Control of glutamate clearance and synaptic efficacy by glial coverage of neurons.

Analysis of excitatory synaptic transmission in the rat hypothalamic supraoptic nucleus revealed that glutamate clearance and, as a consequence, glutamate concentration and diffusion in the extracellular space, is associated with the degree of astrocytic coverage of its neurons. Reduction in glutamate clearance, whether induced pharmacologically or associated with a relative decrease of glial coverage in the vicinity of synapses, affected transmitter release through modulation of presynaptic metabotropic glutamate receptors. Astrocytic wrapping of neurons, therefore, contributes to the regulation of synaptic efficacy in the central nervous system.

A study of the role of neuro-glial remodeling in the oxytocin system at lactation.

Under conditions of strong secretion of neurohypophysial hormone, such as during parturition, lactation and dehydration, the hypothalamic oxytocin-system displays a remarkable morphological plasticity such that astrocytic coverage of its neurones diminishes, their surfaces become directly juxtaposed and contacted by an increased number of synapses. A growing body of evidence indicates that these anatomical changes have an impact on glutamatergic neurotransmission in the supraoptic nucleus, and may be therefore of physiological consequence. We here evaluated the consequences of the inhibition of such plasticity on the overall activity of the oxytocin system during lactation. Remodeling was prevented by performing hypothalamic microinjections in gestating rats of endoneuraminidase, an enzyme that removes polysialic acid from the neural cell adhesion molecule. Our earlier studies established that the presence of polysialic acid is a prerequisite for remodeling of the oxytocin system in the supraoptic and paraventricular nuclei. In dams in which polysialic acid was absent in all magnocellular nuclei after bilateral endoneuraminidase injections, parturition was normal and neither the frequency nor the amplitude of suckling-induced reflex milk ejections was different from vehicle-treated dams. The weight gain of pups was also normal as was water intake by the dams. We then assessed the electrical activity of antidromically identified magnocellular neurones in the polysialic acid-free supraoptic nucleus of isoflurane-anesthetized lactating rats. Basal and bursting activity characteristic of oxytocin neurones before each reflex milk ejection was not significantly different from that recorded in the supraoptic nucleus of rats with normal levels of polysialic acid. Our results indicate that neuro-glial remodeling, despite its role on fine modulation of oxytocin neuronal activity, is not essential to parturition and lactation.

Expression of high levels of the extracellular matrix glycoprotein, tenascin-C, in the normal adult hypothalamoneurohypophysial system.

Glia and neurons of the hypothalamoneurohypophysial system (HNS) undergo reversible morphological changes, which are concomitant with the remodelling of afferents onto the neurons, under different conditions of neurohormone secretion. Here, we show that the adult rat HNS contains high levels of tenascin-C (TN-C), which is an extracellular matrix glycoprotein whose expression is usually associated with neuronal-glial interactions in the developing and lesioned central nervous system (CNS). By using light and electron microscopic immunocytochemical procedures, we visualized TN-C immunoreactivity in the hypothalamic supraoptic (SON) and paraventricular nuclei, where somata of the neurons are localized; in the median eminence, where their axons transit; and in the neurohypophysis, where they terminate. Hypothalamic areas adjacent to the magnocellular nuclei were devoid of immunoreactivity. Electron microscopy of the neurohypophysis showed immunolabelling of perivascular spaces, glial (pituicyte) and axonal surfaces, a type of labelling that also characterized the median eminence. In the hypothalamic nuclei, there was labelling of extracellular spaces and astrocytic surfaces. In normal animals, we detected no cytoplasmic reaction in glia somata, neurons, or endothelial cells. However, in animals treated with the intracellular transport blocker colchicine, there was intracytoplasmic labelling of all HNS glial cells, indicating a glial source for TN-C. Immunoblot analysis revealed TN-C isoforms of apparent high molecular weight (225, 240, and 260 kD) in the SON and median eminence, whereas lower MW forms (190/200 kD) predominated in the neurohypophysis. By using immunocytochemistry and immunoblot analysis, we found no visible differences in TN-C expression in relation to age, sex, or differing neurohypophysial secretion, which suggests that the expression of TN-C is a permanent feature of the HNS.

