RESPIRATORY RHYTHM GENERATION AND SYNAPTIC INHIBITION OF EXPIRATORY NEURONS IN PRE-BOTZINGER COMPLEX - DIFFERENTIAL ROLES OF GLYCINERGIC AND GABAERGIC NEURAL TRANSMISSION

A key distinction between neural pacemaker and conventional network mo dels for the generation of breathing rhythm in mammals is whether phas ic reciprocal inhibitory interactions between inspiratory and expirato ry neurons are required. In medullary slices from neonatal rats genera ting respiratory-related rhythm, we measured the phasic inhibitory inp uts to expiratory neurons with the use of whole cell patch clamp in th e hypothesized rhythm generation site, the pre-Botzinger complex (pre- BotC). Expiratory neurons, which generate tonic impulse activity durin g the expiratory period, exhibited inhibitory postsynaptic potentials (IPSPs) synchronized to the periodic inspiratory bursts of the hypoglo ssal nerve root (XIIn). Bath application of the glycine receptor antag onist strychnine (STR; 5-10 mu M) reversibly blocked these inspiratory -phase IPSPs, whereas the gamma-aminobutyric acid-A (GABA(A)) receptor antagonist bicuculline (BIC; 10-100 mu M) had no effect on these IPSP s. Replacing the control in vitro bathing solution with a Cl--free sol ution also abolished these IPSPs. Respiratory-related rhythmic activit y was not abolished when inspiratory-phase IPSPs were blocked. The fre quency and strength of XIIn rhythmic activity increased and seizurelik e activity was produced when either STR, BIC, or Cl--free solution was applied. Inspiratory-phase IPSPs were stable after establishment of w hole cell patch conditions (patch pipettes contained 7 mM Cl-). Under voltage clamp, the reversal potential of inspiratory-phase inhibitory postsynaptic currents (IPSCs) was -75 mV. The current-voltage (I-V) cu rve for IPSCs shifted to the right when extracellular Cl- concentratio n was reduced by 50% (70 mM) and the reversal potential was reduced to -60 mV, close to the new Cl- Nernst potential. In tetrodotoxin (0.5 m u M) under voltage clamp (holding potential = -45 mV), local applicati on of glycine (1 mM) over pre-BotC induced an outward current and an i ncrease in membrane conductance in expiratory neurons. The effect was blocked by bath application of STR (0.8-1 mu M). Local application of the GABA(A) receptor agonist 4,5,6,7-tetrahydroisoxazolo [5,4-c] pyrid in-3-ol (THIP, 1 mM) induced an outward current and an increase in mem brane conductance that was blocked by BIC (10-100 mM). Under voltage c lamp (holding potential = -45 mV), we analyzed spontaneous IPSCs durin g expiration in expiratory neurons. Bath application of BIC (10 mu M) reduced the IPSC frequency (from 2.2 to 0.3 per s), whereas the inspir atory-phase IPSCs did not change. Bath application of STR (8-10 mu M) abolished both IPSCs. These results indicate that 1) reciprocal inhibi tion of expiratory neurons is glycinergic and mediated by a glycine-ac tivated Cl- channel that is not required for respiratory related rhyth m generation in neonatal rat medullary slices; 2) endogenous GABA and glycine modulate the excitability of respiratory neurons and affect re spiratory pattern in the slice preparation; 3) both glycine and GABA(A ) receptors are found on pre-BotC expiratory neurons, and these recept ors are sensitive to STR and BIG, respectively; 4) glycine and GABA(A) inhibitory mechanisms play different functional roles in expiratory n eurons: both glycine and GABA(A) receptors modulate neuronal excitabil ity, whereas glycinergic transmission alone is responsible for recipro cal inhibition; and 5) intracellular Cl- concentration in these neonat al expiratory neurons is similar to that in adults.