Colocalization of calbindin and GABA in medial nucleus of the trapezoid body of the rat.

Using immunocytochemical methods, both calbindin and GABA were found to be colocalized in the somas of all the cells of the medial nucleus of the trapezoid body (NMTB) of the rat auditory system. In the lateral superior olive (LSO), calbindin was also found in the terminals but not in the cells. Some terminal labelling was found in the medial superior olive (MSO). GABA was also found in the somas of some cells in both LSO and MSO, but most of the labelling was in terminals. In the rat, calbindin appears to be more involved in a pathway that detects interaural intensity differences.

Immunohistochemical evidence for GABAergic cell bodies in the medial nucleus of the trapezoid body and in the lateral vestibular nucleus in the guinea pig brainstem.

The presence of gamma-aminobutyric acid (GABA) in two brainstem nuclei is demonstrated by using a pre-embedding immunohistochemical procedure followed by staining intensification. Firstly, immunoreactivity was found in numerous cell bodies and profiles of the medial nucleus of the trapezoid body (MNTB). Secondly, numerous neurons including giant Deiters' cells, terminals and fibers were strongly labelled within the lateral vestibular nucleus (LVN). These observations suggest that the inhibitory part of the efferent innervation of outer hair cells in the cochlea can originate from the MNTB, and that GABAergic neurons in the LVN may contribute to information processing within this nucleus.

Comparison of synenkephalin and methionine enkephalin immunocytochemistry in rat brain.

Immunocytochemistry using an antiserum to the C-terminal octapeptide of synenkephalin, proenkephalin(63-70), was performed throughout the rat brain and revealed numerous immunopositive fibers and some cell bodies. The morphology and distribution of synenkephalin immunoreactivity was extremely similar to that of a commercial methionine enkephalin (Met-ENK) antiserum. Colchicine pretreatment allowed the immunostaining of cell bodies not otherwise possible without pretreatment, but did not affect the distribution of immunoreactive fibers. Using 6 microns serial sections, we were able to colocalize synenkephalin and Met-ENK immunoreactivities in gigantocellular neurons of the medullary reticular formation. Preabsorption of the antiserum with [Tyr63]proenkephalin(63-70) octapeptide (YEESHLLA) completely eliminated immunoreactivity in the rat brain, while preabsorption with all other peptides used had no detectable effect. We conclude that our antiserum to synenkephalin is specific for enkephalinergic cell bodies, fibers and terminals. The synenkephalin antiserum used in these studies may have advantages over other antisera utilized for immunocytochemical detection of proenkephalin gene expression.

Glutamate-like immunoreactivity in calyces of Held.

The pattern of glutamate-like immunoreactivity was investigated in the medial nucleus of the trapezoid body of adult rats. A monoclonal 'anti-glutamate' antibody was combined with postembedding immunohistochemistry involving a silver-intensified peroxidase-antiperoxidase procedure for light microscopy or an immunogold method for electron microscopy. The most conspicuous glutamate-immunoreactive elements were labelled calyces of Held. Because of the outstanding size of these terminals they may provide an attractive model for studying the details of glutamatergic neurotransmission at a synapse of the mammalian CNS.

Distribution of serotonin 5-HT1C receptor mRNA in adult rat brain.

Based on in situ hybridization histochemistry (ISHH), we describe the anatomical distribution of the serotonin 5-HT1C receptor mRNA. In addition to the very high levels in epithelial cells of the choroid plexus, 5-HT1C receptor mRNA is found throughout the limbic system, in catecholaminergic cells and in serotonergic neurons. Receptor transcripts are also present in the hypothalamus, numerous motor nuclei and the subthalamus. Our results correlate well with serotonin (5-HT) innervation and receptor binding. Receptor mRNA is present in many brain structures in addition to regions previously shown to have 5-HT1C receptor binding. The distribution of this receptor mRNA suggests that the 5-HT1C receptor may mediate a number of the central effects of 5-HT.

Immunohistochemical localization of intracellular plasma proteins in the human central nervous system.