Afferent projections from the mammary glands to the spinal cord in the lactating rat--II. Electrophysiological responses of spinal neurons during stimulation of the nipples, including suckling.

In lactating rats, the milk ejection reflex is evoked and maintained by stimulation of the nipples by the suckling young. In order to understand the processing of the suckling stimulus within the spinal cord, urethane-anaesthetized lactating rats were prepared for electrophysiological recording from the thoraco-lumbar spinal cord during stimulation of the nipples. Single shocks to inguinal or abdominal nipples evoked a cord dorsum potential, consisting of an early (2.6 ms) afferent volley followed by a negative wave (100-200 microV; latency 5-7 ms, duration 5-10 ms). Evoked potentials were also recorded at various depths within the spinal cord, with a maximum amplitude (200-400 microV) at a depth of 400-800 microns, 400-800 microns lateral to the mid-line. At a given recording site, the response was maximal for one particular nipple but submaximal potentials could be evoked from adjacent nipples. Simultaneous stimulation of adjacent nipples caused summation of the response. Unit recordings were made from 35 spinal neurons. Upon electrical stimulation of the nipples, the cells responded with an early train of spikes (latency 5-15 ms), and in 6 cells, a later response (140-180 ms), with a higher stimulation threshold, was also observed. All cells examined showed convergence and summation from different nipples. Twenty out of 27 cells were also activated by stretching of the nipples, which evoked a rapidly adapting response; rhythmical stretching produced a more sustained increase in activity. The cells also responded to other natural stimuli such as touch and pressure or stroking the hair around the nipple and had large receptive fields. Six cells were tested with the suckling stimulus. There was a brisk increase in firing as the pup grasped the nipple and then intermittent (every 18-30 s) episodes of enhanced activity, which directly correlated with the suckling movements. These episodes continued for the duration of the suckling test and were enhanced when a second pup was placed on an adjacent nipple. Finally, from a few experiments when a stimulating electrode was placed within the contralateral antero-lateral funiculus at the level of C2-C3 for antidromic identification, it was seen that some of the cells activated from the nipples projected to higher levels. The short latency responses to nipple stimulation, including suckling, suggest that the suckling stimulus reaches the spinal cord ungated.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for structural plasticity in the supraoptic nucleus of the rat hypothalamus in relation to gestation and lactation.

Supraoptic nuclei of lactating rats present a particular anatomical organization that could serve to facilitate the synchronization of neuronal firing observed during suckling-induced reflex milk ejections. Although magnocellular neurones are usually separated by neuropil elements, particularly glial fibers, in lactating rats, numerous neurosecretory soma and dendritic profiles are in direct apposition, without glial interposition. Concomitantly, there is also a higher incidence of presynaptic terminals contacting two neurosecretory elements in the same plane of section ("double" synapses). In the present study, a quantitative ultrastructural analysis was used to trace the evolution of the structural reorganization of the nucleus at different stages of the reproductive cycle. The percentage of neurosecretory soma and dendritic profiles in direct apposition was low two weeks after the beginning of pregnancy, but the day prior to parturition, as during lactation, over 40% of all neurosecretory profiles were directly in contact and involved about 10% of the total neuronal surface membrane measured (a 5-fold increase over the corresponding frequencies recorded in virgin rats at oestrus). The contiguous neuronal membranes and associated intercellular space appeared unmodified, except for the presence of attachment plaques, that also increased in frequency at late gestation and lactation. The incidence of "double" synapses also increased by late gestation, so that at lactation, they bridged 8% of all the recorded neurosecretory somata and dendrites, (as compared to 1% in the virgin rats). Similar changes were observed during a first and second gestation and lactation. The incidence of direct appositions and "double" synapses then diminished gradually after weaning: 2 months after the end of lactation, the ultrastructure of the nucleus resembled that of virgin animals. These observations demonstrate a plasticity in the structural organization of the supraoptic nucleus that appears closely related to changing physiological states of the animal and that involves both neurone-glial relationships and the neurones' synaptic configuration.

In vitro formation of type 2 astrocytes derived from postnatal rat hypothalamus or cerebral cortex.

