Developmental changes in GABAergic neurotransmission to presympathetic and cardiac parasympathetic neurons in the brainstem

Abstract

Cardiovascular function is regulated by a dynamic balance composed of sympathetic and parasympathetic activity. Sympathoexcitatory presympathetic neurons (PSNs) in the rostral ventrolateral medulla project directly to cardiac and vasomotor sympathetic preganglionic neurons in the spinal cord. In proximity to the PSNs in the medulla, there are preganglionic cardiac vagal neurons (CVNs) within the nucleus ambiguus, which are critical for parasympathetic control of heart rate. Both CVNs and PSNs receive GABAergic synaptic inputs that change with challenges such as hypoxia and hypercapnia (H/H). Autonomic control of cardiovascular function undergoes significant changes during early postnatal development; however, little is known regarding postnatal maturation of GABAergic neurotransmission to these neurons. In this study, we compared changes in GABAergic inhibitory postsynaptic currents (IPSCs) in CVNs and PSNs under control conditions and during H/H in postnatal day 2-5 (P5), 16-20 (P20), and 27-30 (P30) rats using an in vitro brainstem slice preparation. There was a significant enhancement in GABAergic neurotransmission to both CVNs and PSNs at age P20 compared with P5 and P30, with a more pronounced increase in PSNs. H/H did not significantly alter this enhanced GABAergic neurotransmission to PSNs in P20 animals. However, the frequency of GABAergic IPSCs in PSNs was reduced by H/H in P5 and P30 animals. In CVNs, H/H elicited an inhibition of GABAergic neurotransmission in all ages studied, with the most pronounced inhibition occurring at P20. In conclusion, there are critical development periods at which significant rearrangement occurs in the central regulation of cardiovascular function.

Keywords: brainstem; development; neurons; parasympathetic; presympathetic.

Fig. 1.

Schematic drawing of the experimental preparation. A: both bulbospinal presympathetic neurons (PSNs) and parasympathetic cardiac vagal neurons (CVNs) reside in the medulla oblongata. Retrograde labeling of PSNs was performed by injecting of the tracer cholera toxin subunit B bilaterally into T2–T4 spinal segments in P1–P3 rats (A). In another set of experiments, to retrogradely label parasympathetic CVNs, rat pups (P1–P3) received an injection of nontoxic tracer rhodamine into the pericardial sac (A). At the day of experiment, a single medullary slice of 300-μm thickness was obtained, and individual neurons [PSNs in the rostral ventrolateral medulla (RVLM), or, in other sets of experiments, CVNs in the nucleus ambiguous (NA)] were identified by appropriate retrograde labeling and studied using whole cell patch-clamp technique (B).

Fig. 2.

Developmental changes in GABAergic neurotransmission to PSNs and CVNs under control conditions. Examples of GABAergic inhibitory postsynaptic currents (IPSCs), isolated by focal application of strychnine (1 μM), d-2-amino-5-phosphonovalerate (50 μM), and 6-cyano-7-nitroquinoxaline-2,3-dione (50 μM) to block glycine, N-methyl-d-asparate, and non-N-methyl-d-asparate glutamatergic receptors, respectively, are shown in A. An example of abolishment of GABAergic neurotransmission to the patched PSN in P5 animal by application of gabazine (25 μM) is also shown in A. Both the frequency and amplitude of GABAergic IPSCs in bulbospinal PSNs were significantly enhanced in P20 rats (n = 15) compared with P5 (n = 15) and P30 animals (n = 15) under control conditions (125 mM NaCl, 3 mM KCl, 2 mM CaCl₂, 26 mM NaHCO₃, 5 mM glucose, and 5 mM HEPES equilibrated with 95% O₂ and 5% CO_2, pH 7.4). Typical experiments are shown in A, top, whereas the summary data are illustrated in B, top. No significant differences in the amplitude of IPSCs in parasympathetic CVNs were detected between P5 (n = 16), P20 (n = 16), and P30 (n = 16) animals under control conditions. However, the frequency of IPSCs in CVNs was increased in P20 (n = 16) rats compared with P5 animals (n = 16). Typical experiments are shown in A, bottom, and the summary data are demonstrated in B, bottom. Asterisks indicates statistically significant differences, *P < 0.05, **P < 0.01, and ***P < 0.001 (in this and all subsequent figures).

Fig. 3.

Developmental changes in GABAergic neurotransmission to PSNs during H/H. No significant changes in the GABAergic IPSC amplitude in bulbospinal PSNs occur during hypoxia and hypercapnia (H/H) (85% N₂, 6% O₂, and 9% CO₂; pH 7.1) in P5 (n = 15), P20 (n = 15), or P30 (n = 15) animals. However, H/H reversibly diminished the GABAergic IPSC frequency in PSNs in P5 (n = 15) and P30 (n = 15) rats. In contrast, the frequency of GABAergic IPSC in PSNs was unchanged by H/H in P20 animals (n = 15). Typical experiments are shown in A, whereas the summary data are illustrated in B.

Fig. 4.

Developmental changes in GABAergic neurotransmission to CVNs during H/H. A biphasic response to H/H, comprising an initial facilitation following by inhibition of GABAergic IPSC frequency in CVNs, was detected in P5 (n = 16) and P30 (n = 16) animals. In contrast, in P20 rats (n = 16) H/H evoked a gradual inhibition of the GABAergic IPSC frequency. In P20 and P30 animals the significant depression of GABAergic IPSC frequency occurred at 4–5 min of H/H and continued at min 9–10 of H/H, whereas in P5 animals GABAergic IPSC frequency was significantly diminished only at min 9–10 of H/H. The GABAergic IPSC amplitude was not significantly altered by H/H in both P5 (n = 16) and P30 (n = 16) animals. However, H/H diminished GABAergic IPSC amplitude in P20 rats (n = 16). All the changes in GABAergic neurotransmission to CVNs evoked by H/H were reversible. Typical experiments are shown in A, and the summary data are demonstrated in B.