Autonomic basis for the rise in brain temperature during paradoxical sleep.

The rise in brain temperature in the rabbit during paradoxical sleep originates in a temperature rise of the cerebral arterial blood. Heat loss from the ear is a major factor in the regulation of arterial blood temperature in the rabbit, and the primary thermal event in paradoxical sleep is a vasoconstriction of the skin of the ear which results in a rise in arterial blood and brain temperatures. These thermal correlates of paradoxical sleep are not present in a cold environment when the ear skin is already maximally vasoconstricted. The persistence of peripheral vasoconstriction during paradoxical sleep in a hot environment suggests a disturbance in autonomic thermoregulatory control.

Glutamate receptors in the rat hypothalamus and pituitary.

Although several recent anatomical and physiological studies indicate that glutamate receptors are likely to play a role in the regulation of various hypothalamic functions, no attempt has yet been made to specifically characterize glutamate receptor densities, subtypes, or localization in the hypothalamus. To provide this basic information, we have characterized and mapped the binding of [3H]glutamate to N-methyl-D-aspartate (NMDA), non-NMDA, and metabotropic glutamate receptors throughout the diencephalon. Membrane binding assays revealed a [3H]glutamate binding density of 2.6 pmol/mg protein, approximately one third of the hippocampal density. Binding of subtype-specific agonists and antagonists was complex, but clearly indicated that each major glutamate subtype is present in all hypothalamic and preoptic regions in the following approximate relative densities: NMDA > metabotropic Glu receptor > kainate > or = alpha-amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid. Receptor autoradiography confirmed the widespread presence of all major glutamate receptor subtypes with roughly the following relative regional densities: ventromedial, dorsomedial > paraventricular, anterior hypothalamic, supraoptic > arcuate, suprachiasmatic, lateral hypothalamic > preoptic area >> pituitary neural lobe, white matter > pituitary anterior lobe (negligible). Subtype expression varied regionally, with rostral hypothalamic and preoptic regions having proportionally higher levels of non-NMDA vs. NMDA binding. High densities of glutamate receptors in ventromedial and medial hypothalamic regions suggest a prominent role in neuroendocrine and autonomic regulation.

Radioimmunoassay of arginine vasopressin in Rhesus monkey plasma.

Using a new antiserum, an enzymatic radioiodination of arginine vasopressin (AVP), and the methodology of Robertson et al. (1,2), we have developed a sensitive and specific radioimmunoassay for plasma AVP in the monkey. The sensitivity of the assay is 0.5 muU/ml, the cross reaction with oxytocin (OT), minimal. We used this assay to study the effects that variations in blood osmolality have in regulating AVP secretion in unanesthetized, chair-restrained, chamber-isolated, adult female rhesus monkeys. Under water ad lib conditions, plasma AVP and osmolality were relatively constant, averaging 1.7 +/- 0.6 (SD) muU/ml and 298 +/- 3 mosmol/kg, respectively. Water loading decreased plasma AVP and osmolality to 0.6 +/- 0.2 muU/ml and 282 +/- 6 mosmol/kg, respectively. When fluid restriction increased osmolality, plasma AVP rose progressively to twice the baseline after 1 day, and to 6 times the baseline after 3 days. The rise in plasma AVP was linearly correlated with the rise in osmolality (r = 0.93; P less than 0.001). Intravenous infusions of hypertonic saline produced significant rises in plasma osmolality and plasma AVP. There was a dose-related rise in plasma AVP that declined later at the expected rate with the infusion of physiological amounts of synthetic AVP.

Functional and morphological studies of peptide-containing neuroendocrine cells in goldfish hypothalamus.

We report the development of a double-label method which combines intracellular recording, dye-marking, and immunocytochemistry for the study of functional and morphological aspects of peptide-containing neurons in the magnocellular preoptic nucleus of the goldfish hypothalamus. Using multiple techniques, we distinguish three types of peptide-containing neurons: enkephalin (27%), isotocin (49%), and vasotocin (24%). Drawings of serial sections containing the dye-injected cells were the basis for subsequent reconstruction. These peptide-containing cells measure 14-56 micrometer in somata diameter, are unipolar, bipolar, or multipolar in somata shape and lie 6-244 micrometer from the ependymal lining of the preoptic recess of the third ventricle. Their electrophysiological properties match those of other mammalian and fish magnocellular neuroendocrine cells. This report confirms the one neuron, one hormone (peptide) hypothesis, supports synaptic over electrotonic coupling between peptidergic and adjacent hypothalamic neurons, and suggests that chemical and functional classification of single electrophysiologically and neuroanatomically studied central neurons is feasible technologically.

