Karen L. Barnes

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.

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.

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.

Afferent projections of the cervical vagus and nodose ganglion in the dog

The distribution within the brain stem of the afferent projections of the cervical vagus and the nodose ganglion was studied with horseradish peroxidase (HRP) and HRP-wheat germ agglutinin conjugate. Two to eight days after application of tracer into the cervical vagosympathetic trunk or the nodose ganglion the brain stems and ganglia were perfused and processed by the tetramethyl benzidine method. Vagal afferent fibers entered the lateral medulla as a distinct bundle spatially separate from the vagal efferent rootlets which were caudal and ventral to the afferents. Labeled axons in the solitary tract began to enter the nucleus tractus solitarii (nTS) 4.5 mm anterior to obex and were seen throughout the ipsilateral nTS as far as 3.5 mm caudal to obex. Label density varied within the nTS, with heaviest labeling in the dorsal and dorsolateral portions. Label was also seen in the ipsilateral area postrema (ap) and dorsal motor nucleus of the vagus. Labeled fibers crossed in the commissural portions of ap and nTS to enter the contralateral ap and nTS.

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.

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.

A morphological characterization of the canine area postrema

The cellular morphology of the dogs 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 dogs 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.

Projections of the carotid sinus nerve to the medulla in the dog

The afferent and efferent projections of the carotid sinus nerve were examined within the medulla of the dog with axonal transport of horseradish peroxidase (HRP), and compared with the projections of the glossopharyngeal nerve. The carotid sinus nerve was identified electrophysiologically prior to injection of tracer. Carotid sinus nerve afferent fibers entered the medulla as part of the glossopharyngeal nerve root near the caudal limits of the cochlear nuclei. Labeled axons entered the solitary tract and ran caudally to about 3 mm anterior to the obex, where they began to enter the nucleus tractus solitarii (nTS). Carotid sinus afferent fibers and presumptive terminals were discretely localized within limited portions of the ipsilateral dorsal, medial, and lateral nTS as far as 3 mm caudal to the obex. A few fibers entered the dorsolateral area postrema ipsilateral to HRP injection. Labeled fibers in the commissural nTS crossed the midline and entered the contralateral medial nTS. Efferent neurons were observed only in half of the cases, and were limited to one to three labeled perikarya in the periphery of the retrofacial nucleus. Comparison of the carotid sinus distribution with the previously described vagal afferent projections to the canine nTS revealed partially overlapping, but clearly distinctive patterns, which support a viscerotopic organization of the nTS.

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.

Brainstem distribution of neurons with efferent projections in the cervical vagus of the dog

The distribution within the brainstem of cell bodies and efferent fibers projecting in the cervical vagus was studied with retrograde transport of horseradish peroxidase (HRP). Five to eight days after multiple microinjections of HRP into either the cervical vagosympathetic trunk or the nodose ganglion the brainstems and nodose ganglia were perfused and processed by the tetramethyl benzidine method. HRP-positive neurons were found in three brainstem regions: a dorsal cell column comprising the dorsal motor nucleus of the vagus (dmnX), a ventrolateral group in the region of nucleus ambiguus (nA), and scattered cells along a line between these columns. The density of labeled neurons was greatest within dmnX. Axons from cells of the ventrolateral column projected dorsomedially; just ventral to dmnX they turned laterally to exit the medulla in multiple rootlets. Within nA labelled neurons were distributed according to size, with larger cells more medial and smaller ones more lateral. Caudal to nA in nucleus retroambigualis and nucleus dorsalis medialis cell bodies appeared segregated into clusters.