Differential cAMP gating of glutamatergic signaling regulates long-term state changes in the suprachiasmatic circadian clock.

We investigated a role for cAMP/protein kinase A (PKA) in light/glutamate (GLU)-stimulated state changes of the mammalian circadian clock in the suprachiasmatic nucleus (SCN). Nocturnal GLU treatment elevated [cAMP]; however, agonists of cAMP/PKA did not mimic the effects of light/GLU. Coincident activation of cAMP/PKA enhanced GLU-stimulated state changes in early night but blocked light/GLU-induced state changes in the late night, whereas inhibition of cAMP/PKA reversed these effects. These responses are distinct from those mediated by mitogen-activated protein kinase (MAPK). MAPK inhibitors attenuated both GLU-induced state changes. Although GLU induced mPer1 mRNA in both early and late night, inhibition of PKA blocked this event only in early night, suggesting that cellular mechanisms regulating mPer1 are gated by the suprachiasmatic circadian clock. These data support a diametric gating role for cAMP/PKA in light/GLU-induced SCN state changes: cAMP/PKA promotes the effects of light/GLU in early night, but opposes them in late night.

Intrinsic neuronal rhythms in the suprachiasmatic nuclei and their adjustment.

The central role of the suprachiasmatic nuclei in regulating mammalian circadian rhythms is well established. We study the temporal organization of neuronal properties in the suprachiasmatic nucleus (SCN) using a rat hypothalamic brain slice preparation. Electrical properties of single neurons are monitored by extra-cellular and whole-cell patch recording techniques. The ensemble of neurons in the SCN undergoes circadian changes in spontaneous activity, membrane properties and sensitivity to phase adjustment. At any point in this cycle, diversity is observed in individual neurons' electrical properties, including firing rate, firing pattern and response to injected current. Nevertheless, the SCN generate stable, near 24 h oscillations in ensemble neuronal firing rate for at least three days in vitro. The rhythm is sinusoidal, with peak activity, a marker of phase, appearing near midday. In addition to these electrophysiological changes, the SCN undergoes sequential changes in vitro in sensitivities to adjustment. During subjective day, the SCN progresses through periods of sensitivity to cyclic AMP, serotonin, neuropeptide Y, and then to melatonin at dusk. During the subjective night, sensitivities to glutamate, cyclic GMP and then neuropeptide Y are followed by a second period of sensitivity to melatonin at dawn. Because the SCN, when maintained in vitro, is under constant conditions and isolated from afferents, these changes must be generated within the clock in the SCN. The changing sensitivities reflect underlying temporal domains that are characterized by specific sets of biochemical and molecular relationships which occur in an ordered sequence over the circadian cycle.

The organization of the suprachiasmatic circadian pacemaker of the rat and its regulation by neurotransmitters and modulators.

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.

Mesencephalic stimulation elicits inhibition of phrenic nerve activity in cat.

1. Previous work from this laboratory has indicated that the mesencephalon is the anatomical substrate for a mechanism capable of inhibiting central respiratory drive in glomectomized cats for periods of up to 1 h or more following brief exposure to systemic hypoxia; phrenic nerve activity was used as an index of central respiratory drive. 2. The present study was undertaken to further localize the region responsible for the observed post-hypoxic inhibition of respiratory drive. We studied the phrenic nerve response to stimulations of the mesencephalon in anaesthetized, paralysed peripherally chemo-denervated cats with end-expired PCO2 and body temperature servo-controlled. 3. Stimulations of two types were employed. Electrical stimulation allowed rapid determination of sites from which phrenic inhibition could be elicited. Microinjections of excitatory amino acids were used subsequently in order to confine excitation to neuronal cell bodies and not axons of passage. 4. Stimulation of discrete regions of the ventromedial aspect of the mesencephalon in the vicinity of the red nucleus produced substantial inhibition of phrenic activity which lasted up to 45 min. Stimulation of other areas of the mesencephalon either produced no phrenic inhibition or resulted in a slight stimulation of phrenic activity. 5. The results are discussed in the context of the central respiratory response to hypoxia.

Cellular and molecular mechanisms of chemical synaptic transmission.

