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Previous studies have demonstrated that histamine primarily excites unidentified neurons in the rat supraoptic nucleus. We investigated the neuromodulatory effects of histamine on immunohistochemically identified vasopressin neurons in the rat supraoptic nucleus using intracellular recording techniques from the hypothalamo-neurohypophysial explant. Exogenous application of histamine (0.1-100 microM) to vasopressinergic neurons produced a small membrane depolarization accompanied by an increase of up to 100% in the amplitude of the depolarizing afterpotential that follows current-evoked trains of action potentials. The enhancement of the depolarizing afterpotential by histamine did not depend upon the depolarization. Further, histamine enhanced the amplitude of the depolarizing afterpotential when blocking the afterhyperpolarizing potential with d-tubocurarine or apamin, and in the presence of tetrodotoxin and d-tubocurarine or apamin, indicating a postsynaptic action of histamine on the depolarizing afterpotential that is not simply a reflection of a decrease in the afterhyperpolarizing potential. These toxins also had no effect on the histamine-induced depolarization. The enhancement of the depolarizing afterpotential by histamine was mimicked by the histamine H1-receptor agonist 2-thiazolylethylamine and was reduced or blocked by the H1-receptor antagonist promethazine, but was not blocked or reduced in the presence of the histamine H2-receptor antagonist, cimetidine. In summary, these results show that the excitatory effect of histamine on immunohistochemically identified vasopressin neurons in the supraoptic nucleus is due in part to the H1-receptor-mediated enhancement of the depolarizing afterpotential independent of any change in the afterhyperpolarizing potential or membrane potential.

The electrotonic properties of tuberal supraoptic neurons were studied from conventional intracellular recordings made in the hypothalamo-neurohypophysial explant in vitro. The cable parameters electrotonic dendritic length, and the dendritic to somatic conductance ratio, were estimated using the slopes and intercepts of the first two peeled exponentials of the voltage transients generated by current steps. The estimations were made assuming an equivalent cylinder model consisting of a soma and an attached, lumped dendrite of finite length. An equalizing time constant was resolved in 12 of 17 neurons, allowing calculation of both cable parameters. In only one of these 12 was it necessary to assume a somatic shunt to account for the data. The average value of the dendritic electrotonic length was 1.02, and that of the dendritic to somatic conductance ratio, 4.11. In the remaining five neurons, an equalizing time constant could not be peeled and consequently the dendritic cable parameters could not be estimated. The average input resistance of these 12 neurons was 162 M omega and the average membrane time constant was 11.86 ms. Principal Components Analysis revealed that the variance of input resistance and time constant was largely explained by one factor, while that of dendritic electrotonic length and the dendritic to somatic conductance ratio was explained by a separate, independent factor, suggesting a separation of electrical and morphological parameters, respectively. In addition, the variability of the data indicates that considerable differences in the morphology and specific membrane resistivity exist across supraoptic neurons. An analysis of spontaneously occurring postsynaptic potentials revealed that the shapes of these potentials could not be explained simply by assuming that they were determined by their passive decay from some point along the equivalent cable to the soma. In conclusion, dendrites make a significant and previously unappreciated contribution to the electrotonic behavior of supraoptic neurons. These electrotonic properties are similar to those of many other, morphologically diverse, central nervous system neurons.

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 M omega) 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.

Cell area, percentage of cells with more than one nucleolus, and percentage of cells with the nucleolus apposed to the nuclear membrane (marginated) were compared in the medial and lateral magnocellular portions of the paraventricular nucleus (PVN) during water deprivation and voluntary rehydration. Lateral cells were larger and had a greater percentage of cells with multiple nucleoli in all conditions of deprivation and rehydration. In addition laterals were found to have more cells with marginated nucleoli in these cells with only one nucleoli. Medial and lateral cells showed similar responses to deprivation and rehydration.