Local synaptic organization of cholinergic neurons in the basolateral hypothalamus.

A monoclonal antibody to choline acetyltransferase (ChAT) was utilized for immunocytochemical identification of cholinergic neurons in the basolateral hypothalamus. Light and electron microscopic examination revealed a network of cell bodies, dendrites, and axonal processes dorsolateral to the supraoptic nucleus. Within this region the cells immunoreactive for ChAT receive numerous unlabeled terminals which contact dendrites, cell soma, axons and occasional somatic spines. In a few cases, small ChAT-immunoreactive terminals were observed contacting a cholinergic cell soma or large dendrite. Many ChAT-immunoreactive fibers were directed toward the supraoptic nucleus forming a dense local network but very few of these fibers penetrated deeper than approximately 20 micron into the supraoptic nucleus. A total of 63 ChAT-immunoreactive terminals were mapped within the basal hypothalamus, of which the vast majority contacted unlabeled dendrites immediately dorsolateral to the supraoptic nucleus. Labeled terminals were rare or nonexistent in the medial portions of the hypothalamus or deep within the supraoptic nucleus. This pattern of ChAT terminal densities correlates with the distribution of binding for the muscarinic cholinergic probe, [3H]quinuclidinylbenzilate, but not the binding of the putative nicotinic cholinergic probe, [125I]alpha-bungarotoxin, which is high within the supraoptic nucleus. Thus, the cholinergic neurons of the basal hypothalamus appear to form a network of intrinsic connections which probably represent input to muscarinic cholinergic receptors. No evidence was found to suggest that cholinergic presynaptic terminals were colocalized with the alpha-bungarotoxin binding protein within the supraoptic nucleus.

Cholecystokinin in hippocampal pathways.

The distribution of cholecystokininlike (CCK-L) immunoreactive cells and fibers in the rat hippocampal formation and its afferent and efferent connections was studied using the immunoperoxidase technique. In the hippocampal formation CCK-L immunoreactive perikarya were located in the polymorphic zone of the dentate hilus, all layers of Ammon's horn, the subiculum, the presubiculum, and the entorhinal cortex. Cholecystokininlike immunoreactive fibers extended from cell bodies or were located around the cell bodies in the entorhinal cortex, subiculum and stratum pyramidale of Ammon's horn, and among the granule cells and inner molecular layer of the dentate gyrus. The immunoreactive cells in the stratum oriens may be a type of basket cell, since processes from these cells extend into stratum pyramidale and collections of CCK-L immunoreactive fibers are seen around cell bodies in stratum pyramidale. Cholecystokininlike immunoreactive fibers were also observed in the alveus, ventral and lateral fimbria, and ventrolateral lateral septal nucleus. Some of these immunoreactive fibers, therefore, being to either an efferent or afferent hippocampal pathway(s) originating from CCK-L immunoreactive pyramidal cells in the hippocampal formation and/or from the hippocampal subcortical nuclei, the supramammillary nucleus, and the dorsomedial hypothalamic nucleus which contain CCK-L immunoreactive perikarya. The distribution of these immunoreactive fibers in the fimbria and lateral septal nucleus is most consistent with an anteriorly directed efferent hippocampal pathway.

The localization of vasotocin and neurophysin neurons in the diencephalon of the pigeon, Columba livia.

