Neuropharmacological characterization of basal forebrain cholinergic stimulated cataplexy in narcoleptic canines

Abstract

Basal forebrain (BF) cholinergic regulation of cataplexy was investigated in narcoleptic canines. Specific cholinergic agonists and antagonists, and excitatory or inhibitory amino acid neurotransmitter receptor agonists, were perfused through microdialysis probes implanted bilaterally in the BF of narcoleptic canines. Cataplexy was monitored using the food-elicited cataplexy test (FECT) and recordings of electroencephalogram, electrooculogram, and electromyogram. In narcoleptic canines, carbachol and oxotremorine (10(-5)-10(-3) M), but not McN-A-343 or nicotine (10(-4)-10(-3) M), produced a dose-dependent increase in cataplexy. In addition, N-methyl-d-aspartate (10(-4)-10(-3) M) and kainic acid (10(-5)-10(-4) M) did not have any effects, while muscimol (10(-3) M) produced a weak (P < 0.10) increase in cataplexy. In control canines, carbachol (10(-5)-10(-3) M), but not oxotremorine (10(-4)-10(-3) M), produced muscle atonia after the highest concentration in one of three animals. Carbachol (10(-3) M)-induced cataplexy in narcoleptic canines was blocked by equimolar perfusion with the muscarinic antagonists atropine, gallamine, and 4-DAMP but not pirenzepine. These findings indicate that carbachol-stimulated cataplexy in the BF of narcoleptic canines is mediated by M2, and perhaps M3, muscarinic receptors. The release of acetylcholine in the BF was also examined during FECT and non-FECT behavioral stimulation in narcoleptic and control canines. A significant increase in acetylcholine release was found in both narcoleptic and control BF during FECT stimulation. In contrast, simple motor activity and feeding, approximating that which occurs during an FECT, did not affect acetylcholine release in the BF of narcoleptic canines. These findings indicate that BF acetylcholine release is enhanced during learned emotion/reward associated behaviors in canines.

FIG. 1.

Effect of local perfusion of various cholinergic agonists in the basal forebrain on cataplexy in the narcoleptic canine. Local bilateral perfusion with (a) carbachol (10⁻⁵–10⁻³ M), (b) oxotremorine (10⁻⁵–10⁻³ M), (c) McN-A-343 (10⁻⁴–10⁻³ M), (d) nicotine (10⁻⁴–10⁻³ M), (e) atropine (10⁻³–10⁻² M), and (f) muscimol (10⁻³ M) in narcoleptic canines is shown. Drugs were mixed into artificial cerebrospinal fluid and perfused through microdialysis probes in increasing concentrations over the course of a 2- to 4-hour experiment in a stepwise fashion, eg, none during the first hour, 10⁻⁵ M during the second hour, 10⁻⁴ M during the third hour, and 10⁻³ M during the fourth hour. The mean number of cataplectic attacks and elapsed time for two food-elicited cataplexy tests per test period is shown. For the purpose of figure presentation, status cataplecticus (carbachol atonia) was arbitrarily designated as 15 attacks elapsed over 600 s. Each drug perfusion time point prior to status cataplecticus was compared with the basal time points using a Fisher PLSD post hoc test. *P < 0.05 satisfactory for comparison with either basal time point.

FIG. 1.

Effect of local perfusion of various cholinergic agonists in the basal forebrain on cataplexy in the narcoleptic canine. Local bilateral perfusion with (a) carbachol (10⁻⁵–10⁻³ M), (b) oxotremorine (10⁻⁵–10⁻³ M), (c) McN-A-343 (10⁻⁴–10⁻³ M), (d) nicotine (10⁻⁴–10⁻³ M), (e) atropine (10⁻³–10⁻² M), and (f) muscimol (10⁻³ M) in narcoleptic canines is shown. Drugs were mixed into artificial cerebrospinal fluid and perfused through microdialysis probes in increasing concentrations over the course of a 2- to 4-hour experiment in a stepwise fashion, eg, none during the first hour, 10⁻⁵ M during the second hour, 10⁻⁴ M during the third hour, and 10⁻³ M during the fourth hour. The mean number of cataplectic attacks and elapsed time for two food-elicited cataplexy tests per test period is shown. For the purpose of figure presentation, status cataplecticus (carbachol atonia) was arbitrarily designated as 15 attacks elapsed over 600 s. Each drug perfusion time point prior to status cataplecticus was compared with the basal time points using a Fisher PLSD post hoc test. *P < 0.05 satisfactory for comparison with either basal time point.

FIG. 1.

