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We examined intrinsic and extrinsic factors affecting phrenic motoneuron (PMN) excitability in neonatal rats. Using an in vitro brainstem-spinal cord en bloc, 127 PMNs were recorded under whole-cell patch-clamp conditions. Inspiratory synaptic drives and passive membrane properties, including whole-cell membrane capacitance (Cm), input resistance (Rn), and time constant (tau), were measured with either voltage- or current-clamp techniques. On the basis of firing behavior during inspiration, two types of PMNs could be distinguished: active (107/127 = 84%) and silent PMNs (20/127 = 16%). Active PMNs always produced multiple spikes during inspiration, while silent PMNs remained silent for most inspiratory cycles. Compared to silent PMNs, active PMNs had significantly higher Rn, inspiratory drive potential, and more depolarized resting membrane potential (RMP). With respect to inspiratory drive current, no significant difference was observed between the two types of PMN. Analysis of action potential waveforms did not show a significant difference between their threshold levels. Our results suggest that in addition to size-related properties, RMP determines the recruitment of PMNs and consequently, of motor units in the diaphragm.

The purpose of this study was to determine if the medullary neurons that provide the primary excitatory drive to phrenic motoneurons (i.e., rostral ventral respiratory group, rVRG) are a source of double synapse formation in the phrenic nucleus after spinal cord hemisection. The axons of rVRG neurons either ipsilateral or contralateral to the hemisection were labeled by injection of a mixture of HRP and WGA-HRP into the rostral ventral respiratory group. Phrenic motoneurons ipsilateral and caudal to the hemisection were labeled by the retrograde transport of HRP. The ultrastructural results indicated that after hemisection, rVRG neurons from both sides of the medulla formed labelled double synapses in the phrenic nucleus.

The role of N-methyl-D-aspartate (NMDA) receptor-channel activation in the production of respiratory pattern was studied by administration of the NMDA receptor-channel blocker (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801, 1-3 mg/kg, i.v.) to anesthetized adult rats. This dose of MK-801 blocked the excitatory effects of NMDA (applied iontophoretically) on brainstem respiratory neurons. The predominant respiratory response to systemic MK-801 administration was an increase in inspiratory duration and a decrease in amplitude of diaphragm electromyogram and phrenic nerve discharge. Effects on inspiratory timing and amplitude were most pronounced when the rats were vagotomized. Significant changes in arterial blood gases and pH after systemic MK-801 administration in spontaneously breathing rats (vagi intact or cut) indicated that ventilation was depressed by NMDA receptor-channel antagonism. Respiratory timing changes in response to systemic MK-801 administration differed between two rat strains studied. Breathing patterns resembling apneusis, i.e., with irregular inspiratory durations prolonged 2- to 30-fold, occurred in 60% of the vagotomized, spontaneously breathing Sprague-Dawley rats and none of the Wistar rats. Thus, the breathing pattern in Sprague-Dawley rats is more sensitive to interference with NMDA-mediated mechanisms. We propose that respiratory pattern generation and transmission of rhythmic respiratory drive are mediated by synergistic activation of NMDA and non-NMDA receptors at brainstem and spinal cord sites.

Patterns of respiratory neuronal discharge in the pre-Bötzinger complex, hypothesized as a brainstem site generating respiratory rhythm, are described in adult cats. Signals were recorded from neurons in the Bötzinger complex, pre-Bötzinger complex and rostral ventral respiratory group (rVRG) of anesthetized adult cats. The pre-Bötzinger complex, located caudal to expiratory-modulated Bötzinger neurons, contained a mix of neurons with inspiratory-modulated, expiratory-modulated, or phase-spanning patterns of impulse activity, in contrast to the more homogenous neuronal distributions characteristic of adjacent Bötzinger and rVRG regions.

High-performance liquid chromatography (HPLC) was used to detect the presence of excitatory amino acids released from bulbospinal axon terminals projecting to cervical spinal respiratory motoneurons during transmission of inspiratory drive in an in vitro neonatal rat brainstem-spinal cord preparation. Measurements were then repeated under paradigms where transmitter release was decreased by either depression of bulbospinal respiratory drive, or by adding DL-2-amino-4-phosphonobutyrate (AP4) to the solution bathing the spinal cord. The amounts of glutamate, but not aspartate, released decreased significantly with depressed brainstem inspiratory drive or the activation of AP4-sensitive receptors within the cervical (C) spinal cord.

The pontomedullary connections of the rostral division of the ventral respiratory group (rVRG), the largest medullary population of inspiratory bulbospinal and propriobulbar neurons, were identified in the rat by retrograde and anterograde tracing techniques. These experiments revealed that: (i) the Kölliker-Fuse nucleus, portions of the medial and lateral parabrachial nuclei, and all levels of the ipsilateral and contralateral VRG complex have dense reciprocal connections with the rVRG; (ii) the lateral paragigantocellular nucleus has reciprocal but less dense connections with rVRG; (iii) portions of the nucleus of the solitary tract have prominent projections to, but weaker inputs from rVRG; (iv) the raphe, magnocellular tegmental field and spinal trigeminal nuclei have minor projections to rVRG and receive only sparse inputs from rVRG, and; (v) the retrotrapezoid nucleus, pontine lateral tegmental field and area postrema each have only efferent projections to rVRG. These findings are consistent with previous studies of pontomedullary connections of rVRG in the cat, and further document the extensive reciprocal connections between principal respiratory groups. These connections are likely to be important for generation of the respiratory pattern, for the coordination of effector responses of the cranial and spinal respiratory motor neurons to afferent stimuli, and coordination of the central respiratory and cardiovascular control systems.