The regional distribution of plasma protein immunoreactivity was studied in the postmortem central nervous system (CNS) of normal subjects 18 to 78 years old. Samples taken from various areas of brain and spinal cord were processed for peroxidase-antiperoxidase immunocytochemistry using polyclonal antibodies against plasma albumin, prealbumin, alpha 1-acid glycoprotein, alpha 2-macroglobulin, IgG, transferrin, haptoglobin, hemopexin, fibrinogen, as well against the glial fibrillary acidic and S-100 proteins. Many neurons of the spinal cord, cranial nerve nuclei, pontine nuclei, cerebellar dentate nucleus, red nucleus, thalamus and hypothalamus showed strong immunostaining for albumin and moderate to strong staining for alpha 1-acid glycoprotein, IgG, transferrin, haptoglobin, as well as relatively weak immunoreactivity against other plasma proteins. Less intense staining was seen in the nucleus basalis, putamen and Purkinje cells. In contrast, most cerebral cortical neurons were negative except for a few positively stained pyramidal neurons in the hippocampus and in layers III and V of the association neocortex, although more positive pyramidal neurons were observed in the motor and sensory neocortices. Reaction products were also seen in axons of motor and sensory long tracts. These findings suggest that plasma proteins may be transported to spinal cord and brain stem neurons by peripherally projecting nerves and that a series of anterograde and retrograde transneuronal transfers are responsible for the accumulation of plasma proteins in relay nuclei and in other CNS neurons.

Neurofibrillary tangles in cholinergic pedunculopontine neurons in Alzheimer's disease.

The cholinergic neurons located within the pedunculopontine nucleus (Ch5) of patients with Alzheimer's disease (AD; n = 15), Parkinson's disease (PD; n = 2), and neurologically normal (n = 6) subjects were visualized immunohistochemically using choline acetyltransferase, pharmacohistochemically using acetylcholinesterase, or by reduced histochemical methods using nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d). Each histochemical procedure localized a well-delineated, compact lateral group and a more diffuse medial group of neurons within the pedunculopontine nucleus. Co-localization experiments revealed that all three enzymes marked the same population of cholinergic neurons. The extent of pathological alterations associated with the cholinergic neurons within the compact lateral sector of the pedunculopontine nucleus was examined in sections that reacted for NADPH-d, counterstained with thioflavin-S. The average number of neurofibrillary tangles within this portion of the pedunculopontine nucleus was 25.4 (range 0-70) in patients with AD, 1.5 (range 1-2) in those with PD, and 1.2 (range 0-4) in aged control subjects. Of the total number of neurofibrillary tangles counted in AD cases, 72.7% were end-stage ghosts and 27.3% were tangle-bearing neurons. The pathological alteration of cholinergic neurons of the compact lateral aspect of the pedunculopontine nucleus may play a role in some of the behavioral features characteristic of AD.

Noradrenergic neurons with divergent projections to the motor trigeminal nucleus and the spinal cord: a double retrograde neuronal labeling study.

Double retrograde axonal tracing was combined with the indirect immunofluorescence antibody method to determine whether noradrenergic neurons have divergent projections to the motor nucleus of the trigeminal nerve and the spinal cord. Rhodamine-labeled microspheres were injected into the motor trigeminal nucleus and True Blue was deposited into lumbar segments of the spinal cord. After a 10-18-day survival period, brainstem sections were processed for immunofluorescence staining of noradrenergic neurons using antibodies to rat dopamine-beta-hydroxylase. Rhodamine-labeled noradrenergic neurons were observed ipsilaterally throughout the A5 and A7 groups; the contralateral A5 and A7 groups contained few rhodamine-labeled cells. A few rhodamine-labeled noradrenergic neurons were observed in the locus coeruleus and subcoeruleus. True Blue-labeled noradrenergic neurons were identified in the A5 and A7 groups, in the ventral part of the locus coeruleus and in the subcoeruleus. Double retrogradely labeled noradrenergic neurons were observed in the A5 and A7 groups but not in the locus coeruleus and subcoeruleus. Of the total number of rhodamine-labeled noradrenergic cells, a large percentage also contained True Blue: 54% in the caudal A5 group, 59% in the rostral A5 group, and 72% in the A7 group. Of the total number of True Blue-labeled noradrenergic neurons, the percentage of double retrogradely labeled cells was 33% in the caudal A5 group, 46% in the rostral A5 group, and 56% in the A7 group. The findings of this study provide the first anatomic evidence for the existence of a prominent population of noradrenergic cells in the A5 and A7 groups with divergent projections to the motor trigeminal nucleus and the spinal cord. We propose that this subpopulation of noradrenergic neurons in the A5 and A7 groups influences motoneurons at multiple levels of the neuraxis.

