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D J Reis

Publications and source records attributed to D J Reis.

At least 325 records · Page 18Linked to original sources
PubMed1983

Release of [3H]L-glutamine acid (L-Glu) and [3H]D-aspartic acid (D-Asp) in the area of nucleus tractus solitarius in vivo produced by stimulation of the vagus nerve.

We have investigated the effects of bilateral electrical stimulation of the vagus nerves in anesthetized, paralyzed rats on the release of exogenously administered [3H]L-glutamic acid ([3H]L-Glu) or [3H]D-aspartic acid ([3H]D-Asp) from the intermediate portion of the nucleus tractus solitarius (NTS). Electrical stimulation of afferent fibers with the frequency, pulse, duration, and intensity required to activate C-fibers, elicited hypotension and bradycardia. Such stimuli induced the release of [3H]L-Glu, or its stable analogue [3H]D-Asp, from the NTS into perfusate collected through push-pull cannulae. The release of radioactive materials, calculated as a percent of increase in radioactivity above the prestimulation level, was for [3H]L-Glu 114.4 +/- 25.1% (n = 20) during bilateral vagal stimulation, and 45.6 +/- 11.3% (n = 9) (P less than 0.001) during unilateral stimulation. The release of [3H]D-Asp induced by bilateral vagal stimulation was 100.4 +/- 31.9%. The release, which was anatomically specific and restricted to the NTS, was directly related to stimulus (and hence reflex) intensity. Overflow of the inert substances [14C]urea OR [14C]sucrose, co-administered with the [3H] amino acids, did not increase at the same time. Local depolarization of the cells in the NTS by K+ (53mM) increased the overflow of [3H]L-Glu, as well as [14C]urea, and was able to induce the release of [3H]L-Glu when electrical stimulation failed to have an effect. The results are consistent with the hypothesis that L-Glu is a neurotransmitter of neurons in the NTS mediating vasodepressor response from vagal afferents, including those from systemic baroreceptors.

Animals↗
PubMed1983

Immunohistochemical localization of choline acetyltransferase using a monoclonal antibody: a radioautographic method.

Monoclonal antibodies to rat striatal choline acetyltransferase were produced by fusion of sensitized mouse lymphocytes with murine plasmacytoma (NS1) cells. Two stable anti-choline acetyltransferase lines were established by limiting dilution cloning. Specificity of antibody was established by the following criteria: (1) on an enzyme linked immunosorbant assay, antibodies reacted against choline acetyltransferase which was highly purified; (2) by immunoprecipitation, monoclonal antibody bound to its antigen and precipitated choline acetyltransferase activity from solution, when used in conjunction with rabbit antimouse IgG; and (3) monoclonal antibody was shown to specifically localize cholinergic neurons. The monoclonal antibody to choline acetyltransferase was radiolabeled in culture by incubating hybridomas in medium containing 3H-labeled amino acids. This 3H-labeled antibody was used for radioautography on cryostat sections of rat peripheral and central nervous systems. In a sampling of areas, highly specific labeling of cholinergic structures was afforded at both light and electron microscopic levels. Double labeling of tyrosine hydroxylase, a catecholaminergic marker, and choline acetyltransferase was carried out by reacting sections first with the 3H-labeled antibody to choline acetyltransferase and then with rabbit antibody to tyrosine hydroxylase. The choline acetyltransferase label was radioautographically processed and tyrosine hydroxylase was visualized by the peroxidase-antiperoxidase method. The combined techniques of peroxidase and radioautographic histochemistry provide permanent electron dense labels which can be examined simultaneously within a single histologic section.

Animals↗
PubMed1983

Global cerebral vasodilatation elicited by focal electrical stimulation within the dorsal medullary reticular formation in anesthetized rat.

