Focal microdialysis of CO₂ in the perifornical-hypothalamic area increases ventilation during wakefulness but not NREM sleep

Abstract

We investigated whether the perifornical-lateral hypothalamic area (PF-LHA), where the orexin neurons reside, is a central chemoreceptor site by microdialysis of artificial cerebrospinal fluid (aCSF) equilibrated with 25% CO(2) into PF-LHA in conscious rats. This treatment is known to produce a focal tissue acidification like that associated with a 6-7 mm Hg increase in arterial [Formula: see text] . Such focal acidification in the PF-LHA significantly increased ventilation up to 15% compared with microdialysis of normal aCSF equilibrated with 5% CO(2) only in wakefulness but not in sleep in both the dark (P=0.004) and light (P<0.001) phases of the diurnal cycle. This response was predominantly due to a significant increase in respiratory frequency (11%, P<0.001). There were no significant effects on ventilation in the group with probes misplaced outside the PF-LHA. These results suggest that PF-LHA functions as a central chemoreceptor site in the central nervous system in a vigilant state dependent manner with predominant effects in wakefulness.

Figure 1

Anatomical locations of the tip of the microdialysis probe. (A) a photomicrograph of a stained coronal section of the lateral hypothalamus of one representative rat with the tip of microdialysis probe near the fornix indicated in the white rectangle. Scale bar represents 0.5 mm. The schematized anatomical cross sections show in (B) the probe placement within the PF-LHA in the dark (left, N=8) and light (right, N=9) phase groups; and (C) the probe placement outside PF-LHA in the dark (left, N=6) and light (right, N=5) phase groups. The dark filled ovals represent the locations of the microdialysis probe tips. The numbers on the left refer to millimeters caudal to Bregma. Abbreviations: mt, mammillothalamic tract; f, fornix; 3v, third ventricle. The drawings are modified from the atlas of Paxinos & Watson (Paxinos and Watson, 1998).

Figure 2

Illustrative example of one single experiment in one animal with the microdialysis probe located in the PF-LHA. (A) EEG and EMG recordings with breathing tracing before, during and after focal acidification. Scale bar represents 6 min. The open rectangles refer to 6 s. (B) time course of changes in V̇_E before, during and after 25% CO₂ microdialysis in the perfornical hypothalamic area in dark (upper panel) and light (lower panel) phase in two individual rats. Dark circles refer to V̇_E during quiet wakefulness and open circles NREM sleep. The grey rectangles represent the period of microdialysis of 25% CO₂.

Figure 3

V̇_E, f_R and V_T (Mean ± S.E.M.) during CO₂ microdialysis in the groups with the tips located within the PF-LHA in both dark (N = 8, left panel) and light (N=9, right panel) phases. V̇_E and f_R increased significantly during high CO₂/H⁺ (25% CO₂) dialysis compare to normal 5% CO₂ in wakefulness (! ) but not in NREM sleep (! ) during dark and light phase of the diurnal cycle. * P < 0.01, ** P< 0.001, one-way RM ANOVA; Bonferroni post hoc test.

Figure 4

V̇_E, f_R and V_T (Mean ± S.E.M.) during CO₂ microdialysis in the animals with the tips located outside the PF-LHA in both dark (N = 6, left panel) and light (N=4, right panel) phases. V̇_E, f_R and V_T were not significantly changed during high CO₂/H⁺ (25% CO₂) dialysis compare to normal 5% CO₂ in both wakefulness (! ) and NREM sleep (! ) during dark and light phase of the diurnal cycle (P > 0.05, one-way RM ANOVA).