Effect of local perfusion of various cholinergic agonists in the basal forebrain on cataplexy in the narcoleptic canine. Local bilateral perfusion with (a) carbachol (10⁻⁵–10⁻³ M), (b) oxotremorine (10⁻⁵–10⁻³ M), (c) McN-A-343 (10⁻⁴–10⁻³ M), (d) nicotine (10⁻⁴–10⁻³ M), (e) atropine (10⁻³–10⁻² M), and (f) muscimol (10⁻³ M) in narcoleptic canines is shown. Drugs were mixed into artificial cerebrospinal fluid and perfused through microdialysis probes in increasing concentrations over the course of a 2- to 4-hour experiment in a stepwise fashion, eg, none during the first hour, 10⁻⁵ M during the second hour, 10⁻⁴ M during the third hour, and 10⁻³ M during the fourth hour. The mean number of cataplectic attacks and elapsed time for two food-elicited cataplexy tests per test period is shown. For the purpose of figure presentation, status cataplecticus (carbachol atonia) was arbitrarily designated as 15 attacks elapsed over 600 s. Each drug perfusion time point prior to status cataplecticus was compared with the basal time points using a Fisher PLSD post hoc test. *P < 0.05 satisfactory for comparison with either basal time point.

FIG. 2.

Effect of perfusion in the basal forebrain of narcoleptic canines with bilateral carbachol (Carb.) (10⁻³ M) for 60 min alone or combined with an equal concentration (10⁻³ M) of atropine, gallamine, 4-DAMP, or pirenzepine (each at 10⁻⁴ M) on cataplexy. The mean number of cataplectic attacks (a) and elapsed time (b) for two food-elicited cataplexy tests per test period is shown. For the purpose of figure presentation, status cataplecticus (carbachol atonia) was arbitrarily designated as 15 attacks elapsed over 600 s. Each carbachol plus antagonist time point was compared with the corresponding carbachol alone time point using a Neuman-Kewls post hoc test; a, b, and c indicate P < 0.05 satisfactory for comparison of control with the atropine, gallamine, and 4-DAMP groups, respectively.

FIG. 3.

Control tests on the extracellular levels of acetylcholine in the BF of narcoleptic and control canines measured by in vivo microdialysis. The effects of local perfusion with tetrodotoxine (TTX) (10⁻⁵ M) or artificial cerebrospinal fluid (CSF) without Ca²⁺ are shown. Following a 60-min baseline perfusion period with normal CSF the perfusion medium was switched to one containing TTX or no Ca²⁺ for 40 min and samples were collected every 10 min. The control group was perfused with normal CSF for the entire testing period. The control group represents four bilateral sessions in four animals (two control, two narcoleptic), the CSF no Ca²⁺ group represent five bilateral sessions in five animals (two control, three narcoleptic) and the TTX group represents four bilateral sessions from four animals (two control, two narcoleptic). Experimental and control groups were compared at each time point using Neuman-Kewls post hoc tests; a and b indicate P < 0.05 satisfactory for comparison of control with the TTX and CSF no Ca²⁺ groups, respectively.

FIG. 4.

Effects of FECT and control behavioral tests on the extracellular levels of acetylcholine in the basal forebrain of narcoleptic and control canines measured by in vivo microdialysis. In (a) FECT stimulation in narcoleptic canines represents three bilateral sessions in two animals and two bilateral sessions in three animals and in control animals represents two bilateral sessions in three animals. In (b) behavioral stimulation studies were studied on narcoleptic canines only. Motor activity tests represent two bilateral sessions in two animals and one bilateral session in three animals and feeding tests represent two bilateral sessions in one animal and one bilateral session in three animals. During FECT treatment each animal performed two FECT trials per 10 min, during motor activity treatment each animal performed two motor activity trials without having cataplexy per 10 min and during feeding treatment each animal consumed 10 small bites of wet dog food without having cataplexy per 10 min.

FIG. 5.

Photomicrograph showing the positions of the microdialysis probes in the basal forebrain (BF). The BF sections were stained with cresyl violet after bilateral implantation with dummy cannulae at coordinates: AP 29.0, ML 5.0, and DV 46.0 (from dura) used for the microdialysis probes. The black arrows point to the injection cannulae tracts on the left side of the BF near the anterior preoptic area (A) and in the magnocellular region including the vertical (B) and horizontal (C) limbs of the diagonal band of Broca. In (A) and (B) the most ventral aspects of the implantation tracts on the left, extending approximately 3–4 mm along the dorsoventral axis, are indicated. In (C) the most ventral aspects of older tracts from previous implantations (small arrows) as well as dorsal portions of a dummy cannulae tract (large arrows), both extending approximately 4–5 mm along the dorsoventral axis, are shown. Bars, 2 mm..