R. E. J. Dyball

Pharmacological Imposition of Sleep Slows Cognitive Decline and Reverses Dysregulation of Circadian Gene Expression in a Transgenic Mouse Model of Huntington's Disease

Transgenic R6/2 mice carrying the Huntingtons disease (HD) mutation show disrupted circadian rhythms that worsen as the disease progresses. By 15 weeks of age, their abnormal circadian behavior mirrors that seen in HD patients and is accompanied by dysregulated clock gene expression in the circadian pacemaker, the suprachiasmatic nucleus (SCN). We found, however, that the electrophysiological output of the SCN assayed in vitro was normal. Furthermore, the endogenous rhythm of circadian gene expression, monitored in vitro by luciferase imaging of organotypical SCN slices removed from mice with disintegrated behavioral rhythms, was also normal. We concluded that abnormal behavioral and molecular circadian rhythms observed in R6/2 mice in vivo arise from dysfunction of brain circuitry afferent to the SCN, rather than from a primary deficiency within the pacemaker itself. Because circadian sleep disruption is deleterious to cognitive function, and cognitive decline is pronounced in R6/2 mice, we tested whether circadian and cognitive disturbances could be reversed by using a sedative drug to impose a daily cycle of sleep in R6/2 mice. Daily treatment with Alprazolam reversed the dysregulated expression of Per2 and also Prok2, an output factor of the SCN that controls behavioral rhythms. It also markedly improved cognitive performance of R6/2 mice in a two-choice visual discrimination task. Together, our data show for the first time that treatments aimed at restoring circadian rhythms may not only slow the cognitive decline that is such a devastating feature of HD but may also improve other circadian gene-regulated functions that are impaired in this disease.

Central Actions of Peptide and Non-Peptide Growth Hormone Secretagogues in the Rat

Evidence for a central site of action of growth-hormone-releasing peptide (GHRP-6) was sought by (1) counting the number of Fos-positive nuclei within the brain following intracerebroventricular or intravenous injection of peptide and non-peptide GH secretagogues and (2) characterizing the electrophysiological responses of neuroendocrine arcuate neurones (recorded in vivo) following intravenous injection of GHRP-6. Conscious male rates were chronically implanted with intracerebroventricular or intravenous catheters. Dense nuclear Fos staining was induced throughout the ventral arcuate nucleus of rats injected intracerebroventricularly with low doses of GHRP-6 but not in rats injected with the endogenous GH-releasing hormone GHRH or in vehicle-treated controls. The non-peptidyl GH secretagogues L-692,585 and L-692,429 also induced Fos expression in the arcuate nucleus, and the pattern of distribution was similar to that described for GHRP-6. No increase in Fos expression was observed in rats given a systemic injection of a high dose of GHRH. In pentobarbitone-anaesthetized male rats, the effects of intravenous injection of GHRP-6 on the electrical activity of arcuate neurones was predominantly excitatory for putative neuroendocrine cells and inhibitory for the remaining unidentified cells. These results suggest that (1) GHRP-6 and non-peptidyl GH secretagogues have a central site of action involving the activation of a subpopulation of arcuate neurones and (2) this action is not mimicked by the central or peripheral effects of GHRH.

Characterization of hypothalamic noradrenaline receptors in the supraoptic nucleus and periventricular region of the paraventricular nucleus of mice in vitro

In an attempt to determine the basis for apparently conflicting reports of the effects of noradrenaline (NA) on the neurohypophyseal system and its effects on the parvocellular periventricular region of the paraventricular nucleus (PVN), recordings were made from the neurons in the supraoptic nucleus (SON) and the periventricular region in the mouse hypothalamic slice preparation. Of 47 SON neurons, 43 (91%) were excited and two (4%) were inhibited by NA. Seven SON neurons increased the firing rate with increase of NA concentration (10(-7)-10(-4) M). Both the alpha 1-agonists phenylephrine and methoxamine also increased the activity of all SON neurons tested whereas application of the alpha 2-agonist clonidine and the beta-agonist isoproterenol had weak and inconsistent effects. While the alpha 2-antagonist yohimbine had no consistent influence, the alpha 1-antagonist prazosin blocked or reversed the effects of NA. Another group of 37 neurons in the periventricular region of the PVN was also tested; 13 (35%) were excited and 22 (59%) inhibited by application of NA (10(-5) M). When tested with phenylephrine or methoxamine, 6 of the 7 neurons were excited and one inhibited but all the 4 neurons tested were excited by isoproterenol. Clonidine strongly depressed the activity of all 12 neurons tested. The NA-induced excitatory effects were suppressed or reversed by pre-application of prazosin and the beta-antagonist propranolol while the inhibitory ones were suppressed or reversed by yohimbine. Synaptic blockade did not affect the excitatory responses of SON cells to NA nor the inhibitory responses of periventricular neurons to NA or clonidine. We conclude that SON neurons receive adrenergic excitatory effects mainly through alpha 1-receptors. The periventricular neurons receive the excitatory effects through alpha 1- or beta-receptors and receive the inhibitory effects through alpha 2-receptors.

