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Theme : Dreamsc-fos Expression in Mesopontine Noradrenergic and Cholinergic Neurons of the Cat during Carbachol-Induced Active Sleep: A Double-Labeling StudyJack Yamuy, Sharon Sampogna, Francisco R. Morales and Michael H. Chase A large number of c-fos-expressing neurons in the active sleep-carbachol cats whose neurotransmitter phenotype was not identified suggests that non-catecholaminergic, non-cholinergic neuronal populations in mesopontine regions are involved in the generation and maintenance of active sleep. The lack of increased c-fos expression in catecholaminergic neurons during active sleep-carbachol confirms and extends previous data that indicate that these cells are silent during active sleep-carbachol and naturally-occurring active sleep. The finding that cholinergic neurons of the dorsolateral pons were not activated either during wakefulness or active sleep-carbachol raises questions regarding the synaptic mechanisms of activation of these cells during these behavioral states. Current Claim: Non-cholinergic, non-catecholaminergic mesopontine neurons are involved in the mechanisms of generation and maintenance of active sleep-carbachol and naturally-occurring active sleep.
We and others have reported that c-fos expression can be useful to detect neuronal activation during different behavioral states (Merchant-Nancy et al., 1992; Pompeiano et al., 1992, 1994; Shiromani et al., 1992; Yamuy et al., 1993; Tononi et al., 1995). It has been shown that during the active sleep-like state elicited by the microinjection of a cholinergic agonist, carbachol, into the rostrodorsal pontine tegmentum, cats exhibit a larger number of c-fos-expressing neurons in the dorsolateral regions of the rostral pons than do cats that remain awake (Shiromani et al., 1992; Yamuy et al., 1993). It is possible to determine the neurotransmitter phenotype of these activated neurons by double-labeling techniques such as nuclear immunostaining of the proto-oncogene protein product Fos combined with the immunostaining of neurotransmitters (Yamuy et al., 1995a) and enzymes involved in the synthesis of neurotransmitters (Yamuy et al., 1995b). In the present study we sought to determine the pattern of neuronal c-fos expression in mesopontine regions that are densely populated by catecholaminergic and cholinergic cells during the carbachol-induced active sleep state (AS-carbachol), which has been extensively used as a model of active sleep (George et al., 1964; Baghdoyan et al., 1987, 1989; Morales et al., 1987). The present data reveal the existence of a large number of neurons that expressed c-fos during AS-carbachol whose neurotransmitter phenotype was neither catecholaminergic nor cholinergic. In addition, both control and AS-carbachol cats were found to exhibit a small number of c-fos expressing cholinergic neurons in the mesopontine region, which is an intriguing result that raises questions regarding their function and synaptic control during active sleep and wakefulness. Preliminary data of this work have been previously reported (Yamuy et al., 1995b).
Figure 1 Experimental paradigm Two methods were employed to deliver drugs. In five cats, carbachol (4 µg in 0.25 µl of saline, n = 2) or saline (0.25 µl, n = 3) were administered by microinjection, using a 1 µl Hamilton microsyringe. In seven other animals, carbachol (n = 5) and saline (n = 2) were delivered by microiontophoresis (for a detailed description of this method, see López-Rodríguez et al., 1994; Yamuy et al., 1995a). Briefly, for carbachol and saline microiontophoretic ejections, assemblies of five-barrel glass micropipettes were employed. The tips of the micropipettes were broken to a total diameter of 30-40 µm (10-13 µm tip diameter in each micropipette). Four of these pipettes were filled with either a carbachol solution (200 mM) or saline. One pipette of the drug ejection assembly was filled with 2M NaCl for automatic current balancing. Total currents of 300-500 nA for a period of 3 minutes were used for the microiontophoretic injections of carbachol and saline. Carbachol ejections were repeated 5 to 7 times during each experiment in order to induce and then maintain the AS-carbachol state; the control cats received 5 saline ejections. All control and carbachol injections were located in the pontine tegmentum, within the nucleus pontis oralis (NPO). Recording periods lasted between 1 and 