Cholinergic projections from the laterodorsal and pedunculopontine tegmental nuclei to the pontine gigantocellular tegmental field in the cat.

This study demonstrates that the laterodorsal tegmental nucleus (LDT) and pedunculopontine tegmental nucleus (PPT) are sources of cholinergic projections to the cat pontine reticular formation gigantocellular tegmental field (PFTG). Neurons of the LDT and PPT were double-labeled utilizing choline acetyltransferase immunohistochemistry combined with retrograde transport of horseradish peroxidase conjugated with wheat germ agglutinin (WGA-HRP). In the LDT the percentage of cholinergic neurons retrogradely labeled from PFTG was 10.2% ipsilaterally and 3.7% contralaterally, while in the PPT the percentages were 5.2% ipsilaterally and 1.3% contralaterally. These projections from the LDT and PPT to the PFTG were confirmed and their course delineated with anterograde labeling utilizing Phaseolus vulgaris leucoagglutinin (PHA-L) anterograde transport.

Impaired release of atrial natriuretic factor in NaCl-loaded spontaneously hypertensive rats.

Our previous studies demonstrated that NaCl-sensitive spontaneously hypertensive rats (SHR) of the Okamoto strain exhibit increased blood pressure and reduced noradrenergic input to the anterior hypothalamus area when fed high NaCl diets. The current study tested the hypotheses that 1) release of atrial natriuretic factor (ANF) into the plasma is impaired in NaCl-loaded SHR, a defect that would tend to elevate blood pressure, and 2) ANF levels in regions of brain involved in blood pressure regulation, such as the anterior hypothalamic area, are altered in SHR. SHR and control Wistar-Kyoto rats (WKY) were placed on 1% or 8% NaCl diets at age 7 weeks; 2 weeks later, ANF levels were measured in plasma, left and right atria, anterior hypothalamic area, ventral hypothalamic area, posterior hypothalamic area, pons, and medulla by radioimmunoassay. Blood for ANF assay was obtained from intra-arterial cannulas in conscious, unrestrained rats studied in the resting state. The 8% NaCl diet produced an increase in blood pressure in the SHR, but not in the WKY. Plasma ANF levels were significantly greater in WKY fed 8% NaCl than in WKY fed 1% NaCl, but dietary NaCl loading did not produce similar increases in plasma ANF in the SHR. Plasma ANF levels were not significantly different between SHR and WKY fed the 1% NaCl diet. The observation that dietary NaCl loading stimulated ANF release into the plasma in WKY but not in SHR suggests that the exacerbation in hypertension seen in NaCl-loaded SHR may be related to an impairment in ANF release.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical localization of peptides and other neurochemicals in the rat laterodorsal tegmental nucleus and adjacent area.