Vasotocin (VT)- and neurophysin (NP)-synthesizing neurons were demonstrated by immunocytochemistry in the diencephalon of the pigeon, Columba livia. Three diencephalic regions contain VT-NP cells: (1) periventricular preoptic area and hypothalamus, including nucleus periventricularis magnocellularis (PVM); (2) lateral preoptic area and hypothalamus; and (3) dorsal diencephalon. The immunoreactive cells in each of these three regions were divided into groups based on cytology and topography. No differences were found in the location of VT and NP cell groups. The periventricular region contains three continuous cell groups (P1-P3) extending from the posteroventral preoptic area to the anterodorsal hypothalamus and PVM. The lateral region has two cell groups composed of medium- to large-sized cells associated with the quintofrontal tract (L1) or with the optic tract (L2), while a third group (L3) lies between these two cell groups. Two accessory cell groups reside in the dorsolateral hypothalamus; L4 contains scattered cells of varied size, whereas L5 has small- to medium-sized cells clumped together. The dorsal diencephalic cell groups are found in the following locations: (1) lateral and dorsal to the lateral forebrain bundle (DD1); (2) in the area ventral to the dorsomedial anterior thalamic nucleus and dorsolateral to PVM (DD2); and (3) at the dorsolateral border of nucleus rotundus (DD3). To avoid potentially inaccurate mammalian homologies, the cell group nomenclature denotes topographic position. Nevertheless, the presence of VT-NP cells in PVM and projections to the brainstem and spinal cord suggest a homology between PVM and some of the parvocellular subnuclei of the mammalian paraventricular nucleus.

Complications of metrizamide myelography.

Adverse neurobehavioral reactions have not been emphasized as a complication of metrizamide myelography. We encountered six such reactions in approximately 250 metrizamide myelograms. All reactions followed either cervical myelography or panmyelography via lumbar puncture. We also treated a single case of tonic-clonic seizure after intracranial spill of metrizamide in a patient without a history of seizure disorder, and a case of myoclonus following a thoracic metrizamide myelogram that showed a highgrade block. Metrizamide should not be used if an intrathecal block is suspected, or if the location to be studied makes intracranial spill difficult to avoid.

Light- and electron-microscopic characterization of electrophysiologically-identified, horseradish peroxidase-injected magnocellular neuroendocrine cells in goldfish preoptic nucleus.

We recorded intracellularly from neurons in the goldfish preoptic nucleus which were antidromically identified by electrical stimulation of the pituitary gland and marked by intracellular injection of horseradish peroxidase for subsequent localization. At the light-microscopic level, labeled neurons resembled profiles of Golgi-impregnated neurons and lay in the magnocellular portion of the preoptic nucleus. Densely labeled axons and dendrites projected to the lateral forebrain bundle, the medial forebrain bundle, fiber tracts in the preoptico-hypophysial tract, small blood vessels and capillaries, the ependymal lining of the third ventricle and toward the preoptic neurons. Occasionally, a lightly-labeled, large perikaryon lay adjacent to a large, heavily-labeled magnocellular neuron. Ultrastructural examination of these identified cells revealed dense reaction product in neuronal perikarya and processes. Heavily labeled perikarya had elaborate networks of endoplasmic reticulum, extensive Golgi apparatus, occasional somatic spines and infrequent axo-somatic contacts from unlabeled neurons. These labeled perikarya which were frequently in close somatic apposition with unlabeled profiles were sometimes adjacent to a large, lightly-labeled perikaryon. A thin glial sheath separated most labeled neurons and processes from brain capillary endothelium. Labeled dendrites had heavily labeled spines and axo-dendritic contacts from unlabeled neurons. Labeled axons abutted unlabeled-axons and -dendrites. Synaptic boutons innervating labeled structures always contained small clear synaptic vesicles and some boutons also contained large dense-core vesicles. These results demonstrate the complex connections of goldfish preoptic magnocellular neuroendocrine cells with other neurons, fiber systems, brain capillaries, ventricular ependyma and the pituitary and provide further support for non-endocrine as well as endocrine functions of magnocellular neurons.

Light and electron microscopic localization of glutamate immunoreactivity in the supraoptic nucleus of the rat hypothalamus.

The distribution of glutamate immunoreactivity was mapped within the supraoptic nucleus of the rat hypothalamus utilizing a specific anti-glutamate antibody. Magnocellular neuroendocrine cells of the supraoptic nucleus showed intense immunoreactivity for glutamate which varied with the conditions of fixation. Within the perikarya, reaction product was found associated with the endoplasmic reticulum but not the mitochondria, Golgi, dense bodies or neurosecretory granules. A relatively high density of glutamate-immunoreactive terminals was found in the supraoptic nucleus. These terminals were less affected by fixation condition and were generally found contacting large, glutamate-immunoreactive processes within the ventral dendritic neuropil of the supraoptic nucleus. The pattern and characteristics of glutamate immunoreactivity in the supraoptic nucleus suggested the presence of two distinct glutamate pools. The magnocellular neuroendocrine cells may contain a large, labile metabolic pool of glutamate. These cells, in turn, appear to receive glutamate synaptic input from a more stable pool consistent with suggestions that glutamate may be used as a transmitter within this system.

