Journal of Biological Rhythms

The circadian timing system has three principal elements: the retina, the intergeniculate leaflet (IGL) of the thalamus, and the suprachiasmatic nucleus (SCN). Since the human circadian timing system cannot be studied experimentally, we have used another primate, the macaque monkey, to help provide insight into the organization of the human circadian system. The retinohypothalamic tract (RHT) in the monkey projects to the SCN, the anterior and lateral hypothalamic areas, and the retrochiasmatic area in a pattern very similar to that in the rat. The monkey SCN has a population of vasoactive intestinal polypeptide-containing (VIP +) neurons in a zone that overlaps the RHT termination and the termination of neuropeptide Y-containing (NPY +) axons arising in the IGL. This zone is surrounded by a population of vasopressin-containing (VP +) neurons. The human SCN is similar to that of other mammals with populations of VIP+ and VP+ neurons, but it differs in having a large population of neurotensin-containing (NT +) neurons that extends over the entire nucleus, and a moderate population of NPY + neurons located centrally in the nucleus in the presumed area of RHT termination. The lateral geniculate nucleus in the monkey and human is quite different from that in rodents, but contains an area in the pregeniculate nucleus that receives bilateral retinal projections in the monkey and is characterized in both the monkey and human by a population of NPY + neurons and a plexus of enkephalin- and substance P-containing axons. This nucleus appears homologous to the rodent IGL.

Glutamate and γ-aminobutyrate (GABA) are two neurotransmitters that appear to play an important role in the hypothalamic suprachiasmatic nucleus (SCN) and in the adjacent areas of the medial hypothalamus. Converging evidence based on ultrastructural immunocytochemistry, molecular biology, calcium imaging, and electrophysiology suggests not only that GABA and glutamate are used for inhibitory and excitatory activity, respectively, in this region of the rat brain, but that these two fast-acting amino acids may account for the majority of neurotransmission there.

Our recent studies have examined circadian photoreception in mice with hereditary retinal disorders (rd/rd and rds/rds). Despite the loss of visual function in these mice, circadian responses to light remain unaffected. Using c-fos expression within the suprachiasmatic nuclei (SCN) as a marker of neural activation of the circadian entrainment pathway, we have found identical levels of Fos in the SCN of rd/rd and +/+ mice in response to retinal illumination. On the basis of action spectrum studies, and measurements of photopigment retinoids using high-pressure liquid chromatography, we believe that the photopigment mediating circadian responses to light is based upon an opsin, and that 11-cis-retinaldehyde is the photopigment chromophore. Preliminary measurements of mouse rod opsin, blue cone, and green-red cone opsin messenger RNA in retinally degenerate mice suggest that none of these opsins is exclusively used to mediate circadian responses to light. Collectively, our data suggest that circadian photoreception can be maintained by a very small number of rod or cone cells without outer segments, or, alternatively, is performed by an unrecognized class of photoreceptive cell within the mammalian retina.

We have utilized polymerase chain reaction with primers corresponding to conserved amino acid sequences within membrane-spanning regions of known serotonin receptors to identify clones of four putative new indoleamine receptors. We have determined complete amino acid sequences of these four receptors, which fall into three subfamilies; two of these subfamilies are novel. The sites of expression within the brain have been determined for each of the genes. Expression in mammalian cells demonstrates that each new protein is a receptor for serotonin and that each has a distinct pharmacology when compared to known receptors. Two of the new receptors are coupled to cyclic adenosine monophosphate, one negatively (G_i) and one positively (G_s). The latter is a candidate for the serotonin receptor that mediates phase advances in circadian rhythms of the suprachiasmatic nucleus.

