Neural Stalk Research Articles

Increased Sensitivity of Monoamine Release in the Supraoptic Nucleus in Late Pregnancy: Region‐ and Stimulus‐Dependent Responses

Oxytocin neurone activation at birth depends upon noradrenaline-mediated signals from the uterus via a brainstem pathway, as well as on factors within the supraoptic nucleus (SON), including oxytocin itself, and the system adapts during pregnancy to optimise the delivery process. We determined whether noradrenaline release in the SON in response to stimuli activating brainstem inputs or antidromically activating magnocellular neurones is enhanced at term pregnancy. Noradrenaline, serotonin and dopamine concentrations were measured in microdialysis samples collected from the dorsal and ventral SON before, during and after either i.v. cholecystokinin (CCK) or neural stalk stimulation in virgin and late pregnant rats. Each stimulus transiently increased noradrenaline and serotonin but not dopamine concentration in the dorsal SON, and responses were increased on days 21 and 22 of pregnancy compared to day 20 pregnant and virgin rats. Neural stalk stimulation induced sensitisation to subsequent stalk stimulation and so the responses in the dorsal SON were doubled; on day 22 of pregnancy, the area under the curve of monoamine concentration was 3.4-fold greater than in virgins, suggesting that adaptations perinatally enhance responsiveness. In conclusion, there are enhanced responses of noradrenaline and serotonin release in the SON that can generate very high, transient extracellular concentrations at term. This may be a consequence of neuroendocrine adaptations in late pregnancy and probably contributes to optimal oxytocin neurone activation during parturition.

Secretory cells of the supraoptic nucleus have central as well as neurohypophysial projections

Conventional neuroanatomical methods may fail to demonstrate the presence of axons that are finer than 1 microm in diameter because such processes are near or below the limit of resolution of the light microscope. The presence of such axons can, however, be readily demonstrated by recording. The most easily interpreted type of recording for this purpose is the demonstration of antidromic activation of the cell body following stimulation of the region through which the axon passes. We have exploited this technique in the hypothalamus and have demonstrated the presence of double axonal projections or axons branching very near the cell bodies of the secretory cells of the neurohypophysial system in the rat supraoptic nucleus. We found that a small proportion of supraoptic magnocellular cells could be antidromically activated both from the neural stalk and from elsewhere in the hypothalamus, including the suprachiasmatic nucleus (8 cells of a total of 182) and the antero-ventral third ventricular region (AV3V; 4 of 182 cells) near the organum vasculosum of the lamina terminalis (OVLT). Collision of antidromic and orthodromic spikes showed that the cells were clearly antidromically (rather than synaptically, or orthodromically) activated from both sites. A stimulus applied to one of the axons prevented propagation of a spike evoked by a pulse delivered to the other axon until sufficient time had elapsed after the first stimulus for the resultant spike to have propagated from the first stimulus site along one cell process (towards the cell body or branch point), and from this point along the other axonal branch to the second stimulus site (there was also a short additional delay period during which the axon at the site of the second stimulus recovered from its absolute refractory period). If the interval between the stimuli was progressively reduced, there came a point where the second spike failed. Such a clear demonstration of dual projections in a system where the cells were previously thought to have only a single axon raises the possibility that many nerve cells in the CNS have previously unsuspected projections.

Regulation of neuronal nitric oxide synthase mRNA expression in the rat magnocellular neurosecretory system

We examined the activation of nNOS mRNA expression within the supraoptic and paraventricular nuclei (SON and PVN) of the hypothalamus. In salt-loaded rats nNOS mRNA expression was significantly increased in both nuclei. In rats given i.p. injections of 1.5 M NaCl (4 ml/kg), a small but significant increase in nNOS mRNA expression in the SON and PVN was found 6 h after injection; no change was detected 2 or 4 h after injection. In rats in which hyponatraemia had been induced experimentally, nNOS mRNA was downregulated in the SON, and expression levels were not increased within 4 h after intense acute osmotic stimuli. Finally, neurons of the SON were antidromically-activated by neural stalk stimulation for 2 h. No increase of nNOS mRNA expression in the SON was observed 2 h after stimulation. Thus, increased electrical activity is not directly coupled to rapidly increased expression of nNOS mRNA, and hence acute increases in nNOS mRNA expression are unlikely to play a role in short-term adaptation of the magnocellular system to osmotic stimulation.

