GENERATION AND TRANSMISSION OF RESPIRATORY OSCILLATIONS IN MEDULLARY SLICES - ROLE OF EXCITATORY AMINO-ACIDS

1. The involvement of excitatory amino acid (EAA) receptors in the gen eration of respiratory rhythm and transmission of inspiratory drive to hypoglossal (XII) motoneurons was examined in an in vitro neonatal ra t medullary slice preparation. Slices generated rhythmic inspiratory a ctivity in XII nerves. The role of EAAs in rhythm generation was deter mined 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 vent rolateral medulla (central respiratory group), particularly in the pre -Botzinger complex (pre-BotC). The involvement of EAAs in drive transm ission to XII motoneurons was examined by recording perturbations in X II 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 tran smission circuit. 2. Block of non-N-methyl-D-aspartate (non-NMDA) rece ptors by bath application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQ X) reversibly reduced XII nerve burst frequency and amplitude in a con centration-dependent manner, completely blocking respiratory motor out put 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 rec eptor channels by bath application of (+)-5-methyl-10,11-dihydro-5H-di benzo[a,d] cyclohepten-5,10-iminemaleate (MK-801) did not perturb the frequency or amplitude of motor output. Inhibition of EAA uptake in th e slices by bath application of dihydrokainic acid reversibly increase d 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 unila teral microinjection of CNQX produced a dose-dependent, bilateral redu ction in XII nerve burst amplitude without substantial perturbations o f the frequency of respiratory oscillations. Block of non-NMDA recepto rs within the pre-BotC at sites ventral to amplitude altering sites pr oduced a reduction in frequency and ultimately bilateral block of resp iratory network oscillations. 4. Non-NMDA receptor block within the XI I motor nucleus by unilateral microinjection of CNQX produced a dose-d ependent reduction in ipsilateral XII nerve discharge amplitude withou t 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 inspir atory burst amplitude, whereas activation of AP-4 receptors caused a 3 0% reduction in amplitude. 5. Whole-cell recordings of XII motoneuron synaptic currents and potentials indicated XII motoneurons with respir atory-modulated synaptic inputs receive rhythmic inspiratory drive cur rents 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 cur rent 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 aci d hydrobromide (AMPA), and kainate (Kain)] and NMDA receptor agonists caused membrane depolarization or inward current. The postsynaptic act ions of Quis, AMPA, and Kain were potently and competitively blocked b y local application of CNQX. The motoneuron responses to NMDA were irr eversibly blocked by MK-801. 7. Our results indicate that: 1) respirat ory rhythm generation in the in vitro neonatal rat medullary slice is dependent on endogenously released EAAs acting at non-NMDA receptors w ithin the pre-BotC; 2) mutually excitatory, non-NMDA receptor-mediated synaptic interactions between pre-BotC neuron populations on each sid e of the medulla contribute to maintenance of respiratory rhythm; 3) X II motoneurons possess NMDA and non-NMDA (AMPA and Kain) receptors; an d 4) an EAA-like substance acting primarily at non-NMDA receptors medi ates transmission of inspiratory drive to XII motoneurons.