Christopher S. Colwell

Vasoactive intestinal polypeptide mediates circadian rhythmicity and synchrony in mammalian clock neurons

The mammalian suprachiasmatic nucleus (SCN) is a master circadian pacemaker. It is not known which SCN neurons are autonomous pacemakers or how they synchronize their daily firing rhythms to coordinate circadian behavior. Vasoactive intestinal polypeptide (VIP) and the VIP receptor VPAC2 (encoded by the gene Vipr2) may mediate rhythms in individual SCN neurons, synchrony between neurons, or both. We found that Vip−/− and Vipr2−/− mice showed two daily bouts of activity in a skeleton photoperiod and multiple circadian periods in constant darkness. Loss of VIP or VPAC2 also abolished circadian firing rhythms in approximately half of all SCN neurons and disrupted synchrony between rhythmic neurons. Critically, daily application of a VPAC2 agonist restored rhythmicity and synchrony to VIP−/− SCN neurons, but not to Vipr2−/− neurons. We conclude that VIP coordinates daily rhythms in the SCN and behavior by synchronizing a small population of pacemaking neurons and maintaining rhythmicity in a larger subset of neurons.

Linking neural activity and molecular oscillations in the SCN

Neurons in the suprachiasmatic nucleus (SCN) function as part of a central timing circuit that drives daily changes in our behaviour and underlying physiology. A hallmark feature of SCN neuronal populations is that they are mostly electrically silent during the night, start to fire action potentials near dawn and then continue to generate action potentials with a slow and steady pace all day long. Sets of currents are responsible for this daily rhythm, with the strongest evidence for persistent Na+ currents, L-type Ca2+ currents, hyperpolarization-activated currents (IH), large-conductance Ca2+ activated K+ (BK) currents and fast delayed rectifier (FDR) K+ currents. These rhythms in electrical activity are crucial for the function of the circadian timing system, including the expression of clock genes, and decline with ageing and disease. This article reviews our current understanding of the ionic and molecular mechanisms that drive the rhythmic firing patterns in the SCN.

Circadian modulation of learning and memory in fear-conditioned mice.

Endogenous processes referred to as circadian oscillators generate many of the daily rhythms in physiology and behavior of a variety of animals including humans. We investigated the possible circadian regulation of acquisition, recall and extinction in two strains of mice (C-57/6J and C-3H). Mice were trained in either the day or night with a tone and context fear conditioning protocol. The mice were then tested over the course of several days for their ability to recall the training. When comparing the performance of animals in the day and night, the mice acquired the conditioning faster in the day than in the night. Furthermore, the recall for context and tone consistently peaked during the day for at least 3 days after training, irrespective of the time of training. Finally, the loss of this training (or extinction) exhibited a rhythm in that mice trained in night exhibited a greater degree of extinction than mice trained in the day. For all of these rhythms in acquisition, recall, and extinction the phase of the rhythm was controlled by the prior light-dark (LD) cycle. When we reversed the phase of the LD cycle, the phase of the rhythm also reversed. Importantly, all three of the rhythms also continued in constant darkness demonstrating the endogenous, and presumably circadian nature, of the rhythms.

NMDA as Well as Non-NMDA Receptor Antagonists Can Prevent the Phase-Shifting Effects of Light on the Circadian System of the Golden Hamster

The present experiments were designed to evaluate whether the intraventricular administration of excitatory amino acid (EAA) receptor antagonists would prevent light-induced phase shifts of the circadian rhythm of wheel-running activity in the hamster. Administration of the non-N-methyl-D-aspartate (non-NMDA) antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) blocked light-induced phase advances and delays. Similarly, administration of the competitive NMDA receptor antagonist, 3(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), prevented light-induced phase advances and delays. Neither drug by itself caused any consistent effect on the phase of the rhythm. These data provide further evidence that EAA receptors mediate the effects of light on the circadian system, and suggest that both NMDA and non-NMDA receptor types may be involved.

Age-Related Decline in Circadian Output

Disruptions in sleep/wake cycles, including decreased amplitude of rhythmic behaviors and fragmentation of the sleep episodes, are commonly associated with aging in humans and other mammals. While there are undoubtedly many factors contributing to these changes, a body of literature is emerging, suggesting that an age-related decline in the central circadian clock in the suprachiasmatic nucleus (SCN) may be a key element responsible. To explore age-related changes in the SCN, we have performed in vivo multiunit neural activity (MUA) recordings from the SCN of freely moving young (3–5 months) and middle-aged (13–18 months) mice. Importantly, the amplitude of day–night difference in MUA was significantly reduced in the older mice. We also found that the neural activity rhythms are clearly degraded in the subparaventricular zone, one of the main neural outputs of the SCN. Surprisingly, parallel studies indicate that the molecular clockwork in the SCN as measured by PER2 exhibited only minor deficits at this same age. Thus, the circadian output measured at the level of neural activity rhythms in the SCN is degraded by aging, and this decline occurs before the disruption of key components of the molecular clockwork.