2 hours from the beginning of carbachol or saline administration. This period of time was chosen because the c-fos protein product reaches a peak concentration approximately at one or two hours after adequate stimulation (Dragunow and Faull, 1989; Hughes and Dragunow, 1995). At the end of the recording period, the animals were deeply anesthetized with pentobarbital sodium (45 mg/kg, i.p.) and prepared for perfusion. Perfusion, fixation and immunocytochemistry For the first group of cats, following fixation, the brain was dehydrated, cleared in xylene and embedded in "Paraplast Plus" embedding media. Brainstem sections 7 µm-thick were mounted on SuperFrost Plus slides and treated for Fos immunostaining using a rabbit polyclonal Fos antiserum raised against an in vitro-translated gene product, which was kindly provided by Dr. Dennis Slamon (UCLA School of Medicine). The Fos antibody has been employed in previous studies (Menétrey et al., 1989; Yamuy et al., 1993; Yamuy et al., 1995a) and, in Western blot analyses from brainstem tissue obtained two hours following a stimulation paradigm for c-fos expression, it binds to a protein with a molecular weight which is similar to that of Fos (55 kDa). The sections were incubated overnight in the primary antibody which was diluted (1:10,000) in phosphate buffered saline (PBS) Tween. Fos immunoreactivity was visualized by the avidin-biotin procedure using the anti-rabbit ABC "Elite" kit (Vector Laboratories). The reaction was enhanced by adding 0.6% nickel ammonium sulfate to 0.02% diaminobenzidine tetrahydrochloride (Sigma) and 0.015% hydrogen peroxide in 50 ml of 50 mM Tris-buffered saline (TBS), pH 7.5. Following Fos immunostaining, the paraffin sections were treated for TH or ChAT labeling. Non-specific binding sites were saturated during a 1-hour incubation period in 1.5% normal donkey serum (NDS) in PBS-Tween + 1% BSA. The sections were then incubated overnight in a polyclonal antiserum directed against TH (Pel-Freez Biologicals, Rogers, AR; dilution 1:500) or against ChAT (Chemicon, CA; dilution 1:500 in 1.5% PBS-Tween-20-AZ with 1% BSA). After rinsing in PBS-Tween, the tissue was incubated for 90 minutes in biotinylated donkey anti-goat IgG (Jackson Laboratories, PA) at a dilution of 1:300. The sections were then rinsed in PBS-Tween-20 and treated with the ABC complex (Vector standard Elite kit) at a dilution of 1:300 for 90 minutes. After another rinsing, peroxidase activity was visualized by the DAB method, as previously described (Yamuy et al., 1995a). The brainstems from the second group of animals were kept in a solution of sucrose (30%) in 0.1M phosphate buffer at pH 7.4. Forty-eight to seventy-two hours later, the brainstem was frozen and cut in 15 µm-thick sections using a Reichert-Jung cryostat. Thereafter, the sections were stored in 0.1M PBS containing 0.3% Triton X-100 and 0.1% sodium azide. Brainstem sections were treated first for Fos immunostaining. For this purpose, free-floating sections from selected brainstem levels (see below) were incubated in the polyclonal Fos antiserum at a dilution of 1:40,000. The sections were rinsed 4 times in PBS-Triton X for 30 minutes and then incubated for 90 minutes in biotinylated anti-rabbit IgG diluted 1:1,000. After rinsing in PBS-Triton X, free-floating sections were incubated in the ABC complex for 90 minutes at a dilution of 1:400. After rinsing in PBS-Triton X, the tissue was reacted with 0.6% nickel ammonium sulfate, 0.02% DAB and 0.015% hydrogen peroxide in 50 ml of 50 mM Tris buffer, pH 7.5, for 15-30 minutes. For TH and ChAT immunocytochemistry, free-floating sections were treated similarly to paraffin sections. However, they were reacted with lower concentrations of TH and ChAT antisera (1:5,000 and 1:2,000, respectively). Also, for the free-floating sections, PBS-Triton X-100 was the buffer that was employed with the addition to the primary antibody of 1% BSA, 0.1% AZ and, only for TH, 1.5% NDS; 1% BSA in 1.5% NDS was added to the secondary antibody. Data analysis Cell counts for catecholaminergic cells were performed bilaterally in an area of the rostral pons (at P -3, hatched area of Fig. 1A) circumscribed by a line that joined the peripheral TH-immunoreactive neurons. The following nuclei were included in this analysis: the LC, the lateral portion of the NPO in the LPRF (which encompasses nuclei designated by others [Sakai, 1988] as LCalpha and subcoeruleus), and the medial (M) and lateral (L) parabrachial nuclei (PB). Because the limits between the LPRF and the PBM are not clearly demarcated, the number of cells encountered in these regions were pooled and considered as belonging to the LPRF. Cell counts of cholinergic neurons were performed bilaterally in an area circumscribed by a line that joined the peripheral ChAT-immunoreactive neurons (at P -1, hatched area of Fig. 1B). The nuclei included in the analysis of cholinergic neurons were the LDT and PPT (for review, see Jones, 1991). Cell counts from LDT and PPT were pooled because of the poorly demarcated division between these nuclei. In order to be able to pool the number of labeled neurons from 7 µm-thick paraffin sections with that from 15 µm-thick, free-floating sections, TH and ChAT immunoreactive neurons were counted in a sufficient number of superimposed 7 µm-thick, adjacent sections in order to obtain values that were equivalent to those found in one 15 µm-thick section. Comparable numbers of TH- and ChAT-labeled neurons were found in two 7 µm-thick and in one 15 µm-thick sections, so that immunoreactive neurons were counted from two superimposed, adjacent, 7 µm-thick sections and from one 15 µm-thick section. A number of perikarya or nuclei marked in one 7 µm-thick section were readily recognizable in the adjacent section which allowed for an accurate superimposition of the drawings. In order to estimate cell size, measures were taken from the longest diameter of the neuronal somas that exhibited nuclei. Because the soma diameter of medium to large pontine neurons is greater than the thickness of the brainstem sections (Taber, 1961), the range of values provided should be considered only as approximations. The level of statistical significance between the mean number of immunoreactive neurons in control and AS-carbachol animals was evaluated using the Student's t test. The criterion chosen to discard the null hypothesis was p < 0.05.
The pontine microinjection of carbachol elicited the AS-carbachol state with a latency that ranged from 2 to 4 minutes. AS-carbachol lasted between 55 and 122 minutes. The microinjection of saline did not evoke any behavioral change; these control animals remained awake throughout the experimental sessions. Characteristics of Fos, TH and ChAT immunoreactivity
The schematics of Fig. 3 illustrate, in representative control (A) and AS-carbachol (B) cats, the distribution of TH+ (open circles), Fos+ (filled circles) and double-labeled (dotted circles) within the dorsolateral pons. TH+ cells were concentrated in the LC and the dorso-medial portions of the LPRF; they appeared more loosely distributed in more ventral and lateral areas of the LPRF, the PBM and PBL ( Fig. 3A and B). As is evident from Fig. 3, a larger overall number of Fos+ cells was present in the dorsolateral pons of the AS-carbachol cats than in the control animals. These non-TH+, Fos+ cells in AS-carbachol cats were more abundantly located at the dorsomedial portions of the LPRF. It should also be noted that the number of non-TH+, Fos+ neurons was slightly larger in the LPRF ipsilateral to the injection site. In addition, as depicted in Fig. 3, the number of double-labeled neurons was similar in control and AS-carbachol conditions. Fig. 4A illustrates that the mean number per section of TH+ neurons was similar in the dorsolateral pons of control and AS-carbachol cats (200.0 ± 20.2 vs. 193.3 ± 12.0, respectively, P = 0.6). However, the mean number of non-TH+, Fos+ neurons in the dorsolateral pons was larger in AS-carbachol cats than in control cats. This difference was statistically significant (66.7 ± 7.5 vs. 139.0 ± 11.1, control and AS-carbachol cats, respectively, P < 0.005). In addition, the mean number of double-labeled cells was similar in control cats and AS-carbachol cats (12.3 ± 3.2 vs. 11.5 ± 4.4, respectively). Similar mean percentages of double-labeled cells were found in the dorso-lateral pons of control and AS-carbachol cats (7% ± 2% vs. 6% ± 2%, respectively). The percentage of double-labeled neurons was calculated as an index of the proportion of catecholaminergic cells that expressed c-fos. Cell counts of TH-containing and Fos immunoreactive cells for each of the structures are presented in Table 1. The analysis per structure indicates that AS-carbachol animals exhibited a significantly larger mean number of non-catecholaminergic, Fos+ neurons in the LPRF than did the control cats.