We examined the effects of electrical stimulation of a restricted area of the dorsal medullary reticular formation (DMRF) on regional cerebral blood flow (CBF) in anesthetized (by chloralose), paralyzed (by curare) rats. CBF was measured in tissue samples by the Kety principle, with 14C-iodoantipyrine as indicator. Stimulation of DMRF elicited a widespread, significant increase in CBF in 12 of 13 areas. The increase in flow was greatest in cerebral cortex, up to 240% of control. However, it was also substantially increased in selected regions of telencephalon, diencephalon, mesencephalon, and lower brainstem, but not cerebellum. In contrast, electrical stimulation of the midline (interstitial nucleus of the medial longitudinal fasciculus) 1 mm medial to the DMRF did not change CBF. The increase in CBF evoked by DMRF stimulation persisted after transection of the spinal cord at C1 or cervical sympathetic trunk. We conclude that excitation of neurons originating in or passing through the DMRF can elicit a potent and virtually global increase of CBF. The effect appears to be mediated by intrinsic pathways of the central nervous system.

Anesthesia, General↗
PubMed1983

Fastigial stimulation releases vasopressin in amounts that elevate arterial pressure.

Electrical stimulation of the cerebellar fastigial nucleus (FN) in anesthetized, paralyzed, and artificially ventilated rat with a 10-s stimulus train (50 Hz) resulted in a stimulus-locked elevation in arterial pressure (AP) and heart rate, the fastigial pressor response (FPR). Blockade of autonomic effectors by chemosympathectomy (produced by treatment with 6-hydroxydopamine) combined with adrenalectomy, or by spinal cord transection at C1, abolished the FPR but unmasked an elevation of AP with longer latency (10-12 s) and duration (2-4 min), termed the residual FPR. The residual FPR was 1) abolished by midbrain transection, 2) blocked by administration of a specific antagonist of the vasopressor response to arginine vasopressin (AVP) [1,d(CH2)5Tyr(Me)AVP], and 3) was absent in homozygous and attenuated in heterozygous rats of the Brattleboro strain. FN stimulation elevated AVP threefold (from 13 +/- 1 to 38 +/- 8 pg/ml, P less than 0.02; n = 6) in intact rats and sevenfold in rats with combined chemosympathectomy and adrenalectomy (from 14 +/- 1 to 96 +/- 11 pg/ml, P less than 0.001; n = 9). Stimulation of the cerebellar FN can release AVP. In the absence of sympathoadrenal effectors, the amount so released is enhanced and capable of elevating AP.

Animals↗
PubMed1983

Fastigial stimulation in rats releases adrenomedullary catecholamines.

Electrical stimulation of the rostral fastigial nucleus (FN) in anesthetized, paralyzed, and artificially ventilated rats with a 10-s stimulus train elicited a stimulus-locked elevation of arterial pressure (AP) and heart rate (HR) (the fastigial pressor response, FPR) and elevated plasma catecholamines (CA) within 20 s from the onset of stimulus. Norepinephrine (NE) increased from 139 +/- 24 to 280 +/- 43 pg/ml (P less than 0.05, n = 8) and epinephrine (E) from 70 +/- 26 to 360 +/- 107 pg/ml (P less than 0.02, n = 8). Acute adrenalectomy increased basal plasma NE (362 +/- 108 pg/ml, P less than 0.05, n = 6) and reduced E (9 +/- 4 pg/ml, P less than 0.02, n = 6). The magnitude and duration of the FPR and the relative increase of NE were unchanged; however, the elevation of E was abolished. Chemosympathectomy, produced by 6-hydroxydopamine hydrobromide (100 mg/kg iv, 24 h before the experiment), lowered resting AP (from 122 +/- 2 to 77 +/- 1 mmHg, P less than 0.001) and NE (16 +/- 5 pg/ml, P less than 0.01), but not E. After chemosympathectomy, FN stimulation induced a pressor response of greater magnitude and longer latency and duration than in controls, increased NE 3.5-fold (from 16 +/- 5 to 56 +/- 14 pg/ml, P less than 0.05, n = 5) and E 9-fold (from 38 +/- 11 to 336 +/- 88, P less than 0.05, n = 5). The increases in CA were abolished by adrenalectomy. Chemosympathectomy shifted the pressor-dose-response curves of NE and E to the left; thus, the enhanced pressor response to FN stimulation after chemosympathectomy was possibly a consequence of supersensitivity to circulatory CA. Stimulation of cerebellar FN increased plasma CA, as a consequence of coexcitation of both neural and adrenomedullary components of the autonomic nervous system. However, in rats with intact sympathetic nerves the release of adrenomedullary CA did not contribute to the elevation in AP.

