Intermittent activation of peripheral chemoreceptors in awake rats induces Fos expression in rostral ventrolateral medulla-projecting neurons in the paraventricular nucleus of the hypothalamus.

Despite the well-established sympathoexcitation evoked by chemoreflex activation, the specific sub-regions of the CNS underlying such sympathetic responses remain to be fully characterized. In the present study we examined the effects of intermittent chemoreflex activation in awake rats on Fos-immunoreactivity (Fos-ir) in various subnuclei of the paraventricular nucleus of the hypothalamus (PVN), as well as in identified neurosecretory preautonomic PVN neurons. In response to intermittent chemoreflex activation, a significant increase in the number of Fos-ir cells was found in autonomic-related PVN subnuclei, including the posterior parvocellular, ventromedial parvocellular and dorsal-cap, but not in the neurosecretory magnocellular-containing lateral magnocellular subnucleus. No changes in Fos-ir following chemoreflex activation were observed in the anterior PVN subnucleus. Experiments combining Fos immunohistochemistry and neuronal tract tracing techniques showed a significant increase in Fos-ir in rostral ventrolateral medulla (RVLM)-projecting (PVN-RVLM), but not in nucleus of solitarii tract (NTS)-projecting PVN neurons. In summary, our results support the involvement of the PVN in the central neuronal circuitry activated in response to chemoreflex activation, and indicate that PVN-RVLM neurons constitute a neuronal substrate contributing to the sympathoexcitatory component of the chemoreflex.

Synaptic profile of nucleus tractus solitarius neurons involved with the peripheral chemoreflex pathways.

The glomus cells in the carotid bodies (CB) detect alterations in pH and pCO2 and low pO2 level in arterial blood. The carotid sinus nerve conveys the information related to the oxygen level to 2nd-order neurons in the nucleus tractus solitarius (NTS) via tractus solitarius (TS), which is part of the chemoreflex pathways. It has been demonstrated that in 2nd-order NTS neurons receiving inputs from the aortic depressor nerve (ADN), the TS stimulation presents high temporal fidelity. However, the temporal properties of synaptic activity in NTS neurons receiving inputs from CB were not yet fully investigated. Herein using patch-clamp recordings in NTS brainstem slices, we studied TS-evoked excitatory postsynaptic currents (TS-eEPSCs) on morphologically identified 2nd-order NTS neurons that receive afferent inputs from the CB and compared with 2nd-order ADN-NTS neurons recorded in the same experimental conditions. The amplitudes of TS-eEPSCs were similar in both groups, but the latencies and standard deviation (SD) of latency were significantly higher in the CB-NTS neurons (latency: 4±0.2 ms, SD: 0.49±0.03 ms) than in ADN-NTS neurons (latency: 3.3±0.3 ms, SD: 0.19±0.02 ms; P=0.049 for latency and P<0.001 for SD of latency). In a series of double-labeling experiments, we confirmed that some CB-NTS 2nd-order neurons send direct projections to the rostral ventrolateral medulla (RVLM). We conclude that: (a) CB-NTS 2nd-order neurons present temporally distinct postsynaptic currents when compared with ADN-NTS 2nd-order neurons; (b) low SD of latency of TS-eEPSCs is not necessarily a characteristic of all 2nd-order neurons in the NTS; and (c) the presence of direct connections between these 2nd-order neurons in the NTS and RVLM is indicative that these synaptic properties of CB-NTS neurons are relevant for the processing of respiratory and autonomic responses to chemoreflex activation.