Kindled seizures induce a long-term increase in vasopressin mRNA.

Neuroendocrine disturbances are among the significant problems associated with animal and human seizures. To investigate the mechanisms for these disturbances, we examined changes in the expression of vasopressin (VP) mRNA in the hypothalamic magnocellular neuroendocrine cells of rats after amygdala kindled seizures, a model for temporal lobe epilepsy. A prominent increase in VP mRNA was found in the supraoptic nucleus of kindled animals by one week after the last seizure which persisted for at least 4 months. The increase occurred bilaterally in the SON and remained unchanged despite the absence of further stimulation, seizures or change in body fluid homeostasis. Since the VP mRNA change after kindling correlated with the duration of afterdischarge but not the number of amygdala stimuli the change appears to be an effect of the seizure. This chronic increase in VP mRNA appears to reflect a change in neuroendocrine gene expression and may identify an important new mechanism of plasticity that contributes to the neuroendocrine disturbances accompanying epilepsy.

Vasopressin mRNA changes during kindling: the effects of kindling site and stage.

Because of the many anatomical and functional links to the limbic system, the neuroendocrine system is often affected by limbic disturbances. Limbic seizures in humans and animals alter neuroendocrine function and hormone levels. We have shown that in an animal model for partial seizures, the amygdala kindled rat, plasma vasopressin levels are elevated and a sustained increase in vasopressin (VP) mRNA follows stage 5 kindled seizures. In the present experiments we sought to determine when during the course of amygdala kindling the VP mRNA increase occurs and whether specific anatomical pathways mediate this increase. Animals kindled to early seizure stages (stages 1, 2 or 3) had no consistent increase in VP mRNA in the supraoptic nucleus (SON) while animals kindled to generalized seizures, stages 4 or 5, invariably had increased VP mRNA relative to controls. Electrical kindling to stage 5 seizures from two other brain sites, the dorsal hippocampus and the anterior olfactory nucleus, consistently resulted in a significant increase in VP mRNA one week after completing kindling. In all experiments the increase in VP mRNA in the SON showed no differences related to the side or proximity of the electrodes used for kindling. Measures of water balance did not change following kindling. These results indicate that kindled seizure generalization is a prerequisite for the long-term increase in VP mRNA. Furthermore, the VP mRNA increase appears to involve polysynaptic pathways accessible from different limbic kindling sites. These studies support the hypothesis that changes in mRNA regulation may contribute to the neuroendocrine pathophysiology accompanying limbic seizures.

Vasopressin release by nicotine in the cat.

Our goal in this study was to examine where nicotine acted on the neurohypophysial release of arginine vasopressin (AVP). In the chamber-isolated, unanesthetized cat, an IV infusion of nicotine (25--50 micrograms/kg/min for 10 min) produced a 54-fold rise in plasma AVP (65 +/- 12.5 microU/ml) and a behavioral sequence of restlessness, ear twitching, salivation, chewing and retching. Chloralose anesthesia and acute surgical preparation increased plasma AVP (9.4 +/- 3.8 microU/ml) 4-times the control level in the unanesthetized state (2.4 +/- 0.1 microU/ml). In the anesthetized cat, an IV infusion of nicotine produced a 140-fold rise in plasma AVP (802.7 +/- 289.0 microU/ml) and a biphasic response in mean arterial blood pressure (MABP) which rose to 32% above control in the first 5 min and fell to 25% below control during the last 5 min. Bilateral surgical section of the carotid sinus and vagus nerves increased plasma AVP (47.6 +/- 25.8 microU/ml) to 5-times the control level in the anesthetized state (9.4 +/- 3.8 microU/ml). In the anesthetized, baroreceptor-denervated cat, an IV infusion of nicotine produced a 3-fold rise in plasma AVP (141.5 +/- 31.3 microU/ml) and a biphasic MABP response. Hypophysectomy abolished the nicotine-induced rise in AVP but did not modify the biphasic MABP response. These data suggest that in the cat nicotine releases AVP from the neurohypophysis by multiple sites of action within the receptive fields of te carotid sinus and vagus nerves and at unknown loci within the central nervous system.