Knowledge of the neuronal membrane properties and synaptic physiology of the suprachiasmatic nucleus (SCN) is critical for an understanding of the cellular basis of circadian rhythms in mammals. The hypothalamic slice preparation from rodents and a combination of electrophysiological techniques (i.e., extracellular single- and multiple-unit recording, intracellular recording, and whole-cell patch clamp) were used to study (1) the role of excitatory and inhibitory amino acids (i.e., glutamate and γ-aminobutyric acid [GABA] in synaptic transmission, (2) the membrane properties of SCN neurons, and (3) the mechanisms of neuronal synchronization. Antagonists for N-methyl-D-aspartate (NMDA) receptors and non-NMDA receptors blocked excitatory postsynaptic potentials (EPSPs) evoked by stimulation of the optic nerve or other sites when SCN cells were depolarized or at rest, respectively. Bicuculline blocked inhibitory postsynaptic potentials (IPSPs) that were evoked by local stimulation or that occurred spontaneously. The IPSP reversal potential was near the C1^- equilibrium potential, and was shifted to depolarized levels by raising intracellular [C1^-]. Thus, glutamate and GABA appear to mediate fast excitatory and inhibitory synaptic transmission in the SCN. Some SCN neurons, but not all of them, had low-threshold Ca²⁺ spikes and time-dependent inward rectification, thus indicating that the electrical properties of SCN neurons are not homogenous. Neurons with a firing rate of >6 Hz had a regular pattern, and neurons with a rate of <4 Hz had an irregular pattern; since both the firing rate and pattern could be modified with injected currents, SCN neurons with different firing patterns are unlikely to represent distinct classes of cells. Synchronous bursts of action potentials occurred in the SCN after chemical synapses were blocked with [Ca²⁺]-free solutions and with amino acid transmitter antagonists, which indicates that synchronous neuronal activity can occur in the SCN without active chemical synapses and suggests that a different mechanism of communication exists in the SCN. Future in vitro electrophysiological experiments should provide an explanation of how neurotransmitters, local neuronal circuits, and intrinsic membrane properties regulate the electrical activity of SCN neurons during the circadian rhythm.

We review recent studies in our laboratory that have investigated the neural mechanisms underlying photic entrainment of the mammalian circadian system. The results from studies of extracellular single-unit recordings and of photic induction of Fos-like immunoreactivity (Fos-lir) indicate that excitatory amino acid (EAA) transmission, and particularly activation of the N-methyl-D-aspartate (NMDA) receptor subtype, is important for conveying photic information to suprachiasmatic nucleus (SCN) cells. We have also found that a subregion of the SCN still shows Fos-lir after blockade of EAA receptors, and we have evidence suggesting that these cells are innervated by a distinct subdivision of the retinal projection to the SCN. In addition, we have found that photic responses of cells in the intergeniculate leaflet (which projects to the SCN) and of SCN cells are modulated by serotonin (5-HT) via a receptor that resembles the 5-HT_1A subtype.

The aim of the research reported here was to provide information on the neurochemical processes that underlie the generation and entrainment of mammalian circadian rhythms. The studies were centered principally around the in vivo brain microdialysis technique for assessing the daily pattern of neurotransmitter activity in the suprachiasmatic hypothalamus of freely behaving Syrian and Siberian hamsters. This approach yielded several findings related to the activities of serotonergic and excitatory amino acid systems in the region of the suprachiasmatic nuclei (SNC). Specifically, we found that (1) there were daily variations in the extracellular concentrations of 5-hydroxyindoleacetic acid (5-HIAA) and glutamate (GLU) in the SCN region (highest levels of 5-HIAA occurred soon after lights-off, whereas GLU peaked later in the dark phase); (2) the daily rhythm of GLU, but not serotonin, in the SCN region appeared to be circadian in nature and was not driven by an external influence; (3) the rhythm in GLU measured in SCN microdialysate involved a tetrodotoxin-insensitive mechanism and did not appear to be directly linked to the expression of locomotor behavior; and (4) direct application of serotonin receptor agonists via the microdialysis probe suppressed the concentration of extracellular GLU in the SCN region, suggesting that serotonin may modulate GLU release in the SCN.

