The Expression of Voltage-Gated Ca2+ Channels in Pituicytes and the Up-Regulation of L-Type Ca2+ Channels During Water Deprivation

The primary components of the neurohypophysis are the neuroendocrine terminals that release vasopressin and oxytocin, and pituicytes, which are astrocytes that normally surround and envelop these terminals. Pituicytes regulate neurohormone release by secreting the inhibitory modulator taurine in an osmotically‐regulated fashion and undergo a marked structural reorganisation in response to dehydration as well as during lactation and parturition. Because of these unique functions, and the possibility that Ca²⁺ influx could regulate their activity, we tested for the expression of voltage‐gated Ca²⁺ channel α1 subunits in pituicytes both in situ and in primary culture. Colocalisation studies in neurohypophysial slices show that pituicytes (identified by their expression of the glial marker S100β), are immunoreactive for antibodies directed against Ca²⁺ channel α1 subunits Ca_V2.2 and Ca_V2.3, which mediate N‐ and R‐type Ca²⁺ currents, respectively. Pituicytes in primary culture express immunoreactivity for Ca_V1.2, Ca_V2.1, Ca_V2.2, Ca_V2.3 and Ca_V3.1 (which mediate L‐, P/Q‐, N‐, R‐ and T‐type currents, respectively) and immunoblotting studies confirmed the expression of these Ca²⁺ channel α1 subunits. This increase in Ca²⁺ channel expression may occur only in pituicytes in culture, or may reflect an inherent capability of pituicytes to initiate the expression of multiple types of Ca²⁺ channels when stimulated to do so. We therefore performed immunohistochemistry studies on pituitaries obtained from rats that had been deprived of water for 24 h. Pituicytes in these preparations showed a significantly increased immunoreactivity to Ca_V1.2, suggesting that expression of these channels is up‐regulated during the adaptation to long‐lasting dehydration. Our results suggest that Ca²⁺ channels may play important roles in pituicyte function, including a contribution to the adaptation that occurs in pituicytes when the need for hormone release is elevated.     

Fine Structural Changes in Explants of the Neural Lobe of the Rat Hypophysis

To obtain a purified population of pituicytes, pieces of rat neural lobes were dissected free of the pars intermedia, incubated in a variety of media, and fixed at regular intervals between 7 and 56 days of incubation. Many neurosecretory axons survived for up to 21 days without any apparent signs of degeneration. Most axons, however, degenerated and were progressively phagocytosed and subsequently eliminated by pituicytes and microglial cells. Lysed axons that were not eliminated, persisted as dense bodies or paracrystalline inclusions. After 30 days of culture, cluster-forming pituicytes predominated the explants. Pituicytes underwent morphologic changes such as medium-dependent decrease or increase of lipid inclusions, Golgi activation, process extension and interdigitation, formation of gap junctions and bundles of intermediate filaments. At the explant surface in contact with the culture medium, pituicytes differentiated into an epithelial layer of ciliated and microvilli-bearing cells linked by junctional complexes. Long-term neural lobe explants are a relatively pure source of viable pituicytes and should be useful for further studies on the functional significance of these cells.     

Electrophysiological changes in substantia nigra after dynorphin administration

The present experiments were designed to explore the electrophysiological changes induced by dynorphin (Dyn) which was administered by pressure ejection into the zona reticulata of the substantia nigra (SNR). Dyn produced a long-lasting (6.0 +/- 4.5 min) decrease in firing rate in 70% of the cells recorded. Naloxone reversed the Dyn-induced decrease in firing rate in all cases, and also induced an increase in firing frequency on most occasions. Dyn did not alter cell activity in 8 of the 10 cells recorded in zona compacta of substantia nigra. It is concluded that Dyn exerts an inhibitory effect on SNR neurons, which appears to be pharmacologically specific since it is reversed by naloxone.     