Adrenal Medulla↗
PubMed1983

Variations in number of dopamine neurons and tyrosine hydroxylase activity in hypothalamus of two mouse strains.

Mice of the BALB/cJ strain have more neurons and greater tyrosine hydroxylase (TH) activity in the midbrain than mice of the CBA/J strain (Baker, H., T. H. Joh, and D. J. Reis (1980) Proc. Natl. Acad. Sci. U.S.A. 77: 4369-4373). To determine whether the strain differences in dopamine (DA) neuron number and regional TH activity are more generalized, regional TH activity was measured and counts of neurons containing the enzyme were made in the hypothalamus of male mice of the BALB/cJ and CBA/J strains. TH activity was measured in dissections of whole hypothalamus (excluding the preoptic area), the preoptic area containing a rostral extension of the A14 group, the mediobasal hypothalamus containing the A12 group, and the mediodorsal hypothalamus containing neurons of the A13 and A14 groups. Serial sections were taken and the number of DA neurons was established by counting at 50- to 60-microns intervals all cells stained for TH through each area. In conjunction with data obtained biochemically, the average amount of TH per neuron was determined. In all areas, TH activity in CBA/J mice was significantly less (p less than 0.001) than in BALB/cJ mice, ranging from 48% in the mediobasal hypothalamus to 71% in the medial and dorsal hypothalamus. The number of TH-containing neurons was also significantly less in the CBA/J strain (p less than 0.001), ranging from 49% in the preoptic area to 74% in the mediobasal hypothalamus (MBH). With the exception of the MBH, enzyme activity per neuron was similar in both strains. In the MBH, strain differences in TH activity were greater than those for neuron number, resulting in less TH activity per neuron in the CBA/J strain. The results suggest that strain differences in the number of DA neurons are widespread and involve DA systems throughout the brain. Therefore, differences in whole brain TH activity cannot be attributed only to differences in specific regions. Our findings further support the view that the number of neurons of a specific chemical class may be under genetic control.

Animals↗
PubMed1982

Antibodies to rat choline acetyltransferase for immunochemistry and immunocytochemistry.

Choline acetyltransferase was purified from rat striata by several sequential column chromatographic separations on Sepharose 4B, hydroxylapatite, Sephadex G-150 and CM-Sephadex, and finally by elution of enzymatically active protein from gel slices. Conventional rabbit and monoclonal antibodies to the enzyme were produced. Monoclonal antibodies were radiolabeled in vivo and used for specific immunoradioautographic localization of choline acetyltransferase in cholinergic neurons by light and electron microscopy.

Animals↗
PubMed1982

Inhibitory cardiovascular function of neurons in the caudal ventrolateral medulla of the rabbit: relationship to the area containing A1 noradrenergic cells.

Experiments were performed to test the hypothesis that A1 noradrenergic neurons, in the caudal ventrolateral medulla, have an inhibitory cardiovascular function. The lateral portion of the caudal medulla was systematically explored, using focal electrical stimulation, in anesthetized, paralyzed rabbits and arterial pressure and heart rate responses were recorded. Since electrical stimulation activates fibers of passage as well as neuronal cell bodies, we also determined cardiovascular responses to microinjections of L-glutamate, a neuroexcitatory amino acid with minimum effects on fibers of passage. Histological studies of stimulation sites were combined with catecholamine fluorescence histochemical studies to localize the A1 cells. A decrease in arterial pressure and heart rate, restricted to the region containing A1 cells, was observed with low frequency stimulation and with microinjections of L-glutamate. In contrast, when GABA, an inhibitory amino acid, was microinjected into the A1 region, we observed an increase in arterial pressure and heart rate. L-Glutamate and GABA responses were dose-related. The fall in pressure was independent of the fall in heart rate. Results support our hypothesis, providing evidence that neurons in the A1 area tonically inhibit sympathetic vasomotor tone.