The origin and function of type 2 astrocytes in the optic nerve are now well described, but there are few and controversial observations concerning their origin and functional significance in other regions of the mammalian brain. We here describe primary and highly enriched secondary glial cultures obtained from postnatal (P0-P6) rat hypothalami and cerebral cortices that included glial cells with morphological and immunocytochemical characteristics of type 2 astrocytes. The somata of such astrocytes were characteristically small and polygonal; they bore several processes with few branches. They were highly immunoreactive for glial fibrillary acidic protein and the surface antigens, A2B5 and NSP4; they were immunonegative for myelin basic protein and galactocerebroside. They grew on top of a continuous monolayer of much larger, flattened cells, that were glial fibrillary acidic protein-positive but A2B5- and NSP4-negative. In cultures derived from tissues younger than postnatal day 4, their appearance required the addition of adult (horse) serum to the culture milieu; they appeared spontaneously in cultures from older animals. Analysis of the origin of these cells, including experiments using tritiated thymidine incorporation, indicated that these astrocytes resulted from asymmetric divisions of the flat glial cells in the basal layer of the cultures. After their first appearance which varied according to the age of the source tissue, they were continuously generated, with a generation time no longer than 48 h; the life-span of individual cells was found to not exceed one week in neuron-free primary glial cultures. They displayed important process motility but did not show any significant migratory activity. The ready inducibility of glial cells showing many characteristics of type 2 astrocytes, in cultures derived from different brain areas, suggests that type 2-like astrocytes or their committed precursors are not restricted to particular neural structures, but are probably widely distributed within the mammalian brain. Their functional significance within the different brain areas remains to be determined.

Oxytocin neuron activation and Fos expression: a quantitative immunocytochemical analysis of the effect of lactation, parturition, osmotic and cardiovascular stimulation.

As c-fos expression is generally thought to be linked to neuronal activation, we compared Fos immunoreactivity in identified oxytocinergic and vasopressinergic neurons of female rats under various conditions known to elicit particular patterns of electrophysiological and secretory activity in these neurons. In suckled lactating animals, Fos immunoreactivity was visible only in rare oxytocinergic and vasopressinergic neurons of the paraventricular and supraoptic nuclei, even after interruption of suckling for 18-72 h. On the other hand, many Fos-positive cells were visible in the nuclei of parturient rats; they involved about 25% of supraoptic oxytocinergic elements. Even more Fos-positive elements were visible in the nuclei of lactating rats that had also undergone 24 h water deprivation or haemorrhage. This involved about 75% vasopressinergic neurons and 25% oxytocinergic neurons of the supraoptic nucleus. Fos immunoreactivity was particularly conspicuous in oxytocin neurons of the anterior commissural nucleus after haemorrhage. After water deprivation or haemorrhage, Fos-positive oxytocinergic neurons in the supraoptic nucleus were significantly more numerous in virgin rats than in lactating rats. Our observations show that suckling, although a most potent stimulus for oxytocin neuron activation and oxytocin release, is inefficient in inducing Fos synthesis in magnocellular neurons, even after a period of interruption. On the other hand, parturition, water deprivation and haemorrhage were more potent stimuli for both neurosecretory systems. However, under each type of stimulation, only part of the neuronal populations within each nucleus were Fos-positive, suggesting that different stimulus-specific pathways are involved in these regulations. In so far as electrical activity is one possible mechanism for c-fos expression, comparison of the patterns of c-fos activation with the known electrophysiological behaviour of hypothalamic magnocellular neurons suggests that Fos synthesis in these neurons is linked to the number of action potentials generated over a period of time, more than to the pattern of electrical activity, whatever the physiological impact of this pattern. Furthermore, within a group of neurons, the heterogeneity of the response in terms of Fos synthesis may be correlated to the variability of the electrophysiological response within this group.

Afferent projections from the mammary glands to the spinal cord in the lactating rat--I. A neuroanatomical study using the transganglionic transport of horseradish peroxidase-wheatgerm agglutinin.