Amygdala kindling elevates plasma vasopressin.

Acute and chronic effects of epilepsy on endocrine function are known to occur in humans with partial seizures of limbic origin and in animals with limbic kindled seizures. The amygdala, a component of the limbic system, has dense hypothalamic connections and amygdala stimulation in monkeys and cats result in vasopressin release. In the present study we sought to determine if amygdala stimulation in the rats results in an immediate acute release of vasopressin and to determine if acute or chronic changes occur in vasopressin release in the fully kindled animal. Plasma vasopressin, osmolality and hematocrit were measured in blood samples drawn from rats with implanted venous catheters before and after stimulation and at different stages of kindling. Low-frequency (15 Hz) electrical stimulation of the amygdala was followed by an immediate, 3-fold increase in plasma vasopressin concentration. Moreover, although the 60 Hz kindling stimulus did not result in a significant immediate rise in plasma vasopressin prior to kindling, after kindling to stage 5 seizures the 60 Hz kindling stimulus resulted in seizures and a significant immediate rise in plasma vasopressin. In addition, we found that kindling was followed by a significant, though modest, rise in the resting plasma vasopressin without an accompanying change in osmolality or hematocrit. We conclude that kindling results in a persistent alteration in the vasopressinergic neuroendocrine system.

Kindling in spontaneous hypertensive rats.

Vasopressin is a neurohormone and neuromodulator with many effects on behavior. Rats lacking vasopressin have been found to develop kindled seizures more slowly with amygdala stimulation. In the present study the spontaneous hypertensive (SH) rat and rats from the parent strain, the Wistar-Kyoto (WKY) rat received amygdala and pyriform kindling. The SH rat has been reported to have increased plasma vasopressin and increased brain vasopressin release. Plasma vasopressin, osmolality and hematocrit were also measured in blood samples obtained through chronic, indwelling catheters implanted in SH, WKY normal and Sprague-Dawley rats. SH rats were found to kindle with fewer afterdischarges than WKY normal rats with both amygdala and pyriform cortex stimulation. The total afterdischarge duration required to reach each kindling stage was significantly shorter in the SH rat. Plasma osmolality and vasopressin were significantly higher in the SH rats compared to WKY normal rats and Sprague-Dawley rats. These findings provide additional evidence that vasopressin may influence the establishment of enduring behaviors such as kindled seizures.

Accumulation of circulating endogenous and exogenous immunoglobulins by hypothalamic magnocellular neurons.

Rat monoclonal antibodies, used in immunocytochemistry of normal rat brain, result in a granular reaction product within neurons innervating areas lacking a blood-brain barrier. Immunocytochemical characterization shows that the staining is independent of the primary antibody and exclusively dependent on the presence of anti-rat immunoglobulin. This granular staining could be selectively eliminated by pre-adsorption of the anti-rat immunoglobulin with purified rat immunoglobulin or disruption of microtubule retrograde transport systems by intraventricular injection of colchicine. A dependence on retrograde transport and complete independence from local synthesis was further substantiated by the rapid uptake and accumulation of intravenously administered rabbit or rat [125I]immunoglobulins by the supraoptic-neurohypophysial system. Immunoelectron microscopy was used to identify the endogenous rat immunoglobulin within lysosome-like organelles in the cytoplasm of magnocellular neuroendocrine cells. The uptake and incorporation of plasma macromolecules into the lysosomal system of magnocellular and other neurons projecting to regions with a weak blood-brain barrier may represent a novel mode of blood-central nervous system interactions.

Ultrastructural distribution of glutamate immunoreactivity within neurosecretory endings and pituicytes of the rat neurohypophysis.

