Vasopressinergic neurons in rats during ontogenesis: reaction to salt-loading and its modulation by noradrenergic afferents

Salt-loading in adult mammals stimulates vasopressin secretion by vasopressinergic neurons of the supraoptic nucleus that is under control by a number of hormones and neurotransmitters including noradrenalin. This study was aimed to determine at what period of ontogenesis the vasopressinergic neurons begin to respond to salt-loading and when the noradrenergic control of this process is switched on. Rats on the 21st embryonic day (E), the 3rd postnatal day (P) and P13 were salt-loaded, sometimes under simultaneous treatment with prasozin, an inhibitor of al -adrenoreceptors. Thereafter, the hypothalamic nuclei of the animals were processed for immunocytochemistry and in situ hybridization. Salt-loading provoked increased synthesis of vasopressin mRNA and, most probably, vasopressin itself in rats in all studied age groups. Under salt-loading, the intraneuronal content of vasopressin increased significantly at E21 and P3, whereas it did not change at P13. No change in the intracellular contents of vasopressin mRNA and vasopressin was observed in foetuses following salt-loading and treatment with prasozin though the same treatment provoked an increase of both parameters at P3. These data show that noradrenalin provides an inhibitory control of vasopressin expression at least since P3. Thus, vasopressinergic neurons begin to respond to salt-loading at the since P3. Thus, life by the increased expression of vasopressin that is postnatally under the inhibitory control by noradrenalin.     

Evidence for antipyretic vasopressinergic pathways and their modulation by noradrenergic afferents.

The experimental evidence for the antipyretic action of arginine vasopressin (AVP) in guinea-pigs can be summarized as follows: The febrile response to a bacterial pyrogen can be reduced by a microinfusions of exogenous AVP into the ventral septal area of the limbic system. Immunohistochemical studies indicate increased activity of AVP terminals in the ventral septal area (VSA) and in parvocellular AVP neurones of the hypothalamic paraventricular nucleus (PVN) in several stressful situations accompanied by reduced febrile responses (late stage of pregnancy, immobilization, cold adaptation, osmotic stimulation). Also the peripheral and/or central release of AVP measured in some of these situations is increased. Electrical stimulation of the PVN suppresses fever, this suppression can, at least partly, be cancelled by simultaneous intraseptal application of the vasopressinergic V1 receptor antagonist. The documented AVP pathways from the PVN to the septum receive noradrenergic afferents from the lower brainstem. Chronic destruction of these afferents by microinjections of 6-hydroxydopamine (6-OHDA) significantly reduced the fever responses to pyrogen application, while microinfusion of noradrenaline (NA) enhances the fever reaction.     

Response of vasopressin and tyrosine hydroxylase expressing neurons of the rat supraoptic nucleus to chronic osmotic stimulation]

This study has evaluated the dynamic of intracellular vasopressin and tyrosine hydroxylase contents in the neuron cell bodies in the supraoptic nucleus and in the axons of the posterior lobe in rats drinking 2% NaCl for 1, 2, and 3 weeks. The number of vasopressin-immunoreactive neurons increased by the end of the second week of osmotic stimulation that might be explained by the onset of vasopressin synthesis in the neurons which do not synthesize this neurohormone under normal physiological conditions. The concentration of vasopressin fell down continuously during the first two weeks of salt-loading, apparently, due to predominance of the vasopressin release over its synthesis. Over the third week of salt-loading, the intracellular concentration of vasopressin was not changed significantly suggesting the establishment of the dynamic equilibrium between the vasopressin synthesis and release. The number of tyrosine hydroxylase-immunoreactive neurons and the amount of tyrosine hydroxylase in cell bodies and the large axonal swellings, Herring bodies, increased gradually showing that the rate of tyrosine hydroxylase synthesis prevailed over that of its enzymatic degradation. Thus, the chronic stimulation of vasopressin neurons is accompanied by a number of the adaptive reactions; the most important is related to the onset of vasopressin and tyrosine hydroxylase synthesis in the neurons which do not synthetize both of them under normal conditions.     

