Substance P-saporin lesion of neurons with NK1 receptors in one chemoreceptor site in rats decreases ventilation and chemosensitivity

Central chemoreceptors that detect increases in CO2 or H+ and stimulate breathing are widely distributed within the brainstem (Loeschcke, 1982; Coates et al. 1993; Nattie, 1998, 1999, 2000; Li et al. 1999; Solomon et al. 2000; Ballantyne & Scheid, 2001; Nattie & Li, 2001, 2002; Richerson et al. 2001). Chemoreceptor sites have been identified, using in vitro and in vivo approaches, in the regions of: (1) the nucleus tractus solitarius (NTS); (2) the locus ceruleus; (3) the midline medullary raphe; (4) the rostral aspect of the ventral respiratory group; (5) the fastigial nucleus of the cerebellum; and (6) the contiguous regions lying just beneath the ventral medullary surface in the retrotrapezoid nucleus (RTN) and the parapyramidal (Ppy) region as well as more caudally. Why are there so many central chemoreceptor sites? Our work has focused on an in vivo model in which we produce a focal region of acidosis in the brainstem of an unanaesthetized rat by microdialysis of a CO2-rich artificial cerebrospinal fluid (aCSF). An increase in breathing that accompanies this focal stimulation indicates the presence of chemoreception at that site. So far we have identified chemoreceptor responses specific to wakefulness at the RTN region (Li et al. 1999), specific to NREM sleep at the medullary raphe (Nattie & Li, 2001), and present in both wakefulness and sleep in the caudal NTS (Nattie & Li, 2002). These stimulation studies indicate that different sites involved in chemoreception may have different physiological roles dependent on the state of arousal (Nattie, 1998, 1999, 2000, 2001). An alternative approach in the study of central chemoreception in vivo is that of focal inhibition or destruction of neurons. Previous attempts at chemoreceptor disruption have included cooling, neurotoxin-induced lesions and muscimol inhibition (Berger & Cooney, 1982; Loeschcke, 1982; Budzinska et al. 1985; Akilesh et al. 1997; Forster et al. 1997; Nattie & Li, 2000). These have been nonspecific with respect to the cell type affected and, for the most part, the anatomical delineation of the affected region has been imprecise. Admittedly, attempts at cell-specific disruption in the study of central chemoreception have been hampered by a lack of information on the cellular nature of the process. We noted that the overall distribution of NK1R-ir in the rat brainstem (Nakaya et al. 1994) displays some similarity with the distribution of central chemoreceptor sites (Nattie, 2000, 2001). This suggested to us that NK1Rs might be a useful marker for neurons important in chemoreception. The availability of cell-specific toxins for neurons with NK1Rs made this suggestion of practical utility (Wiley & Lappi, 1997). In this paper we test the hypothesis that neurons with NK1Rs located within the RTN/Ppy region of the rostral medulla are involved in central chemoreception. We inject within this region the ribosomal toxin saporin (SAP) conjugated to the natural ligand for the NK1R, substance P (SP; Wiley & Lappi, 1997) and measure breathing in air and in an air mixture containing 7 % CO2 during both wakefulness and sleep in unanaesthetized rats over the subsequent 15 days. This SP-SAP conjugate has proven useful in examination of the role of spinal cord neurons in the response to noxious stimuli (Mantyh et al. 1997) and of pre-Botzinger complex (PBC) neurons in the generation of normal breathing in rats (Gray et al. 1999, 2001; Wang et al. 2002).