Location of the area postrema pressor pathway in the dog brain stem.

Electrical stimulation of the dog's area postrema (AP) induces a response that mimics the pressor response produced by intravertebral infusion of low-dose angiotensin II, which causes an increase in mean arterial pressure associated with transient tachycardia and increased peripheral resistance. The present study investigated in morphine-chloralose anesthetized dogs whether: 1) the characteristics of the AP pressor response are influenced by the presence of carotid sinus afferents; 2) structures rostral to the medulla influence the AP pressor response; and 3) the pressor pathway is initiated by neurons within the AP. Since bilateral cervical sinovagal denervation, which potentiated the phenylephrine pressor response, did not affect the pressor response to AP stimulation, the data provide evidence for an inhibitory influence exerted upon the central baroreflex mechanism by the AP pressor mechanism. The unaltered AP pressor response after midcollicular transection suggests that the efferent pathway is contained within the brain stem caudal to the pons. Finally, the elimination of the pressor response following kainic acid microinjection into the AP provides evidence that the AP pressor mechanism is initiated by neurons within the AP, rather than by fibers of passage from other pressor centers. These results suggest that the AP produces its facilitation of central sympathetic vasomotor outflow via a pathway contained within the medulla.

Neurogenic hypertension produced by lesions of the nucleus tractus solitarii alone or with sinoaortic denervation in the dog.

The cardiovascular effects of bilateral lesions of the nucleus tractus solitarii (NTS) were compared with those of subsequent sinoaortic denervation in the same dogs. Destruction of the lateral but not the medial component of the NTS between + 0.5 and 3 mm anterior to the obex produces mild hypertension and tachycardia, not always sustained for more than 2 weeks. Rises in pressure were accompanied by increased lability which was not present regularly in all dogs but correlated with the baseline level of arterial pressure. On the other hand, sinoaortic denervation following lateral NTS lesions produced the first demonstration of fulminant hypertension in the dog, which led to death within hours. These data suggest that, while NTS lesions in the dog probably only partially interrupt central baroreceptor pathways, the addition of sinoaortic denervation completely disrupts baroreceptor inputs to the central nervous system, thus releasing central sympathetic outflow completely from baroreceptor inhibition.

Neuronal responses to angiotensin II in the in vitro slice from the canine medulla.

The present studies utilized the in vitro slice preparation of the canine dorsomedial medulla, which we have recently developed, to obtain direct evidence for the effects of angiotensin II (Ang II) on the activity of single neurons in this region. Horizontally oriented slices (300 micron) containing the area postrema, nucleus tractus solitarii (NTS), and dorsal motor nucleus of the vagus were perifused with oxygenated artificial cerebrospinal fluid. The effects of microdrop application of Ang II and its antagonist [Sar1,Thr8]Ang II on spontaneous firing rate were determined in 27 extracellularly recorded neurons. Ang II substantially increased the firing rate of 13 neurons located in the medial NTS, but it did not alter the spontaneous activity of the remaining 14 neurons. In most cases Ang II elicited a slowly developing, prolonged excitatory response. The effects of both Ang II and [Sar1,Thr8]Ang II were tested in 13 neurons. [Sar1,Thr8]Ang II produced a short latency, brief excitation in three neurons, marked inhibition of spontaneous firing in two cells, and no effect on the other eight neurons. Administration of [Sar1,Thr8]Ang II blocked the excitatory effects of subsequent administration of Ang II in three neurons. To our knowledge, these observations provide the first evidence for direct actions of both Ang II and [Sar1,Thr8]Ang II on neurons in the canine NTS and for the specificity of the neuronal effects of Ang II as documented by blockade of the excitatory response to Ang II by [Sar1,Thr8]Ang II.

Arginine vasopressin modulates the central action of angiotensin II in the dog.

