1. The involvement of excitatory amino acid (EAA) receptors in the generation of respiratory rhythm and transmission of inspiratory drive to hypoglossal (XII) motoneurons was examined in an in vitro neonatal rat medullary slice preparation. Slices generated rhythmic inspiratory activity in XII nerves. The role of EAAs in rhythm generation was determined by analyzing perturbations of respiratory network activity after bath application of EAA receptor antagonists or local microinjection of antagonists into the main column of respiratory neurons in the ventrolateral medulla (central respiratory group), particularly in the pre-Botzinger complex (pre-BotC). The involvement of EAAs in drive transmission to XII motoneurons was examined by recording perturbations in XII nerve discharge or motoneuron synaptic inputs after microinjection of EAA receptor antagonists into either the XII motor nuclei or sites in the ventrolateral medulla containing interneurons of the drive transmission circuit. 2. Block of non-N-methyl-D-aspartate (non-NMDA) receptors by bath application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) reversibly reduced XII nerve burst frequency and amplitude in a concentration-dependent manner, completely blocking respiratory motor output at concentrations >4 muM. Activation of 2-amino-4-phosphonobutyric acid (AP-4)-sensitive receptors with D,L AP-4 reduced XII nerve burst amplitude by 30% but did not alter burst frequency. Block of NMDA receptor channels by bath application of (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-iminemaleate (MK-801) did not perturb the frequency or amplitude of motor output. Inhibition of EAA uptake in the slices by bath application of dihydrokainic acid reversibly increased the frequency and amplitude of XII motor discharge. 3. Block of non-NMDA receptors at multiple sites along the main column of respiratory neurons in the ventrolateral medulla, including the pre-BotC, by unilateral microinjection of CNQX produced a dose-dependent, bilateral reduction in XII nerve burst amplitude without substantial perturbations of the frequency of respiratory oscillations. Block of non-NMDA receptors within the pre-BotC at sites ventral to amplitude altering sites produced a reduction in frequency and ultimately bilateral block of respiratory network oscillations. 4. Non-NMDA receptor block within the XII motor nucleus by unilateral microinjection of CNQX produced a dose-dependent reduction in ipsilateral XII nerve discharge amplitude without perturbing the frequency of respiratory oscillations. Perturbations of contralateral XII nerve burst amplitude were significantly smaller. NMDA channel block within the XII motor nucleus did not affect inspiratory burst amplitude, whereas activation of AP-4 receptors caused a 30% reduction in amplitude. 5. Whole-cell recordings of XII motoneuron synaptic currents and potentials indicated XII motoneurons with respiratory-modulated synaptic inputs receive rhythmic inspiratory drive currents of 150 +/- 80 pA (mean +/- SD; n = 26) and potentials of 11 +/- 6 mV (n = 25). Exogenous application of CNQX reversibly blocked 90-95% of the rhythmic synaptic inputs. Local application of D,L AP4 reduced inspiratory-modulated synaptic currents by 21 +/- 6%, whereas MK-801 application did not affect inspiratory drive currents or potentials. 6. After blocking action potential-dependent synaptic transmission with tetrodotoxin, local application of L-Glutamate produced an inward current that was reduced to 5% of control by CNQX but was unaffected by D,L AP-4. Local application of non-NMDA receptor agonists [quisqualate (Quis), (R,S)-alpha-amino-3-hydroxy 5 methyl isoxazole-4-propionic acid hydrobromide (AMPA), and kainate (Kain)] and NMDA receptor agonists caused membrane depolarization or inward current. The postsynaptic actions of Quis, AMPA, and Kain were potently and competitively blocked by local application of CNQX. The motoneuron responses to NMDA were irreversibly blocked by MK-801. 7. Our results indicate that: 1) respiratory rhythm generation in the in vitro neonatal rat medullary slice is dependent on endogenously released EAAs acting at non-NMDA receptors within the pre-BotC; 2) mutually excitatory, non-NMDA receptor-mediated synaptic interactions between pre-BotC neuron populations on each side of the medulla contribute to maintenance of respiratory rhythm; 3) XII motoneurons possess NMDA and non-NMDA (AMPA and Kain) receptors; and 4) an EAA-like substance acting primarily at non-NMDA receptors mediates transmission of inspiratory drive to XII motoneurons.