Adrenergic Fibers↗
PubMed1982

Distribution of dopamine-, noradrenaline-, and adrenaline-containing cell bodies in the rat medulla oblongata: demonstrated by the immunocytochemical localization of catecholamine biosynthetic enzymes.

The immunocytochemical localization of the biosynthetic enzymes--tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine-N-methyltransferase (PNMT)--was used to determine the cytological features and precise neuroanatomical location of catecholaminergic neurons in the medulla oblongata of rat. Perikarya labeled with TH were detected in two bilaterally symmetrical columns located in the ventrolateral and dorsomedial medulla. The distribution and the number of neuronal perikarya containing TH were the same as those containing DBH, except in the dorsal motor nucleus of the vagus at the level of the area postrema where the number of neurons immunocytochemically labeled for TH was considerably greater than those labeled for DBH. The detection of perikarya which show immunoreactivity for TH, used in the biosynthesis of dopamine, noradrenaline, and adrenaline, but not DBH, which converts dopamine to noradrenaline, suggests the existence of dopamine-synthesizing neurons in the medulla. Perikarya labeled with PNMT, used in the biosynthesis of adrenaline, were localized in more restricted regions corresponding to rostral subsets of the dorsal and ventral groups labeled for TH and DBH. Counts of neurons immunocytochemically labeled for TH or PNMT were obtained in order to determine the relative ratio of neurons which contain the enzymes necessary for the synthesis of dopamine, noradrenaline, or adrenaline at various levels of the medulla. At the most caudal levels no PNMT labeled neurons were detected. Further rostral, PNMT-labeled neurons were first detected in the ventrolateral medulla. At the level of the area postrema, the number of PNMT-labeled neurons in the ventrolateral medulla was approximately half of the number of cells showing immunoreactivity for TH. In contrast, few PNMT-labeled cells were detected in the dorsomedial medulla at the level of the area postrema compared to many neurons labeled for TH. At rostral medullary levels, in both the ventrolateral and the dorsomedial regions, the number of neurons labeled for TH and PNMT was essentially the same. Thus most, if not all, of the catecholaminergic neurons in the rostral medulla have PNMT, necessary for the synthesis of adrenaline.

Animals↗
PubMed1982

Enkephalin-containing neurons in substantia gelatinosa of spinal trigeminal complex: ultrastructure and synaptic interaction with primary sensory afferents.

The ultrastructural morphology and synaptic associations between enkephalin-containing neurons and sensory afferents was examined in the caudalis portion of rat spinal trigeminal complex, the region corresponding to Rexed's layer II of the dorsal horn. Specific antiserum to Met5-enkephalin was localized by the peroxidase-antiperoxidase technique in normal adult rats and in rats having electrolytic lesions of the trigeminal ganglia at 2, 4 and 6 days prior to sacrifice. In both normal and lesioned animals, the enkephalin-like immunoreactivity (ELI) was diffusely distributed throughout the cytoplasm of neuronal perikarya, dendrites, myelinated and unmyelinated axons, and axon terminals. The axons terminals were characterized by the presence of both small (40-60 nm) clear, and large (60-100 nm), dense-core vesicles and by the formation of predominantly axodendritic synapses. Optimal visualization of degenerating terminals occurred at 4 days after lesions of the trigeminal ganglia. Approximately one-third of the degenerating afferents were in either direct contact or were separated from an enkephalin-containing process by an intervening unlabeled dendrite. The occurrence of a well defined membrane density between a degenerating and enkephalin-labeled bouton was rare. Most commonly, the processes showing ELI were either unassociated or formed a triad arrangement with the degenerating afferents. Within the triad, the degenerating bouton formed a junction with an unlabeled dendrite which in turn was synaptically connected at the same level to an enkephalin-labeled process, usually a dendrite. We conclude that an intermediary neuron may be involved in interactions between primary sensory afferents and enkephalin-containing neurons in spinal trigeminal complex.

Animals↗
PubMed1982

Quantitative immunocytochemistry of tyrosine hydroxylase in rat brain. I. Development of a computer assisted method using the peroxidase-antiperoxidase technique.