Endogenous vasopressin may interact with central autonomic nervous system factors in the regulation of cardiovascular function. In 25 morphine/chloralose-anesthetized dogs, we studied the magnitude of the pressor response produced by an infusion of angiotensin II (AII) into the vertebral arteries (VA), before and after intracisternal (n = 10), intravertebral (n = 9), or intravenous (n = 6) administration of a competitive antagonist of arginine vasopressin (AVP) [d(CH2)5Tyr(Me) AVP]. The dose response curve to vertebral artery infusion of AII (range 2-20 ng/kg/min) was significantly (p less than 0.05) shifted to the right of control after injection of the AVP antagonist (10 micrograms/kg) into the cisterna magna; the ED at 20 mm Hg being almost double after central AVP blockade. This effect of AVP blockade was confined only to the cardiovascular response mediated by AII via the vertebral arteries. When pressor doses of AII were injected into either a vein (i.v.) or the cisterna magna of these same dogs, the increases in mean blood pressure were the same before and after AVP antagonist treatment. In another group of anesthetized dogs, we investigated whether the reduced reactivity to intravertebral AII could be duplicated by giving the AVP antagonist either via the vertebral artery or i.v. Only the cisterna magna route was effective in causing a blunting of the pressor response to vertebral artery AII. These data demonstrate a previously unknown interaction between vasopressin and the centrally mediated pressor response to intravertebral AII.

Functional interactions between angiotensin II and substance P in the dorsal medulla.

Low doses of either angiotensin (Ang) II or substance P (SP) microinjected into the medial nucleus tractus solitarii (NTS) produce hypotension and bradycardia, mimicking activation of the baroreceptor reflex. Anatomical evidence suggests that Ang II binding sites in the medial NTS are located presynaptically on vagal afferent fibers that may contain SP and are codistributed with SP binding sites located postsynaptically on intrinsic medial NTS neurons. To evaluate whether the similar cardiovascular effects of Ang II and SP in the medial NTS could involve Ang II-evoked release of SP, we compared the effects of these peptides on the spontaneous activity of medial NTS neurons recorded in vitro and determined whether Ang II evoked release of SP from rat medulla slices. Both Ang II and SP (1 microM in artificial cerebrospinal fluid) excited 11 of 40 medial NTS neurons. In these cells, the peak response latency was significantly longer to Ang II than to SP (59.5 +/- 4.7 versus 26.5 +/- 2.4 seconds, p less than 0.0001). When rat medulla slices were perfused with Ang II (2 microM in Krebs' bicarbonate), release of SP immunoreactivity was increased by 400% over control perfusion with Krebs' solution alone (p less than 0.05). We have provided the first evidence for an excitatory action of Ang II on neurons in the NTS of the rat and for excitation by both Ang II and SP of a subset of neurons in the medial NTS. Moreover, we have shown for the first time that Ang II can stimulate the release of SP immunoreactivity from the brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurophysiological responses to angiotensin-(1-7).

The aim of this study was to investigate the action of the heptapeptide angiotensin-(1-7) on the spontaneous activity of paraventricular neurons using microiontophoresis. Recent immunocytochemical investigations have shown that this product of angiotensin I is predominantly located in cells and fibers of the forebrain and brain stem. Our results show that most neurons in the paraventricular nucleus are excited by angiotensin-(1-7) at a dose of 50-80 nA. In comparison with angiotensin II or angiotensin III, the onset of response and the occurrence of the maximal effect were significantly delayed. With higher doses of angiotensin-(1-7), there was a decrease in latency and a dose-dependent increase in firing frequency. Of all the angiotensin compounds tested, angiotensin III was the most potent. Preliminary results obtained with an angiotensin antagonist show that the action of angiotensin II, angiotensin III, and angiotensin-(1-7) is blocked by the angiotensin receptor subtype 2 antagonist CGP 42112A. Because the angiotensin-(1-7) system in the brain is associated with central vasopressinergic pathways, vasopressin was tested in a similar way. Neurons in the paraventricular nucleus that were excited by iontophoretically applied angiotensins showed a weak response to vasopressin. Occasionally, a small excitatory action was observed. Our results support the hypothesis that the heptapeptide angiotensin-(1-7) is a biologically active neuropeptide. The data also suggest that amino terminal fragments of angiotensin II are not inactive degradation products.

Pathways of angiotensin formation and function in the brain.