Horseradish peroxidase-wheatgerm agglutinin was injected subcutaneously into one or more nipples of lactating rats to determine the spinal organization of sensory afferents emanating from the mammary glands. After survival periods of 45-96 h, dorsal root ganglia and segments of the spinal cord and/or medulla oblongata were sectioned and reacted histochemically with tetramethylbenzidine to reveal the transganglionically transported tracer. For each nipple injected, the peroxidase reaction product was found in somata, ranging in diameter from 15 to 60 microns, and fibres in 5-11 contiguous dorsal root ganglia. The number of labelled profiles was highest in the 2-4 central-most ganglia of the series and generally decreased progressively rostrally and caudally. After separate injections into each of the six ipsilateral nipples, labelling occurred in all ipsilateral dorsal root ganglia between the 5th cervical and 6th lumbar spinal segments. Substantial overlap of the spinal projections from adjacent mammary glands was seen, a given dorsal root ganglion innervating 2-3 different glands. Label in the spinal cord was restricted to the medial portion of the superficial dorsal horn. It occurred in what appeared to be terminal fields and fibres essentially in the substantia gelatinosa, but was also seen to extend into the marginal zone and sometimes into deeper regions of the dorsal horn. Label was found in both the gracile and cuneate nuclei of the medulla oblongata, though only occasionally and then only very sparsely. The substantial spread and segmental overlap of labelled mammary afferents, and the fact that most labelled afferents terminated in the dorsal horn, suggest that this spinal region may be an important site for the integration of sensory input from the mammary glands that may play a role in the sensory induction of reflex milk ejection.

Mapping of the distribution of polysialylated neural cell adhesion molecule throughout the central nervous system of the adult rat: an immunohistochemical study.

In the nervous system, the neural cell adhesion molecule changes at the cell surface during development, from a form highly enriched in polysialic residues to several isoforms containing much less sialic acid, and is thought to participate in the structuring of neuronal groups and in the establishment of neuronal connections. Recent observations have indicated, however, that it may not be restricted to developing tissues since it is still present in certain adult neuronal centres which can undergo morphological reorganization. In this study, therefore, we examined systematically the distribution of polysialylated neural cell adhesion molecule immunoreactivity throughout the central nervous system of adult male and female rats, using light microscopic immunocytochemistry and immunoblot analysis with an antibody that specifically recognizes the polysialic residues of the molecule. Concomitantly, we compared this immunoreactivity to that due to all isoforms of the neural cell adhesion molecule, detected with a polyclonal serum raised against the NH2-terminal of the protein. Immunoreactivity due to the polysialylated isoform was consistently visualized in several discrete areas of the adult brain and spinal cord. An intercellular punctate immunolabelling characterized the staining in certain hypothalamic and thalamic nuclei, superficial laminae of the dorsal horn of the spinal cord, ventral portion of the dentate gyrus of the hippocampus, lateral geniculate, parabrachial and habenular nuclei, bed nucleus of the stria terminalis, mesencephalic central gray and olfactory bulb. In other areas, such as the piriform cortex, dorsal aspect of the dentate gyrus and fimbria and lamina X of the spinal cord, isolated neuronal-like cells were either completely filled with immunolabel or showed a surface reaction on their cell bodies and processes. Highly immunoreactive isolated glial-like cells were also noted within the ependymal layer of the central canal and lateral ventricles and at times in the peripheral white matter of the spinal cord. In contrast to this discrete localization, staining due to all isoforms of the neural cell adhesion molecule was widespread and diffuse throughout the brain and spinal cord. The expression of the polysialylated isoform in the supraoptic nucleus and hippocampus was confirmed by immunoblot analysis; it occurred together with weakly sialylated isoforms. No obvious differences were detected in the amount or distribution of immunoreactivity due to the polysialylated isoform in relation to the sex or age of the animals (between three and 12 months of age). Our study thus demonstrates that well-defined areas of the central nervous system of the adult rat continue to express the polysialylated isoform of the neural cell adhesion molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

Extracellular GABA concentrations in rat supraoptic nucleus during lactation and following haemodynamic changes: an in vivo microdialysis study.

