Ventrolateral medullary lesions and fastigial cardiovascular response in beagles.

These studies were designed to verify that the putative vasomotor center in the rostral ventrolateral medulla (RVLM) contained the outflow paths for the fastigial nucleus (FN) sympathoexcitatory cardiovascular response. If so, then lesions placed by radiofrequency heating (75 degrees C) or application of kainic acid (40 mM) pledgets would reduce or ablate the pressor-tachycardia response after electrical stimulation of FN. Anesthetized beagles (alpha-chloralose, 115 mg/kg) were used in this study to maintain consistency in medullary brain stem anatomy. A ventral brain stem approach exposed the three chemosensitive zones of Mitchell, Schlaefke, and Loeschcke. In other species the intermediate zone and a portion of the rostral zone underlie the Hokfelt C1 cell group and the putative RVLM vasomotor center. Heart rate, arterial and left ventricular (LV) pressures, and maximal rate of pressure development (LV dp/dt) increased 14-84% above control levels in response to stimulating FN 5-10 times the stimulation threshold. The cardiovascular response was abolished in four of six dogs that received bilateral radiofrequency lesions at a depth of 1-2 mm. In five of seven dogs that received kainate surface lesions, the response was substantially reduced but not abolished. These lesions were effective only in the RVLM, above the corresponding intermediate, but not adjacent rostral or caudal chemosensitive areas. The data support the hypotheses of others that an epinphrine-containing cell group in this region is a final common pathway of sympathoexcitation. Expression of the FN cardiovascular response is primarily mediated through this vasomotor region previously identified by others in the rat, cat, rabbit, and primate.(ABSTRACT TRUNCATED AT 250 WORDS)

Somatic and vagal afferent convergence on solitary tract neurons in cat: electrophysiological characteristics.

Electrophysiological characteristics are described for 67 neurones localized to subnuclei of the solitary tract or the area of the dorsal motor nucleus of the vagus in alpha-chloralose-anesthetized, paralysed cats which received vagal and hindlimb sural or peroneal nerve excitation. The peroneal and sural nerves were stimulated in an exposed hindlimb preparation; the ipsilateral vagus was stimulated at the cervical level. Compound action potentials were recorded from all three nerves. Neurons were recorded with extracellular microelectrodes from the brain stem solitary area contralateral to the stimulated somatic nerves. Ninety-one percent of the recorded neurons were spontaneously active. Eighteen percent and 5% of the neurons received only peroneal or sural excitation, respectively, while 59% of the neurons received convergent peroneal and sural excitation. Thirty-nine of the 67 neurons were also tested for vagal input of which 41% responded with excitation. All of the neurons tested for vagal input also received converging excitation from one or both of the somatic nerves. Thirty-one percent of the vagal-excited neurons received converging input from both the peroneal and sural nerves. The combined mean minimal conduction velocity for peroneal and sural input was 31 +/- 1 m/s (mean +/- 1 S.E., range 9-54 m/s). Thirty-six percent of the peroneal and 31% of the sural afferents were Group II fibers. Significant periods of inhibition of spontaneous neuronal spike activity followed peroneal and sural excitation in 43 and 39% of the neurons, respectively. In many neurons, both excitation and inhibition of spike activity could be elicited at stimulus intensities as low as 1.2 times threshold for the lowest threshold fibers in each nerve. Somatic nerve-induced inhibition of spontaneous neuron activity without prior excitation was also observed. These results suggest that neurons of the solitary tract nuclei receive Group II and Group III somatic afferents which converge on neurons also receiving excitatory vagal input. Consequently, somesthetic and kinesthetic as well as visceral receptor activation may directly modulate solitary tract neurons. A possible conclusion is that the nucleus tractus solitarius is the initial central site of mediation of somatosympathetic reflexes. Modulation of the nucleus tractus solitarius by somatic afferents may then adjust sympathetic tone, via modulation of other medullary centers, in visceral and somatic tissues to match somatic metabolic needs.

Fastigial nucleus modulation of medullary parasolitary neurons.

Input from the cerebellar fastigial nuclei to neurons at the lateral margin of the nuclei of the solitary tract, particularly to the area identified as the nucleus parasolitarius was investigated in acutely prepared, anesthetized dogs. Fastigial nucleus stimulation led to short latency excitation of nucleus parasolitarius units often followed by prolonged inhibition of spontaneous activity. Excitation from deep skeletal muscle afferents, converged on 25% of the spontaneously active units excited from the fastigial nuclei; these afferents originated primarily from the ipsilateral forelimb muscles. This study provides electrophysiological evidence for fastigial modulation of neurons previously demonstrated autoradiographically to receive presumed monosynaptic fastigial nucleus efferents. The convergence of forelimb muscle afferent information tentatively identified as being from Group Ia or Group II pressure stretch receptors suggests that the nucleus parasolitarius may be an integrative area for cerebellar, sensorimotor and/or autonomic information.