Mechanisms of vasopressin secretion

The magnocellular vasopressin system of the rat has been studied intensively in recent years. This review outlines the electrophysiological characteristics of vasopressin neurons, the characteristics of stimulus-secretion coupling in the neural lobe, and describes some of the major features of the neural regulation of this system which underlie physiological regulation of vasopressin release by osmoregulatory stimuli. The major afferent pathways to the magnocellular system are now well characterised. Those involved in osmoregulation have been mapped using expression of the primary response gene c-fos as a marker for neuronal activation.

Synaptic input from the retina to the suprachiasmatic nucleus changes with the light‐dark cycle in the Syrian hamster.

1. Single cell extracellular recordings were made from the suprachiasmatic nucleus (SCN) in urethane‐anaesthetized Syrian hamsters at different times of the light‐dark cycle. Peristimulus time histograms (PSTHs) were created following stimulation of the optic nerve. 2. Both short‐latency (< 50 ms) and long‐latency (> 50 ms) excitatory responses were seen. Almost all inhibitory responses had a short latency. 3. A total of 288 SCN neurones were recorded. Taking all types of response together, 55 (36.9%) of the 149 neurones tested in the dark period responded to optic nerve stimulation while only 23 (16.6%) of the 139 neurones tested in the light period responded. The difference between the proportion of all responsive and non‐responsive neurones in the dark and light periods was highly significant (P < 0.01, Fishers exact probability test). The difference in the proportion of excitatory responses was also significant (P < 0.01). 4. During the dark period, the mean spontaneous firing rate (5.00 +/‐ 0.88 spikes s‐1; mean +/‐ S.E.M., n = 55) of the responsive cells was significantly higher than that of the non‐responsive cells (2.65 +/‐ 0.33 spikes s‐1; mean +/‐ S.E.M., n = 74; P < 0.01; Students unpaired t test). 5. Injection of APV (20 mM, 2 microliters, I.C.V.; n = 6), an antagonist for the NMDA receptor, or CNQX (10 mM, 2 microliters, I.C.V.; n = 5), an antagonist of the non‐NMDA receptor, significantly reduced the responses of all the neurones tested. 6. We conclude that there is daily variation in the firing of SCN neurones in vivo and the variation is restricted to those cells receiving optic nerve inputs. The change in the responsiveness of the SCN to optic nerve stimulation at different times of day suggests that there is a rapidly changing cycle of synaptic function in the SCN. The action of the antagonists suggests that the excitatory retinal projections to the SCN which show this variation are mediated by glutamate and that both NMDA and non‐NMDA receptors are involved.

Leptin modulates spike coding in the rat suprachiasmatic nucleus.

The mammalian circadian pacemaker, the suprachiasmatic nucleus (SCN), contains receptors to the adipose tissue hormone leptin. In the present study, the effects of leptin on the electrophysiological activity of the SCN cells were characterised in vitro in rat brain slices. During extracellular recording, application of 20 nm leptin (n = 36) decreased mean spike frequency (Wilcoxon signed rank test, z = −3.390, P < 0.001) and increased the irregularity of firing measured by the entropy of the log interspike interval distribution (Student’s paired t‐test, t = 2.377, P = 0.023), but had no consistent effect on spike patterning as measured by the mutual information between adjacent log interspike intervals (z = 0.745, P = 0.456). Intracellular current‐clamp recordings (n = 25) revealed a hyperpolarising effect of 20 nm leptin on SCN neurones (z = −2.290, P = 0.022). The hyperpolarisation largely resulted from the effect of leptin on the subgroup of cells (n = 13) that generated ‘rebound’ spikes upon termination of a hyperpolarising current pulse (z = −2.697, P = 0.007). Leptin application also increased the group mean duration of the afterhyperpolarisation (n = 25, t = 2.512, P = 0.023). The effects of leptin on extracellularly recorded spike activity were consistent with the changes in membrane potential and spike shape. They suggest that leptin can directly modulate the electrical properties of SCN neurones and, in this way, contribute to the mechanism by which metabolic processes influence the circadian clock.

Measuring spike coding in the rat supraoptic nucleus

Measuring spike coding objectively is essential to establish whether activity recorded under one set of conditions is truly different from that recorded under another set of conditions. However, there is no generally accepted method for making such comparisons. Measuring firing frequency alone only partially reflects spike patterning. In this paper, novel quantities based on the logarithmic interspike intervals are proposed as useful measures of spontaneous activity. We illustrate the methods by comparing extracellular recordings from magnocellular cells of the rat supraoptic nucleus in vivo and in vitro and between oxytocin and vasopressin cells in vivo. A bimodal Gaussian function fitted to the log interspike interval histogram accurately described the distribution profile for very different types of activity. We introduce the entropy of the log interval distribution as a novel quantity that measures the capacity of a cell to encode information other than a constant instantaneous frequency. Unlike existing entropy measures that are based on spike counts, it quantifies the variability in the interval distribution. In addition, the mutual information between adjacent log intervals is proposed as an objective measure of patterned activity. For cells recorded in vivo and in vitro, there was no significant difference in mean spike frequencies but there were differences in the log interval entropy (t=–4.97, P < 0.001) and the mutual information (z=–2.64, P < 0.01). The differences may result from the disruption of connections in the slice preparation. When a comparison was made between the spike activity of oxytocin and vasopressin cells recorded in vivo, there was a difference in mutual information (z= 5.15, P < 0.001) but not in mean spike frequency. Both comparisons highlight the potential limitations of using mean spike frequency alone as a measure of spike coding. We propose that our novel parameters based on interval analysis constitute informative measures of spontaneous activity under different physiological conditions.

