What really happens in the SCN at night.
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Biomedical subjects
R E Dyball
Publications and source records attributed to R E Dyball.
Neural connections of hypothalamic neuroendocrine nuclei in the rat.
The secretion of many hormones, including oxytocin, vasopressin and growth hormone, is not constant but shows a day-night rhythm. The suprachiasmatic nucleus (SCN) is thought to generate most mammalian biological rhythms and previous studies have reported suprachiasmatic efferents to the paraventricular nucleus (PVN) and the supraoptic nucleus (SON). We used in vivo extracellular electrophysiological techniques to show that the SCN also sends direct and indirect neural projections to the arcuate nucleus (ARC). This projection consisted of both excitatory and inhibitory components and may contribute to the entrainment of the rhythm in growth hormone secretion to the day-night cycle. Some SCN neurones appear to project to both the SON and the ARC. The SCN in turn receives excitatory and inhibitory inputs from the ARC and the peri-nuclear zone of the SON (peri-SON), which may provide feedback information, as well as allowing nonphotic entrainment of the SCN, for example, in response to feeding. Our data thus suggest extensive two-way connections between the SCN and its target nuclei which may contribute to the generation of day-night neuroendocrine rhythms. They also suggest the existence of indirect retinal projections to the ARC and PVN. We further investigated the retinal projection to the SCN. We were unable to demonstrate a significant difference in retinal input to those suprachiasmatic cells which had efferent projections to particular hypothalamic targets (SON and/or ARC), and those which did not.
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Synchronized clusters of action potentials can increase or decrease the excitability of the axons of magnocellular hypothalamic neurosecretory cells.
Extracellular recordings were made from supraoptic nucleus (SON) cells in urethane anaesthetized male rats in vivo. Two stimulating electrodes were positioned to activate the cells antidromically, one in the mid axon region of the cells and the other at the axon terminals. Trains of 5-20 just-subthreshold stimuli at 5 s intervals decreased the threshold for antidromic activation from both sites. Whereas neither single stimuli, nor the stimuli at the beginning of a train of 20 stimuli evoked antidromic action potentials, later action potentials did so. Paradoxically, trains of 20 just-suprathreshold stimuli increased the threshold for activation of both axons and terminals. In recordings from the same cells, stimuli were applied singly at 5 s intervals at an intensity which almost invariably evoked an antidromic action potential. Identical stimuli were then applied in trains of 20 stimuli at 50 Hz. After the first train, the initial stimulus pulses of the trains frequently fell below threshold. Following a conditioning train of five stimuli applied to one electrode, the period of decreased threshold (increased excitability) at the other electrode lasted less than 100 ms and the period of increased threshold (decreased excitability) after 12 trains of 20 stimuli lasted between 5 and 10 s. Both decreased and increased excitability were seen at axons and terminals of both putative oxytocin and vasopressin cells. Since the excitability changes were shown in vivo at frequencies encountered during recordings, it is likely that they influence the probability of spike propagation and hormone secretion under physiological conditions.
Action Potentials↗
GHRP-6-induced changes in electrical activity of single cells in the arcuate, ventromedial and periventricular nucleus neurones [correction of nuclei] of a hypothalamic slice preparation in vitro.
