Abstract
We have proposed that the inspiratory rhythm-generating kernel in the pre-BotC consists of a heterogeneous network of glutamatergic neurons with voltage-dependent pacemaker-like bursting properties. This model is based on our optical imaging [1], electrophysiological [2], and mathematical modeling [3] studies of inspiratory (I) neurons within the pre-BotC of rhythmically active in vitro transverse slice preparations from neonatal rats. For electrophysiological and imaging studies, we have developed novel methods to optically identify I neurons with Ca2+-sensitive dyes combined with IR-DIC imaging for whole-cell current camp (CC) and voltage clamp (VC) analyses of neuronal membrane conductance and synaptic mechanisms. In computational modeling studies we have developed models of single pacemaker neurons and synaptically-coupled networks of these neurons (50–500 cells) with heterogeneous distributions of cellular/network parameters. Model predictions have been tested experimentally from single-cell electrophysiological measurements and from macroscopic recordings of neuron population activity within the pre-BotC [2]. Electrophysiological and optical measurements demonstrate a sub-population of pre-BotC I neurons that exhibit voltage-dependent rhythmic bursting under CC after blockade of non-NMDA gluta-matergic synaptic transmission or after blocking synaptic transmission with Ca2+ channel blockers. The intrinsic bursting frequency of these pacemaker-type cells was a monotonic function of baseline membrane potential, spanning a frequency range of over an order of magnitude (~0.05–1 Hz); the voltage-dependence and frequency range varied for different cells indicating heterogeneity. Under VC with synaptic transmission intact, these pacemaker neurons exhibited glutamatergic synaptic drive currents. Optical imaging and cross-correlation of rhythmic Ca2+ activities of multiple cells demonstrate that the glutamatergic synaptic interactions synchronize bursting within the heterogeneous population, but with a temporal dispersion in neuronal spiking including pre-I spiking patterns [2,3]. Measurements of pre-BotC population activity show network frequency control by changing pre-BotC excitability, like predictions from our network models [3]. We have obtained evidence that a persistent Na+ current (INaP) is the main subthreshold-activating cationic conductance underlying the voltage-dependent pacemaker bursting. We measured Na+ currents in bursting pacemaker and non-bursting pre-BotC I cells. In all cells tested under VC, voltage ramp commands at rates of <100 mV/s inactivated the fast Na+ current and yielded N-shaped IV curves with a negative slope region between -60 and -35 mV. TTX (1 μM) blocked this inward current. The slowest ramp speed tested (3.3 mV/s) failed to fully inactivate the negative slope generating current. The current is therefore a TTX-sensitive INaP. The peak amplitude of INaP ranged from -50 to -100 pA at Em = -20 mV (peak conductance = ~1–2 nS). Boltzmann plots gave half-maximal activation voltage of ~-40 mV and a slope factor of ~5, very similar to values used for INaP in our pacemaker neuron models [3]. In non-pacemaker Icells, the ratio of INaP/Ileak was insufficient to support intrinsic bursting as predicted by our models. Further tests of the role of INaP have been performed using the dynamic clamp to artificially add INaP to non-pacemaker neurons. INaP transformed these neurons into rhythmic bursters with voltage-dependent behavior quantitatively similar to our models and experimental observations. These results generally support our conceptual and computational models for the rhythm-generation kernel as a heterogeneous pacemaker network.
Highlights
To be effective, inspiratory muscles on the left and right sides must contract together
We have found that a prominent gap in the column of ventral respiratory group (VRG) The nucleus tractus solitarii (NTS) relays information from primary related parvalbumin cells [2] likely corresponds to the pBc since visceral receptors to the central nervous system and is critically parvalbumin cells are rare in this zone and never co-localize with involved in the reflex control of autonomic functions
The specific protein(s) necessary for longterm facilitation (LTF) is unknown, we recently found that episodic hypoxia and LTF are associated with elevations in ventral spinal concentrations of brain derived neurotrophic factor (BDNF)
Summary
Inspiratory muscles on the left and right sides must contract together. The left and right halves of the diaphragm are synchronised because a bilateral population of medullary premotor neurones [1] simultaneously excites left and right phrenic motoneurones. Transection studies demonstrate that each side of the brainstem is capable of generating respiratory rhythm independently [2], so that left and right medullary inspiratory neurones must themselves be synchronised. The interconnections and common excitation that accomplish such synchronisation are unknown in rats. The respiratory rhythm of hypoglossal (XII) nerve discharge in transverse medullary slice preparations from neonatal rats is thought to originate in the region of the ventral respiratory group (VRG); generated there by a combination of “pacemaker” neurones [1] and their interactions with other respiratory neurones. Our goal was to discover interconnections between left and right VRG neurones as well as their connections to XII motoneurones
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