New findings from this laboratory suggest that fragments of angiotensin derived from the amino (N-)terminus are biologically active end products of the renin-angiotensin system. In vitro and in vivo experiments revealed that the heptapeptide angiotensin-(1-7) [Ang-(1-7)] is a major endogenous product of the renin-angiotensin system cascade in the brains of rats and dogs. Additional studies with enzyme inhibitors showed that Ang-(1-7) is produced directly from angiotensin I by an enzyme other than the angiotensin converting enzyme. Immunocytochemical fibers within the hypothalamo-neurohypophyseal vasopressinergic system of the rat. Although Ang-(1-7) is as potent as angiotensin II (Ang II) in stimulating release of vasopressin from superperfused hypothalamo-neurohypophyseal explants, the heptapeptide has no dipsogenic or vasoconstrictor activity. In contrast, Ang-(1-7) mimics the effects of Ang II in augmenting the intrinsic discharge rate of neurons within the vagal-solitary complex and in causing monophasic depressor responses after microinjection into the medial region of the nucleus tractus solitarii. The evidence obtained in these experiments suggests novel mechanisms for the generation of angiotensin peptides in the brain. Additionally, the findings suggest that some of the biological actions ascribed to Ang II might be conveyed by the endogenous production of other angiotensin peptides that are generated by enzymatic pathways alternate to those described in the peripheral circulation.

The anteroventral third ventricle region. Participation in the regulation of blood pressure in conscious dogs.

The anteroventral third ventricle (AV3V) region plays an important role in fluid and electrolyte balance and cardiovascular control in the rat; however, experiments in other species have raised questions about the universality of findings in the rat. The effects of discrete lesions placed within the AV3V area on hydromineral balance, the pressor response to angiotensin II given intravenously, and the initiation of a renin-dependent model of hypertension were examined in the dog. A transpharyngeal approach to the optic chiasm enabled us to destroy only the anterior aspects of the AV3V region (aAV3V group) or to include the entire nucleus medianus (NM) as well (aAV3V + NM group). Lesions of the aAV3V caused polydipsia and transient hypernatremia and hyperosmolality. In contrast, adipsia and a sustained increase in plasma sodium levels and osmolality were observed in dogs with lesions of the aAV3V plus the entire NM. Neither lesion altered baseline arterial pressure, heart rate, plasma levels of catecholamines and vasopressin, or total plasma protein levels. Only in aAV3V + NM lesioned dogs was there a tendency for plasma angiotensin II immunoreactivity to be elevated above control values at 2 and 4 days after operation. Neither lesion attenuated the pressor response to intravenous angiotension II or the initiation of renal hypertension induced by aortic coarctation. As observed in other species, structures within the AV3V region participate in hydromineral balance in the dog; however, in the dog portions of the NM dorsal to the AV3V region are essential for the mediation of drinking behavior.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of catecholaminergic neuronal systems in the canine medulla oblongata and pons.

The distribution of catecholamine-containing neurons, fibers, and varicosities in the brainstem of both adult and juvenile dogs was mapped in detail with glyoxylic acid histofluorescence. Four separate groups of catecholamine-fluorescent neurons were identified within the canine medulla and pons in locations comparable to the A1, A2, A5, and A6 regions reported in other species. However, aspects of the pattern and density of the catecholaminergic neuronal systems appeared to be unique to the dog. The A1 neurons of the caudal ventrolateral medulla were much more scattered than in rats or rabbits, but relatively similar to cats. In the A2 region of the dorsomedial medulla, catecholaminergic cells and fibers were uniquely distributed compared to other species: fluorescent neurons were scattered only within the dorsal motor nucleus of the vagus, and a distinctive pattern of fibers and varicosities outlined the nucleus of the solitary tract and dorsal motor nucleus of the vagus. The A5 neurons of the rostral ventrolateral medulla appeared at the rostral limit of the A1 region. Fluorescent A5 cells were more sparse than in rats or primates, and were patterned similarly to cats and rabbits. The canine A6 region contained the most extensive and dense grouping of catecholamine neurons and was similar in pattern to the rabbits but less extensive than that seen in cats or primates. An ascending catecholaminergic fiber pathway was traced through the central tegmental field of the canine medulla and pons, with features similar to the primate. The present study provides the first description of the catecholaminergic neuronal systems of the canine medulla.