Thermal, osmotic and chemical modulation of neural activity in the paraventricular nucleus: In vitro studies

To investigate the functions of the paraventricular nucleus (PVN) which plays an important role as an integration site for the neuroendocrine and autonomic nervous systems, the firing activity of PVN neurons was recorded from hypothalamic slice preparations during thermal, osmotic and chemical stimulation. Neurons responded to environmental factors such as temperature and osmolarity and both warm-responsive and cold-responsive neurons were observed in the PVN. Some PVN neurons were also osmoresponsive and unlike neurons in the supraoptic nucleus, most osmoresponsive PVN neurons decreased their firing rate during hyperosmotic stimulation. One of the classical transmitters, noradrenaline, exerted excitatory effects on PVN neurons through alpha 1- and beta-receptors and inhibitory responses through alpha 2-receptors. Atrial natriuretic polypeptide exerted inhibitory effects on putative parvocellular PVN neurons but it had no effect on putative magnocellular PVN neurons. An endogenous sugar derivative, 2-deoxytetronic acid, thought to be an endogenous satiety factor, elicited inhibitory effects, supporting the possibility that the PVN also may be related to feeding behaviour. Arginine-vasopressin and oxytocin which are synthesised in the magnocellular neurosecretory cells excited PVN neurons, suggesting that the PVN may have short circuits modulating neural activity within the nucleus itself. We conclude that neurons in the PVN may receive multiple information and act as one of the important integrative sites in the brain.

Assessment of spike activity in the supraoptic nucleus.

Novel approaches to the characterization of coding carried by spike trains are discussed. Measuring firing frequency alone may only partially reflect spike patterning, and can only quantify changes of the most obvious kind. We have devised a method that combines probabilistic and information approaches to quantify the variability of the interspike intervals in a way that is independent of spike frequency. To illustrate the technique, the firing of an oxytocin cell and a vasopressin cell were compared before and after osmotic stimulation. A bimodal lognormal function was fitted to the interspike interval histograms. The entropy of the log interval histogram was used to measure the variability of intervals and to reflect the coding capacity of the cell per spike. A perfect metronome shows no variability in interval and thus has no greater coding capacity than is conveyed by its frequency, whereas the variability of intervals of magnocellular neurones means that their irregular activity has greater potential for coding. While the mean spike frequency increased in both the oxytocin and vasopressin cells in response to osmotic stimulation, the changes in their irregularity showed differences. Osmotic stimulation reduced the entropy of the oxytocin cell, reflecting an increase in the regularity of its spike activity. Conversely, osmotic stimulation had little effect on the entropy of the vasopressin cell. Such differences are not evident from a simple inspection of ratemeter activity. The comparison highlights the limitations of mean spike frequency as a measure of spike coding. Parameters based on the interspike intervals constitute informative measures of spike activity that allow objective comparisons to be made between the activity under different physiological conditions.

Spike propagation and conduction failure in the rat neural lobe.

1. Single units were recorded from the rat hypothalamo‐neurohypophysial system in vivo to test the hypothesis that action potential conduction failure might contribute to the relative inefficiency of neurohypophysial hormone release at low frequencies of stimulation, and following prolonged stimulation. 2. Recordings were made from the cell bodies of supraoptic neurones which project to the neural lobe of the pituitary. Stimuli applied to the neural lobe evoked antidromic action potentials (in ten of forty cells) at times when the axonal membrane at the site of stimulation should have been refractory following the passage of a spontaneous, orthodromically conducted action potential. This observation suggests that failure of orthodromic action potentials may occur intermittently in the neural lobe. 3. Recordings from single units in the neural lobe showed similar spontaneous patterns of activity to those seen from cell bodies in the supraoptic nucleus. 4. Stimuli applied to the neural stalk evoked orthodromically conducted spikes in these single units: evoked spikes followed stimulation faithfully at 50‐80 Hz for 1 s or at 20 Hz for 1 min. Such stimulation was accompanied by a reduction in spike height and a prolongation of latency. 5. Comparable changes were seen in the latency and amplitude of evoked potentials recorded from the neural lobe with low‐resistance electrodes. 6. Stalk stimulation at 50 Hz for 1 s was accompanied by a reduction in the threshold for initiation of action potentials, suggesting an increase in the excitability of neural lobe axons. 7. We conclude that, during low‐frequency activation, spike failure occurs intermittently in neurohypophysial axons, and that changes in the excitability of the axons during activation at high frequencies may contribute to the facilitation of neurohypophysial hormone release that occurs with increasing frequencies of stimulation.