Morphological and pharmacological evidence suggest that the dense GABAergic innervation of the supraoptic nucleus is important for regulating the electrical activity of vasopressin and oxytocin neurons. We have employed the technique of intracranial microdialysis to examine extracellular GABA concentrations in the supraoptic nucleus of the anaesthetized rat and questioned whether differences exist in the dynamics of GABA release between virgin and lactating rats, and if events during lactation or following blood pressure manipulation alter endogenous GABA levels in this nucleus. No significant differences were detected between virgin and lactating animals in either basal or 100 mM potassium ion-evoked GABA release. The inclusion of the GABA uptake blocker nipecotic acid (0.5 mM) into the dialysate resulted in a six- to eight-fold increase (P < 0.01) in GABA outflow in both groups of animals. In lactating rats, GABA outflow measured at 4 min intervals was not altered during a 60 min period of suckling by a full litter of pups and no significant change in GABA outflow was detected in relation to individual milk ejections. In virgin rats, removal of 1.5-2 ml of blood resulted in a 30-60 mmHg fall in blood pressure and a non-significant decline in GABA outflow. Replacement of blood resulted in an abrupt 50 mmHg increase in blood pressure and a significant 22% increase in GABA outflow (P < 0.01), but no change in aspartate or methionine concentrations. Repeated intravenous injections of the alpha-adrenoceptor agonist, metaraminol, similarly evoked approximately 50 mmHg increments in blood pressure and a 26% increase in GABA outflow (P < 0.05). Electrical stimulation of the diagonal band of Broca for 10 min produced a two-fold increase in GABA outflow from the supraoptic nucleus (P < 0.05). These results show that the overall profile of basal and potassium-stimulated GABA concentrations in the supraoptic nucleus is not substantially different between lactating and virgin rats. In lactating animals we have found that GABA levels are not altered in response to suckling or at the time of high-frequency firing by oxytocin neurons to induce milk ejection. In contrast, our data further support the hypothesis that GABA inputs to supraoptic neurons are part of a baroreceptor reflex, relaying through the diagonal band of Broca, to signal periods of acute hypertension and inhibit the firing of vasopressin neurons. Such observations suggest the physiological importance of GABA inputs to the supraoptic nuclei and indicate that GABA may be used in a stimulus-specific manner to influence the activity of magnocellular neurons.

Lactation-induced plasticity in the supraoptic nucleus augments axodendritic and axosomatic GABAergic and glutamatergic synapses: an ultrastructural analysis using the disector method.

The disector, an unbiased stereological method for evaluation of synaptic densities, was used to analyse putative GABA and glutamate innervations of the supraoptic nucleus of virgin and lactating rats. The analysis was performed on ultrathin sections labelled for either of the amino acids with a postembedding immunogold technique. Our observations showed that the volume of the nucleus increased by 40% in lactating animals, an increase due to a significant enlargement of dendritic and somatic, but not vascular, volumes. Nevertheless, values of overall synaptic densities in the whole nucleus remained as high as those in virgin rats (37-40 x l0(6) synapses/mm3). About 45% of all synapses were immunoreactive for GABA and 25% for glutamate; there were twice as many GABA- and glutamate-positive synapses on dendrites as on somata. When we estimated synaptic densities in relation to the neuropil (by subtracting the proportion of sampled areas occupied by somatic profiles), we found a significant increase in synaptic density in lactating animals. This affected axodendritic as well as axosomatic synapses, immunopositive and immunonegative for GABA or glutamate. The disector also allowed us to determine that the number of synapses from terminals making contacts on several somata and/or dendrites simultaneously constituted about 9% of all synapses in virgin rats, a proportion which more than doubled in lactating rats. About 50% were immunopositive for GABA and 30% for glutamate. Our data offer further evidence of physiologically-linked structural synaptic plasticity in the supraoptic nucleus and clearly demonstrate that it affects both inhibitory and excitatory inputs on dendrites, as well as on somata, throughout the nucleus.

The polysialylated neural cell adhesion molecule reaches cell surfaces of hypothalamic neurons and astrocytes via the constitutive pathway.