A technique is described which couples the aqueous aldehyde fluorescence (Faglu) method [5,6] with polyethylene glycol (PEG) embedding to obtain sections of brain as thin as 2 microns for light microscopic examination. Blocks from rat brains perfused with Faglu solution were embedded in PEG, sectioned on a rotary microtome and examined for amine fluorescence. Both catecholaminergic perikarya and their terminal arborizations were observed with high resolution in 2 micron sections. Single neurons could be traced in serial sections and varicosities as well as intervaricose segments were visible in terminal regions. This technique offers the further advantage of fixation compatible with electron microscopy, immunohistochemistry and other neuroanatomical methods. PEG-embedding of Faglu-fixed tissue thus provides a useful addition to existing fluorescence histochemical techniques for the visualization of catecholamines.

Projection neurons in the rat hypothalamic paraventricular nucleus (pv) were identified using the retrograde transport of horseradish peroxidase (HRP) following injections of this tracer into the neurohypophysis or jugular vein. The objective was to determine if parvocellular pv neurons might be labelled by either injection in addition to the well known magnocellular contingent. Injections confined to the neurohypophysis resulted in the labelling of only magnocellular pv neurons, while intrajugular injections which produced spread of the HRP to the arcuate nucleus-median eminence region resulted in the labelling of both parvocellular and magnocellular pv elements. Thus parvocellular pv neurons may project to the arcuate nucleus and/or the external layer of the median eminance but not to the neurohypophysis.

Processes of magnocellular neurosecretory cells in the rat supraoptic nucleus which project along the pial surface in the ventral glial lamina were investigated using immunocytochemistry, Golgi stains and electron microscopy. Immunocytochemical studies revealed that although both oxytocin- and vasopressin-containing processes were evident in the ventral glial lamina, vasopressin-containing processes predominated. Ventral processes were thicker and of a different morphology than dorsal axon-like processes which joined the hypothalamo-neurohypophysial tract and exhibited large varicosities along their length or at their apparent termination. Golgi stains revealed that classically defined dendrites of supraoptic neurons projected primarily ventrally and often invaded the ventral glial lamina. No axons were traced to the lamina. Ultrastructurally, processes within the ventral glial lamina characterized as dendrites could be stained immunocytochemically for neurophysin and were post-synaptic to a variety of presynaptic elements. The results suggest that many dendrites from magnocellular neurosecretory cells in the supraoptic nucleus project to the ventral glial lamina and form a restricted, receptive plexus. The previously demonstrated coexistence of catecholamine-containing varicosities and other axon types with these processes in the lamina indicates an important role for supraoptic dendrites in integrating a wide variety of information relevant to neurohypophysial hormone release.

Single unit activity was recorded from 400-500 mu m thick slices of rat hypothalamus, using either NaCl- or horseradish peroxidase-filled glass micropipettes. Spontaneous activity was present in the following hypothalamic loci: anterior hypothalamic-preoptic area, nucleus circularis, nucleus of the diagonal band of Broca, paraventricular accessory nucleus, paraventricular nucleus (all portions), periventricular regions of the anterior hypothalamus, and the suprachiasmatic nucleus. The supraoptic nucleus was the only major cell group studied to exhibit no spontaneous activity. Cells of the paraventricular and circularis nuclei were spontaneously active, displayed firing rates and patterns of activity similar to those recorded in vivo for magnocellular elements of the hypothalamus, and in some cases responded to increases in the osmolality of the bathing medium with altered firing rates and/or patterns of activity. Many cells in these preparations were characterized by phasic, bursting patterns of activity. Slow, irregular and regular, continuous activity was also frequently observed, as is typical in vivo. Median firing rates were in the range of 4-6 spikes/sec, somewhat faster than the rates usually reported for anesthetized in vivo preparations. These rates are more similar to those observed in unanesthetized monkeys or rats with diencephalic islands. Extracellular HRP marking provided a high degree of localization for many of the recorded cells. These results indicate that the hypothalamic slice preparation is useful for studies in which it is desirable to eliminate extrahypothalamic connections and in which it is necessary to exercise a fine degree of control over the extracellular environment of the cells.

