A riot of rhythms: neuronal and glial circadian oscillators in the mediobasal hypothalamus

Background

Proneurotrophins and mature neurotrophins elicit opposite effects via the p75 neurotrophin receptor (p75^NTR) and Trk tyrosine kinase receptors, respectively; however the molecular roles of proneurotrophins in the CNS are not fully understood.

Results

Based on two rare single nucleotide polymorphisms (SNPs) of the human brain-derived neurotrophic factor (BDNF) gene, we generated R125M-, R127L- and R125M/R127L-BDNF, which have amino acid substitution(s) near the cleavage site between the pro- and mature-domain of BDNF. Western blot analyses demonstrated that these BDNF variants are poorly cleaved and result in the predominant secretion of proBDNF. Using these cleavage-resistant proBDNF (CR-proBDNF) variants, the molecular and cellular roles of proBDNF on the CNS neurons were examined. First, CR-proBDNF showed normal intracellular distribution and secretion in cultured hippocampal neurons, suggesting that inhibition of proBDNF cleavage does not affect intracellular transportation and secretion of BDNF. Second, we purified recombinant CR-proBDNF and tested its biological effects using cultured CNS neurons. Treatment with CR-proBDNF elicited apoptosis of cultured cerebellar granule neurons (CGNs), while treatment with mature BDNF (matBDNF) promoted cell survival. Third, we examined the effects of CR-proBDNF on neuronal morphology using more than 2-week cultures of basal forebrain cholinergic neurons (BFCNs) and hippocampal neurons. Interestingly, in marked contrast to the action of matBDNF, which increased the number of cholinergic fibers and hippocampal dendritic spines, CR-proBDNF dramatically reduced the number of cholinergic fibers and hippocampal dendritic spines, without affecting the survival of these neurons.

Conclusion

These results suggest that proBDNF has distinct functions in different populations of CNS neurons and might be responsible for specific physiological cellular processes in the brain.     

PSA-NCAM and B-50/GAP-43 are coexpressed by specific neuronal systems of the adult rat mediobasal hypothalamus that exhibit remarkable capacities for morphological plasticity

The present study was designed to determine whether the mediobasal hypothalamus of adult rats contains neurons that continue to coexpress the highly polysialylated neural cell adhesion molecule (PSA-NCAM) and B-50/GAP-43, two proteins coexpressed by virtually all of the neurons of the fetal and neonatal rat central nervous system. Confocal laser scanning microscopy combined with double- or triple-fluorescence immunostaining was used to identify the hypothalamic neurons that express high levels of both PSA-NCAM and B-50/GAP-43 and to study the possible modifications of their morphological organization following a surgical lesion through the mediobasal hypothalamus. In intact animals, PSA-NCAM and B-50/GAP-43 were found to be colocalized within numerous fibers projecting throughout the external layer of the median eminence that were immunoreactive for either gamma-aminobutyric acid (GABA) or tyrosine hydroxylase (TH). Three to 30 days after a lesion through this region, numerous regenerating axonal sprouts, triple-immunostained for PSA-NCAM, B-50/GAP-43, and either GABA or TH, were detected along the ventricular surface of, and throughout the perivascular layer of, the median eminence. Surprisingly, high levels of PSA-NCAM and B-50/GAP-43 were also associated with numerous supraependymal neurons that exhibited long ramified processes and were immunoreactive for GABA but TH-negative. The use of the proliferation marker, ³H-thymidine, further indicated that the emergence of such supraependymal neurons after median eminence lesion was not related to the proliferation of preexisting quiescent cells. These data indicate that the mediobasal hypothalamus of the adult rat contains two neuronal systems, in which the continued coexpression of PSA-NCAM and B-50/GAP-43 is related to remarkable capacities for postlesional, morphological plasticity. J. Comp. Neurol. 384:181-199, 1997. © 1997 Wiley-Liss, Inc.     