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.

Connections from the subfornical organ to the oxytocin and vasopressin systems in the lactating rat. A study using electrical stimulations, lesions and electrophysiological recordings

The medial septal area has been implicated in the control of the magnocellular neurosecretory cells of the hypothalamus, and in particular, in the regulation of neurons secreting oxytocin. The present study investigated the hypothesis that this medial septal pathway originates in the subfornical organ. Brief electrical stimulation of the subfornical organ or the medial septum both evoked a transient rise in intramammary pressure equivalent to that caused by an i.v. injection of 1 mU oxytocin. The optimal frequency was 5–20 Hz for 5–10 s. Prolonged stimulation also elicited at its onset a single transient response, similar to that evoked by brief stimulation. Extracellular recordings were made from neurosecretory cells of the supraoptic nucleus identified by antidromic stimulation of the neural stalk and further classified as vasopressinergic and oxytocinergic by their reaction at the time of reflex milk ejection induced by suckling. Single-pulse stimulation of the subfornical organ rarely produced excitation, but short trains of stimuli evoked a large excitation in most oxytocinergic and vasopressinergic neurons. To delineate further the pathway from the subfornical organ to the magnocellular neurons, stimulations were combined with various lesions of the medial forebrain. The effects of stimulation of the subfornical organ were abolished after a section immediately rostral to the organ, and in most cases after lesion of the medial septum. Stimulation of the medial septum no longer had an effect after the subfornical organ had been lesioned a week prior to experiments, a period sufficient to allow degeneration of subfornical efferents. This study shows that the excitatory afferent input to the oxytocin and vasopressin-secreting neurons of the hypothalamus from the medial septal area originates in the subfornical organ. This input is not involved in the main afferent control of the milk ejection reflex since lesions of the subfornical organ and of the medial septum had no effect on the reflex. It is suggested, therefore, that the subfornical input to both oxytocin and vasopressin cells intervenes to facilitate synergistic action of both hormones in non-reproductive functions.

Does recurrent inhibition exist in the paraventricular magnocellular neurosecretory cells in the male rat?

Extracellular recordings were performed from the paraventricular (PVN) neurosecretory cells in the anesthetized male rats. After sub- and suprathreshold stimulations to the neural stalk, two of 38 neurons showed inhibitory phases and eleven of the remaining 36 neurons showed inhibitory responses after suprathreshold stimulations. These results indicate that the recurrent inhibitory system exists in the PVN.

Transient extracellular volume reduction in neural lobe of rat hypophysis in response to neural stalk stimulation in vitro and its relationship to extracellular potassium

1. Using ion-sensitive microelectrodes, a transient reduction in the local volume of neural lobe extracellular space was found to accompany the elevation in extracellular potassium induced by stimulation of the neural stalk. The volume decrease and potassium increase had similar stimulus-response curves when stimulus frequency was varied from 1 to 40 Hz, with maximal response at 20 Hz. The curves for stimulus duration diverged, as a near maximal potassium response was reached in 4-16 s with a 20-Hz stimulus, while the extracellular volume decrease was maximal at 64 s. 2. The volume decrease, but not the potassium increase, was strongly inhibited by lowering bath temperature and moderately inhibited by furosemide and by lowering extracellular chloride concentration. Both the volume and the potassium response were enhanced by ouabain. 3. In conclusion, shrinkage of the local extracellular space in neural lobe during nerve activity is mediated by a metabolically active process which is only partially dependent upon extracellular chloride concentration and anion-cation co-transport, but is relatively independent of Na(+)-K+ pump activity. A transient shrinkage in extracellular space during increased neurohypophysial nerve activity would be expected to play a role in hormone diffusion, ion buffering, and extracellular current flow.

Microcirculatory patterns in adult rat cerebral hypophysis: a scanning electron microscope study of replicated specimens.

