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

Abstract

All medullary central chemoreceptor sites contain neurokinin-1 receptor immunoreactivity (NK1R-ir). We ask if NK1R-ir neurons and processes are involved in chemoreception. At one site, the retrotrapezoid nucleus/parapyramidal region (RTN/Ppy), we injected a substance P-saporin conjugate (SP-SAP; 0.1 pmol in 100 nl) to kill NK1R-ir neurons specifically, or SAP alone as a control. We made measurements for 15 days after the injections in two groups of rats. In group 1, with unilateral injections made in the awake state via a pre-implanted guide cannula, we compared responses within rats using initial baseline data. In group 2, with bilateral injections made under anaesthesia at surgery, we compared responses between SP-SAP- and SAP-treated rats. SP-SAP treatment reduced the volume of the RTN/Ppy region that contained NK1R-ir neuronal somata and processes by 44 % (group 1) and by 47 and 40 % on each side, respectively (group 2). Ventilation (.V(E)) and tidal volume (V(T)) were decreased during air breathing in sleep and wakefulness (group 2; P < 0.001; two-way ANOVA) and P(a,CO2) was increased (group 2; P < 0.05; Student's t test). When rats breathed an air mixture containing 7 % CO(2) during sleep and wakefulness, .V(E) and V(T) were lower (groups 1 and 2; P < 0.001; ANOVA) and the Delta.V(E) in air containing 7 % CO(2) compared to air was decreased by 28-30 % (group 1) and 17-22 % (group 2). SP-SAP-treated rats also slept less during air breathing. We conclude that neurons with NK1R-ir somata or processes in the RTN/Ppy region are either chemosensitive or they modulate chemosensitivity. They also provide a tonic drive to breathe and may affect arousal.

Figure 1. Illustration of NK1R-ir in a cross-section of the medulla just caudal to the facial nucleus

A is a low power view with letters B, C and D representing sites magnified in B, C and D. Note extensive labelling of neuronal processes, reticular staining pattern and the small number of neuronal somata. Scale bar in B applies to C and D.

Figure 2. Illustration of RTN/Ppy region of interest

A-D show NK1R-ir in 4 partial cross-sections at rostrocaudal levels marked in millimetres caudal to bregma at lower right. The dotted line in each section outlines our area of interest. Abbreviations: P, pyramid; VII, facial nucleus; Amb, nucleus ambiguus.

Figure 3. Illustration of SP-SAP injection effects on NK1R-ir

Two cross-sections of the medulla are shown at the level of the rostral (A) and mid-facial (B) nucleus taken from control rats. C and D represent cross-sections at the same levels taken from rats with SP-SAP lesions. The area outlined by the dotted line in A and B defines in each section our RTN/Ppy area of interest exhibiting NK1R-ir. This area is much smaller in C and D as a result of the SP-SAP lesion. Scale bar is 1 mm and applies to all panels.

Figure 4. Rostrocaudal extent of lesions

Mean ± s.e.m. (N = 5) values of the area of NK1R-ir in our RTN/Ppy area of interest for every other 50 μm section from 10 to 11.8 mm caudal to bregma in unilateral (group 1; A) and bilateral (group 2; B) experiments. The line marked VII in B shows the rostrocaudal position of the facial nucleus (Paxinos & Watson, 1982). Asterisks shows significance (two-way ANOVA, P < 0.001; Student-Newman-Keuls post hoc test; P < 0.05). In A, results from control uninjected sides are pooled and in B, results from the two sides are averaged to facilitate comparisons.

Figure 5. Specificity of SP-SAP lesions

NK1R-ir (A) and TPOH-ir (B) of adjacent sections taken from a rat injected with SP-SAP. Note loss of NK1R-ir in area of A outlined by dotted line. The same area in B shows TPOH-ir cells. Abbreviations as above.

Figure 6. Effects of unilateral (group 1) control injections of SAP alone on , V_T and f

•, data obtained during wakefulness; ○, data obtained during NREM sleep. Air-breathing data are at the bottom of each panel; 7 % CO₂ data at the top. Mean ± s.e.m. values are shown.

Figure 7. Effects of unilateral (group 1) injections of SP-SAP on , V_T and f

•, data obtained during wakefulness; ○, data obtained during NREM sleep. Air-breathing data are at the bottom of each panel; 7 % CO₂ data at the top. Mean ± s.e.m. values are shown. Asterisks mark significance compared to initial baseline (P < 0.001, one-way repeated measures ANOVA; P < 0.05, Tukey's post hoc test).

Figure 8. Effects of unilateral (group 1) injections of SP-SAP or SAP on the CO₂ response in sleep and wakefulness

The ‘CO₂ response’ expressed as the Δ comparing 7 % CO₂ to air for unilateral (group 1) SP-SAP (top) and SAP control (bottom) during NREM sleep (○) and wakefulness (•). Data are means ± s.e.m. Asterisks mark significance compared to initial baseline (P < 0.001, one-way repeated measures ANOVA; P < 0.05, Tukey's post hoc test).

Figure 9. Effects of bilateral (group 2) injections of SP-SAP or SAP on , V_t and f in wakefulness

Effects during wakefulness of bilateral (group 2) injections of SAP alone (control; ○) and of SP-SAP (•) on , V_T and f. Air-breathing data are at the bottom of each panel; 7 % CO₂ data at the top. Mean ± s.e.m. values are shown. Asterisks mark significant differences between SAP and SP-SAP groups (P < 0.001, two-way ANOVA with treatment and time as factors).

Figure 10. Effects of bilateral (group 2) injections of SP-SAP or SAP on , V_t and f in sleep

Effects in NREM sleep of bilateral (group 2) injections of SAP alone (control; ○) and of SP-SAP (•) on , V_T and f. Air-breathing data are at the bottom of each panel; 7 % CO₂ data at the top. Mean ± s.e.m. values are shown. Asterisks mark significant differences between SAP and SP-SAP groups (P < 0.001, two-way ANOVA with treatment and time as factors).

Figure 11. Effects of bilateral (group 2) injections of SP-SAP or SAP on the CO₂ response in wakefulness and sleep

The ‘CO₂ response’ expressed as the Δ comparing 7 % CO₂ to air for bilateral (group 2) SP-SAP (○) and SAP control (•) during wakefulness (top) and during NREM sleep (bottom). Data are means ± s.e.m. Asterisks mark significance of SAP compared to SP-SAP (P < 0.002 in wakefulness; P < 0.001 in sleep; two-way ANOVA with treatment and time as factors).