Postnatal Changes in Electrophysiological Properties of Rat Nucleus Tractus Solitarii Neurons

Whole-cell recordings in brainstem slices revealed postnatal changes in passive and firing properties in the rat caudal nucleus tractus solitarii (cNTS) neurons. Membrane potential, threshold for Na⁺ spike and degree of sag were unchanged during development. In the adult, the rheobase was twice that found at birth. The input resistance decreased over the period studied, while time constants declined markedly after the third postnatal week. At all postnatal ages, Na⁺-dependent action potentials (APs) were elicited in response to depolarization. Nevertheless, AP duration gradually decreased by 40% over the developmental period studied. Spike amplitude was smaller at birth than at any other ages and reached a peak two weeks after birth. At all ages, Na⁺-dependent APs were blocked by application of tetrodotoxin. Full APs were replaced by an initial slow oscillation in young cells and by oscillations in older cells. The TTX-resistant oscillations were altered by cobalt (2 mM) and cadmium (100 μM). The spike afterhyperpolarization (AHP) was not altered during development, but was observed in less neurons in adult cells when measured at a holding potential of -60 mV. Neurons were subdivided into one of three classes based on their responses to a hyperpolarizing prepulse: 1) post-inhibitory rebound (PIR) cells, 2) delayed excitation (DE) cells and 3) NON cells expressing neither PIR nor DE. The relative proportions of different cell types varied with age. The mean maximum duration of DE increased three times. Voltage-clamp experiments revealed that the DE was due to the activation of an A-current. In addition, a three-fold increase in its inactivation rate was observed postnatally. The physiological significance of these results is discussed.     

Changes in the immunoreactivities of an opioid peptide leumorphin in the hypothalamus and anterior pituitary during the estrous cycle of the rat and their relation to sexual behavior

Leumorphin, an opioid peptide whose functions are unknown, is found in mammalian brain and pituitary and stimulates lordosis behavior in estrogen-treated female rats. To elucidate the role of leumorphin in the physiological control of female sexual behavior, the levels of immunoreactive (ir) leumorphin as well as ir dynorphin (dynorphin A) were measured in the rat brain and pituitary during the estrous cycle. There was a clear variation of ir leumorphin in the hypothalamus and anterior pituitary during the estrous cycle. The levels of ir leumorphin in the hypothalamus and anterior pituitary on the afternoon of proestrus were significantly higher (P less than 0.01) than those on the afternoons of estrus and metestrus. The rise in the hypothalamic levels of ir leumorphin on the afternoon of proestrus was correlated with the receptivity of lordosis during the estrous cycle. Furthermore, there was a close correlation with ir dynorphin levels. These findings are in agreement with studies demonstrating a common precursor for leumorphin and dynorphin. Ir leumorphin in the hippocampus and neurointermediate pituitary did not change significantly during the estrous cycle. Because the leumorphin antiserum used recognizes rimorphin (dynorphin B) 1.78 times more than porcine leumorphin on a molar basis, high performance-gel permeation chromatography was done on pooled extracts of hypothalamus taken at proestrus and estrus. The peak in the leumorphin-like substance in the activation of sexual behavior is discussed.     

慢性酒精性周围神经病的电生理改变

目的探讨慢性酒精性周围神经病的电生理特点和诊断价值.方法检测36例慢性酒精中毒患者与35例正常人肌电图对照,统计分析.结果观察组中确诊为临床或亚临床周围神经病的共30例(83.3 3%).观察组的正中神经、尺神经、胫后神经、腓总神经、腓肠、腓浅神经传导速度(NCV)及波幅明显低于正常对照组,感觉神经异常率高于运动神经,下肢异常率高于上肢.各神经传导速度与酒精摄入总量(TLDE)呈负相关.结论酒精对周围神经的毒性呈剂量依赖性.神经电生理检查能敏感地评价慢性酒精中毒患者的周围神经受损程度.     

Immunoreactive glial fibrillary acidic protein in pituicytes of the rat neurohypophysis

The glial fibrillary acidic protein (GFAP) has been associated with glial filaments. Electron microscopic examination of rat pituicytes in our laboratory has revealed few of these 8–9 nm filaments that are present in other astrocytes. Since the literature is inconsistent on the existence of filaments in pituicytes, we investigated the content of GFAP in these cells. Immunocytochemical methods revealed a strong positivity for GFAP in pituicytes. Furthermore, the primary antiserum dilution required for optimal staining suggests that there may be more GFAP in pituicytes than in other glial elements. The significance of immunoreactive GFAP in pituicytes is discussed in terms of possible functions and embryonic origins.     

糖尿病性周围神经病的临床与电生理改变

目的 探讨糖尿病性周围神经病的电生理特点及其诊断价值.方法 检测52例2型糖尿病患者与50例正常人肌电图进行对照统计分析.结果 观察组中确诊为临床或亚临床周围神经病的共41例(78.85%).观察组的正中神经、尺神经、胫后神经、腓总神经、腓肠神经传导速度(NCV)及波幅(AMP)明显低于正常对照组.各神经传导速度与糖尿病病程及糖化血红蛋白(HbA1c)水平呈负相关.观察组中合并正中神经腕管卡压者明显高于一般人群.结论 神经电生理检查能一定程度地反映糖尿病患者的周围神经受损程度.     