Correlative scanning-immunoelectromicroscopic analysis of neuropeptide localization and neuronal plasticity in the endocrine hypothalamus

Thirty-two Sprague-Dawley rats were divided into four groups, eight rats per group. Animals were hypophysectomized with removal of both the pars distalis and the neural lobe of the neurohypophysis. Groups of eight rats were euthanized 1, 2, 4 and 8 weeks following hypophysectomy and prepared for routine scanning electron microscopy (SEM) and correlative immunoelectron microscopy employing antisera against arginine vasopressin (AVP). Eight normal rats served as controls. In experimental rats that survived one to eight weeks posthypophysectomy, remarkable neuroanatomical alterations were notable in the median eminence and adjacent third cerebral ventricular lumen. In contrast to normal control rats, large numbers of neurites were observed with SEM to insinuate from the lateral recess into the cerebral ventricular lumen and as early as one week following hypophysectomy they overgrew the apical surfaces of ependymal cells that constitute the lining of the cerebral ventricle. Immunoelectron microscopy revealed that a significant proportion of these neurites were magnocellular in origin in that they harbored AVP-positive neurosecretory vesicles. In addition to large numbers of invading magnocellular neurites, neuronal perikayria with apparent axosomatic synapses were observed to emerge upon the thick feltwork of invading axons, the latter of which appeared to freely terminate within the ventricular lumen. AVP-positive axon profiles were, in addition, seen to terminate upon the basal lamina of portal perivascular spaces in the zona externa of the median eminence. These data are consistent with the idea that following hypophysectomy (to include high stalk section of the neurohypophyseal system), that there is rapid, and dynamic sprouting and regrowth of AVP-positive axons into the adjacent third cerebral ventricular lumen and to the contact zone of the median eminence as well. This phenomenon may represent a compensatory physiological response to injury of the neurohypophyseal system characterized by a highly plastic neuroanatomical reorganization of magnocellular elements which appear to utilize the CSF of the third cerebral ventricle as a functional terminus for the neurocisternal secretion of AVP which ultimately enters the systemic circulation.     

Cell surface expression of polysialic acid on NCAM is a prerequisite for activity-dependent morphological neuronal and glial plasticity.

Polysialic acid (PSA) on the extracellular domain of the neural cell adhesion molecule (NCAM) reduces cell adhesion and is considered an important regulator of cell surface interactions. The hypothalamo-neurohypophysial system (HNS), whose glia, neurons, and synapses undergo striking, reversible morphological changes in response to physiological stimulation, expresses high levels of PSA-NCAM throughout life. Light and electron microscopic immunocytochemistry in normal rats and rats in which cell transport was blocked with colchicine showed that PSA-NCAM is expressed in both HNS neurons and glia, particularly at the level of astrocytic processes that envelop neuronal profiles and can undergo remodeling. Moreover, we confirmed that the overall levels of PSA-NCAM were not greatly altered by stimulation (lactation and chronic salt ingestion). Nevertheless, PSA is essential to morphological plasticity. Using comparative ultrastructural analysis, we found that, after specific enzymatic removal of PSA from NCAM by microinjection of endoneuraminidase close to the hypothalamic magnocellular nuclei in vivo, there was no apparent withdrawal of astrocytic processes nor any increase in synaptic contacts normally induced by lactation and dehydration. Our observations demonstrate, therefore, that expression of PSA on cell surfaces in the adult HNS is indispensable to its capacity for activity-dependent morphological neuronal-glial and synaptic plasticity. The carbohydrate PSA on NCAM can thus be considered a necessary permissive factor to allow neuronal and glial remodeling to occur whenever the proper inductive stimulus intervenes.     

Non-uniform release at the frog neuromuscular junction: evidence of morphological and physiological plasticity

The frog neuromuscular junction (NMJ) is a fusiform structure parallel to the muscle fiber with a few secondary and tertiary branches. Both sprouting and regression can occur on the same nerve terminal, suggesting a continuous on-going remodelling of the mature neuromuscular junction. Thus, the frog NMJ is a dynamic structure. Ultrastructural observations of the nerve terminal suggest that the active zones are distributed equally along the mature nerve terminal. Disorganized active zones have however been observed in distal regions. The density of synaptic vesicles is also uniform throughout the whole structure. However, mitochondria appear to be more abundant in the very distal regions of the nerve terminal. The postjunctional folds and the cholinergic receptors are also uniformly distributed along the NMJ. However, during remodelling periods, the distributions of postjunctional folds and of cholinergic receptors are not uniform in the degenerating and regenerating regions. Fig. 1 summarizes these morphological data. The frequency of spontaneous release (MEPPs) at the NMJ is higher in the proximal region than in the distal regions and recent evidence suggests that the mean MEPP amplitude is higher in the proximal than in the distal portions. Evoked transmitter release is also non-uniform along the frog NMJ. As for spontaneous release, it is higher in the proximal regions than in the distal regions. Failures of the active propagation of the PNAP at low safety points, such as the end of the myelinated axon and the branching points, may be one of the mechanisms responsible for unequal evoked release. It is also possible that the PNAP does not actively invade the whole extend of the nerve terminal since Na+ channels are absent from the distal regions. Fig. 2 summarizes these physiological data.     