During the last decade much progress has been made in understanding the cellular and molecular mechanisms by which nerve cells communicate with each other and nonneural (e.g., muscle) target tissue. This review is intended to provide the reader with an account of this work. We begin with an historical overview of research on cell-to-cell communication and then discuss recent developments that, in some instances, have led to dramatic changes in the concept of synaptic transmission. For instance, the finding that single neurons often contain multiple messengers (i.e., neurotransmitters) invalidated the long-held theory (i.e., Dale's Law) that individual neurons contain and release one and only one type of neurotransmitter. Moreover, the last decade witnessed the inclusion of an entire group of compounds, the neuropeptides, as messenger molecules. Enormous progress has also been made in elucidating postsynaptic receptor complexes and biochemical intermediaries involved in synaptic transmission. Here the development of recombinant DNA technology has made it possible to clone and determine the molecular structure for a number of receptors. This information has been used to gain insight into how these receptors function either as a ligand-gated channel or as a G protein-linked ligand recognition molecule. Perhaps the most progress made during this era was in understanding the molecular linkage of G protein-linked receptors to intramembranous and cytoplasmic macromolecules involved in signal amplification and transduction. We conclude with a brief discussion of how synaptic transmission leads to immediate alterations in the electrical activity and, in some cases, to a change in phenotype by altering gene expression. These alterations in cellular behavior are believed to be mediated by phosphoproteins, the final biochemical product of signal transduction.

Two long-lasting central respiratory responses following acute hypoxia in glomectomized cats.

1. Central respiratory response to acute (10 min) hypoxia, as measured by phrenic nerve activity, was determined in peripheral chemo-denervated cats. 2. Hypoxia was induced by ventilating cats for 10 min at reduced inspired oxygen levels (inspired O2 fraction, FI,O2 = 0.06-0.15). The degree of hypoxaemia was determined from an arterial blood sample and ranged from 'severe' (arterial O2 pressure, Pa,O2 less than 26 Torr) to 'mild' (Pa,O2 greater than 35 Torr). The respiratory response was monitored for 1 h following return to ventilation with 100% oxygen. 3. The results confirmed the finding of prolonged (greater than 60 min) inhibition of respiration upon return to hyperoxic conditions following severe hypoxia, as reported previously (Millhorn, Eldridge, Kiley & Waldrop, 1984). A new finding was a long-lasting (greater than 60 min) facilitation of respiration following exposure to less severe (Pa,O2 greater than 35 Torr) hypoxia. 4. Medullary extracellular fluid pH was measured in six cats. Changes in pH could not explain either the prolonged inhibition following severe hypoxia or the long-lasting facilitation observed following mild hypoxia. 5. Ablation studies were performed in order to determine the locations of the neuronal substrates for the inhibitory and facilitatory mechanisms. The results of this series of experiments indicate that the mesencephalon is necessary for activation of the inhibitory mechanism, while the facilitatory mechanism requires the presence of higher brain structures, notably the diencephalon. 6. Following removal of the diencephalon, the inhibitory response was seen following even mild hypoxic insults, i.e. those shown to produce facilitation in animals with intact brains. In the absence of the mesencephalon, neither prolonged inhibition nor prolonged facilitation could be produced following hypoxia. 7. It is proposed that there are two centrally mediated long-lasting responses to acute hypoxia. Facilitation is seen following mild hypoxia. Inhibition is more likely following severe hypoxia. However, both mechanisms appear to be triggered simultaneously and the output of the central respiratory controller reflects the influence of each.

Progesterone stimulates respiration through a central nervous system steroid receptor-mediated mechanism in cat.

We have examined the effect on respiration of the steroid hormone progesterone, administered either intravenously or directly into the medulla oblongata in anesthetized and paralyzed male and female cats. The carotid sinus and vagus nerves were cut, and end-tidal PCO2 and temperature were kept constant with servo-controllers. Phrenic nerve activity was used to quantitate central respiratory activity. Repeated doses of progesterone (from 0.1 to 2.0 micrograms/kg, cumulative) caused a sustained (greater than 45 min) facilitation of phrenic nerve activity in female and male cats; however, the response was much more variable in females. Progesterone injected into the region of nucleus tractus solitarii, a respiratory-related area in the medulla oblongata, also caused a prolonged stimulation of respiration. Progesterone administration at high concentration by both routes also caused a substantial hypotension. Identical i.v. doses of other classes of steroid hormones (17 beta-estradiol, testosterone, and cortisol) did not elicit the same respiratory effect. Pretreatment with RU 486, a progesterone-receptor antagonist, blocked the facilitatory effect of progesterone. We conclude that progesterone acts centrally through a steroid receptor-mediated mechanism to facilitate respiration.