Blockade of NK-1 receptors in the lateral commissural nucleus tractus solitarii of awake rats had no effect on the cardiovascular responses to chemoreflex activation

The neurotransmission of the chemoreflex in the nucleus tractus solitarii (NTS), particularly of the sympatho-excitatory component, is not completely understood. There is evidence that substance P may play a role in the neurotransmission of the chemoreflex in the NTS. Microinjection of substance P (50 pmol/50 nl, N = 12, and 5 nmol/50 nl, N = 8) into the commissural NTS of unanesthetized rats produced a significant increase in mean arterial pressure (101 +/- 1 vs 108 +/- 2 and 107 +/- 3 vs 115 +/- 4 mmHg, respectively) and no significant changes in heart rate (328 +/- 11 vs 347 +/- 15 and 332 +/- 7 vs 349 +/- 13 bpm, respectively) 2 min after microinjection. Previous treatment with WIN, an NK-1 receptor antagonist (2.5 nmol/50 nl), microinjected into the NTS of a specific group of rats, blocked the pressor (11 +/- 5 vs 1 +/- 2 mmHg) and tachycardic (31 +/- 6 vs 4 +/- 3 bpm) responses to substance P (50 pmol/50 nl, N = 5) observed 10 min after microinjection. Bilateral microinjection of WIN into the lateral commissural NTS (N = 8) had no significant effect on the pressor (50 +/- 4 vs 42 +/- 6 mmHg) or bradycardic (-230 +/- 16 vs -220 +/- 36 bpm) responses to chemoreflex activation with potassium cyanide (iv). These data indicate that the activation of NK-1 receptors by substance P in the NTS produces an increase in baseline mean arterial pressure and heart rate. However, the data obtained with WIN suggest that substance P and NK-1 receptors do not play a major role in the neurotransmission of the chemoreflex in the lateral commissural NTS

Cardiovascular responses to microinjection of trans -(+)-ACPD into the NTS were similar in conscious and chloralose-anesthetized rats

The changes in mean arterial pressure (MAP) and heart rate (HR) in response to the activation of metabotropic receptors in the nucleus tractus solitarii (NTS) with trans-(+)-1-amino-1,3-cyclopentanedicarboxylic acid (trans-(+)-ACPD) were evaluated in conscious and anesthetized Wistar, male rats weighing 240-260g (N=8). The responses obtained with trans-(+)-ACPD were compared with the responses to L-glutamate (1 nmol/100 nl), since in a previous study we showed that anesthesia converted a pressor response to L-glutamate microinjected into the NTS of conscious rats to a depressor response in the same rats under urethane or chloralose anesthesia. Microinjection of 3 doses of trans-(+)-ACPD (100, 500 and 1000 pmol/100 nl) produced a dose-dependent fall in MAP (range, -20 to -50 mmHg) and HR (range, -30 to -170 bpm) under both conscious and chloralose anesthesia conditions. These data indicate that the cardiovascular responses to the activation of metabotropic receptors by trans-(+)-ACPD are not affected by chloralose anesthesia while the cardiovascular responses to the activation of excitatory amino acid (EAA) receptors by L-glutamate are significantly altered.

Clycine blocks the pressor response to L-glutamate microinjected into the nucleus tractus solitarii of conscious rats

Microinjection of L-glutamate into the nucleous tractus solitarii (NTS) of conscious freely moving Wistar rats (240-260 g) produces pressor (+48 ñ 4mmHg) and bradicardic (-153 ñ 20 bpm) responses. In the present study L-glutamate (2.5 nmol/100 nl) was microinjected before and after microinjection of increasing doses of glycine (10, 25 and 50 nmol/100 nl, N = 6) or saline (vehicle/100nl, N = 6) into the NTS. Microinjections of increasing doses of glycine into the NTS produced a dose-dependent reduction in the pressor but not in the bradycardic responses to L-glutamate. [10 nmol (+29 ñ 5mmHg and -110 ñ 18 bpm), 15 nmol (+12 ñ 7 mmHg and -88 ñ 21 bpm) and 50 nmol (+4 ñ 2 mmHg and -100 ñ 31 bpm)] The dose-dependent blockade of the pressor response to L-glutamate by glycine suggests an inhibitory neuromodulatory role for this amino acid in the sympatho-excitatory activity produced by L-glutamate microinjection into the NTS