Abstract
Glutamatergic neurons in the retrotrapezoid nucleus (RTN) function as respiratory chemoreceptors by regulating breathing in response to tissue CO2/H+. The RTN and greater parafacial region may also function as a chemosensing network composed of CO2/H+-sensitive excitatory and inhibitory synaptic interactions. In the context of disease, we showed that loss of inhibitory neural activity in a mouse model of Dravet syndrome disinhibited RTN chemoreceptors and destabilized breathing (Kuo et al., 2019). Despite this, contributions of parafacial inhibitory neurons to control of breathing are unknown, and synaptic properties of RTN neurons have not been characterized. Here, we show the parafacial region contains a limited diversity of inhibitory neurons including somatostatin (Sst)-, parvalbumin (Pvalb)-, and cholecystokinin (Cck)-expressing neurons. Of these, Sst-expressing interneurons appear uniquely inhibited by CO2/H+. We also show RTN chemoreceptors receive inhibitory input that is withdrawn in a CO2/H+-dependent manner, and chemogenetic suppression of Sst+ parafacial neurons, but not Pvalb+ or Cck+ neurons, increases baseline breathing. These results suggest Sst-expressing parafacial neurons contribute to RTN chemoreception and respiratory activity.
Highlights
The retrotrapezoid nucleus (RTN) is an important respiratory control center located in the ventral parafacial region of the medulla oblongata [12, 14]
Consistent with our previous work showing that expression of a Dravet syndrome-associated Scn1a mutation disrupted RTN chemoreception and respiratory activity [26], we found that Scn1a is expressed by all parafacial neurons, inhibitory neurons that showed relatively higher levels of expression compared to glutamatergic neurons (Figure 1-figure supplement 3)
We found the inhibitory effect of CO2/H+ on chemosensitive RTN neurons was retained when purinergic signaling was blocked by bath application of pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS; 100 μM) and 192 8-phenyltheophylline (8-PT; 10 μM) (T7 = 0.1515, p > 0.05), or in the presence of ionotropic receptor blockers including CNQX (10 μM) to block AMPA/kainite receptors, gabazine (10 μM) to block GABAA receptors, and strychnine (2 μM) to block glycine receptors (T7 = 3.100, p = 0.02) (Figure 2-figure supplement 2)
Summary
The retrotrapezoid nucleus (RTN) is an important respiratory control center located in the ventral parafacial region of the medulla oblongata [12, 14]. Previous work showed using a Phox2b-eGFP reporter line that RTN chemoreceptors have numerous presumptive inhibitory (symmetric) and glutamatergic synapses (asymmetric) [28] These results are supported by functional evidence showing in urethane-anesthetized rats that unilateral RTN injection of glutamate receptor blockers (AP5 or CNQX) decreased baseline breathing and the ventilatory response to CO2 [47], whereas unilateral RTN injection of the GABAA receptor blocker bicuculline stimulated baseline breathing and blunted CO2-stimulated respiratory activity [46]. In the context of disease, suppression of parafacial inhibitory neural activity in a mouse model of Dravet syndrome caused a disordered breathing phenotype, and at the cellular level increased baseline activity and CO2/H+ dependent firing of RTN chemoreceptors [25] These results suggest loss of inhibitory neural activity disinhibited RTN chemoreceptors and contributed to breathing problems. The CO2/H+ response profile of Sst+ parafacial neurons, together with their preferential contribution to baseline breathing, suggest these cells are important determinants of resting respiratory drive
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