A role for the Kolliker-Fuse nucleus in cholinergic modulation of breathing at night during wakefulness and NREM sleep

Abstract

For many years, acetylcholine has been known to contribute to the control of breathing and sleep. To probe further the contributions of cholinergic rostral pontine systems in control of breathing, we designed this study to test the hypothesis that microdialysis (MD) of the muscarinic receptor antagonist atropine into the pontine respiratory group (PRG) would decrease breathing more in animals while awake than while in NREM sleep. In 16 goats, cannulas were bilaterally implanted into rostral pontine tegmental nuclei (n = 3), the lateral (n = 3) or medial (n = 4) parabrachial nuclei, or the Kölliker-Fuse nucleus (KFN; n = 6). After >2 wk of recovery from surgery, the goats were studied during a 45-min period of MD with mock cerebrospinal fluid (mCSF), followed by at least 30 min of recovery and a second 45-min period of MD with atropine. Unilateral and bilateral MD studies were completed during the day and at night. MD of atropine into the KFN at night decreased pulmonary ventilation and breathing frequency and increased inspiratory and expiratory time by 12-14% during both wakefulness and NREM sleep. However, during daytime studies, MD of atropine into the KFN had no effect on these variables. Unilateral and bilateral nighttime MD of atropine into the KFN increased levels of NREM sleep by 63 and 365%, respectively. MD during the day or at night into the other three pontine sites had minimal effects on any variable studied. Finally, compared with MD of mCSF, bilateral MD of atropine decreased levels of acetylcholine and choline in the effluent dialysis fluid. Our data support the concept that the KFN is a significant contributor to cholinergically modulated control of breathing and sleep.

Fig. 1.

Histochemical and immunohistochemical staining for Nissl substance and muscarinic type-2 (M2) receptors provides anatomic characterization of the pontine respiratory group in goats. A: sagittal sections (from a control goat) 4.9 mm lateral to the midline show the location in the rostral, dorsal pons of the lateral (LPBN) and medial parabrachial nuclei (MPBN) and the Kölliker-Fuse nucleus (KFN) relative to the cerebellar peduncle (SCP). B: transverse Nissl- and M2-stained sections from another control goat and a schematic to further illustrate the location of the LPBN, MPBN, and KFN. The superior and inferior colliculus (IC), trigeminal motor nucleus (5), basal pons (BP), facial nucleus (7), and nucleus ambiguus (NA) are labeled for reference. D, dorsal; V, ventral; R, rostral; C, caudal; M, medial; L, lateral.

Fig. 2.

In the rostrocaudal direction of control goats, there are changes in the anatomy of the LPBN, MPBN, and KFN. A: immunohistochemically stained hemisections for the M2 receptors of a control goat and a schematic illustrating the location of the LPBN, MPBN, and KFN at 7 different rostrocaudal distances from the peak (pKFN) number of neurons in the KFN. B: plots of the number of neurons in the LPBN, MPBN, and KFN of control goats at different rostrocaudal distances relative to the pKFN. Among the 3 subnuclei, note the differences in number of neurons rostrocaudally. Filled symbols indicate unilateral neuron counts from individual goats; open symbols indicate the mean of all control goats.

Fig. 3.

Pulmonary ventilation (V̇i) of 4 different goats during microdialysis (MD) of mock cerebrospinal fluid (mCSF) or atropine in studies during the day in the awake state and in studies at night during the awake and NREM sleep states. The MD was in either the rostral pontine tegmental nucleus (RPTN) or in the LPBN, MPBN, or KFN. Note the relatively stable V̇i throughout the protocol for all goats except that for the nighttime studies in the KFN goat, V̇i decreased during atropine MD in both the awake and NREM sleep states. Shaded boxes indicate periods of MD with either mCSF or atropine.

Fig. 4.

Neither unilateral nor bilateral MD of either mCSF or atropine during daytime studies had any significant effect on ventilatory parameters, with the exception of an increase in expiratory time (Te) following unilateral MD of atropine into the LPBN (n = 3). Data were obtained during MD in the RPTN (n = 3), MPBN (n = 4), or KFN (n = 6). f, Breathing frequency; Ti, inspiratory time. Shaded boxes indicate periods of MD with either mCSF or atropine. *P < 0.05, significant differences from control.

Fig. 5.

Unilateral and bilateral MD of atropine during nighttime studies in KFN animals (n = 6) decreased V̇i and f and increased Ti and Te during both awake and NREM sleep states. There were no consistent and sustained changes in any variable during unilateral or bilateral MD of mCSF or atropine into the RPTN (n = 3), LPBN (n = 3), or MPBN (n = 4). Shaded boxes indicate periods of MD with either mCSF or atropine. *P < 0.05, significant differences from control.

Fig. 6.

During the recovery period from bilateral MD of atropine into the KFN (n = 6), there was a significant (P < 0.05) increase in NREM sleep time and an increase the duration of a NREM sleep epoch. During recovery from unilateral MD of either mCSF or atropine into the KFN, there was an increase in the duration of a NREM sleep epoch. During MD of mCSF or atropine into the RPTN (n = 3), LPBN (n = 3), or MPBN (n = 4), there were no significant effects of NREM sleep time or duration of NREM epoch. Shaded boxes indicate periods of MD with either mCSF or atropine. *P < 0.05, significant differences from control.

Fig. 7.

There were no significant (P > 0.05) changes in the level of genioglossus (GG) muscle activity during MD of mCSF or atropine into the LPBN (n = 3), MPBN (n = 4), or KFN (n = 6) during daytime or nighttime studies. Shaded boxes indicate periods of MD with either mCSF or atropine.

Fig. 8.

The coefficient of variation (CV; SD/mean × 100) for daytime GG muscle activity was increased during unilateral and bilateral MD of atropine into the KFN (n = 6). There was no significant effect on the CV of GG activity during MD into the KFN at night, and there were no significant (P > 0.05) effects with MD into the LPBN (n = 3) or MPBN (n = 4) during daytime or nighttime studies. Shaded boxes indicate periods of MD with either mCSF or atropine. *P < 0.05, significant differences from control.