The origin of somatostatin-containing nerve fibers innervating the hypothalamic supraoptic nucleus

Light and electron microscopic studies were performed to study the connections between somatostatin (SOS)-containing nerve terminals and vasopressin (VP)-containing neurons in the rat supraoptic nucleus (SON). SOS-positive fibers innervate the SON in both the oxytocinergic and vasopressinergic areas. Using double immunostaining symmetric synaptic contacts were visualized between SOS immunoreactive boutons and the soma of VP immunopositive neurons. Surgical transection deafferentiating the SON from all possible directions do not effect the presence of SOS immunopositive fibers. These results suggest a local origin of the SOS fibers. Somatostatin-containing perikarya can indeed be found at the dorsal border of the SON at the rostral and caudal pole of the nucleus--we suggest that these cells innervate the SON. The presence of synaptic contacts between SOS fibers and VP neurons as well as the lack of these fibers in the VP deficient Brattleboro rats indicate a role for SOS in the synthesis and/or release of vasopressin in the SON.     

Median preoptic nucleus projections to vasopressin-containing neurones of the supraoptic nucleus in sheep. A light and electron microscopic study

It has been postulated that osmoreceptors are situated in either or both of two components of the lamina terminalis, the subfornical organ (sfo) and organum vasculosum laminae terminalis (ovlt) and that information from these sites may be relayed to the hypothalamus directly or via a synapse in the median preoptic nucleus (mnpo). We have investigated the nature of projections from the mnpo to vasopressin (AVP)-containing neurones in the hypothalamus. Microinjections of horseradish peroxidase-wheat germ agglutinin (HRP-WGA) have been made into the mnpo and supraoptic nucleus (son) of the sheep. These injections indicated that in the sheep, as in the rat, the mnpo shares a reciprocal innervation with the sfo and ovlt. Furthermore, the most extensive efferent outflow of the mnpo is to the son, with lesser projections directed to the pvn and other hypothalamic sites. When examined at the electron microscopic level, fibres projecting from the mnpo to the son were found to form synapses with immunocytochemically identified AVP neurones. It is suggested that this pathway is one of the major routes by which information from putative osmoreceptors in the lamina terminalis is conveyed to AVP neurones in the hypothalamus.     

Simultaneous detection of tyrosine hydroxylase-immunoreactivity and vasopressin mRNA in neurons of the human paraventricular and supraoptic nucleus

Our purpose was to investigate the proportion of tyrosine hydroxylase (TH)-immunoreactive (IR) neurons expressing vasopressin (VP) mRNA in the human paraventricular and supraoptic nuclei by combining in situ hybridization with immunohistochemistry on the same tissue section. A variability in the proportion of TH-IR neurons synthesizing VP mRNA was observed in adults which was usually more than 50%. In neonates almost all the TH-IR neurons appeared to contain VP mRNA.     

Nerve growth factor receptor-containing cholinergic neurons of the basal forebrain project to the thalamic reticular nucleus in the rat

The origin of nerve growth factor receptor-immunoreactive (NGFr-ir) fibers innervating the thalamic reticular nucleus (Rt) was here investigated in the rat using retrograde tracers in combination with immunocytochemistry. Neurons retrogradely labeled from Rt were scattered ipsilaterally throughout the medial septal nucleus and the other cell groups of the basal forebrain, which contained NGFr-ir cells; 10–20% of these retrogradely labeled neurons were also NGFr-ir. Furthermore, a few retrogradely labeled NGFr-ir cells were detected in the basal forebrain on the contralateral side. Retrograde tracing combined with a double immunocytochemical procedure revealed that all the NGFr-ir neurons labeled from Rt also displayed immunoreactivity for choline acetyltransferase. The present results demontrate that the NGFr-ir neurons of the basal forebrain which project to Rt are cholinergic. The possible functional implications of these findings are discussed.     