Aging and neuronal plasticity: lessons from a model

In spite of many well-documented examples of age-related reductions in neuronal plasticity, the causes of such changes remain largely unknown. One example of age-reduced plasticity involves an aberrant sprouting response of mature rat sympathetic neurons into the CNS (hippocampal formation). This phenomenon has proven to be useful for exploring the relative contribution of target aging (extrinsic influences) versus neuronal aging (intrinsic influences) to reduced sprouting. Aged sympathetic neurons mount a robust growth response when confronted with young target tissue or when exposed to exogenous trophic factor in vivo. In contrast, the aged target tissue (the hippocampal formation in this example) exhibits reduced receptivity for sympathetic sprouting. This change in the target does not appear to be due to alterations in baseline levels of trophic or substrate support for axonal growth. Rather, aging appears to dampen the consequences of target denervation so that the aged target elicits less sprouting. Age-related reductions in neuronal sprouting are speculated to reflect increasing commitment to information storage at the expense of neuronal plasticity.     

Is taurine a hypothalamic neurotransmitter?: A model of the differential uptake and compartmentalization of taurine by neuronal and glial cell particles from the rat hypothalamus

Although taurine has been postulated to be a neurotransmitter or neuromodulator in the mammalian CNS, little is known concerning its role in brain function. Evidence suggesting that taurine may influence endocrine and homeostatic mechanisms via the hypothalamus resulted in our investigations into its function in this brain region. The main objectives of the research were to characterize the specific binding, uptake, and release of taurine in the hypothalamus. A specific aim was to examine the proposed neurotransmitter role for taurine in the hypothalamus. This was accomplished by comparing the characteristics and properties of the binding, uptake, and release of taurine with those for the classical neurotransmitters which satisfy the criteria for a neurotransmitter. On such a comparative basis, the characteristics of taurine uptake satisfy the neurotransmitter criterion of inactivation of taurine in the hypothalamus. However, the observed characteristics of taurine binding and release in the hypothalamus do not satisfy the respective neurotransmitter criteria of specific receptors and Ca2+-dependent evoked release. Therefore, solely on the basis of the experimental observations reported herein, we must conclude that taurine apparently does not function as a neurotransmitter in the hypothalamus. Two uptake systems were found in the P2 fraction, a high affinity uptake system and a low affinity uptake system. Uptake systems for taurine have previously been reported in glial and nerve cell homogenates, and therefore, because of the known contamination of crude synaptosomal preparations with glial particles, we sought to determine the cellular origin of the two taurine uptake systems in our crude preparation. Using a variety of diverse biochemical techniques such as hypo-osmotic shock, release experiments and Arrhenius plots, we determined that physical changes of the media or depolarizing stimuli which would influence neuronal and glial cell particles differently, also had differing effects on high and low affinity taurine uptake or its release from the respective uptake compartments. We conclude that the high affinity taurine uptake system/compartment is located on/in neuronal membranes/particles/particles and that the low affinity taurine uptake system/compartment is located on/in neuronal membranes/particles and that model for the differential cellular transport and compartmentalization of taurine into neuronal and glial cells has important implications concerning its possible role in the CNS.     