Circadian Regulation of Hippocampal Long-Term Potentiation

The goal of this study is to investigate the possible circadian regulation of hippocampal excitability and long-term potentiation (LTP) measured by stimulating the Schaffer collaterals (SC) and recording the field excitatory postsynaptic potential (fEPSP) from the CA1 dendritic layer or the population spike (PS) from the soma in brain slices of C3H and C57 mice. These 2 strains of mice were of interest because the C3H mice secrete melatonin rhythmically while the C57 mice do not. The authors found that the magnitude of the enhancement of the PS was significantly greater in LTP recorded from night slices compared to day slices of both C3H and C57 mice. They also found significant diurnal variation in the decay of LTP measured with fEPSPs, with the decay slower during the night in both strains of mice. There was evidence for a diurnal rhythm in the input/output function of pyramidal neurons measured at the soma in C57 but not C3H mice. Furthermore, LTP in the PS, measured in slices prepared during the day but recorded during the night, had a profile remarkably similar to the night group. Finally, PS recordings were carried out in slices from C3H mice maintained in constant darkness prior to experimentation. Again, the authors found that the magnitude of the enhancement of the PS was significantly greater in LTP recorded from subjective night slices compared to subjective day slices. These results provide the 1st evidence that an endogenous circadian oscillator modulates synaptic plasticity in the hippocampus.

Excitatory synaptic transmission in neostriatal neurons: regulation by cyclic AMP-dependent mechanisms

The purpose of the present study was to examine whether cAMP-dependent mechanisms regulated excitatory synaptic transmission in the neostriatum. A brain slice preparation was utilized for intracellular recordings of the excitatory postsynaptic potentials (EPSPs) evoked by electrical stimulation. Bath application of forskolin, an activator of adenylate cyclase, enhanced the EPSP amplitude and duration. This potentiation was dose dependent and did not occur with the inactive analog 1,9-dideoxyforskolin. Forskolin potentiation was unaltered by treatment with the GABAA receptor antagonist bicuculline. Furthermore, two inhibitors of cAMP-dependent protein kinase (PKA), Rp-cAMPS and IP20-amide, attenuated forskolins enhancement of the EPSP. In addition, the PKA activator Sp-cAMPS enhanced excitatory synaptic transmission. Interestingly, treatment with PKA inhibitors alone depressed while the phosphatase inhibitor okadaic acid enhanced the synaptic response. These results suggest a role for tonic kinase and phosphatase activity in regulating excitatory synaptic transmission in the neostriatum. Finally, forskolin was found to enhance the responses of neostriatal neurons to glutamate receptor agonists. This potentiation, which occurred in the presence of tetrodotoxin, provides at leas part of the explanation for the cAMP/PKA-dependent regulation of the EPSP. Overall, these results suggest a role for the adenylate cyclase cascade in the regulation of excitatory synaptic transmission in the neostriatum.

Circadian modulation of calcium levels in cells in the suprachiasmatic nucleus.

There is reason to believe that resting free calcium concentration [Ca2+]i in neurons in the suprachiasmatic nucleus (SCN) may vary with the circadian cycle. In order to start to examine this hypothesis, optical techniques were utilized to estimate resting Ca2+ levels in SCN cells in a rat brain slice preparation. [Ca2+]i measured from the soma was significantly higher in the day than in the night. Animals from a reversed light–dark cycle were used to confirm that the phase of the rhythm was determined by the prior light–dark cycle. The rhythm in Ca2+ levels continued to be expressed in tissue collected from animals maintained in constant darkness, thus confirming the endogenous nature of this variation. Interestingly, the rhythm in Ca2+ levels was not observed when animals were housed in constant light. Finally, the rhythm in Ca2+ levels was prevented when slices were exposed to tetrodotoxin (TTX), a blocker of voltage‐sensitive sodium channels. Similar results were obtained with the voltage‐sensitive Ca2+ channel blocker methoxyverapamil. These observations suggest a critical role for membrane events in driving the observed rhythm in Ca2+. Conceptually, this rhythm can be thought of as an output of the circadian oscillator. Because [Ca2+]i is known to play a critical role in many cellular processes, the presence of this rhythm is likely to have many implications for the cell biology of SCN neurons.

Do NMDA receptors mediate the effects of light on circadian behavior

We report here the results of experiments designed to evaluate whether a specific NMDA receptor antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]cyclohepten-5,10-imine maleate (MK-801), blocks the phase shifting effects of light on the circadian rhythm of wheel-running activity in golden hamsters. Intraperitoneal administration of (+)-MK-801 produced a dose-dependent blockade of both light-induced phase advances and delays. The effect was stereoselective and treatment with related compounds, phenylcyclidine and ketamine, also blocked light-induced phase shifts. MK-801, by itself, did not cause any consistent effect on the phase of the rhythm. These data, coupled with previous findings, indicate that excitatory amino acid receptors play an important role in the transmission of light information from the retina to the circadian system.

NMDA receptor antagonists block the effects of light on circadian behavior in the mouse

We report here the results of experiments designed to evaluate whether NMDA receptors mediate the phase shifting effects of light on the circadian rhythm of wheel-running activity in mice. Intraperitoneal administration of either the non-competitive NMDA receptor antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]cyclohepten-5,10-imine maleate (MK-801), or the competitive NMDA receptor antagonist, 3(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) blocked light-induced phase advances and delays. Neither drug, by itself, caused any consistent effect on the phase of the rhythm. Furthermore, there was no significant difference between the effects of MK-801 on light-induced phase shifts in a retinally degenerate and retinally normal strain of C57 mouse. These data, coupled with previous findings, indicate that excitatory amino acid receptors play an important role in the transmission of light information from the retina to the circadian system.