Opposite regulation of body temperature by cholinergic input to the paraventricular nucleus and supraoptic nucleus in rats

Hypothalamic cholinergic system plays an important role in the regulation of body temperature and fluid balance. We have previously shown that cholinergic stimulation of the anterior hypothalamus and preoptic area was accompanied by a fall in body temperature, increased water intake, and increased Fos protein in the paraventricular nucleus (PVN) and supraoptic nucleus (SON). In the present study, to estimate the role played by cholinergic input to the PVN and SON in thermoregulation and water intake, we used microdialysis for cholinergic stimulation with neostigmine and analysis of the nucleus, and also investigated immunoreactivity for c-Fos protein in the brain. This stimulation increased extracellular concentration of acetylcholine in these nuclei. Stimulation of the PVN decreased body temperature and increased water intake. On the other hand, stimulation of the SON increased body temperature. Both in PVN-stimulated and SON-stimulated rats, c-Fos-like immunoreactivity (Fos-IR) was evident in the PVN, SON and certain regions including locus coeruleus (LC), area postrema and nucleus of the solitary tract (NTS). Addition of atropine to the dialysis medium attenuated the increase of Fos-IR and suppressed the cholinergic stimulation-induced responses in body temperature and water intake. These results suggest that cholinergic muscarinic mechanisms in PVN and SON play an opposite function in the regulation of body temperature. The same neuronal pathway including LC and NTS may participate in an advance both in hypothermia and in hyperthermia.     

Ultrastructure of cholinergic neurons in the laterodorsal tegmental nucleus of the rat: Interaction with catecholamine fibers

The ultrastructure of choline acetyltransferase (ChAT)-immunoreactive neurons in the laterodorsal tegmental nucleus (TLD) of the rat was investigated by immunohistochemical techniques. The immunoreactive neurons were medium to large in size, with a few elongated dendrites, contained well-developed cytoplasm, and a nucleus with deep infoldings. They received many nonimmunoreactive, mostly asymmetric synaptic inputs on their soma and dendrites. ChAT-immunoreactive, usually myelinated, axons were occasionally seen in TLD. Only one immunoreactive axon terminal was observed within TLD, and it made synaptic contact with a nonimmunoreactive neuronal perikaryon. The synaptic interactions between ChAT-immunoreactive neurons and tyrosine hydroxylase (TH)-immunoreactive fibers in the TLD were investigated with a double immunohistochemical staining method. ChAT-immunoreactivity detected with a beta-galactosidase method was light blue-green in the light microscope and formed dot-like electron dense particles at the electron microscopic level. TH-immunoreactivity, visualized with a nickel-enhanced immunoperoxidase method, was dark blue-black in the light microscope and diffusely opaque in the electron microscope. Therefore, the difference between these two kinds of immunoreactivity could be quite easily distinguished at both light and electron microscopic levels. In the light microscope, TH-positive fibers were often closely apposed to ChAT-immunoreactive cell bodies and dendrites in TLD. In the electron microscope, the cell soma and proximal dendrites of ChAT-immunoreactive neurons received synaptic contacts from TH-immunoreactive axon terminals. These results provide a morphological basis for catecholaminergic regulation of the cholinergic reticular system.     

Postnatal maturation of brain cholinergic systems in the precocial murid Acomys cahirinus: Comparison with the altricial rat

The spiny mouse (Acomys cahirinus) is the only precocial murid species. It has some neuroanatomical peculiarities such as a relatively thin cerebral cortex and a large hippocampus. The levels of choline acetyltransferase, membrane-bound acetylcholinesterase and muscarinic receptor sites (measured as [3H]quinuclidynil benzilate binding) were assessed in the whole brain on days 1, 7, 14, 21, 28 and 80 (adult), and compared with those of Wistar rats of the corresponding ages. At birth choline acetyltransferase was significantly higher in spiny mice than in rats but the adult levels were similar, with an overall increase of about 5.2- and 14-fold for the former and the latter species, respectively. Membrane-bound acetylcholinesterase level and maximal density of muscarinic receptor sites in spiny mice were considerably higher at birth, in contrast adult levels were significantly lower than in rats with a respective overall increase of about 1.5- and over 4.5-fold. The high degree of maturity attained at birth by spiny mice partially depends on the long gestation period. However, if we consider postconception age, the maturation of choline acetyltransferase appears to be delayed at birth in the spiny mice, probably in relation to the lack of external stimulation during intrauterine life. In the cerebral cortex, hippocampus and striatum of adult spiny mice, when compared with the rats, there were similar levels of choline acetyltransferase but lower levels of membrane-bound acetylcholinesterase and, in the cerebral cortex, lower density of muscarinic receptor sites.     

Daily profiles of arginine vasopressin mRNA in the suprachiasmatic, supraoptic and paraventricular nuclei of the rat hypothalamus under various photoperiods

Daily rhythm of arginine vasopressin (AVP) mRNA levels in the suprachiasmatic nucleus (SCN) of rats maintained under a short, LD 8:16 photoperiod differed from that of rats maintained under a long, LD 16:8 photoperiod: under the short photoperiod the morning AVP rise occurred significantly later than under the long one. Daily profiles of AVP mRNA in the supraoptic and paraventricular nuclei were not rhythmic and AVP mRNA levels under LD 8:16 did not differ from those under LD 16:8. The data indicate that photoperiod affects selectively the clock driven AVP gene expression in the SCN.     