Animal models, hypertension, and central nervous system mechanisms.

Accumulating evidence suggests that the central nervous system plays a fundamental role in chronic hypertension. On the other hand, the means by which nervous factors influence the long-term control of blood pressure remain obscure. Animal models that replicate some of the functional aspects of essential hypertension in man have been of value and continue to provide insight into the complexity of the problem. Although none of the experimental models gives definitive answers, separate and combined analyses suggest the multiplicity of sites at which the stimulus must be applied for blood pressure to remain elevated.

Pressor responses of angiotensin II microinjected into the dorsomedial medulla of the dog.

Angiotensin II (Ang II) was injected into regions of the dorsomedial medulla of dogs where both specific Ang II binding and neural elements containing this peptide are found. Picomole amounts of the peptide were delivered simultaneously from a linear array of 3 micropipettes with tips positioned concurrently in either the area postrema (ap), nucleus tractus solitarii (nTS), dorsal motor nucleus of the vagus (dmnX), or hypoglossal nucleus (nXII). Significant increases in blood pressure occurred with Ang II injections into the medial nTS (+12 +/- 2 mm Hg), the ap(+9 +/- 3 mm Hg), and the nXII (+6 +/- 2 mm Hg). In both the medial nTS and the nXII, the pressor responses were accompanied by significant increases in heart rate (+13 +/- 3 beats/min and +8 +/- 3 beats/min, respectively). Ang II injected into the dmnX did not produce consistent effects on blood pressure or heart rate. These data demonstrate that unilateral injections of picomole amounts of Ang II produce changes in blood pressure and heart rate which involve neural elements in the ap and medial nTS.

Angiotensin II receptor localization in the canine CNS.

Specific binding of [125I]angiotensin II [(125I]Ang II) to sections of dog brain was determined by in vitro receptor autoradiography. Highly discrete, dark images representing specific binding of [125I]Ang II were observed in areas corresponding to the nucleus of the solitary tract, dorsal motor nucleus of the vagus, area postrema, ventrolateral medulla, pineal, subfornical organ, nucleus medianus, septum, organum vasculosum of the lamina terminalis and the anterior pituitary. The specific binding was frequently present either as a narrow band or tiny spot within a small portion of the nuclei to which the binding corresponded. The location of these Ang II recognition sites in regions associated with regulation of autonomic and neuroendocrine function provides further evidence for a role of this peptide within the central nervous system.

A morphological characterization of the canine area postrema.

The cellular morphology of the dog's area postrema (AP) was demonstrated with the Golgi-Cox technique. Golgi preparations suggested division of the canine AP into three regions: a periventricular mantle zone, a central region, and a junctional zone adjacent to the nucleus tractus solitarii. The distinctive feature of the dog's AP was arrays of periventricular neurons intermixed with glialoid cells. Additional Bodian sections revealed a commissural fiber network connecting the halves of the AP at the obex. The interconnected three-layer structure implies a polysynaptic pathway by which AP stimulation activates central sympathetic outflow.

Quantitative assessment of cutaneous sensory function in subjects with neurologic disease.

Based upon techniques devised for the behavioral study of cutaneous sensation in monkeys, a method has been developed which studies quantitatively cutaneous sensation in man. The techniques is analogous to the von Békésy method of audiometry and employs a subject-operated stimulus and signalling divice. In tests utilizing electrical stimulation of the skin surfaces the subject serves as his own control for comparison of one cutaneous zone with another and from one trial session to another. A permanent, written record of stimulus and nonverbal perceptual response is produced in this instrumental method which permits statistical analysis of responses. The analysis includes determination of cutaneous sensory thresholds, limits of stimulus intensity during detection, duration of perception, detection cycle rates, and persistence indices. This instrumental method of cutaneous sensory assessment is quantifiable, free of verbal bias, and repeatable in terms of defined stimulus strengths. In applied clinical studies, patients with peripheral nerve lesions show elevations of perceptual thresholds, reduced numbers of detection-disappearance cycles per unit time, prolonged, contorted decay slopes, and occasionally persistence of perception in the absence of stimulation. Patients with central lesions have variable threshold abnormalities, but little slowing of cycle rate or perceptual persistence. These quantitative sensation parameters can be evaluated longitudinally during the course of an illness and its treatment. The method has potential use in the investigation of basic aspects of sensation and its interactions with behavior.

