Brain Research 884 2000 68–76 www.elsevier.com locate bres
Research report
GABAergic neurons of the cat dorsal raphe nucleus express c-fos during carbachol-induced active sleep
Pablo Torterolo, Jack Yamuy, Sharon Sampogna, Francisco R. Morales, Michael H. Chase
Department of Physiology and the Brain Research Institute , UCLA School of Medicine, Los Angeles, CA 90095, USA
Accepted 22 August 2000
Abstract
Serotonergic neurons of the dorsal raphe nucleus DRN cease firing during active sleep AS, also called rapid-eye-movement sleep. This cessation of electrical activity is believed to play a ‘permissive’ role in the generation of AS. In the present study we explored the
possibility that GABAergic cells in the DRN are involved in the suppression of serotonergic activity during AS. Accordingly, we examined whether immunocytochemically identified GABAergic neurons in the DRN were activated, as indicated by their expression of
c-fos, during carbachol-induced AS AS-carbachol. Three chronically-prepared cats were euthanized after prolonged episodes of AS that was induced by microinjections of carbachol into the nucleus pontis oralis. Another four cats controls were maintained 2 h in quiet
wakefulness before being euthanized. Thereafter, immunocytochemical studies were performed on brainstem sections utilizing antibodies against Fos, GABA and serotonin. When compared with identically prepared tissue from awake cats, the number of Fos1 neurons was
larger in the DRN during AS-carbachol 35.965.6 vs. 13.964.4, P,0.05. Furthermore, a larger number of GABA1 Fos1 neurons were observed during AS-carbachol than during wakefulness 24.863.3 vs. 4.061.0, P,0.001. These GABA1 Fos1 neurons were
distributed asymmetrically with a larger number located ipsilaterally to the site of injection. There was no significant difference between control and experimental animals in the number of non-GABAergic neurons that expressed c-fos in the DRN. We therefore suggest that
activated GABAergic neurons of the DRN are responsible for the inhibition of serotonergic neurons that occurs during natural AS.
2000 Elsevier Science B.V. All rights reserved.
Theme : Neural basis of behavior
Topic : Biological rhythms and sleep
Keywords : Active sleep; REM sleep; Dorsal raphe; Fos; GABA; Serotonin
1. Introduction DRN neurons project to brainstem nuclei implicated in the
genesis and modulation of this state, such as the laterodor- Serotonergic 5-HT neurons comprise one of the most
sal and pedunculo-pontine tegmental nuclei LDT-PPT, widely distributed neurochemical systems in the vertebrate
the nucleus pontis oralis NPO and the locus coeruleus central nervous system [19]. The dorsal raphe nucleus
[17,45,46,51,56]. Furthermore, recordings from presumed DRN contains the largest number of serotonin containing
serotonergic neurons in the DRN of chronic unanesthetized neurons in the entire brain [49,60]. This nucleus has been
animals have revealed that the activity of these cells varies implicated in a number of physiological processes, e.g.
during the states of sleep and wakefulness. They exhibit a nociception, analgesia, thermoregulation, as well as being
slow tonic rate of discharge during wakefulness W and involved in motor and autonomic functions [19,57].
their activity decreases and loses its typical regularity Numerous studies have demonstrated that serotonergic
during quiet sleep QS; these neurons cease firing during neurons of the DRN are also involved in the regulation of
AS [7,16,31,47,54]. Decreases in serotonin levels in sever- sleep and wakefulness [19,21,40]. With respect to AS,
al brain regions during AS are correlated with the decrease in firing rate that also occurs during this state reviewed by
[40]. It has been postulated that this decrease in activity
Corresponding author. Tel.: 11-310-206-3499; fax: 11-310-206-
has a ‘permissive’ effect on neuronal systems that generate
3499. E-mail address
: mchaseucla.edu M.H. Chase.
the behavioral and physiological indices of AS [30,41,43].
0006-8993 00 – see front matter
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P . Torterolo et al. Brain Research 884 2000 68 –76
69
This suppression of cellular discharge was reversed follow- maintained with a gas mixture of halothane 1–3 in
ing the iontophoretic application of GABA antagonists
oxygen. Thereafter, the head was positioned in a stereo-
A
into the DRN during AS [25]. In addition, microdialysis taxic frame and the calvarium was exposed. Stainless steel
experiments have demonstrated that GABA release in the screw electrodes were placed in the frontal and parietal
DRN increases during AS, and that microperfusion of the bones for recording the electroencephalogram EEG and
GABA antagonist picrotoxin decreases the time spent in into the orbital portion of the frontal bone to record eye
AS while GABA agonist muscimol increases the duration movements EOG. Deep bipolar electrodes were im-
of this state [37]. These facts suggest that GABAergic planted in both lateral geniculate nuclei in order to monitor
inhibition may be responsible for the cessation of activity ponto-geniculo-occipital waves PGO. A Winchester plug
in serotonergic neurons that accompanies this state. How- connected to these electrodes and a chronic head-restrain-
ever, the origin of the GABAergic control of DRN neurons ing device were bonded to the calvarium with acrylic
during AS is not clear. cement. A small hole, 5 mm in diameter, which was made
We and others have used the expression of c-fos during in the calvarium overlying the cerebellar cortex, was then
AS induced by microinjection of carbachol into the NPO filled with bone-wax. This hole was subsequently used to
of the cat to examine neural activity during this state provide access for drug administration. At the end of these
[9,33,34,48,62–64]. The AS-carbachol state resembles surgical procedures, an analgesic Buprenex , 0.01 mg
naturally occurring AS polygraphically, electrophysiologi- kg, i.m. was administered. Incision margins were kept
cally and behaviorally [3,12,32,55]. Furthermore, in a clean and a topical antibiotic was administered on a daily
similar model, carbachol microinjection into the NPO was basis. After the animals had recovered from these surgical
found to produce a cessation of firing of serotonergic procedures, they were adapted to the head-restraining
neurons in the DRN, which is similar to that which occurs device for 2 weeks.
during natural AS [50]. Using the AS-carbachol model, we previously demonstrated that the DRN has a larger number
2.3. Experimental procedures of c-fos-expressing neurons during AS-carbachol than
during wakefulness and that the majority of these neurons In the experimental animals, carbachol 0.8 mg in 0.2 ml
are not serotonergic [63]. of saline was microinjected into the NPO AP 22 to 23,
In the present study, using a double-labeling procedure L 1 to 2, H 23.5 to 25, according to Berman’s atlas [6]
for GABA and Fos, we found that the number of activated using a 2-ml Hamilton syringe. After the animals had spent
GABAergic neurons in the DRN increases during AS- approximately 2 h in the AS-carbachol state, they were
carbachol. We therefore hypothesize that these cells may be deeply anesthetized with sodium pentobarbital i.p. 60
GABAergic neurons that inhibit serotonergic neurons of mg kg and perfused for immunocytochemistry see
the DRN during this state. Preliminary results of this study below. Within this time period, the c-fos protein product
have been presented in abstract form [53]. Fos is known to reach optimal concentration [9,34,62].
The control animals underwent the same surgical and habituation procedures as the experimental animals. All of
2. Material and methods them remained for 2 h in a state of quiet wakefulness