The blood vascular bed of the cerebral hypophysis in the adult rat was replicated completely or incompletely by arterial injection of different amounts of methacrylate resin, to be observed with a scanning electron microscope. Complete replication confirmed our previous findings (Murakami et al., 1987) on the distribution and structure of the vascular beds in and around the hypophysis of the rat. One long major and several minor portal routes (vide infra) were reproduced sufficiently together with the systemic veins of the posterior lobe. Incomplete replication demonstrated that resin flows: 1) via the long portal vessels from the median eminence and neural stalk to the anterior lobe; 2) via the accessory long portal vessels from the subependyma to the anterior lobe; 3) via the short portal vessels from the posterior lobe to the anterior lobe; 4) via the neuro-intermedial portal vessels from the posterior lobe to the intermediate lobe; 5) via the intermedio-distal portal vessels from the intermediate lobe to the anterior lobe; and 6) via the tuberal portal vessels from the tuberal lobe to the anterior lobe. Incomplete replication also demonstrated that resin in the median eminence and neural stalk is drained preferentially into the anterior lobe via the long portal vessels, and that resin in the posterior lobe is drained mainly into the systemic veins. We were unable to demonstrate a retrograde resin flow from the anterior lobe to the median eminence, subependyma, neural stalk, intermediate lobe and posterior lobe, nor an ascending resin flow from the posterior lobe to the median eminence and subependyma. Also failing to be noted were an ascending resin flow from the hypophysis to the hypothalamus and a descending resin flow from hypothalamus to the hypophysis.

Axonal transport of neuropeptide encoding mRNAs within the hypothalamo-hypophyseal tract of rats.

Hypothalamic vasopressin and oxytocin transcripts have been detected in the posterior pituitary suggesting either transcription of the respective genes in pituicytes or axonal mRNA transport from the hypothalamus to the nerve terminals of the posterior pituitary. The concept of axonal mRNA transport is supported firstly, by Northern blot and in situ hybridization analysis indicating that vasopressin and oxytocin mRNAs are also present in the neural stalk; secondly, by intron analysis and transcription run on experiments demonstrating the absence of primary vasopressin and oxytocin transcripts in non-neuronal cells of the posterior pituitary; thirdly, by embryonic developmental studies showing that appearance of vasopressin transcripts in the hypothalamus and the pituitary anlage is correlated. Furthermore, during axonal transport the respective mRNAs are subject to specific modification at the poly(A) tails.

In vivo Intracellular Recording of Neurons in the Supraoptic Nucleus of the Rat Hypothalamus

Abstract Intracellular recordings were made from cells in the hypothalamic supraoptic nucleus in the urethane-anaesthetized male rat using the ventral surgical approach. Impalements lasted from 5 min to 1 h and recorded cells had an input resistance of 55 to 170 megohms. Spikes of over 50 mV were recorded from 14 cells which could be antidromically activated by stimulation of the neural stalk. The spikes showed a hyperpolarizing afterpotential and the broadening characteristic of rapidly firing magnocellular neurons, which recovered rapidly (<200 ms). When depolarized, the cells showed evidence of a transient potassium current. Recurrent synaptic coupling between the recorded cell and adjacent cells would be expected to alter the hyperpolarizing afterpotential of an antidromic spike as compared with a spontaneous spike; no perceptible difference in the waveforms of the different types of spike could be detected in 11 spontaneously active cells. Application of just subthreshold stimuli to the neural stalk did not evoke depolarizing or hyperpolarizing potentials. Suprathreshold shocks to the neural stalk, when the antidromic spike was prevented by collision, also had no discernible effect on membrane potential. Thus intracellular recordings from magnocellular neurons in vivo revealed electrophysiological properties similar to those seen in vitro. No evidence for synaptic interconnection between magnocellular neurons was found in male rats.

