Presynaptic or postsynaptic location of receptors for angiotensin II and substance P in the medial solitary tract nucleus.

1. Microinjection of angiotensin (Ang) II or substance P (SP) into the medial nucleus tractus solitarii (nTS) produces similar decreases in arterial pressure and heart rate. We previously reported that some medial nTS neurons responsive to SP were also excited by Ang II, and that Ang II increased the release of SP from medulla slices. Both electrophysiological and anatomic data suggest that the cardiovascular effects of these peptides may be mediated by a common neuronal pathway consisting of SP-containing vagal afferent fibers with presynaptic Ang II receptors that innervate medial nTS neurons with SP receptors. To evaluate the validity of this model, we established the presynaptic or postsynaptic location of the receptors for Ang II and SP that mediate excitation of medial nTS neurons by determining the capacity of each peptide to activate the cell before and after blocking synaptic transmission in rat dorsal medulla slices. 2. Extracellular recordings were obtained from 55 medial nTS neurons responsive to Ang II or SP in 400-microns horizontal slices of the dorsal medulla. Neuronal excitation by Ang II and SP was tested before, during, and after reversal of synaptic blockade with low-Ca2+ (0.2 mM), high Mg2+ (5 mM) artificial cerebrospinal fluid (aCSF). Elimination of synaptically evoked short latency responses of the neuron to current pulses applied to afferent fibers in the solitary tract (TS) documented blockade of synaptic transmission by low-Ca2+ aCSF. In most cases, the basal firing rate of the cell increased slowly during perfusion with low-Ca2+ aCSF and stabilized after approximately 30 min at a higher level of spontaneous activity. Responses to the peptides and TS stimulation were also documented after synaptic blockade had been reversed by adding aCSF containing 2-mM Ca2+. 3. Of the 55 medial nTS neurons, 41 were responsive to Ang II; whereas, 50 of the 55 cells were responsive to SP. The neurons were divided into three subgroups on the basis of their responsiveness to Ang II and SP. Although most neurons were responsive to both Ang II and SP (n = 36), five other cells were excited only by Ang II, and 14 neurons were activated only by SP. Of the 55 neurons, 26 were also responsive to L-glutamate: 14 of 17 cells responsive to both Ang II and SP, all 5 neurons excited by Ang II but not by SP, and 7 of 10 neurons responsive only to SP were also excited by L-glutamate. The latency of the action potentials evoked by TS stimulation was much shorter in those neurons responsive only to Ang II (3.6 ms) than in cells excited by both Ang II and SP (6.8 ms) or responsive only to SP (7.4 ms). 4. In 21 of the 36 medial nTS neurons responsive to both Ang II and SP, Ang II continued to excite the cell when synaptic responses to TS stimulation were prevented by low-Ca2+ aCSF, but had no effect on the firing rate of the other 15 neurons during synaptic blockade. Excitation induced by Ang II was also prevented in two of the five medial nTS neurons responsive only to Ang II when synaptic transmission in the slice was blocked. Low-Ca2+ aCSF failed to prevent excitation by SP or L-glutamate in all medial nTS cells responsive to these agonists (n = 50 and n = 26, respectively). In contrast to these observations in medial nTS neurons, Ang II-induced excitation was not altered during synaptic blockade in any of the six dmnX cells studied. No responses to SP or L-glutamate were blocked in dmnX neurons, as also seen in the medial nTS. 5. When all medial nTS neurons responsive to Ang II were examined, the latencies of the response to TS stimulation were significantly shorter in those neurons with presynaptic Ang II receptors than in the group of cells with postsynaptic receptors. In addition, neurons with presynaptic Ang II receptors were distributed differently within the medial nTS than cells with postsynaptic Ang II receptors.(ABSTRACT TRUNCATED)

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.

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.