Fastigial nucleus projections in the midbrain and thalamus in dogs.

Efferent connections to midbrain and thalamus from portions of the cerebellar fastigial nucleus were investigated using autoradiographic techniques. Bipolar stimulating electrodes were placed in the fastigial nucleus of anesthetized beagles and the area which produced maximal increases in blood pressure and heart rate was localized in each dog. A mixture of [3H]leucine and [3H]proline (4:1) was injected into that area and autoradiograms were prepared. Injections filled the rostral and various parts of the caudal fastigial nucleus. The rostral-caudal extent of injection sites were mapped in the horizontal plane from sequential coronal, thionin-stained sections and "primary" and "secondary" injection zones were defined according to specific criteria. Labeled axons reached the mesencephalon via the contralateral uncinate fasiculus. Ascending fibers assembled in a diffuse contingent at the prerubral level adjacent to the ventrolateral periaqueductal gray. The heaviest projections were contralateral to the injection site, but ipsilateral terminals were observed as well. In the midbrain, axons entered the contralateral and ipsilateral superior colliculus to branch repeatedly and terminate in the deep and intermediate layers. Additional terminals were observed bilaterally in the nuclei of the posterior commissure and pretectal areas at the midbrain-diencephalic junction. In the thalamus, labeled axons formed into three groups which terminated in: the contralateral paraventricular complex and medial dorsal nucleus; the contralateral central medial, paracentral, parafasicular and central lateral nuclei, and the contralateral ventral medial and ventral lateral nuclei. There was a sparse projection to the ipsilateral ventral lateral nucleus. The contralateral projection to the ventral medial and ventral lateral nuclei was marked by dense clusters of label ventral to the internal medullary lamina extending, in the dorsal ventral lateral nucleus, to its rostral pole. Projections to specific somesthetic thalamus or the hypothalamus were not observed. These ascending projections in the canine brain generally conform to those described in other nonprimate mammals. The fastigial nucleus presumably provides information concerning equilibrium and body proprioception to the superior colliculus and to thalamic nuclei including both specific motor relay and "nonspecific" midline and intralaminar nuclei, much the same as reported in the cat. The projection to the ventral medial and ventral lateral thalamic nuclei terminate in areas known to participate in the control of axial and proximal limb muscle activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Fastigial nucleus cardiovascular response and brain stem lesions in the beagle.

Changes in the excitatory cardiovascular response (heart rate, arterial blood pressure, left ventricular pressure, and LV dP/dt as an index of myocardial contractility) resulting from electrical stimulation of the cerebellar fastigial nucleus (FN) were recorded after placement of DC or radio-frequency lesions or after microinjections of kainic acid into brain stem areas that receive FN projections and have been shown to be involved in central cardiovascular control. FN-induced increases in heart rate, blood pressure, and contractility were reduced or abolished by lesions made in the restiform body or the A5 area, which is homologous to the catecholamine-containing region in cats and rats. Lesions in the paramedian reticular nucleus, rostral and caudal to obex, failed to reduce the FN cardiovascular response. Nucleus of the solitary tract lesions augmented the FN pressor response and tachycardia. Kainic acid (1 microliter of 100 mM solution) caused profound depression of heart rate, blood pressure, and contractility and reduced or eliminated the FN-induced cardiovascular response when injected into the A5 area, previously identified by the pressor response following electrical stimulation. We concluded from these observations that a descending fastigiobulbar sympathoexcitatory pathway courses through a previously identified A5 pressor area that is also capable of a depressor response when the cell bodies alone are activated.

Fastigial nucleus projections to the brain stem in beagles: pathways for autonomic regulation.

