Robert Y. Moore

Destruction of the hamster serotonergic system by 5,7-DHT: effects on circadian rhythm phase, entrainment and response to triazolam

The role of the serotonergic system in the regulation of hamster circadian rhythms was analyzed using intraventricular injection of the selective neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT). Sixty days after 5,7-DHT administration, immunoreactive serotonin in the forebrain, particularly the suprachiasmatic nuclei and intergeniculate leaflets, was severely depleted in 16 animals, moderately depleted in four and only slightly affected in four. 5,7-DHT produced an immediate and sustained advance of the onset of running wheel activity relative to the 24 h light-dark (LD) cycle. Activity onset occurred 0.7 +/- 0.07 h before lights out among 5,7-DHT-treated animals compared with 0.18 +/- 0.04 h after lights out for vehicle-infused controls. This new, advanced phase angle of entrainment was maintained throughout the 60-day period of the study while the animals remained in a LD cycle, including after an 8-h phase advance of the light cycle. 5,7-DHT treatment also delayed the offset of wheelrunning in 16 of 24 animals and reduced the likelihood of a smooth pattern of reentrainment to the shifted LD cycle. The drug treatment did not affect circadian period in constant darkness, the rate of reentrainment to an 8-h phase advance or the amount of wheelrunning activity per day. In addition, 5,7-DHT treatment had no effect on the ability of triazolam, a short-acting benzodiazepine, to accelerate the rate of reentrainment to an 8-h phase advance. These observations show that ascending projections of midbrain raphe serotonin neurons participate in the regulation of the circadian activity phase but are not required for triazolam-induced acceleration of reentrainment to a phase-advanced LD cycle.

Melatonin inhibits metabolic activity in the rat suprachiasmatic nuclei.

The pineal hormone melatonin has been implicated in the regulation of circadian rhythms and in photoperiodic control of reproduction. The effects of melatonin require the hypothalamic suprachiasmatic nucleus (SCN), a principal pacemaker controlling circadian rhythms. To determine whether SCN activity was directly affected by exogenous melatonin, rats received either melatonin or saline injections 15 min before administration of 2-deoxy-[1-14C]glucose (2-DG) at two times of day, circadian time (CT) 10 and CT14, and the brains of these rats were processed for autoradiographic determination of 2-DG uptake within the SCN. We report that SCN 2-DG uptake was inhibited by melatonin at CT10, when 2-DG uptake is normally high, and unaffected at CT14, when 2-DG uptake is normally low. This indicates that the SCN may be neural substrates through which melatonin exerts at least some of its effects on mammalian physiology.

Two brain nuclei controlling circadian rhythms are identified by GFAP immunoreactivity in hamsters and rats

The intergeniculate leaflet (IGL) of the lateral geniculate complex is marked by the presence of neuro-peptide Y-containing neurons that project to the suprachiasmatic nuclei (SCN) of the hypothalamus. In the present study, we demonstrate that both the IGL and SCN in the hamster and rat are specifically delineated by the presence of glial fibrillary acidic protein-like immunoreactivity. This is significantly greater than in most other diencephalic regions and is particularly dense in the hamster brain. These observations suggest that glial-neuronal interactions may participate in circadian rhythm generation and regulation.

GABAA/benzodiazepine receptor localization in the circadian timing system

gamma-Aminobutyric acid (GABA) and exogenous benzodiazepines are thought to play a role in the neural regulation of circadian rhythms. Because binding sites for the benzodiazepines and GABAA ligands are functionally coupled as part of the GABAA/benzodiazepine receptor complex (GABAA/BZR), we analyzed the localization of GABA neurons and GABAA/BZR within 3 nuclei involved in circadian rhythm regulation using autoradiographic and immunohistochemical techniques. Glutamic acid decarboxylase-immunoreactive axons are present in the suprachiasmatic nuclei (SCN), intergeniculate leaflet (IGL), and dorsal raphe nucleus (DR). Immunoreactivity for the GABAA/BZ receptor complex is absent from the SCN and the IGL whereas the DR shows a dense, uniform immunoreactivity. Semiquantitative analysis of autoradiograms for [3H]diazepam and [3H]flunitrazepam binding reveals a moderate level of binding in the SCN, a low level of binding in the IGL, and the highest level of the DR. Based on both the pattern of benzodiazepine binding and of receptor immunoreactivity the DR would appear to be a likely target site for GABAA and benzodiazepine action. The SCN would also appear to be a possible target site. The results suggest the IGL is not a site for direct GABAA and benzodiazepine action, but do not exclude a role for the IGL in the neural circuitry mediating GABA and benzodiazepine interactions with the circadian system.

Paraventricular nucleus projections mediating pineal melatonin and gonadal responses to photoperiod in the hamster.

Knife cuts were placed around the paraventricular nucleus of the hypothalamus (PVN) in order to identify the pathways mediating photoperiodism and pineal melatonin production in male golden hamsters. Cuts in the coronal plane caudal to the PVN, have no effect on photoperiodic control of the testes unless they actually damage the PVN. Bilateral parasagittal cuts at the medial border of the lateral hypothalamus block short photoperiod-induced gonadal regression. Nighttime levels of pineal melatonin are reduced by these cuts, but unaffected by caudal cuts. Projections from the lateral PVN region descending towards the spinal cord appear to be critical for the control of pineal melatonin production and the control of the testicular function by short photoperiod.