Understanding how neurons and glia sort and deliver cell adhesion molecules to their cell surface should provide important clues as to how such molecules participate in dynamic neuronal functions in the developing and adult brain. The present study examines translocation of polysialylated neural cell adhesion molecule (PSA-NCAM), a negative regulator of cell adhesion, in cells of the rat hypothalamo-neurohypophysial system in which it is expressed throughout life and which undergo morphological remodelling in response to stimulation. PSA-NCAM expression in this system does not vary markedly in relation to different conditions of regulated neurosecretion, suggesting that the glycoprotein reaches cell surfaces via the constitutive pathway. To study this more directly, we here used immunofluorescence for PSA on NCAM in live, unpermeabilized cells to monitor PSA-NCAM surface expression in organotypic slice cultures from postnatal rat hypothalami. Subsequent immunolabelling for oxytocin confirmed that the cultures included magnocellular oxytocinergic neurons displaying many properties of adult neurosecretory neurons in situ. In the cultures, immunoreaction for PSA-NCAM was visible on the surface of oxytocinergic and non-oxytocinergic axons. This reaction disappeared after exposure of the cultures to endoneuraminidase, an enzyme which specifically cleaves alpha-2-8-linked PSA from NCAM. PSA-NCAM reappeared on axonal surfaces 4h after enzyme washout. Such reexpression was visibly not affected by neuronal activity inhibition (blockade of Ca(2+) channels with Mn(2+), of Na(+) channels with tetrodotoxin, or of glutamate receptors with 6-cyano-7-nitroquinoxaline-2,3-dione or D-2-amino-5-phosphonopentanoic acid) or facilitation (K(+) depolarization or GABA-A receptor blockade with bicuculline). In contrast, PSA-NCAM surface translocation was inhibited reversibly by cooling the cultures at 20 degrees C, a procedure which blocks constitutive secretion and which resulted in accumulation of PSA-NCAM in the cytoplasm of oxytocinergic and non-oxytocinergic neurons. This treatment also revealed PSA-NCAM in the cytoplasm of underlying astrocytes. Our observations provide direct evidence that PSA-NCAM reaches the cell surface of hypothalamic neurons and astrocytes via the constitutive pathway, independently of Ca(2+) entry and enhanced neuronal activity. Thus, PSA-NCAM in the hypothalamo-neurohypophysial system would be continuously available to permit its cells to undergo remodelling whenever the proper stimulus intervenes.

Structural plasticity in the hypothalamic supraoptic nucleus at lactation affects oxytocin-, but not vasopressin-secreting neurones.

Magnocellular neurones in the supraoptic nucleus of the hypothalamus are usually separated by neuropil and glial elements. In lactating animals, however, the surface membranes of many neurosecretory somata and dendrites are frequently in direct apposition, without any glial interposition. A significant number of such neurones are also bridged by the same presynaptic terminal ("double synapses"). As the supraoptic nucleus is composed of two types of neurosecretory cell, secreting either oxytocin or vasopressin, we carried out comparative quantitative analyses on identified supraoptic neurones of virgin and lactating rats to determine which neurones were affected by the structural changes and to what extent. The neurones were identified in: (i) normal and Brattleboro homozygote rats by electron microscopic immunocytochemistry (pre- and post-embedding procedures) using antisera raised against oxytocin, vasopressin and oxytocin-related neurophysin I, and (ii) in homozygous Brattleboro rats by their neuronal content of approximately 170 nm neurosecretory granules. We report here that, in virgin animals under normal conditions, a small proportion of both types of neurone show neuronal appositions. At lactation, neuronal appositions are far more numerous and extensive, as are "double synapses". These changes affect exclusively the oxytocinergic neurones. The increased appositions cannot result solely from glial retraction because the hypertrophied oxytocin cells have a greater absolute, though smaller proportional, coverage by glial processes than cells in the control animals. From the present observations, and those obtained in chronically dehydrated animals (see accompanying article), it is clear that the plastic changes in the supraoptic nucleus are closely related to the activity of its oxytocinergic neurones. During lactation, these structural modifications may serve to facilitate and maintain the characteristic synchronized electrical activity of these neurones at milk ejection. On a few occasions, we also found appositions between one oxytocinergic and one vasopressinergic neurone, which may account for the rare cases of electrophysiological interactions between the two types of cell.

Osmotic stimulation causes structural plasticity of neurone-glia relationships of the oxytocin but not vasopressin secreting neurones in the hypothalamic supraoptic nucleus.