Interrelationships between tyrosine hydroxylase-immunoreactive dopaminergic afferents and somatostatinergic neurons in the rat central amygdaloid nucleus

 Interrelationships between dopaminergic afferents and somatostatinergic neurons of the rat central amygdaloid nucleus were studied using tyrosine hydroxy-lase/somatostatin double immunolabeling for light and electron microscopy. Additionally, morphological features of somatostatin neurons in different subnuclei of the central nucleus were studied, and the results were complemented by codistribution studies of somatostatin and D₁ and D₂ dopamine receptor mRNA expression. Dense axonal immunolabeling for tyrosine hydroxylase was colocalized with somatostatin-immunoreactive or somatostatin mRNA-reactive neurons in the medial and the central lateral part of the central nucleus. The number of somatostatinergic neurons detected was higher using in situ hybridization than using immunolabeling. Somatostatin-immunoreactive neurons of the medial central nucleus possessed deeply indented nuclei, and immunoreaction product was confined to the Golgi apparatus and its vicinity. On the other hand, those in the central lateral subnucleus possessed nuclei without indentations and showed diffuse staining of the cytoplasm and/or in large vesicles. Double labeling showed that in the central lateral central nucleus, somatostatin-immunoreactive neurons were contacted by tyrosine hydroxylase-immunoreactive terminals, and on the electron microscopic level synaptic contacts between differently labeled structures were observed. D₁ and D₂ receptor mRNA-reactive neurons were differentially distributed in central nucleus subnuclei. D₁ receptor mRNA-expressing neurons were found only in the medial subnucleus, while D₂ receptor mRNA was expressed by a number of neurons in the lateral central and a few in the medial one. Thus, the study proves that somatostatin-immunoreactive neurons of the central lateral central nucleus are directly innervated by dopaminergic afferents and may express the D₂ dopamine receptor. Accepted: 2 July 1996     

Activity-dependent feedback modulation of spike patterning of supraoptic nucleus neurons by endogenous adenosine

Neuropeptide secretion from the dendrites of hypothalamic magnocellular supraoptic nucleus (SON) neurons contributes to the regulation of neuronal activity patterning, which ultimately determines their peptide output from axon terminals in the posterior pituitary gland. SON dendrites also secrete a number of other neuromodulators, including ATP. ATP degrades to adenosine in the extracellular space to complement transported adenosine acting on pre- and postsynaptic SON A1 receptors to reduce neuronal excitability, measured in vitro. To assess adenosine control of electrical activity in vivo, we made extracellular single-unit recordings of the electrical activity of SON neurons in anesthetized male rats. Microdialysis application (retrodialysis) of the A1 receptor antagonist, 8-cyclopentyl-1,3-dimethylxanthine (CPT) increased phasic vasopressin cell intraburst firing rates progressively over the first 5 s by 4.5 +/- 1.6 Hz (P < 0.05), and increased burst duration by 293 +/- 64% (P < 0.05). Hazard function plots were generated from interval interspike histograms and revealed that these effects were associated with increased postspike excitability. In contrast, CPT had no effect on the firing rates and hazard function plot profiles of continuously active vasopressin and oxytocin cells. However, CPT significantly increased clustering of spikes, as quantified by the index of dispersion, in oxytocin cells and continuously active vasopressin cells (by 267 +/- 113% and 462 +/- 67%, respectively, P < 0.05). Indeed, in 4 of 5 continuously active vasopressin cells, CPT induced a pseudophasic activity pattern. Together, these results indicate that endogenous adenosine is involved in the local control of SON cell activity in vivo.     

Tyrosine hydroxylase expression in differentiating neurons of the rat arcuate nucleus: inhibitory effect of serotoninergic afferents]

In vivo studies, serotonine synthesis in the rat fetal brain was inhibited by p-chlorphenylalanine from the 11th to the 20th embryonic day. Serotonine depletion significantly decreased thyrosine hydroxylase content in the neurones of males and females on the 21st embryonic day and in males--on the 35th postnatal day. In vitro, a co-culture of arquate nucleus' and raphe nucleus' embryonic neurones resulted in a sex-specific increase of the thyrosine hydroxylase level in the former neurones. The raphe nucleus' neurones manifested an increased level of serotonine. The findings suggest an activating long-lasting effect of serotonine afferents on the thyrosine hydroxylase expression in differentiating neurones of the arquate nucleus in rats during prenatal ontogenesis.     