The effects of histamine on the firing of supraoptic neurosecretory neurons in the rat were examined in vitro using acutely prepared, hypothalamo-neurohypophysial explants perifused with an artificial cerebrospinal fluid. Extracellular action potentials meeting the criteria of antidromic invasion from neurohypophysial stalk stimulation were recorded from 135 neurons in the tuberal portion of the supraoptic nucleus, which lies superficially along the tuber cinereum and consists of mostly vasopressin-containing neurons. Units could be classified as slow/silent (76.3%), phasic (21.5%) or continuous (2.2%) on the basis of their spontaneous activity. Histamine applied briefly to the perifusate excited approximately one-third of the slow silent neurons and approximately two-thirds of the phasic neurons, with a wide range (10(-3)-10(-9)) in the effective concentration across neurons. The H1-receptor agonists 2-pyridylethylamine and 2-thiazolylethylamine mimicked these excitations in 10 of 12 and 3 of 6 neurons tested, respectively. The H2-receptor agonists dimaprit (4 neurons) and impromidine (5 neurons) failed to excite any of the tested neurons previously excited by histamine. The H1-receptor antagonist promethazine antagonized histamine's excitatory effect in 8 of 9 cells, while the H2-receptor antagonist cimetidine had little effect on the 9 cells tested. Histamine also modified bursts of activity induced in some slow/silent neurons by antidromic stimulation without having an observable effect in the absence of an antidromic burst. In 10 of 18 neurons histamine produced an elongation of burst duration and a modest increase in intraburst firing rate when applied during an antidromically evoked burst. In an additional 5 of 17 neurons, which had neither previously responded to histamine nor shown an antidromically-evoked burst, the pairing of histamine application and antidromic shocks resulted in an antidromically evoked burst. The effects of histamine on evoked bursts also appeared to be mediated by an H1-receptor. Histamine's excitation of supraoptic neurons is thus dependent on the electrical activity expressed by the neuron at the time of testing. Conductances activated by depolarization of the neuron may be modified by histamine or this compound may alter the threshold for burst generation. Considered with data showing H1-receptor localization and histamine-immunoreactive fibers within the supraoptic nucleus, the present results, as well as those showing the potency of centrally applied histamine in releasing vasopressin, suggest histamine may act physiologically by altering the electrical activity of vasopressin-secreting neurons.

Subcutaneous injections of isotonic saline induced nucleolar proliferation in supraoptic neurons in animals sacrificed approximately 5 min postinjection. The magnitude of this proliferation was sustained 4 and 8 hr postinjection. Polyethylene glycol (PG) injections depleted blood volume 4 and 8 hr after the injection, but the percentage of SON cells with multiple nucleoli in these animals was not different from saline-injected controls. The anterior (SOa) portion of the SON in rats given 2% NaCl to drink instead of water for three days contained more cells with multiple nucleoli than controls. This effect was enhanced after five days ingestion, and accompanied by a similar response in the tuberal portion of SON (SOt). Rehydration for ten days after three days of 2% NaCl intake brought the percentage of cells with multiple nucleoli down to control levels. Cell area in SON cells paralleled nucleolar responses during dehydration and rehydration. The results demonstrate the sensitivity of nucleolar proliferation in SON to environmental changes ranging from osmotic to neurogenic stress.