Analysis of spine morphological plasticity in developing hippocampal pyramidal neurons

Dendritic spines are targets of most excitatory inputs in the central nervous system (CNS) and are morphologically heterogeneous. Ultrastructural studies have traditionally classified spines into four major categories (filopodia, stubby, thin, and mushroom) based on their distinct morphologies. The recent discovery of rapid morphological plasticity of spines has raised the possibility that those categories, rather than being intrinsically different populations of spines, represent instead temporal snapshots of a single dynamic phenomenon. We examined this question with two-photon time-lapse imaging of developing hippocampal pyramidal neurons, transfected with E-GFP in cultured slices. After blind scoring to morphologically classify spines into the four traditional groups, we analyzed the fate of populations of spines over a period of 2-4 h. We found considerable morphological conversions among all categories, although systematic trends were detected. While most stubbies and spines (defined for our analysis as the combination of thin and mushroom protrusions) retained their basic morphologies, most filopodia transformed into stubbies and spines, although they could also extend out of existing spines. Our results suggest that in developing hippocampal pyramidal neurons, traditional morphological distinctions are stable over short (<4 h) periods of time, but that at the same time, considerable mixing among these groups takes place.     

Phenotyping of nNOS neurons in the postnatal and adult female mouse hypothalamus

Neurons expressing nitric oxide (NO) synthase (nNOS) and thus capable of synthesizing NO play major roles in many aspects of brain function. While the heterogeneity of nNOS‐expressing neurons has been studied in various brain regions, their phenotype in the hypothalamus remains largely unknown. Here we examined the distribution of cells expressing nNOS in the postnatal and adult female mouse hypothalamus using immunohistochemistry. In both adults and neonates, nNOS was largely restricted to regions of the hypothalamus involved in the control of bodily functions, such as energy balance and reproduction. Labeled cells were found in the paraventricular, ventromedial, and dorsomedial nuclei as well as in the lateral area of the hypothalamus. Intriguingly, nNOS was seen only after the second week of life in the arcuate nucleus of the hypothalamus (ARH). The most dense and heavily labeled population of cells was found in the organum vasculosum laminae terminalis (OV) and the median preoptic nucleus (MEPO), where most of the somata of the neuroendocrine neurons releasing GnRH and controlling reproduction are located. A great proportion of nNOS‐immunoreactive neurons in the OV/MEPO and ARH were seen to express estrogen receptor (ER) α. Notably, almost all ERα‐immunoreactive cells of the OV/MEPO also expressed nNOS. Moreover, the use of EYFP^Vglut2, EYFP^Vgat, and GFP^Gad67 transgenic mouse lines revealed that, like GnRH neurons, most hypothalamic nNOS neurons have a glutamatergic phenotype, except for nNOS neurons of the ARH, which are GABAergic. Altogether, these observations are consistent with the proposed role of nNOS neurons in physiological processes.     

5,7-Dihydroxytryptamine injections increase glial fibrillary acidic protein in the hypothalamus of adult rats

The distribution and levels of glial fibrillary acidic protein (GFAP) were determined in the adult rat hypothalamus following axotomy of serotonin (5-HT) neurons. Seven days after unilateral intrahypothalamic injection of the 5-HT neurotoxin, 5,7-dihydroxytryptamine, there was a marked increase in the number of GFAP-labelled astrocytes in the ipsilateral hypothalamus of 5,7-DHT-treated as compared to sham-treated rats. In addition, levels of GFAP were significantly increased 7 days after 5,7-DHT injection.     

Seasonal regulation of structural plasticity and neurogenesis in the adult mammalian brain: Focus on the sheep hypothalamus

To cope with variations in the environment, most mammalian species exhibit seasonal cycles in physiology and behaviour. Seasonal plasticity during the lifetime contributes to seasonal physiology. Over the years, our ideas regarding adult brain plasticity and, more specifically, hypothalamic plasticity have greatly evolved. Along with the two main neurogenic regions, namely the hippocampal subgranular and lateral ventricle subventricular zones, the hypothalamus, which is the central homeostatic regulator of numerous physiological functions that comprise sexual behaviours, feeding and metabolism, also hosts neurogenic niches. Both endogenous and exogenous factors, including the photoperiod, modulate the hypothalamic neurogenic capacities. The present review describes the effects of season on adult morphological plasticity and neurogenesis in seasonal species, for which the photoperiod is a master environmental cue for the successful programming of seasonal functions. In addition, the potential functional significance of adult neurogenesis in the mediation of the seasonal control of reproduction and feeding is discussed.     