Projections from the nucleus accumbens to cholinergic neurons of the ventral pallidum: a correlated light and electron microscopic double-immunolabeling study in rat

A correlated light- and electron microscopic double-immunolabeling study combining choline acetyltransferase immunocytochemistry with anterograde tracing ofPhaseolus vulgaris leucoagglutinin (PHA-L) revealed that axons of the nucleus accumbens terminate on cholinergic neurons of the ventral pallidum. These findings are discussed with respect to the possibility that these cholinergic neurons may be part of parallel circuits, providing feedback to the same cortical and amygdaloid areas which innervate the nucleus accumbens.     

Synaptogenesis in the rat suprachiasmatic nucleus: a light microscopic immunocytochemical survey

MabQ155, a monoclonal antibody against synaptophysin, has been used to conduct a light microscopic survey of synaptogenesis in the suprachiasmatic nucleus of the perinatal rat. Synaptophysin is an integral component of synaptic vesicle membranes which is expressed in growth cones and growth cone filopodia as well as in mature synapses. With the light microscope, mabQ155 immunoreactivity in growth cones can be distinguished from that in presynaptic terminals on the basis of the size of immunoreactive puncta. The current study presents a qualitative and quantitative analysis of synaptogenesis from the day of birth (P0) to postnatal day 10 (P10). In our quantitative analysis we have used daily intervals during the first postnatal week, distinguished between growth cones and presynaptic terminals, and divided the suprachiasmatic nucleus into sampling regions that are related to the progress of synaptogenesis. Our data demonstrate regional differences in synaptogenesis within the suprachiasmatic nucleus (SCN), document the temporal progression from the penetration of growth cones to the appearance of mature synapses, and provide information about gradients of synaptogenesis in the nucleus during development.     

Cholinergic neurons expressing substance P receptor (NK1) in the basal forebrain of the rat: a double immunocytochemical study

Cholinergic neurons expressing substance P receptor (SPR, NK₁) were examined in the rat brain using double immunofluorescence. The distribution of SPR-like immunoreactive (SPR-LI) neurons completely overlapped with that of choline acetyltransferase (ChAT)-LI neurons in the medial septal nucleus, the nucleus of diagonal band of Broca, the magnocellular preoptic nucleus, the substantia innominata of basal forebrain, the caudate-putamen, and the ventral pallidum of the basal ganglia. In the mesopontine tegmentum and the cranial motor nuclei of the brainstem, the distribution of SPR-LI and ChAT-LI neurons was partially overlapping. Neurons showing both SPR-like and ChAT-like immunoreactivities, however, were predominantly found above basal forebrain regions and 82–90% of these ChAT-LI neurons displayed SPR-like immunoreactivity, in addition to the confirmatory observation that 100% of the ChAT-LI neurons exhibit SPR-like immunoreactivity in the basal ganglia. In contrast, neurons double-labeled for SPR-like and ChAT-like immunoreactivities were hardly detected in aforementioned regions of the brainstem. The present study has provided morphological evidence for direct physiological modulation of cholinergic neurons by tachykinins through substance P receptor in the basal forebrain of the rat.     

Gene expression in central cholinergic neurons in response to axotomy and deafferentation

Although the molecular and cellular responses to injury in the central nervous system (CNS) have been widely investigated, few studies have examined the potential variations between direct and indirect neuronal injury. To differentiate between the response to axotomy and deafferentation, two central cholinergic populations were analyzed: the horizontal limb of the diagonal band of Broca (HLDB) and the interneurons in the corpus striatum (CS). At time points from one hour to eight weeks postinjury the levels of choline acetyltransferase (ChAT) mRNA and protein were assessed by in situ hybridization and immunohistochemistry. Also examined was the expression of the immediate early gene product, c-fos. One week post axotomy, neurons in the HLDB exhibited an increase in the levels of ChAT mRNA without a concomitant increase in ChAT protein, followed by a steady decrease reaching a nadir in both parameters at eight weeks. In contrast, a transient increase occurred at one week postdeafferentation in the levels of both ChAT mRNA and protein in the interneurons of the CS. Axotomized neurons in the HLDB did not exhibit either c-fos mRNA or protein expression, while robust fos induction occurred after one hour in deafferented neurons in the CS. These data demonstrate that the molecular and cellular responses differ following direct and indirect neufenal injury. Furthermore, they suggest that in these central cholinergic populations deafferentation may result in cellular hyperactivity and cell survival while axotomy, results in decreased cellular activity and subsequent cellular regression. © 1994 Wiley-Liss, Inc.     

