GABA in nucleus tractus solitarius participates in electroacupuncture modulation of cardiopulmonary bradycardia reflex

Abstract

Phenylbiguanide (PBG) stimulates cardiopulmonary receptors and cardiovascular reflex responses, including decreases in blood pressure and heart rate mediated by the brain stem parasympathetic cardiac neurons in the nucleus ambiguus and nucleus tractus solitarius (NTS). Electroacupuncture (EA) at P5-6 stimulates sensory fibers in the median nerve and modulates these reflex responses. Stimulation of median nerves reverses bradycardia through action of γ-aminobutyric acid (GABA) in the nucleus ambiguus, important in the regulation of heart rate. We do not know whether the NTS or the neurotransmitter mechanisms in this nucleus participate in these modulatory actions by acupuncture. We hypothesized that somatic nerve stimulation during EA (P5-6) modulates cardiopulmonary inhibitory responses through a GABAergic mechanism in the NTS. Anesthetized and ventilated cats were examined during either PBG or direct vagal afferent stimulation while 30 min of EA was applied at P5-6. Reflex heart rate and blood pressure responses and NTS-evoked discharge were recorded. EA reduced the PBG-induced depressor and bradycardia reflexes by 67% and 60%, respectively. Blockade of GABAA receptors in the NTS reversed EA modulation of bradycardia but not the depressor response. During EA, gabazine reversed the vagally evoked discharge activity of cardiovascular NTS neurons. EA modulated the vagal-evoked cardiovascular NTS cellular activity for 60 min. Immunohistochemistry using triple labeling showed GABA immunoreactive fibers juxtaposed to glutamatergic nucleus ambiguus-projecting NTS neurons in rats. These glutamatergic neurons expressed GABAA receptors. These findings suggest that EA inhibits PBG-evoked bradycardia and vagally evoked NTS activity through a GABAergic mechanism, likely involving glutamatergic nucleus ambiguus-projecting NTS neurons.

Keywords: glutamic acid; phenylbiguanide; vagal excitation.

Fig. 1.

A: site of retrograde tracer injection (red spot) of an original brain of the rat medulla (bregma −13.32 mm) according to Paxinos and Watson's rat brain atlas (30). The bright red area indicated by the arrow demonstrates the injection site of rhodamine-labeled fluorescent microspheres in the nucleus ambiguus. Scale bar in A represents 1 mm. B–E: confocal microscopic images demonstrate triple-fluorescent labeling in the nucleus tractus solitarius (NTS) (bregma −13.56 mm) of a rat. Arrows indicate neurons stained with vesicular glutamate transporter 3 (VGLUT3), GABA_A receptors, retrograde tracer originating from nucleus ambiguus, and colabeling of VGLUT3, GABA_A, and tracer, respectively. Scale bars represent 20 μm.

Fig. 2.

Confocal microscopic images showing triple-fluorescent labeling in the NTS (bregma −13.56 mm) of a rat. Arrows in A, C, and D indicate neurons containing VGLUT3, the retrograde tracer originating from nucleus ambiguus, and colabeling of VGLUT3 and tracer, respectively. Arrowheads in B and D indicate neural processes containing GAD67. In D, the neural processes labeled with GAD67 (green) are in close proximity to the perikarya stained with VGLUT3 and the retrograde tracer indicated with an arrow. Scale bars represent 20 μm.

Fig. 3.

Bar histograms display decreases in mean blood pressure (ΔMAP) and heart rate (ΔHR) to phenylbiguanide (PBG) before and after microinjection of kainic acid (KA) in the intermediate NTS. KA transiently reduced parasympathoexcitatory hemodynamic and negative chronotropic reflex responses. Baseline blood pressure and heart rate are shown above each bar as means ± SE. *Significant difference compared with control PBG responses.

Fig. 4.

Decreases in mean arterial pressure (MAP) and heart rate (HR) were reduced with electroacupuncture (EA). Bar histograms display consistent responses to repeated PBG (every 10 min). EA reduced the decrease in MAP and HR for at least 70 min. Microinjection of saline into the NTS did not influence the inhibitory cardiovascular reflex responses. Baseline blood pressure and HR are shown above each bar as means ± SE. *Significant difference compared with control PBG responses.

Fig. 5.

EA modulation of the PBG-induced bradycardia (right) but not hypotension (left) is transiently reversed by gabazine. *Significant difference compared with baseline PBG responses; †significant difference from preceding EA PBG-EA response. ‡End of gabazine's activity and the effect of EA. Letters in a-h shown in the bars correspond to the original tracings above showing decreases in MAP and HR. Baseline MAP and HR are shown above bars as means ± SE.

Fig. 6.

Characterization of NTS neurons. A and B, respectively, display time and frequency (coherence of 0.87 and frequency 2.4) domain analyses to show cardiac rhythmicity. C: decreased NTS activity during a nitroprusside-evoked decrease in BP, demonstrating that it was barosensitive. Discharge activity of the NTS neuron also was increased in response to intravenous PBG (D, closed arrow indicates time of NTS activity shown with neurogram). Antidromic stimulations show that the neuron projects to the nucleus ambiguus (E). The NTS neuron, activated antidromically by 2 Hz, 11 μA, and 0.5 ms NAmb stimulation (↓), collided with the orthodromically median nerve (MN, P5–6, *) evoked spike (E, middle). #Antidromic spike that is absent during collision (middle). Critical interval was 13.5 ms and refractory period was 4.86 ms. PSD, power spectral density.

Fig. 7.

Bar histograms display NTS neuronal-evoked activity during repeated stimulation of vagal afferents every 10 min. A: consistent evoked activity with stimulation of vagal afferents. B and C: EA reduced evoked activity for at least 60 min through GABA mechanism. Blockade with gabazine (C) reversed the effect of EA (c) compared with preblockade (b) in contrast to saline (B). Letters a-c in bars correspond with peristimulus histograms (C).

Fig. 8.

Composite map displays the sites of microinjections, iontophoresis, and extracellular recordings in the intermediate NTS of cats. For ease of representation, all recording sites are displayed on the right and microinjections on the left. Microinjections with KA at 1 and 3 mm lateral to midline also are shown on the left NTS. *Sites located within NTS. ○, control site outside intermediate NTS. Coronal section is 0 to 0.6 mm rostral to obex.