Ultrastructural demonstration of neurohaemal contacts in the internal zone of the median eminence of the Mongolian gerbil (Meriones unguiculatus): correlation with synaptophysin immunohistochemistry

Electron microscopy of the median eminence (ME) of the Mongolian gerbil (Meriones unguiculatus) revealed that, unlike most other mammalian species, abundant neurohaemal contacts were present not only in the external zone (EZ), but also in the internal zone (IZ) up to the subependymal layer. In the IZ, nerve terminals with dense core vesicles and/or small clear vesicles abutted on the outer basal lamina of the perivascular space of portal capillaries, alternating with tanycyte processes. In addition to these neurohaemal contacts, several layers of vesicle-filled varicosities surrounded the portal vasculature. An analysis of serial thin sections showed that the latter varicosities could also reach the perivascular basal lamina or contact it through small extensions in other planes of section. Apparently at least some of the nerve terminals making neurohaemal contacts were en passant in nature. A correlative investigation of synaptophysin (a major integral membrane protein of small synaptic vesicles) immunoreactivity at the light microscopical level demonstrated a conspicuously dense immunostaining around portal capillaries in both EZ and IZ of the proximal and distal ME (neural stalk). Since this perivascular accumulation of immunoreactivity coincides precisely with the ultrastructural accumulation of vesicle-filled axons which establish numerous neurohaemal contacts, it is concluded that this pattern of synaptophysin immunostaining indicates sites of neurohaemal contacts at the light microscopical level. During postnatal development, the perivascular concentration of synaptophysin immunoreactivity in the IZ appeared concomitantly with the early postnatal expansion of long portal capillary loops into the IZ.(ABSTRACT TRUNCATED AT 250 WORDS)

Intraterminal recordings from the rat neurohypophysis in vitro.

1. Intracellular recordings were obtained from neurosecretory terminals (endings of Herring bodies), axons and pars intermedia cells in the isolated neuro-intermediate lobe of the rat. Responses to current injection and stimulation of the neural stalk (NS) were examined at 34 degrees C. Cellular identity was verified following injection of Lucifer Yellow. 2. Neurosecretory terminals were identified by a constant-latency action potential response to NS stimulation and an appropriate collision test. This response was not blocked by Ca2(+)-free solutions. Hyperpolarization of the terminals could block the generation of a local spike. Injection of Lucifer Yellow into eleven units confirmed that such responses were recorded from neurosecretory terminals. 3. Neurohypophysial nerve terminals had a resting potential of -60.4 +/- 1.1 mV and displayed a spike amplitude of 72.4 +/- 1.9 mV. The local spike threshold was -41.6 +/- 1.9 mV. Voltage-current relations were linear near resting potential, but displayed strong outward rectification positive to -55 mV. While terminals could fire at high frequencies during NS stimulation, repetitive activity could not be evoked by prolonged depolarizing current pulses. 4. During the initial 1-3 s of a train of brief depolarizing pulses or NS stimuli, nerve terminals showed a progressive broadening of their action potentials. At steady state, the duration of these impulses increased logarithmically with firing rate, showing a maximum near 25 Hz. Spike broadening in nerve terminals was reversibly abolished by superfusion of the neural lobe with Ca2(+)-free, Mn2(+)-containing solutions. In the absence of external Ca2+, action potentials were smaller, and lacked a prominent shoulder on their repolarizing phase. 5. Sustained (greater than 5 s) repetitive stimulation at 10-20 Hz led to a gradual increase in the latency for invasion and eventual failure of the spike-generating mechanism within the terminal. This effect required several seconds to recover. In contrast, action potentials recorded within the axon followed continuous repetitive stimulation and did not show any frequency-dependent changes in duration (0.7 +/- 0.1 ms). 6. Cells of the pars intermedia (PI) displayed an input resistance of 215.7 +/- 47.4 M omega and fired a single action potential in response to current injection. The amplitude of this current-evoked spike decreased during repetitive stimulation, but its duration was not affected. In 87% of the PI cells tested, stimulation of the NS evoked a Ca2(+)-sensitive synaptic response which reversed near -40 mV, but no cell was directly activated by the stimulus.