Ultrastructural and morphometric studies of nonsecretory neurons of the rat supraoptic nucleus

Ultrastructural and morphometric studies were made on nonsecretory and complementarily neurosecretory neurons of the rat supraoptic nucleus. 6% of perimeter of nonsecretory neuronal perikarya was covered by presynaptic endings. The value was well in agreement with that of interneurons elsewhere. The covering ratio of neurosecretory neurons was 12%. The perikarya of nonsecretory and neurosecretory neurons bear an average of 14 and 49 axon terminals, respectively. Nonsecretory neurons should be interneurons, receiving much less information than neurosecretory neurons.     

Electrical properties of neurons recorded from the rat supraoptic nucleus in vitro

The electrical properties of neurons in the supraoptic nucleus (so.n.) have been studied in the hypothalamic slice preparation by intracellular and extracellular recording techniques, with Lucifer Yellow CH dye injection to mark the recording site as being the so.n. Intracellular recordings from so.n. neurons revealed them to have an average membrane potential of -67 +/- 0.8 mV (mean +/- s.e.m.), membrane resistance of 145 +/- 9 M omega with linear current-voltage relations from 40 mV in the hyperpolarizing direction to the level of spike threshold in the depolarizing direction. Average cell time constant was 14 +/- 2.2 ms. So.n. action potentials ranged in amplitude from 55 to 95 mV, with a mean of 76 +/- 2 mV, and a spike width of 2.6 +/- 0.5 ms at 30% of maximal spike height. Both single spikes and trains of spikes were followed by a strong, long-lasting hyperpolarization with a decay fitted by a single exponential having a time constant of 8.6 +/- 1.8 ms. Action potentials could be blocked by 10(-6) M tetrodotoxin. Spontaneously active so.n. neurons were characterized by synaptic input in the form of excitatory and inhibitory postsynaptic potentials, the latter being apparently blocked when 4 M KCl electrodes were used. Both forms of synaptic activity were blocked by application of divalent cations such as Mg2+, Mn2+ or Co2+. 74% of so.n. neurons fired spontaneously at rates exceeding 0.1 spikes per second, with a mean for all cells of 2.9 +/- 0.2 s-1. Of these cells, 21% fired slowly and continuously at 0.1 - 1.0 s-1, 45% fired continuously at greater than 1 Hz, and the remaining 34% fired phasically in bursts of activity followed by silence or low frequency firing. Spontaneously firing phasic cells showed a mean burst length of 16.7 +/- 4.5 s and a silent period of 28.2 +/- 4.2 s. Intracellular recordings revealed the presence of slow variations in membrane potential which modified the neuron's proximity to spike threshold, and controlled phasic firing. Variations in synaptic input were not observed to influence firing in phasic cells.     

Lesion of the perinuclear zone attenuates cardiac sensitivity of vasopressinergic supraoptic neurons

Discrete stretch of the caval-atrial junction decreases the activity of vasopressin-secreting neurons in the supraoptic nucleus (SON). The perinuclear zone (PNZ) of the SON is necessary for inhibition of vasopressin neurons following an increase in blood pressure. To determine whether the PNZ is necessary for cardiopulmonary regulation of vasopressin neurons, male rats received three unilateral injections of the excitotoxin ibotenic acid (n = 9) or phosphate-buffered saline vehicle (n = 10) into the PNZ. Extracellular activity of antidromically identified phasic vasopressin neurons in the ipsilateral SON was recorded. Of the 26 neurons recorded from vehicle-injected rats 26 were inhibited by an increase in blood pressure and 22 of those neurons were sensitive to caval-atrial distension. Of the neurons recorded from PNZ-lesion rats, only 12 of 29 were inhibited by an increase in blood pressure (P < 0.05), and only 11 neurons were sensitive to caval-atrial stretch (P < 0.05). Functional lesion of the PNZ significantly attenuates both arterial and cardiopulmonary baroreceptor-mediated inhibition of supraoptic vasopressin neurons, suggesting that the PNZ is a necessary component of both pathways.     