Beta-Adrenergic Stimulation Decreases Glial and Increases Neural Contact with the Basal Lamina in Rat Neurointermediate Lobes Incubated in vitro

Physiological stimuli such as dehydration and lactation/suckling of the young have been found to induce reversible changes in the relationships of neural and glial elements with the basal lamina of the neurohypophysis. Such stimulation is associated with a decline in the extent of basal lamina occupied by glial processes and an increase in nerve terminals abutting the lamina. One possible mechanism playing a role in these changes is the activation of beta-adrenergic receptors on the neurohypophysial glia (the pituicytes), since such cells in primary culture from adult rats undergo dramatic morphological transformation when stimulated with beta-adrenergic agonists. We sought to determine if changes similar to those seen in vivo and predicted from the responses of cultured cells would occur with beta-adrenergic stimulation of the isolated neurointermediate lobe in vitro. Neurointermediate lobes from adult male rats were incubated in artificial cerebrospinal fluid containing ascorbic acid alone (control) or in the same medium with either 10^?8, 10^?7 or 10^?5 M isoproterenol for 15 min after a 30-min period of preincubation in control medium. Quantitative ultrastructural analysis revealed significant decreases in pituicyte and, corresponding increases in neural, membrane abutting the basal lamina at the lower two drug concentrations. These results support the findings of other studies suggesting a beta-adrenergic mediation of pituicyte morphology and a role for beta-receptors in control of posterior pituitary function.     

Changes in lymphocyte beta-adrenergic receptors in depression and mania.

The beta-adrenergic receptors were studied in vitro in lymphocytes obtained from patients with major affective disorders and controls. Specific L-[3H]-dihydroalprenolol binding was decreased in both depressed and manic patients compared to controls and euthymic patients. Isoproterenol-stimulated, but not prostaglandin El-stimulated, cyclic adenosine-3',5'-monophosphate production was decreased in manic and depressed patients. These results suggest decreased lymphocyte beta-receptor functioning in depression and mania. This decrease may be an index of changes in brain beta-receptors in mania and depression, or may simply reflect homeostatic regulation of peripheral beta-receptors in response to stress-induced increases in circulating catecholamines.     

Adenovirus-mediated gene delivery to cells of the magnocellular hypothalamo-neurohypophyseal system

The objective of the present study was to define the optimum conditions for using replication-defective adenovirus (Ad) to transfer the gene for the green fluorescent protein (GFP) to the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei and cells of the neurohypophysis (NH). As indicated by characterizing cell survival over 15 days in culture and in electrophysiological whole cell patch-clamp studies, viral concentrations up to 2 x 10(7) pfu/coverslip did not affect viability of transfected PVN and NH cultured cells from preweanling rats. At 2 x 10(7) pfu, GFP gene expression was higher (40% of GFP-positive cells) and more sustained (up to 15 days). Using a stereotaxic approach in adult rats, we were able to directly transduce the PVN, SON, and NH and visualize gene expression in coronal brain slices and in the pituitary 4 days after injection of Ad. In animals receiving NH injections of Ad, the virus was retrogradely transported to PVN and SON neurons as indicated by the appearance of GFP-positive neurons in cultures of dissociated cells from those brain nuclei and by polymerase chain reaction and Western blot analyses of PVN and SON tissues. Adenoviral concentrations of up to 8 x 10(6) pfu injected into the NH did not affect cell viability and did not cause inflammatory responses. Adenoviral injection into the pituitary enabled the selective delivery of genes to the soma of magnocellular neurons. The experimental approaches described here provide potentially useful strategies for the treatment of disordered expression of the hormones vasopressin or oxytocin.     

Ultrastructural localization of enkephalin immunoreactivity in the substantia nigra of the monkey

Localization of enkephalin-like immunoreactivity in the substantia nigra of the Japanese red-faced monkey was investigated by light and electron microscopy using the peroxidase-anti-peroxidase method. Immunoreactive precipitates were predominantly distributed in the nerve fibers of the substantia nigra pars reticulata but not in the perikarya, and a low density of immunoreactive enkephalin was found in the pars compacta of this nucleus. Electron microscopic observations showed that enkephalin-immunoreactive precipitates were located in axon terminals containing numerous small clear vesicles either with or without labeled large granular vesicles and formed predominantly axo-dendritic synapses.     