Tuberal supraoptic neurons—I. Morphological and electrophysiological characteristics observed with intracellular recording and biocytin filling in vitro

Previous studies of the tuberal, or retrochiasmatic, portion of the supraoptic nucleus suggest its functional similarity to the more densely populated anterior supraoptic nucleus, but the basic electrophysiological and morphological features of tuberal supraoptic nucleus neurons have not been described. Using the hypothalamo-neurohypophysial explant preparation in the rat, intracellular recordings and biocytin injections were made in tuberal supraoptic nucleus neurons and the results indicate that the two parts of the nucleus are similar. The generally oval-shaped somata of tuberal supraoptic nucleus neurons exhibited short, irregularly shaped appendages, and possessed 2–5 varicose, sparsely branching dendrites oriented in the horizontal plane. Many tuberal supraoptic nucleus neurons could be antidromically stimulated (mean latency = 6.4 ms). Filled neurons had varicose axons which were traced to the median eminence and even as far as the neural stalk, but which did not bifurcate. Both axons and dendrites were sparsely invested with short, hair-like appendages. The input resistance of the recorded neurons (mean = 177.7 MΩ) was positively correlated with the membrane time constant (mean = 13.1 ms; r = 0.83). Tuberal supraoptic nucleus neurons displayed a prominent afterhyperpolarization following individual spikes or bursts of spikes, as well as firing frequency adaptation in response to positive current pulses. Although numbering far fewer than those of the anterior supraoptic nucleus, tuberal supraoptic nucleus neurons have axons which are more often intact in this preparation, and a dendritic tree which radiates within the plane of the explant. Thus these neurons should provide a useful model for further study of the electrophysiological and morphological characteristics of mammalian neurosecretory neurons.

Quantitative Comparisons Between the Electrical Activity of Supraoptic Neurons and Vasopressin Release in vitro

Abstract The firing rate of antidromically identified neurons in the tuberal portion of the rat supraoptic nucleus and vasopressin release were compared in an in vitro preparation of the hypothalamo-neurohypophysical system. Extracellular potentials were isolated and held while the perifusing medium was collected for radioimmunoassay of vasopressin. Neurons in the tuberal portion of the supraoptic nucleus were induced to first bursts of action potentials by injecting supra-threshold concentrations (0.01, 0.1 and 1 mM) of the alpha(1)-agonist phenylephrine into the perifusing line. Phenylephrine caused a dose-dependent release of vasopressin into the perifusate and a dose-dependent increase in the peak firing rate, initial burst duration and the total number of action potentials (compared to a 10 min control period) of the recorded neurons. Peak firing rate and peak vasopressin release both increased linearly with the log of the phenylephrine dose, but the efficiency ratio of the increase in vasopressin release to the increase in firing rate was greater from the middle to the highest dose than it was for the lowest to the middle dose, indicating a possible facilitation of hormone release with higher firing frequencies. The total amount of vasopressin released in 10 min post-injection and the total number of evoked action potentials in the same period also increased linearly, but in this case there was no change in the efficiency ratio between the low to middle and the middle to high doses of phenylephrine. Release of vasopressin to phenylephrine was dependent on an intact neural stalk, and in a separate group of isolated neural lobes, 1 mM phenylephrine did not significantly alter the peak or total amount of vasopressin released to an electrical stimulus given in the pattern of an evoked burst. In another group of explants the temporal relationship between firing rate and vasopressin release was examined after a single dose of 1 mM phenylephrine. In these explants the true time-course of release was estimated using a deconvolution procedure which corrected for diffusion of the hormone. Mean vasopressin release and spike activity were still elevated above baseline 4 to 5 min after the first elicited burst, suggesting that the latter parts of the initial burst and/or the after-discharges contribute to the prolonged vasopressin release. However, there was a dramatic drop in the efficiency ratio (vasopressin release/peak firing rate) from the first to the second minute following stimulation. Thus, changes in the frequency of action potentials generated by supraoptic neurons can be directly related to simultaneous changes in the rate of vasopressin release in the same preparation. The results suggest that elevations in firing rate are accompanied by an increased rate of vasopressin release, but as has been demonstrated in isolated neural lobes, this relationship is probably not constant across different stimulation strengths or within a single burst.

The role of interconnection between supraoptic nucleus and anterior third ventricular region in osmoregulation in the rat.