The long-term goal of our research is to understand how cells of the suprachiasmatic nucleus (SCN) are organized to form a 24-hr biological clock, and what roles specific neurotransmitters and modulators play in timekeeping and resetting processes. We have been addressing these questions by assessing the pattern of spontaneous neuronal activity, using extracellular and whole-cell patch recording techniques in long-lived SCN brain slices from rats. We have observed that a robust pacemaker persists in the ventrolateral region of microdissected SCN, and have begun to define the electrophysiological properties of neurons in this region. Furthermore, we are investigating changing sensitivities of the SCN to resetting by exogenous neurotransmitters, such as glutamate, serotonin, and neuropeptide Y, across the circadian cycle. Our findings emphasize the complexity of organization and control of mammalian circadian timing.

Photic stimulation during the subjective night induces the expression of Fos among a discrete population of cells in the suprachiasmatic nuclei (SCN) region of the Syrian hamster. Light appears to stimulate Fos expression only when administered at circadian times (CTs) at which exposure causes a phase shift. Different populations of SCN cells express Fos in response to light pulses that result in phase advances versus phase delays, raising the possibility that different cell populations in the suprachiasmatic hypothalamus participate in light-induced phase advances and delays of the circadian oscillator. Microinjection of excitatory amino acid (EAA) antagonists into the region of the SCN attenuates light-induced phase advances of the free-running activity rhythm and light-induced Fos expression in the hamster SCN. However, injection of N-methyl-D-aspartate (NMDA) at CT 18, which results in a widespread pattern of Fos expression in the hypothalamus that includes the retinorecipient zone of the SCN, does not produce phase advances of the circadian oscillator. The results demonstrate that both light-induced Fos expression and light-induced phase advances are dependent upon EAA neurotransmission within the SCN region. However, expression of Fos in the SCN induced by the EAA agonist NMDA is not sufficient to cause phase advances of the SCN oscillator.

The primary objective of this research was to examine expression of the immediate–early gene c-fos within the suprachiasmatic nucleus (SCN) for evidence of circadian regulation by photic stimuli. In vivo and in vitro analyses demonstrate that photic signals have an inductive effect on c-fos expression in the SCN, but only at critical times when light is capable of phase-shifting circadian rhythms. This evidence for correlative relations between the effects of light signals in inducing c-fos gene expression in the SCN and modulating the circadian period of the SCN pacemaker suggests that immediate-early genes may be components of the signal transduction cascade by which light entrains circadian rhythms. In addition, dual-immunostaining methods were utilized to examine neurochemical identity of SCN cells that exhibit this circadian induction of c-fos expression in response to light. Within the ventrolateral SCN, the photic induction of Fos expression occurred in neurons expressing gastrin-releasing peptide (GRP), but not in those containing vasoactive intestinal polypeptide (VIP). This finding suggests that SCN neurons containing GRP may be involved in the transduction of photic signals mediating circadian entrainment.

Amniote circadian organization derives from the interactions of circadian oscillators and photoreceptors located in the hypothalamic suprachiasmatic nuclei (SCN), the pineal gland, and the eyes. In mammals, circadian organization is dominated by the SCN, which serve as “master pacemakers” in the control of a wide array of behavioral and physiological rhythms (including locomotion, sleep-wake, thermoregulation, cardiovascular function, and many endocrine processes). Among the rhythms under SCN control in mammals are the circadian synthesis and secretion of the pineal hormone melatonin, which relies on a multisynaptic pathway via the sympathetic nervous system to maintain and entrain rhythmicity in this hormone. Several studies have indicated that pineal melatonin feeds back on SCN rhythmicity to modulate circadian patterns of activity and other processes. However, the nature and system-level significance of this feedback are unknown. Recently published work indicate, that although pinealectomy does not affect rat circadian rhythms in light-dark cycles or constant darkness, wheel-running activity rhythms are severely disrupted in constant light. These data suggest that either (1) pineal feedback regulates the light sensitivity of the SCN, and/or (2) it affects coupling among circadian oscillators within the SCN or between the SCN and its output. Research in our laboratory is currently addressing each of these hypotheses.