A comparative quantitative analysis was carried out on identified supraoptic neurones of male and female Wistar and Long Evans rats under normal conditions and after chronic osmotic stimulation, and in homozygous Brattleboro rats suffering from diabetes insipidus. The neurones were identified by immunocytochemical or morphological means. Osmotic stimulation resulted in significant increases in the number and extent of direct neuronal appositions and in the number of presynaptic terminals contacting two neurosecretory cells simultaneously ("double" synapses). In the supraoptic nuclei of both sexes these increases were restricted to the oxytocin secreting neurones. In Brattleboro homozygous rats treated with vasopressin, the proportion of oxytocinergic neurones in apposition was not modified, but the number of appositions per soma profile decreased as did the incidence of "double" synapses. In nuclei of osmotically stimulated rats, increase in cell volume affected both types of neurosecretory cell and was accompanied by an increase of the absolute extent of glial coverage. However, the extent of glial coverage of the oxytocinergic neurones did not match the hypertrophy of the cells, resulting in a decrease in their relative glial coverage, compared to normal hydrated animals. The increased neuronal appositions, therefore, cannot result simply from a retraction of glial processes. The structural reorganization of the oxytocinergic system observed during chronic osmotic stimulation was as extensive as that observed at lactation. Moreover, the changes were as extensive in Wistar as in Brattleboro lactating rats, although the latter have an added osmotic stimulus. This implies that lactation and osmotic stimulation do not produce additive effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural visualization and neurochemical characterization of spinal projections of primary sensory afferents from the nipple: combined use of transganglionic transport of HRP-WGA and glutamate immunocytochemistry.

We used transganglionic transport of the neuronal tracer horseradish peroxidase coupled to wheat germ agglutinin (HRP-WGA) and post-embedding immunogold staining to determine the spinal projections and neurochemical identity of sensory afferents originating from a discrete cutaneous area. After SC injection of tracer into the nipple of lactating rats and reaction with tetramethylbenzidine stabilized with diaminobenzidene (TMB-DAB) or DAB and cobalt (TMB-DAB-Co), we found labeled terminals in the internal part of the first two layers of the dorsal horn where they formed axodendritic synapses and, at times, central elements of glomeruli, synaptic complexes believed to be involved in the integration of sensory messages. Immunogold staining of ultra-thin sections of tissue reacted with TMB-DAB-Co revealed that many mammary afferents contained glutamate as putative neurotransmitter. This combined approach thus offers the possibility of marking a limited set of primary afferents, after capture of tracer by sensory receptors of restricted peripheral areas, to visualize their projections at the spinal level and to determine their neurochemical nature with electron microscopy.

Recurrent mammary gland contractions induced by a low tonic release of oxytocin in rats.

In urethane-anaesthetized lactating rats, intramammary pressure occasionally displayed recurrent variations or oscillations having a slow rise time, low amplitude, long duration and a periodicity of 1-4 min. These oscillations differed from changes in intramammary pressure characteristic of reflex milk ejections induced by suckling, and were also observed in unsuckled rats. They were suppressed by lesions of the pituitary stalk or by stimulating the septum, a structure that inhibits the activity of the magnocellular system. They could be induced by long-term low frequency stimulation of the pituitary stalk, lesions of the septum or long-term infusions of oxytocin at a low rate of 0.05-0.3 mu./min. We suggest that the recurrent oscillations in intramammary pressure constitute a particular mode of response of the mammary gland to a tonic release of oxytocin resulting from a moderate but sustained increase in the basal level of electrical activity of the oxytocin-secreting neurones.

Structural plasticity in the rat supraoptic nucleus during gestation, post-partum lactation and suckling-induced pseudogestation and lactation.

In the supraoptic nucleus (SON) of parturient and lactating rats, large portions of the surface membranes of almost all oxytocinergic neurons are directly juxtaposed with no glial interposition. A significant number of the same neurones are also contacted by the same presynaptic terminal ('double' synapses). Our present observations have revealed that direct appositions between adjacent neurons in the SON increase quite rapidly during the day before parturition. 'Double' synapses also become visible during late gestation, but they appear more progressively. Earlier studies have shown that 1 month after weaning, as in virgin rats, there are again very few appositions and 'double' synapses in the nucleus. We show here that the SON can remain structurally modified, and to the same degree, beyond normal weaning time so long as lactation is prolonged by renewing suckling litters. However, if the mothers are deprived of their pups immediately after birth, neuronal appositions disappear within 2 days and 'double' synapses by 10 days. In non-pregnant primiparous rats, continuous exposure to suckling litters leads to pseudogestation and eventually lactation (in 16-22 days). Examination of the SON in such animals revealed that the oxytocinergic system is already modified by day 12 of dioestrus; during suckling-induced lactation, the anatomical changes are identical to those seen during a normal post-partum lactation. These observations indicate that neither gestation nor suckling alone are indispensable for the anatomical reorganization of the SON apparent at lactation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adhesion molecules and structural plasticity of the adult hypothalamo-neurohypophysial system.