Calcitonin gene-related peptide (CGRP): immunocytochemical identification of a neuropeptide synthesised by ventral paraventricular magnocellular neurones in the sheep

The distribution of calcitonin gene-related peptide (CGRP)-immunoreactive neurones was examined in the hypothalamus and pituitary gland of the short-term ovariectomised ewe. A large number of magnocellular CGRP-immunoreactive neurones were identified in the ventral paraventricular nuclei (PVN); few were found in the dorsal PVN and supraoptic nuclei. Parvicellular CGRP-immunoreactive neurones were identified in low density scattered throughout the preoptic region, anterior and basal hypothalamus and region of the stria terminalis. A dense CGRP innervation of the median eminence and neural lobe of the pituitary was observed. These observations reveal substantial species differences in CGRP immunoreactivity compared with the rat and show that magnocellular CGRP-synthesising neurones in the sheep are essentially restricted to, and define, the ventral PVN. This suggests a functionally distinct role for this previously neglected division of the PVN within the ovine hypothalamo-neurohypophyseal system.     

Compartmental ordering of cholinergic innervation in the mediodorsal nucleus of the thalamus in human brain

The cholinergic innervation of the mediodorsal (MD) nucleus of the thalamus was visualized immunohistochemically in human brain postmortem, using an antibody against human choline acetyltransferase (ChAT). The ChAT staining of the MD nucleus was more intense than in the surrounding thalamic nuclei but weaker than that of the striatum. No ChAT-positive cell bodies were detected. The ChAT-positive neuropil was unevenly distributed, with patches of dense immunoreactivity contrasting with a weaker surrounding matrix. In adjoining sections stained for ChAT immunoreactivity and for acetylcholinesterase (AChE) activity, the zones enriched in ChAT-immunostained neuropil corresponded to AChE-rich regions. The three-dimensional reconstruction of the richest zone in AChE/ChAT activity evidenced a cylindrical organization throughout the rostrocaudal axis of the MD nucleus. Counts of ChAT-positive varicosities confirmed an inhomogeneous distribution; the density of varicosities was 30% higher in ChAT-rich regions than in surrounding matrix. These findings suggest that the activity of intrinsic neurons within the nucleus may be differentially regulated by cholinergic systems.     

Choline's phosphorylation in rat striatal slices is regulated by the activity of cholinergic neurons

The mechanism by which populations of brain cells regulate the flux of choline (Ch) into membrane or neurotransmitter biosynthesis was investigated using electrically stimulated superfused slices of rat corpus striatum. [Me-¹⁴C]Ch placed in the superfusion medium for 30 min during a 1-h stimulation period was incorporated into tissue [¹⁴C]phosphorylcholine (PCh) and [¹⁴C]phosphatidylcholine (PtdCh). Stimulation also caused a profound inhibition of PCh synthesis and a 10-fold increase in [¹⁴C]ACh release into the medium; it failed to affect tissue [¹⁴C]ACh levels. This effect was not explained by changes in ATP levels nor in the kinetic properties of Ch kinase (E.C. 2.7.1.32) or Ch acetyltransferase (ChAT) (E.C. 2.3.1.7). To investigate the mechanism of these effects, Ch uptake studies were performed with and without hemicholinium-3 (HC3), a selective inhibitor of high affinity Ch uptake. A two-compartment model accurately fit the observed data and yielded aK_m for Ch uptake of 5 μM into cholinergic structures and 72 μM into all other cells. Using this model it was estimated that cholinergic neurons account for 60% of observed uptake of Ch at physiologic Ch concentrations, even though they represent fewer than 1 % of the total cells in the slice. The model also predicts that an increase in Ch uptake within cholinergic neurons, reported to be associated with depolarization [4,27,32], would significantly inhibit Ch uptake into all other cells, and would account for the observed decrease in PCh synthesis.     

