Inhibitory control of ascending glutamatergic projections to the lamprey respiratory rhythm generator

Highlights

• We found neurons projecting to the pTRG in vagal, glossopharyngeal and facial motor regions as well as in the sensory OLA.

• Projecting neurons are glutamatergic, surrounded by GABA-immunoreactive structures and associated with glycinergic cells.

• Blockade of GABA_A and glycine receptors within these regions increases the respiratory frequency.

• The results indicate that disinhibition can be one important mechanism subserving ascending pathway activation.

• Projecting neurons are possibly involved in sensory-mediated respiratory regulation.

Abstract

Neurons within the vagal motoneuron region of the lamprey have been shown to modulate respiratory activity via ascending excitatory projections to the paratrigeminal respiratory group (pTRG), the proposed respiratory rhythm generator. The present study was performed on in vitro brainstem preparations of the lamprey to provide a characterization of ascending projections within the whole respiratory motoneuron column with regard to the distribution of neurons projecting to the pTRG and related neurochemical markers. Injections of Neurobiotin were performed into the pTRG and the presence of glutamate, GABA and glycine immunoreactivity was investigated by double-labeling experiments. Interestingly, retrogradely labeled neurons were found not only in the vagal region, but also in the facial and glossopharyngeal motoneuron regions. They were also present within the sensory octavolateral area (OLA). The results show for the first time that neurons projecting to the pTRG are immunoreactive for glutamate, surrounded by GABA-immunoreactive structures and associated with the presence of glycinergic cells. Consistently, GABA_A or glycine receptor blockade within the investigated regions increased the respiratory frequency. Furthermore, microinjections of agonists and antagonists of ionotropic glutamate receptors and of the GABA_A receptor agonist muscimol showed that OLA neurons do not contribute to respiratory rhythm generation. The results provide evidence that glutamatergic ascending pathways to the pTRG are subject to a potent inhibitory control and suggest that disinhibition is one important mechanism subserving their function. The general characteristics of inhibitory control involved in rhythmic activities, such as respiration, appear to be highly conserved throughout vertebrate evolution.

Introduction

The isolated brainstem of the adult lamprey, a lower vertebrate that diverged from the main vertebrate line ∼560 million years ago (Kumar and Hedges, 1998), spontaneously generates a stable and regular respiratory neuronal activity in vitro (fictive respiration) for at least 12 h; this activity is very similar to that underlying respiration in intact animals (Rovainen, 1977, Rovainen, 1983, Thompson, 1985, Russell, 1986, Bongianni et al., 1999, Bongianni et al., 2002, Bongianni et al., 2006, Mutolo et al., 2007, Mutolo et al., 2010, Mutolo et al., 2011, Martel et al., 2007, Cinelli et al., 2013, Cinelli et al., 2014). The lateral walls of its central nervous system consist of two longitudinal zones or plates: the ventral primarily motor basal plate and the dorsal primarily sensory alar plate that comprises the octavolateral area (OLA; see e.g. Nieuwenhuys, 1972, Villar-Cerviño et al., 2008). Respiratory motoneurons are located in the facial, glossopharyngeal and, especially, in the vagal nuclei (Rovainen, 1974, Rovainen, 1977, Rovainen, 1979, Guimond et al., 2003), while the putative central neural mechanisms generating the respiratory rhythmic activity are located in the paratrigeminal respiratory group (pTRG), rostral to the trigeminal motor nucleus (Mutolo et al., 2007, Mutolo et al., 2010, Mutolo et al., 2011, Cinelli et al., 2013, Cinelli et al., 2014, Bongianni et al., 2016).

Endogenously released excitatory amino acids, but not GABA and glycine, have been shown to have an essential role in the respiratory rhythmogenesis (Rovainen, 1983, Martel et al., 2007, Bongianni et al., 1999, Bongianni et al., 2006, Bongianni et al., 2016, Cinelli et al., 2013, Cinelli et al., 2014). Only GABAergic influences have a modulatory role at the pTRG level. On the other hand, GABAergic and glycinergic inputs to neurons within the vagal motoneuron region mediate changes in respiratory frequency through ascending excitatory projections to the pTRG (Cinelli et al., 2014). However, an extensive characterization of the facial, glossopharyngeal and vagal motoneuron regions is still lacking with regard to projections to the pTRG and related neurochemical markers such as glutamate, GABA and glycine.

The present study was performed on in vitro brainstem preparations of the lamprey to investigate whether retrogradely labeled neurons could be identified within the whole respiratory motoneuron column by injections of Neurobiotin into the pTRG. The presence of glutamate, GABA and glycine immunoreactivity within the projection areas was ascertained by double-labeling experiments and the responses to microinjections of bicuculline or strychnine into the same regions were investigated.

In addition, since retrogradely labeled neurons were found in the alar plate within the OLA, the distribution of glutamate, GABA and glycine immunoreactivity was investigated and microinjections of bicuculline and strychnine were also performed in this region. An attempt was made to disclose a possible role of this portion of the OLA in respiratory rhythm generation by using microinjections of ionotropic glutamate receptor agonists and antagonists as well as of the GABA_A receptor agonist muscimol.