Previously, we demonstrated that systemic injection of the growth hormone secretagogue, growth hormone-releasing peptide (GHRP)-6, selectively activated cells in the hypothalamic arcuate nucleus, as reflected by increased electrical activity and induction of the immediate early gene c-fos. The growth hormone secretagogue receptor distribution is not confined to the arcuate nucleus, suggesting that additional sites of action may exist. In the present study we characterized the electrophysiological responses of cells in the arcuate nucleus, ventromedial nucleus and periventricular nucleus in an in-vitro hypothalamic slice preparation, following bath application of GHRP-6. Additionally, since central somatostatin administration has been shown to attenuate the induction of the c-fos gene by GHRP-6, we sought to determine whether the arcuate cells activated by GHRP-6 are also somatostatin-sensitive. Male Wistar rats (100-150 g body weight (BW)) were anaesthetized (urethane; 1.2 g/kg BW) and the brains removed. Coronal sections (400 microm thickness) were cut through a block of hypothalamus and were transferred to a slice chamber perfused with artificial cerebrospinal fluid. Forty-one arcuate nucleus cells were tested with bath application of 15 microm GHRP-6 for 10 min, 16 of which were tested subsequently (>30 min later) with application of 10 microM somatostatin. Following GHRP-6 administration, 19 cells (46. 3%) showed a significant increase in firing rate during the 15-min period after GHRP-6 application (P<0.001), 17 cells (41.5%) did not respond and the remaining five cells (12.2%) were significantly inhibited. Six of the eight arcuate nucleus cells that were excited by GHRP-6 were significantly inhibited by somatostatin. By contrast, five of the six arcuate nucleus cells that were unresponsive to GHRP-6 were also unresponsive to somatostatin. In the ventromedial nucleus, of 19 cells tested, eight cells (42.1%) were excited by GHRP-6, eight cells (42.1%) were unresponsive and the remaining three cells (15.8%) were significantly inhibited. Of 19 cells recorded in the periventricular nucleus, 13 (68.4%) were unresponsive to GHRP-6 and six (31.6%) were significantly inhibited. Thus, electrophysiological studies in vitro suggest that: (1) neurones in the hypothalamic arcuate nucleus, ventromedial nucleus and periventricular nucleus show changes in electrical activity in response to GHRP-6; and (2) the arcuate nucleus cells excited by GHRP-6 are also subject to inhibitory control by somatostatin.
Action Potentials↗
Neurones in the supraoptic nucleus of the rat are regulated by a projection from the suprachiasmatic nucleus.
1. In the rat, projections from the suprachiasmatic nucleus (SCN) to the supraoptic nucleus (SON) of the hypothalamus were characterized in vivo using extracellular recordings and in slice preparations using both extracellular and whole-cell patch clamp recording. 2. Of 117 magnocellular neurones recorded in the SON in vivo, fifteen (13%) displayed a short latency excitation, sixty-eight (58%) a short latency inhibition, six (5%) were unresponsive and twenty-eight (24%) gave long latency responses following SCN stimulation. 3. The responses of putative vasopressin cells in the SON to SCN stimulation in vivo (4 out of 61 cells, 7% excited; 49 out of 61 cells, 80% inhibited) were significantly different from those of putative oxytocin cells (10 out of 50 cells, 20% excited and 16 out of 50 cells, 32% inhibited; P < 0.02, test for differences between proportions). 4. Recordings in vitro using patch technology in whole-cell mode showed both inward and outward currents in SON cells at holding potentials near resting membrane potential following stimulation of the SCN region. The outward currents could be blocked by bicuculline (10 microM; n = 7) and the inward currents were blocked by the non-NMDA antagonist 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2,3-dione (5 microM; n = 4). 5. We conclude that there is a strong projection from the SCN to the SON with both inhibitory (GABAergic) and excitatory (glutamatergic) components which may regulate the daily changes in neurohypophysial hormone secretion.
2-Amino-5-phosphonovalerate↗
Electrophysiological evidence for retinal projections to the hypothalamic supraoptic nucleus and its perinuclear zone.
Secretion of vasopressin (VP) and oxytocin (OT) displays a daily rhythm. Using electrophysiological methods, we investigated the projections from the optic nerve to the supraoptic nucleus (SON) and its perinuclear zone (PNZ) which might underlie the rhythm. Extracellular recordings were made from magnocellular cells in the SON and its PNZ in 22 urethane-anaesthetized female Wistar rats while stimulating the optic nerve. The responses of magnocellular and PNZ cells were classified as orthodromic excitatory (OD+) or inhibitory (OD-) after creating peri-stimulus time histograms (PSTHs). Twenty-six of 73 (35.6%) VP and OT cells and 16 of 42 (38.1%) PNZ cells were excited by optic nerve stimulation. PNZ cells displayed both short (for 7 cells 30 ms or less) and long (> 60 ms) latency responses. Most (6/7) short latency responses had a short duration but longer latency responses were longer. No magnocellular cells showed responses with both short latency and short duration. Short latency responses with a short duration probably reflect direct monosynaptic inputs whereas longer latency responses with longer duration may reflect complex inputs. Thus the retina projects to the PNZ and to the SON but the PNZ receives a stronger direct input. Such projections might provide a light-related input to SON cells and suggest a role for the PNZ in this input.
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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, Fisher's 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; Student's 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.
