Disinhibition of the cardiac limb of the arterial baroreflex in rat: a role for metabotropic glutamate receptors in the nucleus tractus solitarii

Abstract

The nucleus tractus solitarii (NTS) is the first site of integration for primary baroreceptor afferents, which release glutamate to excite second-order neurones through ionotropic receptors. In vitro studies indicate that glutamate may also activate metabotropic receptors (mGluRs) to modulate the excitability of NTS neurones at pre- and postsynaptic loci. We examined the functional role of metabotropic glutamate receptors (mGluRs) in modulating the baroreceptor reflex in the rat NTS. Using the working heart-brainstem preparation, the baroreflex was activated using brief pressor stimuli and the consequent cardiac (heart rate change) and non-cardiac sympathetic (T8-10 chain) baroreflex gains were obtained. Microinjections of glutamate antagonists were made bilaterally into the NTS at the site of termination of baroreceptor afferents. NTS microinjection of kynurenate (ionotropic antagonist) inhibited both the cardiac and sympathetic baroreflex gains (16 +/- 5% and 59 +/- 11% of control, respectively). The non-selective mGluR antagonist MCPG produced a dose-dependent inhibition of the cardiac gain (30 +/- 3% of control) but not the sympathetic gain. Selective inhibitions of the cardiac gain were also seen with LY341495 and EGLU suggesting the response was mediated by group II mGluRs. This effect on cardiac gain involves attenuation of the parasympathetic baroreflex as it persists in the presence of atenolol. Prior NTS microinjection of bicuculline (GABA(A) antagonist) prevented the mGluR-mediated attenuation of the cardiac gain. These results are consistent with the reported presynaptic inhibition of GABAergic transmission by group II mGluRs in the NTS and constitute a plausible mechanism allowing selective feed-forward disinhibition to increase the gain of the cardiac limb of the baroreflex without changing the sympathoinhibitory component.

Figure 1. Injection site in the NTS

Photomicrograph of a histological section from a representative experiment in which bilateral NTS microinjection (100 μm rostral to calamus scriptorius, 300 μm lateral to the midline and 550 μm deep to the dorsal surface) of isoguvacine (60 pmol) inhibited both baroreflex sympathoinhibition and bradycardia without significant effect on the chemoreflex. Subsequent unilateral injection of pontamine sky blue (1 μl) was used to mark the injection site as being within the dorsomedial NTS. The overlaid line drawing (right) shows the nuclear boundaries at this level of the medulla (bregma −14.08 mm; from Paxinos & Watson, 1986). Abbreviations: CC, central canal; NTS, nucleus tractus solitarii; sol, solitary tract; Cu and Gr, cuneate and gracile nuclei; amb, nucleus ambiguus; 12, hypoglossal nucleus; AP, area postrema.

Figure 2. Blockade of ionotropic glutamate transmission in the NTS inhibits baroreflex-mediated sympathoinhibition and bradycardia

A, control baroreflex response with bradycardia and sympathoinhibition evoked by the increase in perfusion pressure. B, bilateral microinjection of kynurenate (600 pmol) into the NTS inhibits both the baroreflex bradycardia (to 19% of control) and sympathoinhibition (to 52% of control). C, the baroreflex responses recover after 15 min. D, group data showing that both the cardiac baroreflex gain and the sympathetic baroreflex gain (both normalized to control) were significantly attenuated by kynurenate (Student's t test, *P < 0.05, n = 6).

Figure 3. The baroreflex bradycardia is selectively inhibited by mGluR antagonist

A, control baroreflex bradycardia and sympathoinhibition evoked by an increase in perfusion pressure. B, bilateral NTS microinjection of the non-selective mGluR antagonist MCPG (600 pmol) inhibits the baroreflex bradycardia but not the baroreflex sympathoinhibition. C, the effect is reversible after 20 min. D, log dose–response relationship for MCPG (30 pmol to 3 nmol) inhibition of the baroreflex bradycardia (*P < 0.01 compared to control, Student's paired t test, n = 7).

Figure 4. Broad-spectrum and group II selective mGluR antagonists inhibit the cardiac baroreflex

Pooled data showing the inhibitory effect of MCPG (non-selective mGluR antagonist, 600 pmol, n = 5) (A) and LY341495 (Group II selective mGluR antagonist, 6 pmol, n = 7) (B) on the cardiac baroreflex gain but not the sympathetic baroreflex gain (normalized to control, *P < 0.05, Student's t test).

Figure 5. MCPG in the NTS attenuates the cardiac baroreflex by an action on the parasympathetic outflow

The sympathetic contribution to the cardiac baroreflex was blocked by the addition of a β-adrenoceptor antagonist to the perfusate (atenolol, 5 μg ml⁻¹). A, control baroreflex bradycardia. B, 10 min after the addition of atenolol when a stable, slower heart rate was achieved, activation of the baroreflex produced a smaller bradycardia (largely because of the fall in baseline heart rate). This bradycardia is mediated through the parasympathetic limb of the cardiac baroreflex. C, NTS microinjection of MCPG (600 pmol) abolished this vagal baroreflex bradycardia indicating an effect on the parasympathetic limb of the reflex. D, pooled data from naive WHBP (n = 7) and preparations β-blocked with atenolol (n = 3) showing the reduction in cardiac baroreflex gain after the NTS microinjection of MCPG (600 pmol, data expressed as percentage of control). There was no significant difference in the effect of MCPG on the baroreflex gain between the naive and atenolol-treated groups.

Figure 6. Inhibitory action of MCPG on the cardiac baroreflex requires GABA_A receptors in the NTS

A, control baroreflex bradycardia. B, microinjection of MCPG (600 pmol) to the NTS inhibits the baroreflex bradycardia. C, recovery to control (20 min). D, subsequent microinjection of the GABA_A antagonist bicuculline (0.6 pmol) followed by MCPG (600 pmol) prevented the attenuation of the cardiac baroreflex. E, after washout of bicuculline (20 min) a repeat application of MCPG again attenuates the baroreflex bradycardia. F, group data showing the initial significant reversible inhibition of the baroreflex by MCPG. There is no significant attenuation of the cardiac gain by bicuculline nor by the subsequent injection of MCPG. After washout of bicuculline, MCPG once again attenuates the bradycardia (n = 3, *P < 0.01, Student's t test).