Neuropeptidomics of the supraoptic rat nucleus

The mammalian supraoptic nucleus (SON) is a neuroendocrine center in the brain regulating a variety of physiological functions. Within the SON, peptidergic magnocellular neurons that project to the neurohypophysis (posterior pituitary) are involved in controlling osmotic balance, lactation, and parturition, partly through secretion of signaling peptides such as oxytocin and vasopressin into the blood. An improved understanding of SON activity and function requires identification and characterization of the peptides used by the SON. Here, small-volume sample preparation approaches are optimized for neuropeptidomic studies of isolated SON samples ranging from entire nuclei down to single magnocellular neurons. Unlike most previous mammalian peptidome studies, tissues are not immediately heated or microwaved. SON samples are obtained from ex vivo brain slice preparations via tissue punch and the samples processed through sequential steps of peptide extraction. Analyses of the samples via liquid chromatography mass spectrometry and tandem mass spectrometry result in the identification of 85 peptides, including 20 unique peptides from known prohormones. As the sample size is further reduced, the depth of peptide coverage decreases; however, even from individually isolated magnocellular neuroendocrine cells, vasopressin and several other peptides are detected.     

Inhibitory effects of propofol on supraoptic nucleus neurons of rat hypothalamus in vitro

To investigate the effects of novel intravenous general anesthetic propofol on membrane electrophysiological characteristics and action potential (AP) of the supraoptic nucleus (SON) neurons and possible ionic mechanisms, intracellular recordings were conducted in SON neurons from the coronal hypothalamic slice preparation of adult male Sprague Dawley (SD) rats. The results showed that bath application of 0.1 mmol/L propofol induced a significant decline in resting potential (P < 0.01), and higher concentrations of propofol (0.3 and 1.0 mmol/L) decreased time constant and slope resistance of cell membrane (P < 0.01). Under the hyperpolarizing current pulses exceeding 0.5 nA, an anomalous rectification was induced by hyperpolarization-activated cation channel (I(h) channel) in 11 out of 18 tested SON neurons. Bath of propofol reversibly decreased the anomalous rectification. Moreover, 0.1 mmol/L propofol elevated threshold level (P < 0.01) and decreased Max L. slope (P < 0.05) of the spike potential in SON neurons. Interestingly, 0.3 and 1.0 mmol/L propofol nullified APs in 6% (1/18) and 71% (12/17) tested SON neurons, respectively. In the SON neurons where APs were not nullified, propofol (0.3 mmol/L) decreased the amplitude of spike potential (P < 0.05). The higher concentrations of propofol (0.3 and 1.0 mmol/L) decreased firing frequencies evoked by depolarizing current pulses (0.1-0.7 nA), and shifted the current intensity-firing frequency relation curves downward and to the right. These results suggest that propofol decreases the excitability of SON neurons by inhibiting I(h) and sodium channels.     

Choline acetyltransferase immunocytochemical staining of the rat supraoptic nucleus and its surroundings

Summary Two different monoclonal antibodies raised against choline acetyltransferase were used, together with preembedding immunocytochemical techniques, to visualize the possible cholinergic innervation of the supraoptic and paraventricular nuclei of the rat hypothalamus. Light microscopy confirmed the presence of a group of bipolar and multipolar immunoreactive neurones in the hypothalamus dorsolateral to the supraoptic nucleus as well as numerous immunopositive fibers. Electron microscopy showed that the immunopositive cell bodies contained the usual perikaryal organelles while most immunoreactive fibers appeared dendritic; immunonegative terminals made synaptic contact onto these profiles. Immunopositive terminals making synaptic contact onto dendritic profiles were also noted in this area. In contrast, light microscopy showed no immunoreactivity to choline acetyltransferase in the magnocellular nuclei themselves. Electron microscopy revealed some immunopositive profiles along the boundaries of both nuclei, along the optic chiasm adjacent to the supraoptic nucleus and in the ventral glial lamina but not within the nuclei proper. Surprisingly, these immunopositive profiles appeared dendritic and were often contacted by one or more immunonegative synapses. Our observations thus indicate that cell bodies and dendrites in the supraoptic and paraventricular nuclei are not directly innervated by cholinergic synapses. The functional significance of the putative cholinergic dendrites in close proximity to magnocellular neurones remains to be determined.     