1. To determine the importance of the region of the hypothalamus anterior and ventral to the third ventricle (AV3V) in the osmotic regulation of neurohypophysial hormone release, extracellular recordings were made in the urethane-anaesthetized rat from neurones in the supraoptic nucleus (SON) and in the AV3V using a ventral surgical approach. 2. In the first series of experiments, recordings were made from SON cells (n = 41) which could be antidromically activated from the neural stalk in control rats and rats 2 weeks after electrolytic destruction of the AV3V (n = 74). Destruction of AV3V significantly (P less than 0.01) reduced the responsiveness of SON cells to an osmotic challenge (1 ml of 1.5 M-NaCl I.P.). 3. In the second series of experiments recordings were made in the AV3V region while stimulus pulses were applied to the region of the SON. The cells recorded could be divided into five groups; those which were inhibited (presumably synaptically; OD-; n = 97), those which were excited (also presumably synaptically; OD+; n = 73), those which could be antidromically activated (AD; n = 50), those which showed a complex cycle of excitation and inhibition after the stimulus (n = 40) and those which did not respond to the stimulus (n = 47). 4. The characteristic response of OD- cells to an osmotic challenge was inhibition (six out of ten cells) and that of OD+ cells was excitation (thirteen out of seventeen cells). No AD cells showed excitation following I.P. NaCl injection (nine out of nine). 5. Our experiments provide clear evidence that the AV3V region is important for the normal responsiveness of the cells of the SON to raised plasma osmolality and for an interaction between the SON and the AV3V region. 6. Our results also suggest, contrary to our expectation, that the SON exerts both excitatory and inhibitory influences on the AV3V. Since the cells which project from the AV3V to the SON region are not osmoresponsive, the importance of the direct projection from the AV3V to SON may be to maintain the responsiveness of SON neurones rather than to provide osmotic drive.

Oxytocin release evoked by electrical stimulation of the medial forebrain in the rat: Analysis of stimulus parameters and supraoptic neuronal activity

The role of the medial forebrain area (vertical limb of the diagonal band, medial septum and medial nucleus accumbens) in the control of oxytocin secretion in lactating rats was investigated. Electrical stimulation of the medial forebrain evoked a reproducible rise in intramammary pressure, equivalent to that caused by i.v. injection of 1 mU oxytocin. No presser effect accompanied this response. Radio-immunoassay of plasma samples showed that stimulation caused a significant rise in the concentration of circulating oxytocin. The effects of changing the parameters of stimulation to the medial forebrain were compared with those evoked by stimulation of the neural stalk. The optimal frequency for stimulation of the forebrain was found to be four-fold lower (10–20 Hz) than that for stimulation of the neural stalk (50 Hz). During continuous prolonged stimulation of the forebrain (20 Hz; 2 min) only a single transient response was obtained, whereas a protracted response was obtained as a result of prolonged stimulation of the stalk. Recordings were made from antidromically identified neurosecretory cells in the supraoptic nucleus. Electrophysiological responses to electrical stimulation of the medial forebrain were characterized by two main features. (1) Single-pulse stimulation produced only a small excitation (one or two action potentials), while high-frequency trains produced a profound facilitation of this response, with each pulse evoking short-duration ‘bursting’ behaviour in the supraoptic neurons. (2) During long trains of stimulation this frequency-dependent facilitation declined and could only be renewed after a period of rest. The study indicates that the medial forebrain provides a major excitatory afferent input to the oxytocin-secreting neurons of the supraoptic nucleus, and that the pulse-like secretion of oxytocin following medial forebrain stimulation results from the complex facilitatory and adaptive characteristics of this input.

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.

Golgi-like immunostaining of pituicytes and tanycytes positive for glial fibrillary acidic protein in the neurohypophysis of the Mongolian gerbil (Meriones unguiculatus).

Glial cells that contain the glial fibrillary acidic protein (GFAP; the major protein constituent of glial filaments) were stained immunohistochemically in thick frozen sections of the neurohypophysis of the Mongolian gerbil (Meriones unguiculatus). The resulting Golgi-like images provided informations on cytological features and distributional patterns of tanycytes and pituicytes. In the proximal median eminence, numerous bundled processes of tanycytes were revealed. They emerged from the ependymal and sub-ependymal layer and mostly reached the brain surface. Several tanycytic processes extended into the anatomical neural stalk. In the whole neural lobe, a dense network of GFAP-immunoreactive pituicyte processes was visualized. Stained pituicytes were highly asymmetric and exhibited a great morphological variability. Immunopositive fibers which were encountered in the intermediate lobe might be derived from pituicytes. Electron-microscopically further evidence was obtained that GFAP-positive pituicytes correspond to filament-rich fibrous pituicytes at the ultrastructural level.