Chronic haloperidol and clozapine differentially affect dynorphin peptides and substance P in basal ganglia of the rat

The effect of chronic neuroleptic treatment, using haloperidol or clozapine, on immunoreactive dynorphin peptide and substance P levels in basal ganglia of rats was examined. The drugs were administered i.p. in daily doses for 10 days (haloperidol 1 mg/kg and clozapine 10 mg/kg). Dynorphin A, dynorphin B and substance P were measured in substantia nigra, striatum, globus pallidus and hypothalamus using specific radioimmunoassays. The most prominent effects were observed with with clozapine which increased levels of all measured peptides in substantia nigra. Haloperidol only affected nigral substance P levels which declined, while nigral dynorphin peptide levels remained unchanged. In striatum, haloperidol slightly reduced dynorphin peptides while substance P was unaffected. Clozapine increased striatal substance P but the dynorphin peptides were not affected. Minor changes in dynorphin peptides found in globus pallidus and hypothalamus were not statistically reliable. Substance P was not changed in these structures after either of the two drugs. High molecular weight fragments (greater than or equal to 5,000) from the dynorphin precursor, proenkephalin B, were measured in substantia nigra and striatum using trypsin digestion and subsequent analysis of generated Leu-enkephalin-Arg6. These high molecular weight fragments were found to be affected in the same manner as the dynorphin peptides. This study indicates that the two types of neuroleptic drugs have different modes of interaction on peptide systems in basal ganglia of rats. Dynorphin peptides and substance P were also differentially affected.     

Effects of morphine and naloxone on the K-stimulated release of methionine-enkephalin from slices of rat corpus striatum

Methionine-enkephalin (ME) released from superfused slices of rat corpus striatum was estimated by radioimmunoassay (RIA). The basal release of2.5 ± 0.2pmol/g/min (0.15% of content per min) was increased approximately 3-fold upon exposure of tissue to 30 mM K⁺ for 5 min. This increase in release was not observed in the absence of Ca²⁺. Both morphine (10⁻⁵ M) and (−)-naloxone (10−⁵ and 10−⁶ M) significantly depressed the release of ME evoked by 30 mM K⁺ but did not alter basal release. The +-isomer of naloxone, which lacks opiate antagonist activity, did not affect basal or evoked release. A consistent depression of release was not observed when 47 mM K⁺ was used to evoke the release of ME. The issue of whether a feedback mechanism controls the release of ME from the striatum cannot be resolved until it is known whether the effect of morphine and naloxone on ME release are mediated by opiate or non-opiate mechanisms.     

The Cellular Electrophysiological Properties Underlying Multiplexed Coding in Purkinje Cells

Neuronal firing patterns are crucial to underpin circuit level behaviors. In cerebellar Purkinje cells (PCs), both spike rates and pauses are used for behavioral coding, but the cellular mechanisms causing code transitions remain unknown. We use a well-validated PC model to explore the coding strategy that individual PCs use to process parallel fiber (PF) inputs. We find increasing input intensity shifts PCs from linear rate-coders to burst-pause timing-coders by triggering localized dendritic spikes. We validate dendritic spike properties with experimental data, elucidate spiking mechanisms, and predict spiking thresholds with and without inhibition. Both linear and burst-pause computations use individual branches as computational units, which challenges the traditional view of PCs as linear point neurons. Dendritic spike thresholds can be regulated by voltage state, compartmentalized channel modulation, between-branch interaction and synaptic inhibition to expand the dynamic range of linear computation or burst-pause computation. In addition, co-activated PF inputs between branches can modify somatic maximum spike rates and pause durations to make them carry analog signals. Our results provide new insights into the strategies used by individual neurons to expand their capacity of information processing.SIGNIFICANCE STATEMENT Understanding how neurons process information is a fundamental question in neuroscience. Purkinje cells (PCs) were traditionally regarded as linear point neurons. We used computational modeling to unveil their electrophysiological properties underlying the multiplexed coding strategy that is observed during behaviors. We demonstrate that increasing input intensity triggers localized dendritic spikes, shifting PCs from linear rate-coders to burst-pause timing-coders. Both coding strategies work at the level of individual dendritic branches. Our work suggests that PCs have the ability to implement branch-specific multiplexed coding at the cellular level, thereby increasing the capacity of cerebellar coding and learning.     