Receptor subtype that mediates the neuronal effects of angiotensin II in the rat dorsal medulla.

We have shown previously that many neurons in the caudal medial nucleus tractus solitarii (nTS) are excited by angiotensin (Ang) II. The selective Ang II receptor antagonists losartan (AT1; DuP 753) and CGP 42112A or PD 123177 (AT2) were used to evaluate the receptor subtype that mediates excitation of medial nTS neurons by Ang II (1 microM) in rat medulla in vitro slices. Neither losartan nor the AT2 antagonists altered the baseline firing of either Ang II-sensitive or Ang II-unresponsive neurons. However, in six cells with low-frequency spontaneous activity that remained above baseline after excitation by Ang II, subsequent administration of losartan reversed the firing pattern to the initial low-frequency activity. Losartan (10 microM) blocked the excitation by Ang II in 29 medial nTS neurons. The Ang II-induced excitation recovered from Type I blockade in 1 h. In contrast, both CGP 42112A (10 and 100 microM, n = 12) and PD 123177 (100 microM, n = 7) failed to block excitation by Ang II in all neurons tested. Furthermore, the AT2 antagonists were ineffective in preventing Ang II-induced neuronal excitation both when they were the first antagonist tested and when they were evaluated after the neuron had recovered from AT1 receptor blockade. These studies suggest that the Ang II-induced excitation of caudal medial nTS neurons is mediated by AT1 Ang II receptors.

Contribution of the vagus nerve to angiotensin II binding sites in the canine medulla.

Specific angiotensin II (Ang II) binding sites are present in the dorsal medulla of several species, and dose-related cardiovascular effects are produced by microinjection of the peptide into this region. Because the anatomical location of Ang II binding sites in the area postrema (ap), nucleus tractus solitarii (nTS), and dorsal motor nucleus of the vagus (dmnX) coincides with the topography of vagal afferent fibers and efferent motor neurons, the effect of either nodose ganglionectomy or cervical vagotomy on Ang II binding sites in the dorsomedial medulla was investigated in dogs by in vitro receptor autoradiography. Two weeks after unilateral ganglionectomy, there was a marked reduction in the density of specific Ang II binding sites in the ipsilateral ap, nTS, and dmnX, and an absence of binding sites in the region where vagal afferent fibers course through the rostral medulla. Unilateral cervical vagotomy, which has been shown to spare central processes of afferent fibers, resulted in a loss of binding only in the ipsilateral dmnX. We also show that Ang II binding sites are present in the nodose ganglion and central and peripheral processes of the vagus nerve. The data indicate that medullary Ang II binding sites are associated with both vagal afferent fibers and efferent motor neurons.

Light microscopic localization of angiotensin II binding sites in canine medulla using high resolution autoradiography.

Angiotensin II (Ang II) produces dose-related, site-specific cardiovascular effects in the canine and rat dorsal medulla. Our previous studies suggested that Ang II binding sites are associated with presynaptic vagal afferent fibers in the nucleus tractus solitarii (nTS) and vagal efferent neurons in the dorsal motor nucleus of the vagus (dmnX). High resolution autoradiography now establishes the relationship of putative Ang II receptors to the cytoarchitecture of these nuclei. Sections of the canine medulla oblongata were processed for film or emulsion autoradiography with 0.3-1 nM 125I-Ang II. Quantitative densitometry of films before and after processing sections for emulsion coating confirmed no selective alteration in labeling. In emulsion coated sections, dense labeling was seen over the majority of the large perikarya and surrounding neuropil in the ventral dmnX. Bands of label overlaid vagal efferent fibers coursing ventrolaterally to exit the medulla. In the nTS, Ang II binding was restricted to regions with heavy vagal afferent innervation. In the dorsal nTS, label was distributed over both cell bodies and neuropil, with highest density capping the solitary tract. In the medial nTS, label was concentrated over perikarya, with scattered grains over the intervening neuropil. The discrete subnuclear association of Ang II binding sites in the dorsal medulla with vagal cells and fibers documents that Ang II receptors are present on both afferent vagal fibers and intrinsic medullary neurons, and reveals an anatomical substrate for the autonomic effects of Ang II in this region.