2-Amino-5-phosphonovalerate↗
Retrogradely labelled neurosecretory neurones of the rat hypothalamic arcuate nucleus express Fos protein following systemic injection of GH-releasing peptide-6.
Previously, we demonstrated that the synthetic hexapeptide GH-releasing peptide (GHRP-6) activates a subpopulation of arcuate neurones, as reflected by increased electrical activation and by the detection of Fos protein in cell nuclei. Here we set out to determine (1) what proportion of the cells activated by GHRP-6 are neurosecretory neurones and (2) whether the cells activated by GHRP-6 contain tyrosine hydroxylase (TH; a marker of dopaminergic cells in this region) or beta-endorphin. In the first study, adult male rats were injected i.v. with the retrograde tracer, Fluorogold, to detect cells which project outside the blood-brain barrier (and are therefore likely to be neurosecretory neurones). Three days later the conscious rats were injected i.v. with 50 micrograms GHRP-6 and the brains processed for the immunocytochemical detection of Fos protein. Between 68% and 82% of the arcuate neurones expressing Fos protein following GHRP-6 injection were retrogradely labelled with Fluorogold. In the second study, conscious male rats, bearing a chronically implanted jugular catheter, were killed 90 min following an i.v. injection of 50 micrograms GHRP-6 and the brains were processed for the double immunocytochemical detection of Fos protein and either TH or beta-endorphin. Less than 7% (mean +/- S.E.M. = 6.7 +/- 2.6% nuclei/section per rat) of the arcuate neurones expressing Fos protein following GHRP-6 injection were TH-containing cells. Of 143 beta-endorphin-containing arcuate cells detected only four cells were identified as containing Fos protein. Thus, the majority of arcuate neurones activated by GHRP-6 (1) project outside the blood-brain barrier (and are therefore likely to be neuro-secretory neurones) and (2) were not identified as TH- or beta-endorphin-containing cells.
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Electrophysiological evidence for mutual excitation of oxytocin cells in the supraoptic nucleus of the rat hypothalamus.
1. Using the ventral surgical approach in vivo, extracellular recordings were made from seventy-nine cells in the supraoptic nucleus of urethane-anaesthetized male, virgin female or lactating female rats while stimulating the pituitary stalk. Cells were classed according to their spontaneous firing activity as: continuous (putative oxytocin), phasic (putative vasopressin) and silent. 2. Stimulation of the neural stalk produced an excitation (up to 25 ms poststimulus) in eleven of the seventy-nine antidromically identified magnocellular neurones, consistent with the existence of excitatory collaterals or dendritic contacts between such cells. In these recordings a second spike could frequently be seen, following the antidromic spike, with a variable latency. Such spikes consistently collided with subsequent antidromically evoked spikes. Poststimulus excitation was only seen in silent and continuously firing (putative oxytocin) cells, suggesting that oxytocin and vasopressin cells have different connections. 3. Excitatory connections were seen more frequently in lactating females (8 out of 22 cells) than in males (1 out of 15 cells) or virgin females (2 out of 10 cells), and thus may make an important contribution to the bursts of firing which precede reflex milk ejection.
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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.
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Osmoresponsiveness of the rat supraoptic nucleus in vivo depends on glutamatergic inputs.
Intracellular recording from supraoptic nucleus (SON) neurones in hypothalamic slices revealed that a 40 mOsm change in osmolality had little effect on membrane conductance. However, intracellular recordings in vivo revealed a significant increase in EPSP frequency after the plasma osmolality had been raised by approximately 10 mOsM. We recorded extracellularly in vivo from 18 antidromically identified SON neurones in urethane-anaesthetised male Wistar rats, while hypertonic saline was infused intravenously (1.05M NaCl, 50 microliters/min for 35 minutes). In 9 experiments an intracerebroventricular (ICV) injection of 5 microliters 10mM kynurenic acid was given 5 minutes prior to start of the infusion. Kynurenate significantly reduced the osmotic response (P < 0.001, Student's t-test); the rat of increase in spike frequency was reduced from 0.089 +/- 0.004 Hz/min (n = 9) to 0.035 +/- 0.003 Hz/min (n = 9). Kynurenic acid did not reduce the basal firing rate of SON neurones, but in osmotically stimulated neurones, it reduced the firing rate to basal levels. Hence the osmoresponsiveness of SON neurones depends on a glutamatergic input that is independent of mechanisms that maintain basal electrical activity.