Changes in markers of neuronal and glial plasticity after cortical injury induced by food restriction

The regenerative capacity of the adult central nervous system is limited. We investigated whether short-term food restriction (FR; 50% of the daily food intake lasting 3 months) modulates processes of brain plasticity after cortical injury. Quantitative changes of growth-associated protein 43 (GAP-43) and synaptophysin (SYP) mRNA levels in the ipsilateral cortex of the adult rat during the recovery period (from 2 to 28 days) after injury were investigated by real-time RT-PCR. Using Western blot and immunohistochemical analyses we examined the levels and localization of proteins involved in neuronal plasticity, SYP and GAP-43, as well as glial fibrillary acidic protein (GFAP), a marker of glial plasticity. A marked rise in GAP-43 and SYP immunoreactivity observed in the FR group on the 7th day after injury pointed to increases in axonal branching and synapses in the cortex surrounding the lesion. The appearance of reactive astrocytes was accompanied by the absence of immunoreactivity for GAP-43 and SYP in ad libitum fed animals. This finding supports the hypothesis that morphological hypertrophy of astrocytes associated with GFAP synthesis is responsible either directly or indirectly for the inhibitory role of activated glia on axonal regeneration. Examination of the effects of FR on serum corticosterone and glucose concentrations and GAP-43, SYP and GFAP expression revealed that FR facilitated recovery of the injured region by attenuating reactive astrogliosis and enhancing the expression of neuronal plasticity markers.     

Differences in the regenerative response of neuronal cell populations and indications for plasticity in intraspinal neurons after spinal cord transection in adult zebrafish

In zebrafish, the capacity to regenerate long axons varies among different populations of axotomized neurons after spinal cord transection. In specific brain nuclei, 84-92% of axotomized neurons upregulate expression of the growth-related genes GAP-43 and L1.1 and 32-51% of these neurons regrow their descending axons. In contrast, 16-31% of spinal neurons with axons ascending to the brainstem upregulate these genes and only 2-4% regrow their axons. Dorsal root ganglion (DRG) neurons were not observed to regrow their ascending axons or to increase expression of GAP-43 mRNA. Expression of L1.1 mRNA is high in unlesioned and axotomized DRG neurons. In the lesioned spinal cord, expression of growth-related molecules is increased in a substantial population of non-axotomized neurons, suggesting morphological plasticity in the spinal-intrinsic circuitry. We propose that locomotor recovery in spinal-transected adult zebrafish is influenced less by recovery of ascending pathways, but more by regrowth of descending tracts and rearrangement of intraspinal circuitry.     

Is taurine a hypothalamic neurotransmitter?: a model of the differential uptake and compartmentalization of taurine by neuronal and glial cell particles from the rat hypothalamus

Although taurine has been postulated to be a neurotransmitter or neuromodulator in the mammalian CNS, little is known concerning its role in brain function. Evidence suggesting that taurine may influence endocrine and homeostatic mechanisms via the hypothalamus resulted in our investigations into its function in this brain region.The main objectives of the research were to characterize the specific binding, uptake, and release of taurine in the hypothalamus. A specific aim was to examine the proposed neurotransmitter role for taurine in the hypothalamus. This was accomplished by comparing the characteristics and properties of the binding, uptake, and release of taurine with those for the classical neurotransmitters which satisfy the criteria for a neurotransmitter. On such a comparative basis, the characteristics of taurine uptake satisfy the neurotransmitter criterion of inactivation of taurine in the hypothalamus. However, the observed characteristics of taurine binding and release in the hypothalamus do not satisfy the respective neurotransmitter criteria of specific receptors and Ca²⁺-dependent evoked release. Therefore, solely on the basis of the experimental observations reported herein, we must conclude that taurine apparently does not function as a neurotransmitter in the hypothalamus.Two uptake systems were found in the P₂ fraction, a high affinity uptake system and a low affinity uptake system. Uptake systems for taurine have previously been reported in glial and nerve cell homogenates, and therefore, because of the known contamination of crude synaptosomal preparations with glial particles, we sought to determine the cellular origin of the two taurine uptake systems in our crude preparation. Using a variety of diverse biochemical techniques such as hypo-osmotic shock, release experiments and Arrhenius plots, we determined that physical changes of the media or depolarizing stimuli which would influence neuronal and glial cell particles differently, also had differing effects on high and low affinity taurine uptake or its release from the respective uptake compartments. We conclude that the high affinity taurine uptake system/compartment is located on/in neuronal membranes/particles and that the low affinity taurine uptake system/compartment is located on/in glial membranes/particles. Such a model for the differential cellular transport and compartmentalization of taurine into neuronal and glial cells has important implications concerning its possible role in the CNS.     