We sought to develop a quantitative immunocytochemical procedure using the peroxidase-antiperoxidase (PAP) technique to analyze amounts of neurotransmitter biosynthetic enzyme tyrosine hydroxylase (TH) within the nucleus locus coeruleus (LC) of rat brain. Rats were perfused with 4% paraformaldehyde. The brains were embedded in paraffin, sectioned at 5 micrometers in the sagittal plane and immunocytochemically stained with antibodies to bovine adrenal TH. Staining intensity, measured by a TV image analysis system was reproducible within +/- 5%. Reaction conditions required so that the intensity of the PAP reaction product was directly and linearly related to the amount of TH enzyme protein in tissue were obtained by reacting tissues with saturating concentrations (2.5 mM) of diaminobenzidine (DAB) substrate, a constant dilution of antibody, and incubation time adjusted so that the darkest elements in tissue were below saturation. Variations in staining intensity of serial sections through the LC were found to be insignificant when compared to variations in staining between animals. In order to increase the amount of immunoreactive TH in the LC, groups of rats were treated with reserpine. Immunocytochemical and biochemical analyses were performed in parallel groups of control and reserpine animals allowed to survive 1-3 days following a single injection (10 mg/kg s.c.) of reserpine. A close correlation was found to exist between the amount of TH enzyme protein determined biochemically and the density of staining for TH. The maximal increase in TH measured immunocytochemically was 2.2-fold which was about 80% of the maximal induction determined biochemically. We conclude that the PAP method can be used for quantitative immunocytochemistry of brain TH providing that optimal reaction conditions are established.

3,3'-Diaminobenzidine↗
PubMed1982

Quantitative immunocytochemistry of tyrosine hydroxylase in rat brain. II. Variations in the amount of tyrosine hydroxylase among individual neurons of the locus coeruleus in relationship to neuronal morphology and topography.

We sought to determine by a computer assisted quantitative immunocytochemical method using the peroxidase-antiperoxidase (PAP) technique if individual neurons of the nucleus locus coeruleus (LC) varied with respect to the amount of tyrosine hydroxylase (TH) within each cell and, if so, whether the amount of TH is related to morphometric characteristics and/or location of the neuron within the LC. Appropriate immunohistochemical reaction conditions were used so that the intensity of DAB reaction product formed in the PAP reaction was directly and linearly related to the amount of TH enzyme protein present in the tissue. The distribution of the average optical density of 1425 individual LC neurons obtained from 5 animals and standardized to an overall mean density varies unimodally over a 3-fold range. The relationship between staining intensity and morphological and topographic characteristics of individual neurons was examined. There was no correlation between staining intensity and morphological features of individual neurons, including cytoplasmic area. However, differences in the amount of TH in individual neurons correlated significantly (P less than 0.01) with the location of the cell in LC: the lightest staining neurons were located in the central core of the LC. The density of staining did not differ significantly in the other 4 traditionally defined regions of LC (the anterior, posterior, and ventral poles of the LC and the nucleus subcoeruleus) which form a rim around the central core. Similarly, cells which are closest to the center of mass of the LC showed a lighter staining intensity than cells located progressively further from the center. These lighter staining cells in the center were also packed more closely together (P less than 0.01). Therefore dense packing of neurons correlates with light staining intensity (P less than 0.01). We conclude that neurons of the LC vary with respect to the amount of TH, which in turn relates to the location of the cell in the LC and possibly its packing density, but not its morphometric characteristics. Since in noradrenergic neurons, the amount of TH is directly influenced by firing rate, the results suggest that the biochemical heterogeneity of LC neurons reflects differences in their functional activities. In turn, this may relate to heterogeneity of LC with respect to projection fields.

Animals↗
PubMed1982

Destruction of noradrenergic neurons in rabbit brainstem elevates plasma vasopressin, causing hypertension.

When A1 noradrenergic neurons in the caudal ventrolateral medulla of rabbits are destroyed electrolytically or by local injection of the neurotoxin kainic acid, the concentration of vasopressin in plasma increases, causing hypertension. The A1 neurons may tonically inhibit the activity of vasopressin-secreting neuroendocrine cells through a direct hypothalamic projection.

Adrenergic Fibers↗