In transplantation studies using the tau mutation in the golden hamster, it has been demonstrated that suprachiasmatic nucleus (SCN) pacemaker cells and mechanisms of communication with the host brain are retained even after tissue dissociation and maintenance for many weeks in primary cell culture. Brain grafts of cultured SCN cells are capable of restoring overt rhythms of locomotor activity, and preliminary studies where cells from two tau genotypes are combined in a single graft demonstrate that pacemaker cells may communicate with each other to produce coherent rhythms with intermediate periods. The opportunity is presented, therefore, to study pacemaker-pacemaker communication in circadian chimeras produced by SCN transplantation.

Immunocytochemical analysis of graft-host interactions requires the positive identification of host versus donor cells. Although grafted blocks of tissue are easily recognized during immunocytochemical analysis, implants of dissociated and cultured cells may be more diffusely located and are not as readily identified. Unless distinct strain- or species-specific markers are available, it is difficult to identify connections that may carry timing information to the host organism. We have taken an anatomical approach that utilizes cell-labeling techniques for hamster tissue along with foreign protein expression in transgenic mice to identify patterns of communication among graft and host cells, focusing specifically on SCN-SCN communication. The data indicate the usefulness of these transgenes as markers in transplantation studies where communication between graft and host is addressed.

It is well established that the mammalian suprachiasmatic nucleus (SCN) is a biological pacemaker that entrains the activity of organisms to their environment and controls circadian rhyth-micity. However, neither the nature of these coupling signal or signals from the SCN, nor their target or targets in the brain, are well understood. Fiber efferents from the SCN reach nearby hypothalamic regions, suggesting a coupling role for neural efferent pathways. The SCN produces diffusible signals that reach nearby hypothalamic sites and the cerebrospinal fluid, suggesting a role for a diffusible efferent pathway. We consider the possibility of redundant coupling signals of the SCN, and review evidence suggesting that diffusible elements may be sufficient to sustain locomotor rhythmicity in adult animals and to restore locomotor rhythmicity in lesioned hamsters bearing SCN grafts. We also provide data for the occurrence of signals that synchronize oscillators, regardless of initial phase. The distinct role of neural and diffusible SCN coupling signals, and the role of SCN-driven rhythmic systems (pineal melatonin rhythms, body temperature), remain to be explored.

Fetal suprachiasmatic nucleus (SCN) tissue transplanted into the third ventricle of hamsters bearing complete SCN lesions restores the circadian locomotor rhythm with a period that depends exclusively on the genetically determined period of the tissue donor. If the host is only partially lesioned and thus retains rhythmicity with its own genetically determined period, an implant from an animal of a different genotype can induce a second rhythm with a period determined by the donor genotype. Both rhythms can be present simultaneously in the record of such a “temporal chimera,” interacting only superficially (i.e., not at the level of the pacemaker). Our data support the interpretation that under such circumstances the graft is able to capture part of the locomotor output of the circadian system, but does not make functional connections with the host SCN pacemaking system.

The aim of the present research was to determine the magnitude and direction of immediate phase shifts of human rhythms following a single exposure to a 3-hr pulse of bright light or physical activity. The pulse of light or activity was presented under “constant-routine” conditions, and measurements of the resultant phase shifts were performed under the same constant-routine conditions on the first day following pulse presentation. Four overt rhythms that are strongly dependent on circadian timing-namely, the rhythms of plasma Cortisol, plasma thyroid-stimulating hormone (TSH), plasma melatonin, and body temperature-were monitored. The analysis of the TSH profiles indicated that exposure to light at about the time of the minimum of body temperature resulted in phase advances averaging less than 1 hr in magnitude. Exposure to light approximately 3 hr before the time of the minimum of body temperature resulted in phase delays of 1–2 hr. Preliminary analyses of the melatonin profiles have confirmed these observations. Our findings regarding the effects of exercise are still inconclusive.