Localization of parvalbumin mRNA in rat brain by in situ hybridization histochemistry.

Parvalbumin mRNA was localized in rat brain by in situ hybridization using a 35S labelled rat parvalbumin cDNA and a synthetic oligodeoxyribonucleotide (corresponding to base sequences 140 to 183 of rat parvalbumin cDNA). Strongest hybridization signals were detected in the Purkinje cells of the cerebellum and in neurones of the reticular nucleus of the thalamus. Signal was also detected in the cerebral cortex, hippocampus, basal ganglia and brain stem in agreement with the distribution of parvalbumin immunoreactivity.

Restoration of the corticostriatal projection in rat neostriatal grafts: electron microscopic analysis.

The corticostriatal projection in rat neostriatal grafts was studied by using the axonal transport of Phaseolus vulgaris-leucoagglutinin. The neostriatal primodia from 15-18-day embryos were used to make a cell suspension which was implanted unilaterally into the rat neostriatum 3-5 days after kainic acid lesion. Two to four months later, regions of the frontal cortex ipsilateral to the grafts were injected iontophoretically with Phaseolus vulgaris-leucoagglutinin.

Neuropeptide expression in rat dorsal root ganglion cells and spinal cord after peripheral nerve injury with special reference to galanin.

The temporal course of changes in peptide expression in the dorsal root ganglia L4 and L5 and in the dorsal horn of the spinal cord has been studied in rats subjected to a sciatic nerve transection at a mid-thigh level following different survival times. Galanin-, substance P-, vasoactive intestinal polypeptide-, peptide histidine-isoleucine- and calcitonin gene-related peptide-like immunoreactivities have been studied both by immunohistochemistry and radioimmunoassay. Galanin messenger ribonucleic acid has also been studied by in situ hybridization in the dorsal root ganglia of normal and lesioned animals.

Hybridization histochemistry or in situ hybridization in the study of neuronal gene expression.

1. Currently popular techniques of in situ hybridization histochemistry for the detection of cellular nucleic acids (DNA or RNA) are reviewed. 2.

Loss of calbindin-28K immunoreactive neurones from the cortex in Alzheimer-type dementia.

An antibody raised against chick intestinal calbindin D28K was used to study the number and size of calbindin immunoreactive neurones in postmortem human brains from neurologically normal controls and from patients with neuropathologically diagnosed Alzheimer-type dementia (ATD). In the controls, calbindin immunoreactive neurones were observed in all cerebral cortex areas examined including the frontal, temporal and parietal cortices. When compared with the controls, the number and size of calbindin immunoreactive neurones were significantly reduced in the cortices of patients with ATD.

In vitro and in vivo release of neurokinin A-like immunoreactivity from rat substantia nigra.

In vivo and in vitro perfusion techniques have been used to study the release of neurokinin A-like immunoreactivity from the rat substantia nigra. Potassium depolarization and electrical field stimulation evoked calcium-dependent release from nigral slices. Potassium depolarization was also effective in vivo.

Localization of vasopressin mRNA in the rat brain using in situ hybridization with short cRNA probes made from synthetic DNA templates.

Short antisense and sense RNA sequences were transcribed from annealed double stranded oligodeoxynucleotide templates containing the T7 RNA polymerase promotor sequence and its complementary sequence, together with 5' overhanging sequence corresponding to either the sense or antisense sequences of part of the rat vasopressin gene to produce after transcription 35S-labelled sense or antisense RNA for use in situ hybridization histochemistry. Labelled antisense RNA coding for a vasopressin sequence visualized sites of vasopressin mRNA expression (as did the 35S-labelled antisense oligodeoxynucleotide sequence), whereas sense RNA sequences revealed no specific sites of hybridization. This method represents an accessible, convenient and general method for the generation of cRNA probes suitable for use in situ hybridization.

Insulin-like growth factor II in the rat choroid plexus.

In situ hybridization histochemistry has been used to demonstrate the expression of the mRNA for insulin-like growth factor II (IGF-II) in the adult rat choroid plexus. IGF-II mRNA was found in the choroid plexus cells by using two different probes specific for different parts of IGF-II sequence. Parallel studies on consecutive sections showed that message for a choroid plexus marker transthyretin mRNA was also localized in the same choroid plexus cells.

Localization of vasopressin mRNA-containing neurones in the hypothalamus of the monkey.