Action Potentials↗
Induction of c-fos expression in hypothalamic magnocellular neurons requires synaptic activation and not simply increased spike activity.
Magnocellular neurons of the hypothalamic supraoptic nucleus have been shown to express the immediate-early gene c-fos in a number of experimental and physiological circumstances. In each case the induction of the immediate-early gene followed the increase in the spike activity of the cells. Since an increase in the intracellular concentration of calcium following influx through voltage-sensitive calcium channels is a known stimulus for c-fos expression and since the action potentials of these neurons have a large calcium component, we hypothesized that c-fos induction in these neurons could be attributed to calcium influx during spike activity. In the present experiments we use extracellular recording and immunocytochemistry for Fos, the protein product of c-fos, to demonstrate the activation of the cells following intracerebroventricular administration of the muscarinic agonist, carbachol. Fos expression following carbachol injection was then compared with that induced by a similar number of antidromically evoked action potentials. Antidromic activation, unlike the activation induced by carbachol, did not lead to the induction of Fos. We conclude that Fos induction in these neurons requires receptor activation rather than spike activity.
Action Potentials↗
Induction of c-fos in magnocellular neurosecretory neurons. A link between electrical activity and peptide synthesis?
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Interaction between magnocellular neurons and cells of the perinuclear zone involves NMDA receptors.
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Inputs from the nucleus tractus solitarii to the magnocellular neurosecretory system.
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Naloxone potentiates the release of oxytocin induced by systemic administration of cholecystokinin without enhancing the electrical activity of supraoptic oxytocin neurones.
Studies performed in conscious female rats confirmed that iv injection of cholecystokinin octapeptide (CCK; 20 mu/kg) increased the circulating concentration of oxytocin but not that of vasopressin, and confirmed that the stimulation of oxytocin release was markedly facilitated after iv administration of naloxone (1 mg/kg), indicating attenuation of oxytocin release by endogenous opioids. To investigate the site of action of the endogenous opioids, the electrical activity of putative oxytocin neurones in the supraoptic nucleus was recorded in urethane-anaesthetised female rats. Oxytocin neurones responded to CCK injection with an increase in firing rate lasting 5-15 min, but this response was not facilitated by prior injection of naloxone. The results suggest that the opioid influence upon CCK-induced oxytocin release operates at the level of the neurosecretory terminals in the neurohypophysis rather than centrally. Since CCK does not elevate vasopressin release, it appears unlikely that dynorphin, the opioid peptide co-existing with vasopressin, is responsible in these circumstances for the cross-inhibition of oxytocin release. It is suggested that products of proenkephalin A, the met-enkephalin precursor present in the supraoptic nucleus and in the neurohypophysis itself, may be active in the regulation of oxytocin release.
Anesthesia↗
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.
Afferent Pathways↗
Expansion of plasma volume by intragastric isotonic saline inhibits supraoptic neurones in rats.
To determine whether an increase in plasma volume might directly influence supraoptic neurones, single cell extracellular recordings were made from magnocellular neurones of the supraoptic nucleus in urethane-anaesthetized rats as plasma volume was expanded by intragastric injection of isotonic saline. Continuous ratemeter records taken before, during and after intragastric injections of 10 ml isotonic saline showed that the firing rate of putative vasopressin cells was reduced by 2.21 spikes/s (P < 0.02; n = 9; paired t-test) after 50 min. Putative oxytocin cells, after an initial increase in firing rate which lasted approximately 30 min, showed a decrease of 0.98 spikes/s (P < 0.02; n = 6; paired t-test). A population of 93 control cells of both types had a median firing rate of 4.69 spikes/s, a comparable group of 65 cells recorded 1 h after intragastric injection had a median firing rate of 3.15 spikes/s and another group of 68 cells recorded 1 h after a second injection had a median firing rate of 2.5 spikes/s. These differences were significant (P < 0.04 and P < 0.01; Mann-Whitney U test). The haematocrit of plasma samples taken from five similarly anaesthetized control animals was 49.7%. One hour after one intragastric injection the value was significantly (P < 0.02; paired t-test) reduced to 46.7% and 1 h after a second injection it was further reduced to 42.1% (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)
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