An ultrastructural analysis of post-embedding glutamate immunocytochemistry within the neural lobe of the pituitary was used to explore the possible role of glutamate within the magnocellular neuroendocrine cells. Relative densities of a colloidal gold marker associated with various cellular and subcellular compartments of the neural lobe were quantified by computer analysis of electron micrographs. Robust glutamate immunoreactivity was observed in both pituicytes (cytoplasm, mitochondria and nucleus) and neurosecretory endings. Within the neurosecretory endings, glutamate staining was specifically localized to the microvesicles with no overlap into the neurosecretory granule population. Stimulation of the vasopressin/oxytocin neurosecretory system by water deprivation increased glutamate content in pituicytes and mitochondria within neurosecretory endings but had little influence on microvesicle glutamate content. The results are consistent with the existence of multiple functional pools of immunoreactive glutamate in both pituicytes and neurosecretory endings. Microvesicles within the neurosecretory endings exhibit many properties of secretory vesicles, appear to be functionally independent of the neurosecretory granules, and have sufficient glutamate immunoreactivity to suggest that this amino acid may be compartmentalized for release in the neural lobe.

Quantitative mapping of glutamate presynaptic terminals in the supraoptic nucleus and surrounding hypothalamus.

Although the hypothalamus is generally regarded to have low levels of glutamate receptors, anatomical and physiological studies have provided consistent evidence implicating glutamate as a potential transmitter for the control of neuroendocrine cell activity. To clarify the extent of the contribution of synapses utilizing glutamate for control of vasopressin/oxytocin neuroendocrine cells, we mapped the density and location of glutamate immunoreactive terminals in the supraoptic nucleus and surrounding hypothalamus. Colloidal gold particle densities in presynaptic terminals were measured from electron micrographs of: (1) the magnocellular neuroendocrine cell perikarya (main body of the supraoptic nucleus), (2) the dendritic field of the magnocellular neuroendocrine cells (ventral dendritic neuropil) and (3) the hypothalamic perinuclear zone dorsal to the supraoptic nucleus. In addition, serial sections were stained, alternatively, for glutamate or GABA to determine glutamate staining in GABA cells. Terminals with high glutamate immunoreactivity were clearly distinguished from the glutamate precursor staining found in GABA terminals and were abundant at all rostral-caudal levels within each region. The number of glutamate terminals identified in each region was similar but represented a very high proportion of all terminals in the ventral dendritic neuropil (38%) vs. the main body of the supraoptic nucleus and the perinuclear zone (20-22%). The regional variation in the relative proportion of glutamate terminals was determined largely by differences in the number of non-glutamate terminals within each region. Glutamate and GABA terminals together accounted for over two-thirds of the innervation of vasopressin/oxytocin neuroendocrine cells. No systematic relationship was observed between excitatory and inhibitory inputs on the same cell. These results suggest that glutamate is the predominant excitatory transmitter used for control of vasopressin/oxytocin cells. The relative contribution of glutamate neurotransmission to a particular region will depend, in part, on the number and type of competing non-glutamate terminals.

Dye-marked paraventricular neuroendocrine cells in vivo in cat hypothalamus.

In antidromically identified neurons in the cat hypothalamus we recorded and injected fluorescent dye-markers (Lucifer Yellow, LY; Procion Yellow, PY) intracellularly. The dye-filled neurons lay in the rostral portion of the magnocellular paraventricular nucleus of the hypothalamus. We observed two morphological cell types of similar size based on the intracellular injections of LY or PY: a bipolar cell type with fusiform perikaryon and a multipolar cell type with a polygonal perikaryon. These morphological cell types correspond to previous descriptions of immunocytochemically identified vasopressinergic and oxytocinergic magnocellular neurons in mammals. This study demonstrates the feasibility of in vivo intracellular dye-marking and electrophysiological recordings from mammalian hypothalamic neurons. We have here a basis for correlating morphological characteristics with the physiological traits of single magnocellular neuroendocrine cells.

Ultrastructural immunocytochemical localization of enkephalin in the goldfish preoptic nucleus.

This study describes the ultrastructural localization of the opioid peptide enkephalin (ENK) in the preoptic nucleus of the goldfish. Using immunocytochemical techniques, ENK could be seen in neurosecretory granules and throughout the cytoplasm of magnocellular neurons with an agranular distribution. ENK was also associated with small clear vesicles and large dense-core vesicles within certain axon terminals in the preoptic nucleus. In this report, we discuss the cellular and synaptic relationships of ENK neurons in the preoptic nucleus of the teleost.