The schematics of Fig. 6 illustrate the distribution of ChAT+ and Fos+ cells in the LDT and PPT of representative control (A) and AS-carbachol (B) cats. The AS-carbachol cats exhibited a larger number of Fos+ neurons than did the control cats. However, in both control and AS-carbachol groups of animals, there were only a few examples of double-labeled cells. In fact, it was exceptional to find any double-labeled ChAT-containing neurons in the large number of sections from different levels of the LDT and PPT. For example, in one control and three AS-carbachol cats there were no double-labeled ChAT+, Fos+ neurons in the LDT and PPT. Another control animal exhibited a cluster of 4 double-labeled neurons in the caudal pole of the LDT adjacent to the site of saline injection; there were also two double-labeled neurons in the LDT at approximately P -2. One of the AS-carbachol cats exhibited seven double-labeled cells, and another exhibited two double-labeled cells in a section that corresponded approximately to P -1. Figs. 7A and B depict one of these exceptional cases of a large-sized, ChAT-containing neuron, which exhibits a heavily Fos-labeled nucleus, from the LDT of an AS-carbachol animal. As illustrated in the bar chart of Fig. 4B, cell counts from the LDT and PPT showed that the mean number of ChAT+ neurons was similar in the dorsolateral pons of control and AS-carbachol cats (232.5 ± 24.5 vs. 247.2 ± 9.6, respectively); however, the mean number of non-cholinergic Fos+ neurons in the dorsolateral pons was significantly larger in AS-carbachol than in control cats (142.2 ± 21.9 vs. 36.5 ± 13.5, respectively, P < 0.05). Because of the paucity of double-labeled cholinergic neurons in both control and AS-carbachol animals, further quantitative analyses were not indicated.
TH and Fos immunoreactivity These data agree with and extend the results of previous electrophysiological studies which indicate that noradrenergic neurons of the LC are virtually silent during AS-carbachol and naturally-occurring active sleep (Hobson et al., 1975; Aston-Jones and Bloom, 1981; Rasmussen et al., 1986; Sakai, 1988, 1991). In these studies, the recorded units were characterized as noradrenergic by indirect evidence, using criteria such as their location, the duration of their action potential (usually more than 2 ms), their slow and regular pattern of discharge, and a cessation of discharge following the systemic administration of clonidine, an alpha2-receptor agonist (Svensson et al., 1975; Rasmussen et al., 1986). The present data demonstrate that the increased number of Fos+ neurons in the dorsolateral pons of AS-carbachol cats are non-catecholaminergic. Most of these non-catecholaminergic, Fos+ neurons were small-sized. The presence of large numbers of non-catecholaminergic neurons in the LC complex of the cat confirms and extends the notion that this region is populated by heterogeneous neuronal types (Léger and Hernandez-Nicaise, 1980). Although there are cholinergic neurons intermingled with noradrenergic neurons at this level of the dorso-lateral pons of the cat (Jones and Beaudet, 1987), these non-catecholaminergic, Fos+ neurons were not ChAT-containing cells (see Results). The dorsolateral pontine tegmentum is populated, in addition to catecholaminergic and cholinergic cells, by neurons that contain diverse putative neurotransmitters and neuromodulators (Romagnano et al., 1991; Sakai, 1991). For example, the existence of GABAergic and a small number of glycinergic neurons in the dorsolateral pontine tegmentum has been reported (Kosaka et al., 1987; Fort et al., 1993). It is possible that a portion of the small-sized, non-catecholaminergic, Fos+ cells are local interneurons (Léger and Hernandez-Nicaise, 1980). Based upon the existence of neurons that contain cholinergic receptors in the dorsolateral pons (Baghdoyan et al., 1994), some of the c-fos-expressing cells in the vicinity of the injection site may have been cholinoceptive and directly activated by carbachol. In this regard, excitatory actions of carbachol on non-noradrenergic cells of the LC complex have been reported (Sakai, 1991). On the other hand, Fos immunoreactive neurons located distant to the injection site were likely trans-synaptically activated following carbachol microapplication. Although it is possible that a portion of the c-fos-expressing neurons may not be involved in AS-carbachol (i.e., false-positive neurons), we suggest that the majority of the non-catecholaminergic, Fos+ neurons in the dorsolateral pons participate in the control of AS-carbachol and active sleep. Neurons in the dorsomedial and lateral pontine reticular formation (LPRF) and peribrachial areas of the pons increase their rate of discharge in conjunction with the occurrence of PGO waves and the autonomic changes that characterize the states of AS-carbachol and active sleep (Katayama et al., 1984; Rasmussen et al., 1986; Sakai, 1988; Shiromani et al, 1992; Yamuy et al., 1993; for review, see Vertes, 1984). In addition, studies that utilized anatomical tracing techniques have shown that areas of the medulla that are involved in the mechanisms of motor suppression of AS-carbachol and active sleep receive projections that originate in the dorsolateral pontine tegmentum (Shiromani et al., 1990). It is therefore likely that a portion of the non-catecholaminergic, Fos+ neurons may be responsible for certain aspects of the state of AS-carbachol.