Neuronal perikarya containing vasopressin mRNA were detected in cryostat sections of cynomolgus monkey brains by using an in situ hybridization technique. The neurones were observed in hypothalamic regions (supraoptic nucleus, paraventricular nucleus, suprachiasmatic nucleus and accessory supraoptic nucleus). These findings are in agreement with previous reports using immunohistochemical methods.

In situ hybridization histochemistry: localisation of vasopressin mRNA in rat brain using a biotinylated oligonucleotide probe.

A biotinylated antisense oligonucleotide probe specific for the glycopeptide sequence of arginine vasopressin mRNA has been used with amplified detection for visualisation of arginine vasopressin mRNA in the rat hypothalamus. RNAase pretreatment to destroy arginine vasopressin mRNA and use of excess complementary oligonucleotide (sense) to absorb the biotinylated antisense oligonucleotide demonstrated the reaction is specific for arginine vasopressin mRNA. Further, dehydration of rats using 2% saline resulted in an increase in specific staining.

Cell-specific immuno-probes for the brain of normal and mutant Drosophila melanogaster. I. Wildtype visual system.

We have screened antibodies for immunocytochemical staining in the optic lobes of the brain of Drosophila melanogaster. Seven polyclonal antisera and five monoclonal antibodies are described that selectively and reproducibly stain individual cells and/or produce characteristic staining patterns in the neuropile. Such antisera are useful for the cellular characterization of molecular and structural brain defects in visual mutants.

Effect of neurotensin and its fragments neurotensin-(1-6) and neurotensin-(8-13) on dopamine release from cat striatum.

At low concentrations, neurotensin (10(-9) M) enhanced electrically evoked release of dopamine. At higher concentrations, neurotensin (10(-7) M) also enhanced basal release of dopamine. The carboxy-terminal sequence of neurotensin-(8-13) was fully active and enhanced both electrically evoked and basal release.

Presence of a substance P-like immunoreactive neurone system from the parabrachial area to the central amygdaloid nucleus of the rat with reference to coexistence with calcitonin gene-related peptide.

An ascending neurone system containing substance P-like immunoreactivity (SPI) from the lateral parabrachial nucleus (PBL) to the central amygdaloid nucleus (AC) was detected. Destruction of the external subdivision of the PBL resulted in a marked ipsilateral reduction of SPI fibres in the AC, which suggests that SPI neurones project mainly ipsilaterally to the AC. This was supported by the findings that injection of biotin-wheatgerm agglutinin into the AC labelled many neurones in the ipsilateral external subdivision of the PBL.

Loss of matrix calcium-binding protein-containing neurons in Huntington's disease.

In post-mortem brain specimens from patients dying with a clinical diagnosis of Huntington's disease (HD) immunohistochemistry showed a substantial loss from the neostriatum of neurons containing the calcium-binding protein calbindin 28K. These calbindin neurons, and the straital compartment in which they are sited, are particularly damaged in HD, suggesting that a failure of calcium buffering or homeostasis may contribute to cell death in HD.

Evidence for release of calcitonin gene-related peptide and neurokinin A from sensory nerve endings in vivo.

Sensitive radioimmunoassays for calcitonin gene-related peptide and the tachykinin, neurokinin A, have been used to show that acute administration of the sensory neurotoxin capsaicin (10 mg/kg i.p.) to normal adult rats, causes a substantial release of calcitonin gene-related peptide immunoreactivity (15-fold increase) and neurokinin A immunoreactivity (4- to 5-fold increase) into the plasma.

In vitro release of somatostatin from cerebral cortical slices: characterization of electrically evoked release.

Calcium-dependent, tetrodotoxin (1 microM)-sensitive release of somatostatin-like immunoreactivity (SRIF-LI) could be evoked by electrical field stimulation of vibratome-cut cerebral cortical slices superfused in vitro. The release of SRIF-LI from cortical slices was frequency-dependent, and showed facilitation between 5 and 25 Hz. Release was also current-dependent and above a threshold of 10 mA increased to plateau at 80 mA.

Pterygopalatine ganglion cells contain neuropeptide Y.

By immunohistochemistry, neuropeptide Y (NPY) localizes to neurons in the rat pterygopalatine ganglion. These cells also are intensely or moderately reactive with acetylcholinesterase histochemistry. In contrast, both tyrosine hydroxylase immunohistochemistry and glyoxylic acid-induced fluorescence for catecholamines stain smaller clustered cells, similar in appearance to small intensely fluorescent (SIF) cells and clearly distinct from the NPY-immunoreactive cells.

