Cuneiform neurons activated during cholinergically induced active sleep in the cat

Abstract

In the present study, we report that the cuneiform (Cun) nucleus, a brainstem structure that before now has not been implicated in sleep processes, exhibits a large number of neurons that express c-fos during carbachol-induced active sleep (AS-carbachol). Compared with control (awake) cats, during AS-carbachol, there was a 671% increase in the number of neurons that expressed c-fos in this structure. Within the Cun nucleus, three immunocytochemically distinct populations of neurons were observed. One group consisted of GABAergic neurons, which predominantly did not express c-fos during AS-carbachol. Two other different populations expressed c-fos during this state. One of the Fos-positive (Fos(+)) populations consisted of a distinct group of nitric oxide synthase (NOS)-NADPH-diaphorase (NADPH-d)-containing neurons; the neurotransmitter of the other Fos(+) population remains unknown. The Cun nucleus did not contain cholinergic, catecholaminergic, serotonergic, or glycinergic neurons. On the basis of neuronal activation during AS-carbachol, as indicated by c-fos expression, we suggest that the Cun nucleus is involved, in an as yet unknown manner, in the physiological expression of active sleep. The finding of a population of NOS-NADPH-d containing neurons, which were activated during AS-carbachol, suggests that nitrergic modulation of their target cell groups is likely to play a role in active sleep-related physiological processes.

Fig. 1.

c-fos expression in neurons of the Cun nucleus in an experimental cat. A, Diagram of a brainstem section of an experimental animal to show the location of the Cun nucleus and surrounding structures. B, The photomicrograph was taken at low magnification (10×) to illustrate the concentration of Fos⁺ nuclei in this nucleus. The histological section in the photomicrograph was processed for both ChAT and Fos immunostaining. Neurons expressing ChAT are those dorsal to the brachium conjunctivum; Fos-labeled nuclei are located dorsal to this population of cells. c-fos-expressing cells were not ChAT-labeled neurons, and ChAT-labeled neurons did not expressc-fos. The fibers of the IV cranial nerve were transversely cut and can be observed in the top left corner of the photomicrograph. C, Higher magnification photomicrograph of the ChAT-labeled cells.D, Higher magnification photomicrograph of the Fos⁺ cells. Scale bars: B, 100 μm;C, D, 60 μm. bc, Brachium conjunctivum; PAG, periaqueductal gray;IVn, trochlear nerve.

Fig. 2.

Examples of Cun nucleus neurons illustrating the morphology of their cell bodies. Photomicrographs in the left column illustrate neurons from a section of an experimental animal that was processed for Fos immunostaining and counterstained by Pyronin Y. Right column, as left column, but with tissue obtained from a control animal. The arrows point to triangular cell bodies. The rest of the cell bodies exhibit a fusiform shape. Note that the cytoplasm surrounding the nucleus of the neurons is very thin. Scale bars, 5 μm.

Fig. 3.

Localization of Fos-labeled neurons of the Cun nucleus at four levels of the cat brainstem. The schematics are from an experimental animal and correspond to coronal sections of the brainstem taken at 540 μm intervals. The most posterior section corresponds approximately to posterior −2.1 (Berman, 1968). Thecircles represent Fos⁺ nuclei; tissue was obtained immediately after a long episode of AS-carbachol and processed for Fos protein immunostaining and counterstained by Pyronin Y. Aq, Aqueduct; bc, brachium conjunctivum; bp, brachium pontis; LLD, dorsal nucleus of the lateral lemniscus; ml, medial lemniscus; P, pyramidal tract; PAG, periaqueductal gray; ll, lateral lemniscus.

Fig. 4.

c-fos expression in control and experimental cats. The bar chart represents the mean of the average number of Fos⁺ neurons per section per cat within the Cun nucleus. These data were obtained from five experimental and six control cats. Mean ± SEM values: control, 12.5 ± 2.0; AS-carbachol, ipsilateral, 96.4 ± 10.5; AS-carbachol, contralateral, 78.2 ± 12.3 (* and +, p < 0.0001).

Fig. 5.

Fos⁺ Cun nucleus neurons did not exhibit glutamate-like immunoreactivity. The histological sections in the photomicrographs were processed for both glutamate and Fos immunostaining. A is a photomicrograph of the Cun nucleus, B is from the inferior colliculus, andC is from mesencephalic trigeminal neurons. Scale bars:A, 50 μm; B, C, 25 μm.

Fig. 6.

Top Left, GABAergic neurons in the Cun nucleus. A is a diagram of one section immunostained for both GABA and Fos. Open circles represent GABA⁺, Fos⁻ neurons;filled circles represent Fos⁺, GABA⁻ neurons; and large circleswith a dot represent double-labeled GABA⁺, Fos⁺ neurons. The histological sections were obtained from an experimental cat and processed for both GABA and Fos immunostaining in B and for GAD and Fos immunostaining in C andD. B and C are examples of putative GABAergic neurons that express c-fos. InD are examples of GAD⁺ neurons that do not express c-fos. Scale bars, 25 μm.bc, Brachium conjunctivum; IV, fourth ventricle; Vt, mesencephalic tract.