Efferent connections from a portion of the cerebellar fastigial nucleus were investigated using autoradiography. Bipolar stimulating electrodes were placed in the fastigial nucleus of anesthetized beagles and the area that produced increases in blood pressure and heart rate was localized. A mixture of [3H]leucine and [3H]proline (4:1) was injected into the area and autoradiograms of transported material were prepared. Injections filled the rostral and various parts of the caudal fastigial nucleus. Labeled axons reached the brain stem via two routes, the ipsilateral juxtarestiform body and the contralateral uncinate fasciculus. Ventral portions of the lateral vestibular nucleus were labeled bilaterally, projections to the inferior vestibular and medial vestibular nuclei are contralateral. Nucleus tractus solitarius was heavily labeled on the side opposite the injection. The contralateral medial reticular formation contained many labeled terminals and axons. Label was found in the nucleus reticularis ventralis, lateral reticular nucleus, nucleus gigantocellularis, nucleus pontis caudalis and the paramedian reticular nucleus. No terminal labeling was found in nucleus parvocellularis or nucleus ambiguous. Stimulation of the rostral fastigial nucleus produces increases in blood pressure and heart rate by generalized sympathoexcitation. Many cell groups which facilitate the activity of preganglionic sympathetic neurons do not receive direct fastigial input. It is suggested that that sympathoexcitation resulting from stimulation of the fastigial nucleus occurs through multisynaptic connections in the brain stem.

Effect of central catecholamine depletion on 3H-dexamethasone binding in the dog.

Dogs were bilaterally adrenalectomized (Adx) or sham adrenalectomized 2 weeks after the administration of 6-hydroxydopamine (6-OHDA) or saline-ascorbic acid vehicle directly into the third ventricle (3V). Hypothalamic and hippocampal cytosols were assayed in vitro for high affinity binding of 3H-dexamethasone (3H-DM). 6-OHDA treatment resulted in a significant reduction of norepinephrine concentration in the hypothalamus, but not in the hippocampus, when measured 2 weeks after the second dose. Treatment with this neurotoxin also caused a decrease in 3H-DM binding in the hypothalamus that was detectable after adrenalectomy. A statistically significant reduction in bound DM was not observed in the hypothalamus after 6-OHDA treatment of dogs with intact adrenals, perhaps because of a masking effect of endogenous corticosteroids. No change was observed in the hippocampus. Saturation analysis of binding data revealed the total maximum number of available binding sites in hypothalamic cytosol was lower after 6-OHDA treatment, compared to saline-injected controls. Calculated values for dissociation constants revealed no differences between dogs treated with Adx, saline and Adx, and 6-OHDA. The data support the suggestion that catecholamines may act, in part, by altering the specific binding of a glucocorticoid to its hypothalamic receptor.

Depression of spontaneous and ionophore-induced transmitter release by ruthenium red at the neuromuscular junction.

The effects on spontaneous and ionophore-induced transmitter release of the inorganic dye, ruthenium red (RuR), a known inhibitor of calcium binding sites, were observed at the frog sartorius neuromuscular junction using intracellular recording techniques. Both crude and purified RuR, at concentrations of 1 and 5 micron depressed or blocked spontaneous release of acetylcholine (ACh) and reduced postsynaptic sensitivity to ACh, the crude dye being more potent than the pure. Pretreatment of muscles with RuR prevented the catastrophic reaction of junctions to 100 micron X537A ionophore. Increased levels of Ca2+ restored spontaneous transmitter release to control levels after depression or blockade by RuR. It was concluded that RuR blocks a critical membrane-bound binding site for calcium which is necessary for quantal release of transmitter.

Depression of spontaneous and ionophore-induced neurotransmitter release by Salmonella.

Exposure of frog neuromuscular junctions to heat-killed, lyophilized Salmonella typhimurium (SR 11) produces an early increase in spontaneous transmitter release followed by depression of release and blockade of the obligatory release usually induced by ionophore X537A.

Bacterial lipopolysaccharide depresses spontaneous, evoked, and ionophore-induced transmitter release at the neuromuscular junction.

The neurotoxocity of RNA-free lipopolysaccharide (LPS) extracted from Salmonella Typhimurium (SR-11) was tested at the frog neuromuscular junction using intracellular recording techniques. Spontaneous miniature endplate potential (MEPP) frequency was reduced to 45% of control after 60 minutes in the presence of 10 and 50 micrograms LPS/ml Ringer's solution. Elevation of extracellular [Ca] to 10 mM converted the MEPP frequency response to a biphasic pattern of early acceleration followed by late depression. Evoked endplate potentials (EEPs) were reduced in quantal content until phasic release of transmitter was abolished, while MEPP amplitude and endplate resting potential remained constant. Effects of the potent cation ionophore X537A on MEPP frequency were blocked by 45 minutes of pre-exposure to LPS. Because of its extremely lipophilic character, LPS apparently alters the physical structure of the presynaptic terminal membrane, eventually reducing resting and phasic Ca influx, and isolating the presynaptic terminal from ionophore action.

Endotoxin alters spontaneous transmitter release at the frog neuromuscular junction.