Immunohistochemical evidence for the presence of calcium-binding proteins in enteric neurons.

Immunoreactivity for vitamin D-dependent calcium-binding protein (CaBP) has been localized in nerve cell bodies and nerve fibres in the gastrointestinal tracts of guinea-pig, rat and man. CaBP immunoreactivity was found in a high proportion of nerve cell bodies of the myenteric plexus, particularly in the small intestine. It was also found in submucous neurons of the small and large intestines.

Distribution and characterisation of neuromedin U-like immunoreactivity in rat brain and intestine and in guinea pig intestine.

Neuromedin U-8 (NMU-8) is a peptide isolated from porcine spinal cord which contracts blood vessels and the uterus. Antisera were raised against NMU-8 and used in a radioimmunoassay (RIA) together with HPLC to characterize NMU-like immunoreactivity (NMU-LI) in tissues extracts of rat brain and gut and guinea pig gut. Samples of duodenum, ileum and distal colon were taken from both species, and processed for detection of NMU-LI by fluorescence immunohistochemistry.

Subcellular distribution of mammalian tachykinins in rat basal ganglia.

A combined differential and density gradient centrifugation procedure was used to study the subcellular localisation of the mammalian tachykinins in rat caudateputamen and substantia nigra. Substance P, neurokinin A, neuropeptide K, and neurokinin B were found to be concentrated in the synaptosomal fractions and in fractions containing heavy synaptic vesicles in both regions studied. In contrast, the catecholamines dopamine and noradrenaline had a more widespread distribution throughout the gradient.

Projection of neurotensin-like immunoreactive neurons from the lateral parabrachial area to the central amygdaloid nucleus of the rat with reference to the coexistence with calcitonin gene-related peptide.

The origin of neurotensin-like immunoreactive (NTI) fibers in the central amygdaloid nucleus (AC) in the rat was examined using indirect immunofluorescence and retrograde tracing combined with immunocytochemistry. Destruction of the external subdivision of the lateral parabrachial nucleus, which contains a group of NTI neurons, resulted in a marked reduction of these fibers in the ipsilateral AC, which suggests that most of these fibers are of extrinsic origin. This was also supported by the finding that injection of fast blue dye into the AC labeled many neurons in the external subdivision of the lateral parabrachial nucleus ipsilaterally, and that simultaneous treatment with antiserum against NT stained some of these neurons.

Further analysis of presence of peptides in dopamine neurons. Cholecystokinin, peptide histidine-isoleucine/vasoactive intestinal polypeptide and substance P in rat supramammillary region and mesencephalon.

Double-labeling combined with elution-restaining immunofluorescence techniques were used to analyze the extent of coexistence among the peptides cholecystokinin (CCK), peptide histidine-isoleucine (PHI)/vasoactive intestinal polypeptide (VIP), substance P and the catecholamine-synthesizing enzyme tyrosine hydroxylase in neurons of the supramammillary region and mesencephalon of the rat. Approximately 50% of the PHI/VIP-containing perikarya and about 25% of the CCK-positive cell bodies in the supramammillary region exhibited coexistence of both peptides. Only a very minor portion of these double-labeled neurons were also found to contain immunostaining for tyrosine hydroxylase (indicative of dopamine in these cells).

Transmitter expression and morphological development of embryonic medullary and mesencephalic raphé neurones after transplantation to the adult rat central nervous system. III. Grafts to the striatum.

Implants have been made of dissociated embryonic mesencephalic or medullary raphé cells into the adult rat striatum, previously depleted of its 5HT innervation. The transmitter complement and fibre outgrowth of the grafted neurones were analysed immunocytochemically. Serotonin-containing cells were found in both types of transplant, and the proportionate survival of the potential number of implanted 5HT cells was similar for each type of graft.

Transmitter expression and morphological development of embryonic medullary and mesencephalic raphé neurones after transplantation to the adult rat central nervous system. II. Grafts to the hippocampus.

Suspensions of cells taken from the mesencephalic or medullary raphé regions of the 13-14 day old embryonic rat brain were injected into the hippocampus of adult rats which had previously been denervated of its serotoninergic input by 5,7-dihydroxytryptamine. At periods of up to 14 months after implantation, the brains were taken for immunohistochemical analysis of 5-hydroxytryptamine (5HT)-, substance P (SP)- and thyrotropin releasing hormone (TRH)-like immunoreactivity. Surviving grafts were found in all animals.