Synaptic and neuronal-glial plasticity in the adult oxytocinergic system in response to physiological stimuli

Magnocellular oxytocinergic neurons in the hypothalamus offer a striking example of a mammalian neuronal system whose basic architecture and synaptic circuitry can be reversibly modified in adulthood. During parturition, lactation and prolonged osmotic stimulation, glial coverage of oxytocinergic neurons markedly diminishes and their surfaces are left in extensive juxtaposition, concurrently, there is formation of new synapses, which are predominantly GABAergic and which couple two or more oxytocinergic neurons simultaneously. These structural changes do not permanently modify the anatomy of the system since upon cessation of stimulation, neuronal juxtapositions and shared synapses disappear, to reappear upon new stimulation. At present, we can only speculate about the cellular mechanisms and factors responsible for these reversible neuroanatomical changes. However, oxytocin itself appears to be of primary importance since it can induce similar anatomical changes when chronically infused into the third ventricle.     

Sex differentiation of growth hormone-releasing hormone and somatostatin neurons in the mouse hypothalamus: an immunohistochemical and morphological study

We examine sexual dimorphism in growth hormone-releasing hormone (GHRH) in the arcuate nucleus (ARC), and somatostatin (SS) in the periventricular nucleus (PeN) of the hypothalamus, and investigate when it becomes evident. Using immunohistochemical staining and morphometry, we observed ARC GHRH-immunoreactive (ir) neurons, ARC SS-ir neurons and PeN SS-ir neurons in male and female mice at 5, 20, 30, 40 and 60 days old. The number of ARC GHRH-ir neurons was significantly higher in males than females, after 20 days old. ARC SS-ir neurons showed no significant differences between sexes. On the other hand, PeN SS-ir neurons were significantly more numerous in males at 30, 40 and 60 days than in females. During postnatal development, these GHRH- and SS-ir neurons changed in different patterns from ages 20 to 60 days. The number of ARC GHRH-ir neurons in both sexes decreased from 5 to 20 days, increased until day 40, and then decreased at day 60, while ARC SS-ir neurons in both sexes increased from day 5 to day 60. PeN SS-ir neurons in both sexes increased from days 5 to 20 to 116% in males and 189% in females. Furthermore, in male mice, the increase continued until 40 days of age, while in females, there was no significant difference from days 20 to 60. There were no apoptotic cells; a few proliferating cell nuclear antigen (PCNA) stained cells were found in the ARC and PeN. Our results suggest that the sex difference of ARC GHRH neurons and PeN SS neurons appears by stimulation with testosterone during the development life. The developmental fluctuation in the number of ARC GHRH-ir neurons may not be modulated by testosterone, but by ARC SS neurons.     

A new model of the blood--brain barrier: co-culture of neuronal, endothelial and glial cells under dynamic conditions

Developing in vitro blood-brain barrier (BBB) models that closely mimic the natural state is important for theoretical and practical applications, including drug development. We previously developed an in vitro BBB model based on co-culturing endothelial cells with glia in the presence of flow on hollow fiber tube culture substrates. We now report that this dynamic in vitro BBB (DIV-BBB) can be successfully used to co-culture differentiated serotonergic neurons in the presence of a BBB. These neurons demonstrated fluoxetine-sensitive serotonin (5HT) uptake and depolarization-induced release of [3H]5HT. Our results demonstrate that the DIV-BBB is a suitable model for culturing of neurons in a quasi-physiological microenvironment and in the presence of a high-resistance, stereoselective BBB.     

Immunohistochemical demonstration of plasticity in GABA neurons of the adult rat dentate gyrus

The distribution of GABA fibers within the dentate gyrus was immunohistochemically examined following lesions of the entorhinal cortex in the adult rat. A major change in the pattern of the GAD immunoreactive fibers within the molecular layer, characterized by a marked increase in the density of fibers in the outer molecular layer, was observed. This change in the lamination of the dentate GABA fibers following entorhinal lesions appeared very similar to the changes which occur in acetylcholinesterase staining following entorhinal denervation of the dentate. These results provide morphological support for the sprouting of GABA fibers in the dentate gyrus in response to perforant path destruction.