Neuropeptide Y in the Circadian Timing System

Circadian rhythms are adaptations to the solar cycle of light and dark. In mammals, circadian rhythms express a temporal organization of physiological processes and behavior that results in optimal animal adaptation. The essential features of circadian rhythms are that they are generated by endogenous pacemakers and continuously reset, a process termed “entrainment,” by the light-dark cycle. This generation and entrainment of circadian rhythms is accomplished by a set of central neural structures, the circadian timing system (CTS). The principal components of the CTS are visual pathways mediating entrainment, pacemakers and efferent projections from the pacemakers to effector systems that exhibit circadian function (FIG. I ) . Over the last twenty years there has been a rapid accumulation of information about the organization and function of the CTS (cf. Rusak and Zuckerl and Meijer and Rietveld’ for reviews that reflect this progress). The essential features of the CTS, as we now understand it, are as follows. The lateral eyes are necessary for entrainment. Light activates retinal photoreceptors which are coupled through the usual retinal circuitry to a specific set of ganglion cells, probably a subgroup of W cells,3 responding predominantly to changes in luminous flux. These cells appear to project in both the lateral geniculate complex and to the suprachiasmatic nuclei (SCN) of the hypotha lam~s .~ .~ The projection to the SCN has been designated the retinohypothalamic tract (RHT). Early studies using the autoradiographic tracing method6-* found only projections to the SCN but more recent studies, using sensitive anterograde lectin transport methods, have shown projections to the anterior hypothalamus, lateral hypothalamus and retrochiasmatic area as well as to the SCN.9 The function of the projections beyond the SCN is unclear at this time but there is substantial evidence to establish that the RHT to the SCN is sufficient to maintain entrainment in the absence of other visual projections. The SCN is a circadian pacemaker (FIG. 2). Three lines of evidence sup rt that conclusion. First, ablation of the SCN results in a loss of circadian rhythms.’.’‘There are individual studies from which it is concluded that one or another rhythm, usually the

Serotonin regulation of circadian rhythmicity.

The suprachiasmatic nuclei (SCN) of the hypothalamus generate electrophysiological and metabolic cycles which repeat about every 24 hours.’-3 Ordinarily, this rhythm is synchronized to the environmental photoperiod that also has a period of about 24 hours. The phase angle of entrainment of the endogenous rhythms to the photoperiod remains relatively constant. The circadian clock of the SCN is coupled to the locomotor system and hierarchically controls a circadian rhythm of running activity. Under a typical 14:lO 1ight:dark cycle (LD), a hamster will often run 7-10 km during the dark phase, starting its activity 15-20 min after the lights go off. Thus, under LD conditions, the entrained locomotor rhythm has a period of 24 h and a fixed phase relationship to the photoperiod as well as a relatively constant rhythm amplitude. True circadian rhythmicity is demonstrated when, in the absence of a time-giving stimulus (e.g., under constant light or dark), the measured event oscillates in a “freerunning” pattern. The freerunning rhythm is self-sustained and repeats approximately every 24 h according to the dictates of an endogenous clock in the S C N . Elimination of photic input to the brain results in a freerunning rhythm; destruction4 of the SCN causes loss of circadian locomotor rhythmi~i ty .~ Given that there are no extraretinal photoreceptors in adult mammals, entrainment of the hamster locomotor rhythm could be accomplished through a retinohypothalamic tract (RHT) that conveys photic information directly to the SCN6.’ or through an indirect route consisting of primary optic tract afferents to the lateral geniculate complex and a secondary visual input to the SCN through the geniculohypothalamic tract (GHT).h.8.9 Recent research has shown that the RHT is necessary for circadian rhythm entrainment.’” The G H T is not necessary for entrainment, but does modulate circadian period under constant light, the phase angle of entrainment, as well as the rate at which rhythm reentrainment is accomplished after a phase shift of the 1ight:dark cycle.’’-’3

The central nervous system of Bulla gouldiana: Peptide localization

In the present study, we describe the structure of the central nervous system (CNS) of the marine gastropod Bulla gouldiana, and compare it with the structure of the CNS of the related mollusc, Aplysia californica. In addition, we performed an immunohistochemical analysis of a series of peptides, and the synaptic vesicle protein, synapsin I, in the central nervous system of B. gouldiana. The most common peptide in the B. gouldiana nervous system is the molluscan cardioexcitatory peptide (FMRFamide), which is present in a significant proportion of B. gouldiana neurons. A smaller number of neurons exhibit immunoreactivity to antisera raised against the calcitonin gene related peptide, vasopressin, vasoactive intestinal peptide, cholecystokinin, galanin and enkephalin. In some instances there is colocalization of two or more peptides. Very few neurons or axons exhibit synapsin I-like immunoreactivity. The patterns of immunoreactivity to these antisera is quite similar to the patterns that have been described in other gastropods, including Lymnaea stagnalis and Aplysia californica. These observations emphasize the importance of FMRFamide-like compounds in phylogenetically old nervous systems and indicate that compounds similar to mammalian peptides are present in the gastropod. Thus, the production of a wide variety of peptide molecules and their use in neuronal function appears to be a highly conserved phylogenetic process.