The direct neurotoxic effects of E. coli endotoxin (ETX) on spontaneous transmitter release were tested at the frog sartorius muscle neuromuscular junction. Spontaneous transmitter release was monitored by intracellularly recording miniature end-plate potentials (MEPPs). Junctions were continuously exposed to standard concentrations of 10 microgram/ml of 3 ETX samples, 2 of which produced a significant elevation of MEPP frequency followed by a decline of frequency to very low rates. The third ETX sample, known to have a decreased canine lethality, was without effect on MEPP frequency. No significant changes in MEPP amplitude were evident. The rate of change in MEPP frequency, but not the peak frequency, was reduced by lowering ETX concentrations. Alterations in MEPP frequency induced by ETX were prevented by removing Ca++ and antagonized by high [K+]out. The results suggest that ETX alters ion conductance channels, particularly those for Ca++, in the presynaptic terminal membrane.

Uptake and binding of [3H]hydrocortisone by various pig brain regions.

The cytosol fraction of septum, hypothalamus, and hippocampus of intact and adrenalectomized pigs possessed greater concentrations of radioactivity than the cytosol fraction of amygdala, pituitary, and cortex after an intraventricular injection of [1,2-3H]hydrocortisone. Nuclear extracts from the same brain regions possessed higher concentrations of radioactivity than nuclear extracts of the other brain regions of intact pigs. Nuclear extracts of amygdala, pituitary and hypothalamus from adrenalectomized pigs exhibited the greatest increase over intact pigs in labeled hormone concentration. When adrenalectomized pigs were administered dexamethasone prior to [3H]hydrocortisone, uptake of label was most depressed in hippocampal cytosol and cuclear extract. Also sensitive to the competitive effects of dexamethasone were septal and pituitary nuclear extracts. In intact pigs, pituitary, hippocampus and cortex exhibited higher ratios of bound to total hormone in the cytososl fraction than the other brain regions. Hippocampal and amygdala cytosol possessed the greater magnitude of increase in the ratio of bound to total hormone in adrenalectomized versus intact pigs. The pituitary, septum, amygdala, and cortex of intact and adrenalectomized pigs possessed a ratio of bound to total hormone in nuclear extract 5-10 times greater than that in hippocampus and hypothalamus. However, the latter two regions exhibited a greater increase in bound: total hormone after administration of labeled hormone to adrenalectomized pigs than nuclear extracts of the other brain regions.

Effects of hippocampal and amygdalar stimulation on uptake and binding of 3H-hydrocortisone in the hypothalamus of the cat.

Stimulation of the hippocampus or amygdala of adrenalectomized cats occurred for 10 sec followed by a 50 sec period of no stimulation, beginning 30 min prior to and ending 30 min after administration of 100 muCi of 3H-hydrocortisone into a lateral ventricle. Sixty min after administration of labeled hormone, the hypothalamus was excised and homogenized. Cytosol and nuclear extract fractions were obtained and analyzed for radioactivity and protein content. Separation of bound from free hydrocortisone was achieved by charcoal and adsorption assay. Results reveal that stimulation of the hippocampus resulted in a greater concentration of 3H-hydrocortisone taken up into hypothalamic cells. Also, a greater percentage of total hormone found in the nuclear extract was assayed as bound 3H-hydrocortisone, and the concentration of bound radioactivity in the nuclear extract was increased over control values. Amygdalar stimulation, in general, yielded results similar to those obtained from control cats. However, although a lesser percentage of total hormone in the hypothalamic cytosol was assayed as bound hormone, there was a greater concentration of nuclear bound hormone than in controls, but less than that determined in the hippocampal stimulation group. These results add to the evidence that hippocampus and amygdala have a modulating influence upon the hypothalamo-hypophyseal axis. They also suggest that one manner in which these limbic structures may influence hypothalamic function is to modulate the uptake and binding of hydrocortisone in hypothalamic cells.

Metyrapone inhibition of 3H-hydrocortisone uptake and binding in various brain regions of the pig.

Metyrapone (MET) inhibition of hydrocortisone (3H-HC) uptake binding in the cytosol and nucleus of various brain regions of the pig was demonstrated in vivo. The hippocampus, hypothalamus and septum were the regions most sensitive to the inhibition by MET. The hippocampus exhibited the greatest reduction in 3H-HC concentration in whole homogenate, bound in cytosol and in nuclei. The anterior pituitary and cerebral cortex were the least sensitive to MET. In vitro incubation of hypothalami with 3H-HC and MET substantiated the results obtained in vivo with regard to the inhibitory action of MET on 3H-HC uptake and binding. These results were interpreted to indicate the MET may act directly in certain brain regions and that this inhibitory aciton has important implications for both experimental and clinical uses of this drug.