Bilateral electrolytic lesions and unilateral tracer injections were performed in lactating rats in order to study the participation of the mesencephalic lateral tegmentum in the milk-ejection reflex. The release of oxytocin was detected as a rise in intramammary pressure during each milk ejection. In animals with lesions, the lateral part of the deep grey layers of the superior colliculus, the intercollicular area and the rostromedial portion of the external nucleus of the inferior colliculus were destroyed. The mesencephalic lateral tegmentum of animals in which the milk-ejection reflex was blocked sustained a larger damage than in rats where the frequency of the milk-ejection response was only slowed down. Solutions of True Blue, horseradish peroxidase or horseradish peroxidase coupled to wheat germ agglutinin were injected in the mesencephalic lateral tegmentum of rats with and without lesions. Retrogradely labelled cells were found in several nuclei of the somatosensory pathways: the principal sensory and spinal parts of the trigeminal complex, the cuneate and gracile nuclei, the lateral cervical nucleus and the nucleus proprius of the spinal cord. Labelled cells were also found in the ventral nucleus of the lateral lemniscus, the ventral parabrachial nucleus, the gigantocellular reticular nucleus, the lateral nucleus of the substantia nigra, the prerubral nucleus of the thalamus, the hypothalamic ventromedial nucleus, the zona incerta and in the anterior and lateral hypothalamic areas. Labelled fibres and "terminal-like" labelling were found in the anterior pretectal area, in the thalamic parafascicular nucleus, in the posterior nucleus and the ventroposterior complex, in the zona incerta and in the fields of Forel, but none were observed in the supraoptic or paraventricular nuclei. Injections made in the area of the lateral cervical nucleus and in the cuneate and gracile nuclei labelled fibres and "terminal-like" fields in the external nucleus of the inferior colliculus, the intercollicular area, the deep grey layers of the superior colliculus and in the mesencephalic lateral tegmentum. After injections in the posterior nucleus and ventroposterior complex of the thalamus, retrogradely labelled cells were found in the lateral tegmentum, the intercollicular area and the external nucleus of the inferior colliculus. These results indicate that bilateral lesioning of the mesencephalic lateral tegmentum, which disrupts the milk-ejection response, could damage somatosensory projections originating from the dorsal horn of the spinal cord, the lateral cervical nucleus, the dorsal column nuclei and the sensory and spinal trigeminal nuclei.(ABSTRACT TRUNCATED AT 400 WORDS)

Bilateral electrolytic lesions of some somatosensory structures in the thalamus and spinal cord were made in order to assess their participation in the afferent limb of the milk-ejection reflex in the rat. Lesions involving the lateral cervical nucleus and a part of the dorsolateral funiculus region blocked the milk-ejection reflex, whereas animals with lesions in the dorsal column of the spinal cord or in the ventroposterior complex of the thalamus displayed milk-ejection reflexes similar to those of control animals. Unilateral injections of horseradish peroxidase coupled to wheat germ agglutinin were made after lesioning the lateral cervical nucleus and part of the dorsolateral funiculus. Anterograde labelling was seen mainly contralateral to the injection site: in the external nucleus of the inferior colliculus, the intercollicular zone, the brachium of the inferior colliculus, the lateral reticular nucleus of the thalamus and in the thalamic ventroposterior complex. Sparse projections ipsilateral to the injection site were also observed. These results, combined with our previous observations, suggest that the projection of the lateral cervical nucleus on the mesencephalon is part of the pathway which conveys the sensory information from the suckling stimulus.

To detect neuronal cell bodies whose axon projects to the hypothalamic supraoptic nucleus, small volumes (10-50 nl) of 30% horseradish peroxidase or 2% fast blue solutions were pressure-injected into the area of one supraoptic nucleus of rats. Both dorsal and ventral approaches to the nucleus were used. In animals where the injection site extended beyond the limits of the supraoptic nucleus, retrogradely labelled cell bodies were found in many areas of the brain, mainly in the septum, the nucleus of the diagonal band of Broca and ventral subiculum in the limbic system; the dorsal raphe nucleus, the locus coeruleus, the nucleus of the dorsal tegmentum, the dorsal parabrachial nucleus, the nucleus of the solitary tract and the catecholaminergic A1 region in the brain stem; in the subfornical organ and the organum vasculosum of the lamina terminalis, as well as in the median preoptic nucleus. In contrast, when the site of injection was apparently restricted to the supraoptic nucleus, labelling was only clearcut in the two circumventricular organs, the median preoptic nucleus, the nucleus of the solitary tract and the A1 region. Injections of wheat germ agglutinin coupled with horseradish peroxidase (60-80 nl of a 2.5% solution) made in the septum and in the ventral subiculum anterogradely labelled fibers coursing in an area immediately adjacent to the supraoptic nucleus but not within it. In contrast, labelling within the nucleus was found following anterograde transport of tracer deposited in the A1 region and in an area that includes the nucleus of the solitary tract. Neurones located in the perinuclear area were densely labelled by small injections into the supraoptic nucleus; they may represent a relay station for some afferent inputs to the supraoptic nucleus. These results suggest that the supraoptic nucleus is influenced by the same brain areas which project to its companion within the magnocellular system, the paraventricular nucleus.