The canine nucleus tractus solitarii: light microscopic analysis of subnuclear divisions.

The nucleus tractus solitarii (nTS) is a complex structure situated in the dorsal medulla oblongata. This region receives primary visceral and gustatory sensory afferent fibers and has widespread interconnections with brainstem structures, hypothalamus, and limbic forebrain. In both rat and cat distinct subnuclei correlate with specific functions of the nTS. Since the canine model is used extensively for physiological study and evidence from this laboratory supports a critical role for the canine nTS in cardiovascular function, we examined its morphological organization. Light microscopic analysis of cellular and fiber patterns of the nTS revealed nine discrete regions based on cytoarchitecture: the commissural, lateral, ventral, dorsal, intermediate, interstitial and medial subnuclei, the subnucleus gelatinosa, and the dorsal parasolitary region. Analysis of each subnucleus revealed that both the lateral and ventral subnuclei contained two distinct neuronal groups based on cell size. Neurochemical and functional correlates are being provided by ongoing analyses of each subnucleus of the nTS.

Angiotensin II and angiotensin (1-7) excite neurons in the canine medulla in vitro.

Our group showed previously that the heptapeptide angiotensin (1-7) [Ang-(1-7)] is a bioactive product of the renin-angiotensin system, and produces dose-dependent cardiovascular effects similar to those evoked by Ang II when microinjected into the nucleus tractus solitarii (nTS) of the rat. The effects of Ang II were compared with those of Ang-(1-7) on single neuron activity recorded from the medial nTS or dorsal motor nucleus of the vagus (dmnX) in perifused horizontal slices of the canine dorsomedial medulla. Ang II excited 48% of 31 medial nTS neurons, but only activated 14% of 22 dmnX cells. Ang-(1-7) also excited half of the medial nTS cells and 14% of the dmnXl neurons. Although most medial nTS neurons excited by Ang II were also activated by Ang-(1-7), two cells were excited by Ang II but not by Ang-(1-7), and one cell was excited by Ang-(1-7) but not by Ang II. Because Ang-(1-7) lacks direct vasoconstrictor effects, neurons in the dorsomedial medulla may have different receptor characteristics than peripheral tissues. The observation of a few medial nTS neurons excited by only one Ang peptide suggests that there may be a separate Ang-(1-7) receptor that participates in the physiological effects of Ang peptides mediated by the brain.

Morphology and projections of neurobiotin-labeled nucleus tractus solitarii neurons recorded in vitro.

The suitability of the anterograde tracer neurobiotin to provide information about the morphology and projections of extracellularly or intracellularly recorded medial nucleus tractus solitarii (nTS) neurons was evaluated in horizontally oriented rat dorsal medulla in vitro slices. After responsiveness to angiotensin (Ang) II, substance P (SP), and L-glutamate was evaluated, neurons were labeled by electrophoresis of neurobiotin at the recording site. Extracellular application (2 microA for 2 min) produced discrete injection sites (40-70 microns) with a small group of labeled neurons. Ejections into the solitary tract documented that the tracer was not taken up by axons traversing the injection site. Neuronal perikarya, primary and secondary dendrites, and axons exhibited a dense Golgi-like appearance, with well-defined dendritic spines and axonal varicosities. Dendritic or axonal processes could be followed for more than 1 mm from the cell soma in a 50 microns thick section, documenting the horizontal architecture of the medial nTS. Intracellular electrophoresis filled the soma, primary and secondary dendrites, and axons of neurons characterized for responsiveness to peptides, L-glutamate and solitary tract stimulation. The location within the nTS and axonal projections of neurons responsive to Ang II and SP appeared to differ from those of cells responsive to Ang II and L-glutamate. Thus, either extracellular or intracellular application of neurobiotin in the in vitro slice can reveal differences in axonal or dendritic targets of neuronal subgroups responsive to different neurotransmitters or peptides and provide evidence for the likely autonomic significance of the neurons.