Increase of plasma methionine-enkephalin level in patients at the acute stage of cerebral infarction

We investigated the participation of endogenous opioid peptide in cerebral ischaemia. In 13 patients with supratentorial infarction, the plasma immunoreactive methionine-enkephalin concentration at the acute stage (30.77 ± 3.54 pg/ml) was significantly higher than that at the chronic stage (20.37 ± 2.07 pg/ml) or that of the control subjects (19.64 ± 1.71 pg/ml). However; it was unrelated to infarct size or patient severity. The increased methionine-enkephalin, which appears to originate from the sympatho-adrenal system or infarcted brain, may play a role in the evolution of cerebral ischaemia.     

Role of the ventromedial hypothalamus in the regulation of adenohypophyseal immunoreactive dynorphin in the rat

In this study, we have examined the role of the dorsomedial (DMH), ventromedial (VMH) and arcuate (ARH) nuclei of the hypothalamus in the control of hypothalamic and pituitary immunoreactive (ir) dynorphin (Dyn) A and ir-Dyn B in the rat, by evaluating the effect of discrete, bilateral radiofrequency lesions in these structures. Lesions limited to the VMH reduced the content of ir-Dyn in the anterior pituitary but not in the neurointermediate lobe or in the hypothalamus. Gel chromatographic analysis of anterior pituitary extracts confirmed that ir-Dyn is mainly associated with high molecular weight forms containing Dyn A and Dyn B in their sequence. Anterior pituitary extracts of VMH-lesioned rats displayed a clearly lower proportion of these forms. Destruction of the DMH affected only the hypothalamic content of ir-Dyn; ablation of the ARH did not cause any significant change. Our results suggest that ablation of the VMH may disrupt critical neuronal connections to the median eminence originating in this nucleus or crossing it and participating in control of the adenohypophyseal pool of ir-Dyn.     

Decreased glutamate release correlates with elevated dynorphin content in the hippocampus of aged rats with spatial learning deficits.

The effects of aging on extracellular glutamate and tissue dynorphin content in the hippocampus were examined in Fischer-344 rats. Young adult (4-month-old) and aged (24-month-old) rats were trained to find a hidden platform in the Morris water task. Aged rats were unable to acquire the spatial learning task as rapidly as young controls. Following behavioral testing, an in vivo microdialysis perfusion method was used to determine extracellular glutamate levels in the hippocampus. There was a 25-35% reduction in extracellular glutamate concentration in both dorsal and ventral hippocampus of aged rats compared to young rats, in the absence of any change in tissue glutamate levels. Radioimmunoassay showed an increase in dynorphin A(1-8)-like immunoreactivity [DYN-A(1-8)LI] in both dorsal and ventral hippocampus, but not striatum, of aged rats. Immunocytochemistry indicated that this increase was localized to the dentate granule cells and mossy fibers. Furthermore, among the aged rats the increase in DYN-A(1-8)LI was inversely correlated with the decrease in extracellular glutamate. These results suggest that the disregulation of dynorphin observed in cognitively impaired aged rats is related to reduced excitatory transmission within the hippocampal formation.     

The Adaptive Brain: Glenn Hatton and the Supraoptic Nucleus

In December 2009, Glenn Hatton died, and neuroendocrinology lost a pioneer who had done much to forge our present understanding of the hypothalamus and whose productivity had not faded with the passing years. Glenn, an expert in both functional morphology and electrophysiology, was driven by a will to understand the significance of his observations in the context of the living, behaving organism. He also had the wit to generate bold and challenging hypotheses, the wherewithal to expose them to critical and elegant experimental testing, and a way with words that gave his papers and lectures clarity and eloquence. The hypothalamo‐neurohypophysial system offered a host of opportunities for understanding how physiological functions are fulfilled by the electrical activity of neurones, how neuronal behaviour changes with changing physiological states, and how morphological changes contribute to the physiological response. In the vision that Glenn developed over 35 years, the neuroendocrine brain is as dynamic in structure as it is adaptable in function. Its adaptability is reflected not only by mere synaptic plasticity, but also by changes in neuronal morphology and in the morphology of the glial cells. Astrocytes, in Glenn’s view, were intimate partners of the neurones, partners with an essential role in adaptation to changing physiological demands.