The laterodorsal tegmental nucleus (ntdl) contains a cluster of cells located just medial to the locus coeruleus in the pontine brainstem. The ntdl has been shown to project both rostrally to the forebrain and diencephalon and caudally to the spinal cord. In an effort to characterize this region neurochemically, the present study was conducted to identify a variety of neurochemicals localized within perikarya and fibers of the ntdl and surrounding nuclei. Rats were perfused with formalin, and brain sections were processed for fluorescence immunocytochemistry and acetylcholinesterase (AChE). Of the neurochemicals screened, atrial natriuretic factor (ANF), choline acetyltransferase (ChAT), cholecystokinin (CCK), calcitonin gene-related peptide (CGRP), dynorphin B (Dyn B), galanin, somatostatin, substance P, neurotensin (NT), neuropeptide Y (NPY), vasopressin, vasoactive intestinal polypeptide (VIP), serotonin (5HT), glutamic acid decarboxylase (GAD), and tyrosine hydroxylase (TH) were studied. AChE and ChAT staining revealed that the ntdl contains mostly cholinergic neurons. In addition, brightly reactive substance P and galanin and paler staining CRF, ANF, CGRP, NT, VIP, and Dyn B cell bodies were found within the ntdl. Varicose fibers in this nucleus also contained these peptides in addition to CCK, GAD, TH, 5HT, and NPY. The dorsal tegmental nucleus, dorsal raphe nucleus, locus coeruleus, and the parabrachial region contained a dense and varied assortment of peptides with distinct positions and patterns. This multiplicity of neurochemicals within this area suggests a possible influence on a variety of functions modulated by the ntdl and other closely associated tegmental nuclei.

Morphology, number, and distribution of putative GABA-ergic neurons in the basilar pontine gray of the monkey.

We used an antibody raised against the inhibitory transmitter gamma-aminobutyric acid (GABA) in the basilar pontine gray (bpg) of the monkey. Somata, dendrites, and a plexus of probably axonal fibers exhibited GABA-like immunoreactivity. Labeled neurons were small with oval, triangular, or circular soma shape. They gave rise to 2 to 4 dendrites with little branching. No axons were seen issuing from the soma. Occasionally, appendages consisting of spheroidal bodies positioned at the distal end of tenuous stalks and (in 1 cell) axonlike processes were observed to originate from dendrites. According to their morphology, we suggest that these putative GABA-ergic neurons may correspond to the type II neurons observed in Golgi material. The average number of putative GABA-ergic cells in 40-micron sections was about 30/mm2. When compared with Nissl-stained sections, these amounted to about 5% of all cells. There was no substantial variation in average density in different parts of the bpg. However, labeled cells tended to lie in clusters, perhaps related to the well-established input-output compartments of the bpg. The demonstration of a significant population of putative inhibitory neurons challenges the traditional view of the bpg, which suggests that this brainstem cell group functions as a simple relay exchanging signals between cortex and cerebellum.

Distribution of neurotensin binding sites in the caudal brainstem of the rat: a light microscopic radioautographic study.

Specific high-affinity neurotensin binding sites were labeled in sections of the rat caudal brainstem using a monoiodinated ligand, and their distribution was examined by light microscopic radioautography after fixation with glutaraldehyde. In the medulla, labeled binding sites were mainly concentrated within the dorsal motor nucleus of the vagus, the nucleus of the solitary tract, the external cuneate nucleus, the lateral reticular nucleus, the medial vestibular nucleus, the retrofacial nucleus, the linearis nucleus, the paragigantocellular nucleus and the nucleus raphe pallidus. Within the pons, neurotensin binding sites were detected in the reticulotegmental nucleus, the pontine nuclei, the dorsal tegmental nucleus, the laterodorsal and pedunculopontine tegmental nuclei and the nuclei raphe dorsalis and medianus. Most nuclei found here to contain high densities of neurotensin binding sites have been shown to stain intensely for acetylcholinesterase, suggesting a possible association between this enzyme and neurotensin receptors.

Localization of thyrotropin-releasing hormone prohormone messenger ribonucleic acid in rat brain in situ hybridization.

We studied the distribution of pro- TRH mRNA in rat brain by in situ hybridization histochemistry using radiolabeled single stranded cRNA probes to confirm the hypothesis that the TRH precursor is distributed beyond regions that contain immunoreactive TRH. All regions of the central nervous system previously recognized to contain TRH showed hybridization. Hypophysiotropic neurons in the medial parvocellular division of the paraventricular nucleus showed more intense hybridization than anterior parvocellular division cells, suggesting regional differences in expression. In addition, regions not previously recognized to contain TRH in neuronal perikarya by immunocytochemistry showed specific hybridization for pro-TRH mRNA. These include cells in the olfactory bulbs, dorsal motor nucleus of the vagus, ventrolateral periaqueductal gray, reticular nucleus of the thalamus, and anterior commissural nucleus. Only a single hybridizing band was observed on Northern blots of RNA extracts of the periaqueductal gray and reticular nucleus, identical to that seen in extracts of the paraventricular nucleus. The appearance of pro-TRH mRNA in neurons not previously recognized to contain TRH but which contain the prohormone suggests that non-TRH peptides within the TRH precursor may be preferentially expressed in certain regions of the brain.