The first finding appears difficult to reconcile with the concept that cholinergic neurons of the LDT and PPT are responsible for the generation of active sleep (for review, see Steriade and McCarley, 1990; Jones, 1991). Mesopontine cholinergic cells have been implicated in the generation of EEG desynchronization and PGO waves (Hu et al., 1988, 1989; Sakai, 1988; Steriade et al., 1990a, 1990b; Lydic et al., 1991; Shouse and Siegel, 1992; Williams et al., 1994; Leonard and Lydic, 1995) and in the activation of nucleus pontis oralis (NPO) cholinoceptive neurons that are involved in atonia and the autonomic phenomena of active sleep (Sakai et al., 1979, 1981; Chase, 1983; Chase et al., 1989; Chase and Morales, 1990; Yamamoto, 1990a, 1990b; Yamuy et al., 1993). Data from unitary recording studies from the LDT and PPT have indicated that presumptive cholinergic neurons discharge during EEG desynchronization and in conjunction with PGO waves (El Mansari et al., 1989), and in vitro recordings from identified cholinergic cells have disclosed pharmacological and electrophysiological properties that are proposed to be the basis for the production of PGO waves (Leonard and Llinás, 1990; Luebke et al., 1993). Shiromani et al. (1996) have recently reported that, during the state of active sleep induced by carbachol, 11.2% of cholinergic neurons expressed c-fos whereas during control conditions no ChAT-containing neuron expressed c-fos. These authors propose that these c-fos-expressing, cholinergic neurons may play an important role in the active sleep process (ibid.). However, in the present study it was found that AS-carbachol and control cats exhibited a similar but extremely small number of ChAT-containing neurons that also expressed c-fos. It is possible that methodological differences between the studies explain the discordant results, such as the site of injection of carbachol, the specificity of the Fos antibody and/or the post-injection time of analysis. In any event, the important fact remains that in spite of the many functions that have been attributed to mesopontine cholinergic neurons during active sleep, only a minority of these cells (Shiromani et al., 1996) or an insignificant number (in the present report) express c-fos during this state. It was also anticipated that a portion of cholinergic neurons would express c-fos during control conditions because neurons of the LDT and PPT have been implicated in the mechanisms of arousal, attentional processes (Koyama et al., 1994), EEG desynchronization (as a part of the classic "ascending reticular activating system", Shute and Lewis, 1967; for review, see Jones, 1991) and movement (the PPT encompasses the mesencephalic locomotor region; Coles et al., 1989; Lai and Siegel, 1990; Skinner et al., 1990). In fact, recent microdialysis studies have shown that there is an increase of acetylcholine release in the lateral geniculate nuclei both during wakefulness and REM sleep (Williams et al., 1994). Various alternative explanations can be considered vis-à-vis the lack of c-fos expression in cholinergic neurons during AS-carbachol. For example, because carbachol was applied to the NPO, which is "downstream" in the cascade of neuronal stages that lead to the generation of active sleep, the cholinergic mesopontine system may have been bypassed and therefore not activated during AS-carbachol. If this hypothesis is correct, then the states of active sleep and AS-carbachol may differ in that, in the latter, the mesopontine cholinergic system is not activated. However, this notion does not appear to be supported by the study of Leonard and Lydic (1995), who have shown, using microdialysis techniques, that there is an increased release of Ach in the medial pontine reticular formation (MPRF) during the state of carbachol-induced active sleep. Another possibility that should be considered is that cholinergic neurons of the LDT and PPT were indeed activated during AS-carbachol, but that they either lack the ability to express c-fos or that their c-fos expression was not detected by Fos immunocytochemistry (i.e., ChAT-containing cells were "false-negatives"). In this regard, it has been emphasized that the use of c-fos expression as a functional marker must be carefully evaluated (Dragunow and Faull, 1989; Sharp et al., 1989; Yamuy et al., 1993). However, it should be noted that in the present study, although very small in number, a few ChAT+ cells did express c-fos ( Fig. 7A and B). This demonstrates that at least some cholinergic neurons are capable of expressing this immediate early gene at a level which can be clearly detected by our immunocytochemical techniques. In addition, the study of Robertson and Staines (1994) demonstrates that cholinergic neurons in the LDT are capable of expressing c-fos under