Regulation of the milk ejection reflex in the rat.

Extracellular recordings were made from neurones in or near the supraoptic nucleus in suckled lactating rats under urethane anaesthesia to investigate the mechanism by which the firing of oxytocin cells is synchronized during reflex milk ejection. Cells synaptically driven but not antidromically activated by neural stalk stimulation, which thus probably receive an afferent input from supraoptic neurones, were classified as 'regular' or 'bursters' on the basis of their spontaneous electrical activity. The majority (twelve out of eighteen) of synaptically excited cells (o.d.+) were bursters and the majority of inhibited (o.d.-) cells (eleven out of nineteen) were regular, but only one o.d.+ burster showed any change of activity (inhibition) before milk ejection. Putative oxytocin cells in suckled lactating rats showed a firing pattern between milk-ejection bursts which could not be distinguished from that of putative oxytocin cells in male animals. The mode interspike interval between milk ejections was 47.1 +/- 3.1 ms (mean +/- S.E. of mean) compared with 47.3 +/- 3.3 ms in male rats, and fewer than 1.4% of interspike intervals were less than 20 ms in duration. By contrast, within milk-ejection bursts 40% of interspike intervals were in the range 8-20 ms. Short trains (10 or 20) of pulses applied to the neural stalk at regular (5 min) intervals, in an attempt to simulate the initial part of the milk ejection burst, failed to trigger bursts. In only 2 of 150 tests was the interval between train and milk-ejection burst less than 10 s, and after the pulse train all but one cell showed reduced activity for 1-3 s. The trains of pulses were however not without effect: they significantly (P less than 0.01) enhanced the chance of a milk-ejection burst occurring within the next 2.5 min. Our observation that pulse trains do not trigger bursts suggests that local positive feed-back mechanisms are not responsible for orchestrating the activation of oxytocin cells during the milk-ejection reflex. Moreover, because spontaneous tiring pattern is the same in lactating and non-lactating rats, we found no evidence that the anatomical changes in the synaptic organization within the supraoptic nuclei in lactation have any influence on the firing of oxytocin cells. It is likely, however, since pulse trains alter the timing of milk ejections, that oxytocin released locally in the region of the supraoptic nucleus can influence reflex milk ejection.(ABSTRACT TRUNCATED AT 400 WORDS)

Single ion channel activity in peptidergic nerve terminals of the isolated rat neurohypophysis related to stimulation of neural stalk axons

Scanning electron micrographs revealed that the cut face of the sagitally bisected rat neural lobe was characterized by fine fibres bearing multiple rounded swellings (0.5–5 μm in diameter) presumed to be axonal swellings and peptidergic nerve terminals. Patch-clamp electrodes sealed (seal resistance >10 10 Ω) on to the cell membrane in such regions revealed (36 out of 43 patches) opening of channels conducting inward current following stimulation of the neural stalk at stimulus frequencies between 5 and 20 Hz. Channel opening did not occur immediately on stimulation (delay 5–15 s) and persisted 1–25 s after stimulation of the axons ceased, implying the possible existence of a depolarization-evoked intracellular messenger. Addition of TTX (5–20 μM) to the bath to block spike propagation abolished stalk stimulation-evoked channel opening but TTX in the patch electrode did not prevent opening. Opening was, however, prevented by addition of 4nMM Co 2+ or 4 mM Mn 2+. The slope conductance of this channel in medium with 10 mM Ca 2+wwas approximately 7 pS which was increased to 19 pS in medium containing 95 mM Ba 2+. A second inward channel with a slope conductance of 56 pS in medium with 10 mM Ca 2+ was also characterized and outward channels were noted in four patches. The electrical properties of nerve terminals are difficult to study because of their small size. The demonstration that ion channel activity can be recorded from mammalian peptidergic nerve terminals thus offers wide scope for future studies on the relation between stimulus and secretion.