The adult hypothalamo-neurohypophysial system, responsible for the secretion of the neurohormones, oxytocin, and vasopressin, undergoes reversible neuronal-glial and synaptic changes in response to stimulation (parturition, lactation, and osmotic stimulation). In the hypothalamus, these changes result in reduced astrocytic coverage of oxytocinergic somata and dendrites and concomitant increases in their GABAergic synapses; in the neurohypophysis, they lead to an enlarged neurovascular contact area. We discuss the possible role played by certain cell adhesion molecules, such as the highly sialylated isoform of the neural cell adhesion molecule, PSA-NCAM, the F3 glycoprotein, and the extracellular matrix molecule, tenascin, in such plasticity. The hypothalamo-neurohypophysial system continues to express high levels of these molecules during adulthood and they may serve as permissive factors to allow stimulus-induced structural remodelling to occur.

Maternity leads to morphological synaptic plasticity in the oxytocin system.

The oxytocinergic system, which plays a major role in the control of different aspects of maternity, undergoes extensive synaptic and neuronal-glial remodelling during parturition and lactation and has thus become a remarkable example of activity-dependent morphological synaptic plasticity in the adult mammalian brain. The use of different comparative ultrastructural analyses on the rat supraoptic and paraventricular nuclei, together with identification of pre- and post-synaptic elements, has allowed us to show that there is a significant increase in the number of GABAergic, glutamatergic and noradrenergic synapses impinging on oxytocin neurons, concomitant with a reduction of glial coverage of the neurons. This synaptic plasticity involves axo-dendritic and axo-somatic contacts originating from terminals making one or several synaptic contacts in one plane of section. While noradrenergic afferents arise from medullary catecholaminergic neurons, our recent in vitro observations indicate that GABAergic and glutamatergic afferents derive, at least partly, from local intrahypothalamic neurons, in close proximity to oxytocin neurons. The cellular mechanisms underlying this morphological synaptic plasticity remain to be determined but it is highly likely that they depend on increased activity in both pre- and post-synaptic elements. Moreover, the oxytocin system continues to express 'embryonic' molecular features that may allow the morphological plasticity to occur. In particular, it expresses high levels of cell surface adhesion molecules currently thought to intervene in synaptic remodelling in the developing and lesioned central nervous system, including the weakly adhesive polysialylated isoform of the Neural Cell Adhesion Molecule, the axonal glycoprotein F3 and its ligand, the extracellular matrix glycoprotein, tenascin-C.

Further Evidence that the Septum is not part of the Main Pathway of the Milk Ejection Reflex in the Rat.

Abstract The possible role of the medial septum in the control of oxytocin release and of the milk ejection reflex induced by suckling was investigated in lactating rats by using electrical stimulations and lesions. In anaesthetized animals, brief electrical stimulation of the medial septum at 5 to 50 Hz elicited a single brief milk ejection similar to natural reflex milk ejections, whereas prolonged low frequency stimulations (5 to 10 Hz) induced a prolonged inhibition of the reflex. In acute experiments under anaesthesia, lesions of the medial septum did not impair the amplitude and pattern of reflex milk ejections. In chronic experiments, lesions of the medial septum resulted first in a loss of body weight of the mothers and a parallel reduction in growth of the litters. After a few days, the litters gained weight normally, and the pattern of milk ejections was normal. Thus, the pathways which pass through or originate from the medial septum and which are excitatory for oxytocin release appear not to be involved in the regulation of the milk ejection reflex. In view of these results and those from our previous study on the lateral septum, we conclude that the whole septum is not essential to the milk ejection reflex. However, the effects of septal stimulation suggest that the medial and lateral septum may be involved in a secondary neural circuitry that can inhibit the reflex.