Mapping of the excitatory, inhibitory, and modulatory afferent projections to the anatomically defined active expiratory oscillator in adult male rats

Abstract

The lateral parafacial region (pF L ; which encompasses the parafacial respiratory group, pFRG) is a conditional oscillator that drives active expiration during periods of high respiratory demand, and increases ventilation through the recruitment of expiratory muscles. The pF L activity is highly modulated, and systematic analysis of its afferent projections is required to understand its connectivity and modulatory control. We combined a viral retrograde tracing approach to map direct brainstem projections to the putative location of pF L , with RNAScope and immunofluorescence to identify the neurochemical phenotype of the projecting neurons. Within the medulla, retrogradely-labeled, glutamatergic, glycinergic and GABAergic neurons were found in the ventral respiratory column (Bötzinger and preBötzinger Complex [preBötC], ventral respiratory group, ventral parafacial region [pF V ] and pF L), nucleus of the solitary tract (NTS), reticular formation (RF), pontine and midbrain vestibular nuclei, and medullary raphe. In the pons and midbrain, retrogradely-labeled neurons of the same phenotypes were found in the Kölliker-Fuse and parabrachial nuclei, periaqueductal gray, pedunculopontine nucleus (PPT) and laterodorsal tegmentum (LDT). We also identified somatostatin-expressing neurons in the preBötC and PHOX2B immunopositive cells in the pF V , NTS, and part of the RF. Surprisingly, we found no catecholaminergic neurons in the NTS, A5 or Locus Coeruleus, no serotoninergic raphe neurons nor any cholinergic neurons in the PPT and LDT that projected to the pF L. Our results indicate that pF L neurons receive extensive excitatory and inhibitory inputs from several respiratory and nonrespiratory related brainstem regions that could contribute to the complex modulation of the conditional pF L oscillator for active expiration.

Differential modulation of active expiration during hypercapnia by the medullary raphe in unanesthetized rats

“…Furthermore, 5-HT neurons can increase the activity of chemosensitive RTN neurons [42,72], which provide excitatory inputs to the pFRG and generate active expiration [75]. In the light of the anatomical [6] and functional [31] evidence we suggested that indirect interactions between serotoninergic medullary raphe and the pFRG (via RTN; [6]) might explain the inhibitory effect on abdominal activity produced by dialysis of 8-OH-DPAT in the medullary raphe.…”

“…The RTN and pFRG neurons are found partially overlapped in the ventromedullary surface, and until recently, it was unclear whether medullary raphe serotonergic neurons connect monosynaptically to pFRG neurons or indirectly via RTN neurons. However, a recent anatomical study provided evidence that serotonergic neurons of the medullary raphe do not project to the pFRG [6]. It is known that administration of exogenous 5-HT in the pFRG/ RTN region generates active expiration through a 5-HT 2 receptor-dependent mechanism [31].…”

“…The role of this inhibitory raphe circuitry is still unknown, but we speculate that the excitatory effects on inspiratory and expiratory motor activities produced by the dialysis with muscimol in the medullary raphe could involve this GABAergic population. Anatomical evidence is now available about the sparse projections from GABAergic cells in the medullary raphe region to pFRG [6,64]. Therefore, these inhibitory cells of the medullary raphe could indeed suppress other brain regions that provide excitation to inspiratory and expiratory motor pools [10].…”

“…Therefore, our results suggest that outputs from the medullary raphe have both excitatory and inhibitory effects on respiratory motor output, and that the balance of these differential effects determines the extent of the expression of active expiration. The specific mechanisms of how the inhibitory and excitatory pathways (direct or indirect, via RTN, NTS, and/ or other brain regions [6]) act on the expiratory oscillator, the pFRG, during hypercapnic conditions remains open for further investigation, especially in freely moving animals in different states of vigilance.…”

“…Currently, from the perspective of neural control of breathing, active expiration has received increasing attention, mainly after the identification of a group of neurons in the rostral medullary surface, confined in the parafacial respiratory group (pFRG), that acts as a conditional expiratory oscillator [1,25,26,52]. Accumulating evidence indicates that the pFRG expiratory oscillator (also referred to as the lateral parafacial (pFL); [6,19,20]) is functionally and anatomically distinct from (but coupled to) the inspiratory oscillator in the preBötzinger Complex [19,20]. Contemporary views suggest that the expiratory neurons of the pFRG are synaptically suppressed under resting conditions but become rhythmically active during periods of increased respiratory drive, such as hypercapnia [1,8,75].…”