Developmental regulation of tyrosine hydroxylase expression in primary sensory neurons of the rat

The regulation of transmitter phenotype in primary sensory neurons remains poorly understood. However, recent studies of catecholaminergic (CA) sensory neurons suggest that expression of this particular phenotype may be related to innervation of specific peripheral tissues. In the glossopharyngeal petrosal ganglion (PG) of adult rats, for example, the vast majority of CA sensory neurons innervate a single target, the carotid body. The present study was undertaken, therefore, to begin investigating factors that underlie CA differentiation in sensory neurons, using the rat PG as a model system. Immunocytochemical, biochemical, and morphometric methods were used to investigate the normal time course of CA development in the PG in vivo, employing tyrosine hydroxylase (TH) as a phenotypic marker. These studies revealed two temporally distinct waves of TH expression during embryogenesis. TH immunoreactivity was initially detectable on Embryonic Day (E) 11.5; the number of stained cells increased markedly by E12.5 and then fell off sharply to near 0 by E15.5. Simultaneous immunostaining for TH and neurofilament proteins revealed a high proportion of double-labeled perikarya on E12.5, indicating that the transiently TH-positive cells are neurons. A second, sustained phase of TH expression began on E16.5, and by Postnatal Day 1 adult numbers of TH-containing ganglion cells were present. Western blot analysis demonstrated that TH levels per cell rose 3.5-fold in the perinatal period, indicating that maturation of this particular catecholaminergic trait in PG sensory neurons is highly regulated around birth. Morphometric techniques were used to define the relationship between neurons that transiently exhibit TH immunoreactivity early in gangliogenesis and those that maintain enzyme expression in the mature PG. These studies revealed separate and distinct growth curves for the early and late TH cells, respectively, demonstrating that the appearance, disappearance, and reappearance of immunoreactive cells reflects the differentiation of two separate populations of PG neurons. Moreover, these data indicate that TH expression in the population of CA cells that persists in the mature PG begins around E16.5. This is after peripheral target innervation has begun, raising the possibility that neuron-target interactions regulate biochemical differentiation of these CA sensory neurons.     

A population of neurons expressing tyrosine hydroxylase and migrating from olfactory placode into forebrain during ontogenesis in rats

Olfactory placodes, that give rise to the olfactory and respiratory epithelia during ontogenesis, are a source of many neurons migrating into forebrain in the direction of growth of the olfactory nerves. The neurons expressing gonadotropin releasing hormone (GnRH) are among the best studied in the population in question. This hormone is responsible for the central regulation of reproduction in adult animals. It was already shown that, in addition to the GnRH-immunoreactive neurons, a small amount of neurons expressing tyrosine hydroxylase (TH), the first enzyme of catecholamine synthesis, migrates into the forebrain. Such a transient population of TH-immunoreactive neurons was shown by means of single and double immmunohistochemical labeling. The TH neurons were first found on branches of the olfactory, terminal, and vomeronasal nerves, along the trajectory of migration of GnRH-immunoreactive neurons on day 15 of embryogenesis, which preceded the appearance of GnRH-immunoreactive neurons. On days 17-21 of embryogenesis, both populations of neurons were found in almost the same areas and on day 21 single neurons contained both GnRH and TH. There were no neurons expressing decarboxylase of aromatic acids (DAA), the second enzyme of catecholamine synthesis, among TH-immunoreactive neurons, thus suggesting noncatecholaminergic nature of these neurons. However, single nonenzymatic DAA-immunoreactive neurons were found in the area of anterior olfactory nuclei in the forebrain, which suggests their involvement in local cooperative synthesis of catecholamines in the area where GnRH-immunoreactive neurons penetrate in the forebrain. Thus, the neurons expressing TH, TH and GnRH, and DAA were found in rats during prenatal period in the nasal part of the head along the nerves projecting into the forebrain and in the rostral part of forebrain. The origin and functional significance of these neurons are discussed.