Activation of supraoptic magnocellular neurons by gamma 2-melanocyte stimulating hormone (gamma 2-MSH)

The effects of intracarotid infusions of the peptide gamma₂-melanocyte stimulating hormone (γ₂-MSH) on electrophysiologically and immunohistochemically identified supraoptic nucleus (SON) units were investigated Over a wide dose range this agent always excited SON units, while control infusions of vehicle had no effect. Because neural responses invariably preceded blood pressure elevation, it appears that γ₂-MSH excitation of the magnocellular system was due to a direct effect on the central nervous system and was not a result of systemic cardiovascular responses. These results suggest a forebrain γ₂-MSH sensitive site in the activation of SON magnocellular neurons.     

A1 neurons and excitatory amino acid receptors in rat caudal medulla mediate vagal excitation of supraoptic vasopressin cells

Extracellular recordings from the supraoptic nucleus of the rat established that vasopressinergic neurosecretory cells were excited by stimulation of cervical but not abdominal vagal afferents. This response was absent or significantly attenuated after microinjection of gamma-aminobutyric acid into a region of the caudal medulla known to contain the A1 noradrenaline cell group. Consistent with the possible involvement of the A1 group, vagal stimulation approximately doubled the frequency of proto-oncogene expression in A1 noradrenaline neurons, as indicated by the occurrence of nuclear Fos-like immunoreactivity in tyrosine hydroxylase-positive neurons of the caudal ventrolateral medulla. Finally, A1 region microinjection of either the N-methyl-D-aspartic acid (NMDA) receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV), or the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), significantly reduced vasopressin cell responses to vagal stimulation. These findings suggest that: (i) the A1 group is an essential component in a pathway which relays facilitatory vagal input of cardiopulmonary origin to neurosecretory vasopressin cells, and (ii) the activation of A1 neurons in this pathway involves both NMDA and non-NMDA excitatory amino acid receptors, an observation consistent with an input to A1 cells which generates 'mixed' excitatory postsynaptic potentials.     

The effects of catecholamines on electrical activity of neurons in the guinea pig supraoptic nucleus in vitro

The role of the central noradrenergic system in the supraoptic neuroendocrine regulation was investigated using slices of the guinea pig hypothalamus. Noradrenaline produced a complex membrane effect comprising two distinct depolarizations: one, associated with a moderate increase in input resistance and resulting in an augmentation of the spontaneous firing rate; the other, unaccompanied by a detectable change in input resistance and resulting in depression of the firing rate. The former depolarization was reproducible by applying specific α-agonist, phenylephrine, whereas the latter was induced by a β-adrenergic agonist, isoproterenol. The actions of phenylephrine and isoproterenol were blocked by phentolamine and propranolol, respectively. Amplitude of the phenylephrine-induced depolarization was voltage-dependent with the estimated reversal potential of about −115 mV and changed as a function of [K⁺]_o. On the contrary, amplitude of the isoproterenol-induced depolarization was voltage-independent and was insensitive to changes in external concentrations of K⁺, Na⁺, Cl⁻ and Ca²⁺. We conclude that catecholamines directly modulate the activity of supraoptic neurons through two functionally distinct adrenoceptive sites on neurosecretory cells. The activation of α-receptors may increase cellular excitability through suppression of membrane K⁺ conductance while the activation of β-receptors would depress neuronal firings, possibly through some mechanism which is not directly linked to ionic channels.     

Vestibular and cochlear efferent neurons in the monkey identified by immunocytochemical methods

Attempts were made to identify vestibular (VEN) and cochlear (CEN) efferent neurons in the squirrel monkey using retrograde transport of horseradish peroxidase (HRP) and immunocytochemical methods. HRP implants in the ampulla of the lateral semicircular duct retrogradely labeled cells of VEN bilaterally and some cells of CEN. VEN located lateral to the rostral part of the abducens nucleus formed a compact collection of cells, all of which were immunoreactive only to antisera for choline acetyltransferase (ChAT). CEN, identified by immunoreactivity to ChAT were located at the hilus of the lateral superior olive (LSO), along the lateral border of the LSO and sparsely near lateral parts of the ventral trapezoid nucleus (VTN). A small number of cells and fibers near the border of the VTN and lateral to the LSO were immunoreactive for leucine enkephalin (L-ENK). Fibers immunoreactive for L-ENK also were identified in the hilus of the LSO. No cells of the superior olivary complex were immunoreactive for antisera to ChAT, L-ENK, substance P, gamma-aminobutyric acid or glutamic acid decarboxylase. Cells of VEN and CEN can be identified by their immunoreactivity to ChAT, and some cells and fibers of CEN also contain L-ENK.