appropriate conditions. One explanation for the lack of c-fos expression of cholinergic neurons may be that the synaptic drive for their activation during AS-carbachol was not adequate for triggering the intracellular cascade of events that induce the expression of this proto-oncogene in these cells. In order to express c-fos, neurons need to be driven by orthodromic excitatory stimuli which are capable of evoking the influx of calcium into the postsynaptic cell (Dragunow and Faull, 1989; Morgan and Curran, 1991; Hughes and Dragunow, 1995). If, for example, disinhibitory processes are key to the activation of cholinergic neurons, c-fos expression may not be triggered. Based upon the fact that many neurons discharge in bursts of action potentials triggered by calcium-dependent slow depolarizations, calcium entry during repetitive bursts may result in c-fos expression. According to Steriade et al. (1990a, 1990b), the LDT and PPT contain different types of neurons, based upon their discharge patterns across the sleep-wakefulness cycle. The majority (75%) of those that are presumably related to the EEG desynchronization of wakefulness and active sleep exhibit tonic high frequency firing rates during both states and do not show high frequency spike bursts (ibid.). Other LDT-PPT neurons, although capable of bursting (preceding the occurrence of PGO waves), exhibit a high frequency background firing rate (ibid.). It is possible that these neuronal types, which have been proposed to be cholinergic, may not express c-fos under these conditions. Since neurons of the LDT and PPT have been implicated in diverse modes of behavior, other experimental paradigms (alert wakefulness during motor tasks, for example) may be necessary to disclose their capability for c-fos expression. The second major finding is that AS-carbachol cats exhibited a larger number of non-cholinergic, Fos+ neurons than did control cats. The existence of such a large number of non-cholinergic neurons being activated during AS-carbachol or active sleep has not been previously reported. This may be due to the fact that most of these non-cholinergic neurons of the LDT and PPT were small-sized, which may not be surveyed in electrophysiological studies because of a bias towards recording from larger (cholinergic) neurons. A subpopulation of the small, non-cholinergic, Fos+ neurons may act as local interneuronal relays. In favor of this concept, the existence of GABA-containing interneurons in the LDT and PPT has been reported (Kosaka et al, 1987), and Shiromani et al. (1996) have presented preliminary data that indicate that a portion of the LDT-PPT Fos-expressing neurons are GABAergic. It has been proposed that these GABAergic neurons behave as REM-ON, PGO-OFF units, and that the cessation of their activity disinhibits neighboring PGO-ON neurons (Steriade et al., 1990b). Non-cholinergic, Fos immunoreactive neurons in mesopontine reticular regions may contain neuropeptides which have been proposed to act as neuromodulators. Various peptides, including enkephalin, neurotensin, dynorphin, galanin, substance P and members of the natriuretic peptide family have been reported to be present in neurons of this region (Romagnano et al., 1991; Lechner et al., 1993; Langub et al., 1995). Yet another subpopulation of non-cholinergic, Fos+ neurons may be glutamatergic and implicated in the generation of AS-carbachol. In this regard, Sakai and Koyama (1995) have recently demonstrated that REM-ON cells in the NPO could be activated by kainate, which suggests that glutamatergic neurons may also be involved in the generation of active sleep. Moreover, in vitro studies have disclosed that the LDT and PPT contain small, non-cholinergic neurons which exhibit, following membrane hyperpolarization, a low-threshold, calcium-dependent, slow depolarizing potential that triggers bursts of fast action potentials (Leonard and Llinás, 1990). The behavior of these cells resembles that of the PGO-ON neurons classically described in the literature (Sakai, 1988; Steriade et al., 1990b; for review, see Steriade and McCarley, 1990). In the cat, neurons with similar burst activity project to the lateral geniculate nuclei and discharge preceding PGO waves (Steriade et al., 1990b). Accordingly, these cells may constitute a portion of the population of non-cholinergic, Fos+ neurons present in the LDT and PPT that participate in the generation of PGO waves during AS-carbachol and active sleep. This hypothesis is based upon their soma size, the activation of a calcium conductance that is correlated with the occurrence of PGO waves, and the fact that calcium is a known second messenger capable of inducing the expression of c-fos in neurons (for review, see Morgan and Curran, 1991).
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