Loss of pirenzepine regional selectivity following solubilization and partial purification of the putative M1 and M2 muscarinic receptor subtypes.

Pirenzepine inhibition of [3H]N-methylscopolamine ([3H]NMS) binding was studied in membranous, digitonin-solubilized, and partially purified muscarinic receptors from bovine cortex, an area of the brain rich in the putative M1 muscarinic receptor subtype, and from pons-medulla, an area rich in the putative M2 subtype. In accord with previous work, we found that pirenzepine bound to membranous receptors from cortex with an IC50 that was over one order of magnitude lower than to receptors from pons-medulla. After digitonin solubilization, however, this regional selectivity was significantly reduced. In receptors from pons-medulla, the IC50 for pirenzepine inhibition of [3H]NMS was reduced from 2.1 +/- 0.7 X 10(-6) M in membrane-bound receptors, to 4.3 +/- 0.3 X 10(-7) M after solubilization, whereas in receptors from cortex, the IC50 remained unchanged after solubilization. The solubilized receptors from both brain areas maintained their binding characteristics after partial purification over an ABT-Sepharose affinity column and a hydroxylapetite column. These findings raise the possibility that the different pirenzepine binding characteristics used to define M1 and M2 receptor subtypes are not inherent in the receptor protein itself, but may be due to coupling factors such as effector proteins, phospholipids or cytoskeletal proteins which could be associated with the membranous receptor and become dissociated from the receptor after solubilization.

Changes in neurotransmitter amino acids content in several CNS areas from aggressive and non-aggressive bull strains.

Neurotransmitter amino acid levels (glutamate, aspartate, GABA, and glycine) of crude synaptosomal fractions from several non-limbic CNS areas (pons, ventral tegmentum, midbrain reticular formation, fastigial nucleus of cerebellum, posterior colliculus and anterior colliculus) showed significant differences between aggressive Spanish fighting-bull and non-aggressive Friesian strains. The most unequal distribution was observed in neurotransmitter amino acids, while the non-transmitter amino acids (serine, isoleucine, leucine, threonine and alanine) showed minor and uneven changes. The results suggest a possible relationship between changes in amino acid neurotransmitter levels and the aggressive behavior observed in the aggressive breed.

Ontogenesis of serotoninergic nuclei in the rat stem.

The development of central serotoninergic neurons has been investigated with immunohistochemical techniques using the indirect peroxidase-antiperoxidase (PAP) method in 16-and 19-day-old rat embryos, in 1, 10 and 26 days old young and in adult animals. Immunoreactive neurons were present on embryonic day 16 in the subventricular area of the brain stem. First the countour of nucleus raphe dorsalis became distinct in the subventricular cell mass of the lower midbrain. In the ventral part of the tegmentum, cells were grouped along the midline in bilateral columns from which the nucleus centralis superior, the nucleus raphe pontis and the nuclei pontis differentiated. These nuclei were well defined in the newborn on either side of the midline, and the nucleus centralis superior and nucleus raphe pontis were fused on the midline in 10-day old rat. In the ventral part of the pons and medulla, a bilateral cell mass was also found along the midline. A number of immunoreactive cells moving off the midline constituted the nucleus raphe magnus which was formed on 19. embryonis day. Another contingent of immunoreactive cells remained at the midline and formed the nuclei raphe obscurus and pallidus. In newborn rat, these nuclei were well separated from the nucleus raphe magnus. They would later fuse on the midline, whereas the nucleus raphe magnus would remain a bilateral structure.