Role of glutamate and substance P in the amphibian respiratory network during development

Abstract

This study tested the hypothesis that glutamatergic ionotropic (AMPA/kainate) receptors and neurokinin receptors (NKR) are important in the regulation of respiratory motor output during development in the bullfrog. The roles of these receptors were studied with in vitro brainstem preparations from pre-metamorphic tadpoles and post-metamorphic frogs. Brainstems were superfused with an artificial cerebrospinal fluid at 20-22 degrees C containing CNQX, a selective non-NMDA antagonist, or with substance P (SP), an agonist of NKR. Blockade of glutamate receptors with CNQX in both groups caused a reduction of lung burst frequency that was reversibly abolished at 5 microM (P<0.01). CNQX, but not SP, application produced a significant increase (P<0.05) in gill and buccal frequency in tadpoles and frogs, respectively. SP caused a significant increase (P<0.05) in lung burst frequency at 5 microM in both groups. These results suggest that glutamatergic activation of AMPA/kainate receptors is necessary for generation of lung burst activity and that SP is an excitatory neurotransmitter for lung burst frequency generation. Both glutamate and SP provide excitatory input for lung burst generation throughout the aquatic to terrestrial developmental transition in bullfrogs.

Fig. 1

Respiratory-related neural activity recorded from tadpole brainstem preparations. Effects of CNQX (control, 1, 5 μM) followed by washout with aCSF are shown as integrated activity from CN X and XII (A; pre-metamorphic brainstems) and CN VII and CN XII (B; post-metamorphic brainstems). In both preparations, lung burst activity (high amplitude, infrequent bursts) was abolished with CNQX and resumed upon washout. The low amplitude, more frequent gill bursts (pre-metamorphic) and buccal bursts (post-metamorphic) were unaffected by CNQX.

Fig. 2

Summary of effects of CNQX (0, 0.1, 0.5, 1.0, 5.0 μM) on pre-metamorphic (unfilled circles) and post-metamorphic (filled circles) brainstems measured as gill or buccal burst frequency (min⁻¹) (A) and lung burst frequency (B). There were significant increases in gill and buccal burst frequency and a complete abolition of lung bursts in both groups. *P<0.01 compared with control (Dunnett’s test).

Fig. 3

Summary of effects of CNQX (0, 0.1, 0.5, 1.0, 5.0 μM) on gill or buccal burst amplitude (% control) (A) and lung burst amplitude (B) for pre-metamorphic (unfilled circles) and post-metamorphic (filled circles) brainstems. There were significant increases of buccal and lung burst amplitudes compared with control for post-metamorphic brainstems. *P<0.01 compared with control (Dunnett’s test).

Fig. 4

Effects of Substance P on respiratory-related neural activity from tadpole brainstem preparations. Responses of pre-metamorphic (A) and post-metamorphic (B) brainstems are shown for the highest concentration of SP used (5 μM) followed by washout with aCSF. Note the increase in lung burst frequency with SP.

Fig. 5

Summary of effects of SP (control, 0.1, 1, 5 μM) on gill and buccal burst frequency (min⁻¹) (A) and lung burst frequency (B) for pre-metamorphic (unfilled circles) and post-metamorphic (filled circles) brainstems. Gill and buccal burst frequency was unaffected by SP whereas lung burst frequency increased significantly. *P<0.05 compared with control (Dunnett’s test).

Fig. 6

Summary of effects of SP (control, 0.1, 1, 5 μM) on gill and buccal burst amplitude (% control) (A) and lung burst amplitude (B) for pre-metamorphic (unfilled circles) and post-metamorphic (filled circles) brainstems. There were significant increases in buccal and lung burst amplitudes for post-metamorphic brainstems at the highest SP concentration (5 μM) which persisted throughout the washout period. *P<0.05 compared with control (Dunnett’s test).