus vulgaris-leucoagglutin, into nuclei of the pretectum (medial, commissural, posterior, olivary, anterior, nucleus of the optic tract, posterior limitans), into the superior colliculus, or into the visual thalamic nuclei (lateral posterior, dorsal lateral geniculate, intergeniculate leaflet, ventral lateral geniculate). Retrogradely labeled cell bodies identified nuclei with afferents projecting to the site of injection, whereas the presence of anterogradely labeled fibers with terminals revealed brain nuclei targeted by neurons at the site of injection. The IGL projects bilaterally to all nuclei of the visual shell except the lateral posterior and dorsal lateral geniculate nuclei. The IGL also has afferents from the same set of nuclei, except the nucleus of the optic tract. The extensive bilateral efferent projections distinguish IGL from the ventral lateral geniculate nucleus. The superior colliculus, commissural pretectal, olivary pretectal, and posterior pretectal nuclei also project bilaterally to the majority of subcortical visual nuclei. The IGL has a well-established role in circadian rhythm regulation, but there is as yet no known function for it in the larger context of the subcortical visual system, much of which is involved in oculomotor control.

Neuropeptide Y and enkephalin immunoreactivity in retinorecipient nuclei of the hamster pretectum and thalamus.

This investigation was stimulated by the historical confusion concerning the identity of certain pretectal nuclei and by large differences reported between species with respect to which nuclei receive retinal innervation. Subcortical visual nuclei were studied using immunohistochemistry to identify retinal projections labeled following intraocular injection of cholera toxin, b fragment. In addition, neuropeptide Y (NPY) or enkephalin (ENK) immunoreactive cells and fibers were also evaluated in the retinorecipient pretectal and thalamic areas. The results confirm the established view that the retina directly innervates the nucleus of the optic tract (NOT), posterior (PPT), and olivary pretectal (OPT) nuclei. However, the retina also innervates the hamster medial (MPT) and anterior (APT; dorsal division) pretectal nuclei, results not previously reported in rodents. A commissural pretectal area (CPT) sparsely innervated by retina is also described. The data show for the first time that the posterior limitans nucleus (PLi) receives a moderately dense, direct retinal input. The PLi does not project to the cortex and appears to be a pretectal, rather than thalamic, nucleus. All retinal projections are bilateral, although predominantly contralateral. The PLi contains a moderately dense plexus of NPY- and ENK-IR fibers and terminals. However, peptidergic fibers also traverse the ATP and connect with the dorsomedial pretectium. The OPT contains ENK- and NPY-IR neurons and fibers, but is specifically identifiable by a moderately dense plexus of ENK-IR terminals. Numerous ENK-IR neurons are found in the NOT and PPT. The latter also has moderate numbers of ENK-IR fibers and terminals, but few NPY-IR neurons or fibers. The MPT contains modest numbers of ENK-IR fibers. The APT has no NPY-IR neurons or terminals, but an occasional ENK-IR neuron is seen and there is sparse ENK-IR innervation. Peptidergic innervation of the visual nuclei does not appear to be derived from the retina. The results show a set of retinally innervated, contiguous nuclei extending from the thalamic ventrolateral geniculate nucleus dorsomedially to the midbrain CPT. These nuclei plus the superior colliculus comprise a dorsal "visual shell" embracing a central core of caudal thalamus and rostral midbrain.

The serotonergic projection from the median raphe nucleus to the suprachiasmatic nucleus modulates activity phase onset, but not other circadian rhythm parameters.

The suprachiasmatic nucleus (SCN) is densely innervated by serotonergic fibers originating in the median raphe nucleus (MR). Serotonin (5-HT) specific lesions of the MR alter entrainment and eliminate 5-HT fibers in the SCN, as well as in all other MR-recipient areas. The present study used 5-HT specific lesions of the SCN or the MR to determine the role of 5-HT in the SCN as a regulator of entrainment. Neurotoxic lesions of the MR significantly reduced 5-HT cell bodies in that nucleus and eliminated essentially all 5-HT innervation of the SCN. As previously demonstrated, these anatomical changes were associated with an advance in activity onset, delay in offset and expansion of the activity phase (alpha). Neurotoxin directly applied to the SCN caused an advance in the average activity onset, but had no effect on offset or alpha. About half of the SCN lesion animals had onsets equivalent to the MR lesion group, whereas onsets of the remaining animals were normal. Loss of SCN 5-HT innervation was severe for all SCN lesion animals, but significantly greater for those with advanced activity onsets. These results suggest that although the 5-HT projection to the SCN is likely to be responsible for modulating activity onset, the timing of activity offset appears to be regulated by a MR projection to an area outside the SCN. Furthermore, surprisingly few 5-HT fibers in the SCN are sufficient to maintain the normal phase angle of entrainment.