INTRODUCTION

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THE NUCLEUS BASALIS magnocellularis lies in the basal forebrain, and its most caudal part is represented by the nucleus basalis of Meynert (NBM), a cell cluster that contains the cholinergic neurons projecting to the neocortex (14, 37). In the rat, the ventral globus pallidus and the substantia innominata are equivalent to the more precisely delineated primate NBM, and cholinergic projection neurons represent only a fraction of the total cell population, which also consists of GABAergic and peptidergic neurons (8, 34). The NBM is known to be involved in the control and maintenance of arousal and sleep states. Many data suggest that sleep-promoting and arousal-related mechanisms coexist within the NBM (for review, see Refs. 11, 31, 34).

Glutamatergic fibers represent a major component of the input to the NBM. It has been shown that noncholinergic projections to the NBM from the mesopontine cholinergic nuclei, namely the peduncolopontine tegmental nucleus and the laterodorsal tegmental nucleus, use glutamate as a neurotransmitter (12, 24, 27). Moreover, other researchers have described glutamatergic afferents to the NBM from the pyramidal neurons of the prefrontal and piriform cortex (38) as well as from the amygdala (39), the thalamus, and the hypothalamus (5).

Consistent with the presence of glutamatergic afferents, it has been shown that the NBM is rich in glutamate receptors (17, 40), and it has been demonstrated that the application of glutamate strongly excites cortically projecting NBM neurons (13, 18, 33). Taken together, these findings indicate that glutamate may participate in the regulation of neuronal activity in the NBM and eventually affect the state of vigilance.

The purpose of the present study was, therefore, to assess the effects of a glutamatergic manipulation of the rat NBM on sleep and wakefulness. The hypothesis that excitatory amino acid receptors in the NBM play a significant role in the modulation of sleep and wakefulness was tested by in vivo electroencephalographic (EEG) recordings of naturally sleeping-waking rats that were intra-NBM injected with the excitatory amino acid agonists N-methyl-D-aspartic acid (NMDA) and -amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) hydrobromide. The results show the different effects of AMPA and NMDA on sleep and wakefulness.

	MATERIALS AND METHODS

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Animals and recording apparatus. The experiments involving animals were performed in accordance with national and international laws and policies (EEC Council Directive 86/609, 03 L 358, Dec 12, 1987; Guide for the Care and Use of Laboratory Animals, NIH Publication no. 86-23, 1985). Male albino rats (CD-COBS, Charles River, Calco, Italy; 250-300 g) were anesthetized with chloral hydrate (350 mg/kg ip), positioned in a stereotaxic apparatus (David Kopf, Tujunga, CA), and surgically provided with EEG and nuchal electromyographic (EMG) electrodes to monitor states of sleep and wakefulness. A stainless steel guide cannula (length 1.5 cm; outer diameter 0.5 mm) with an indwelling stylet was stereotaxically and unilaterally implanted so that its tip was placed 2 mm above the NBM to minimize cellular damage on the injection site (1.4 mm posterior to bregma, 2.4 mm lateral from the midline, and 5 mm below the surface of the dura mater; coordinates according to Paxinos and Watson, 1986). The EEG and EMG electrodes were connected through insulated leads to an integrated circuit socket attached to the skull with dental acrylic. On completion of the surgical procedures, the rats were left undisturbed in a Plexiglas box within a large sound-attenuated and electrically shielded recording chamber for a minimum of 5 days before being connected to a flexible tether and slip ring. The experiments were initiated after 2-3 days of habituation to the cable. The animals, individually housed on a 12:12-h light-dark cycle (lights on 9:00 AM) at 23 ± 1°C had free access to food and water.

The drugs were administered through a stainless steel needle inserted into the guide cannula and connected via polyethylene tubing to a 0.5-µl Hamilton microsyringe. The needle extended 2 mm past the tip of the guide cannula, its tip resting in the NBM. Polygraphic recordings (obtained by means of a Grass model 7 polygraph) were visually scored in 30-s epochs subdivided into wakefulness (W), slow-wave sleep (SWS), and desynchronized sleep (DS). The following parameters were taken into account: the percentage of duration of each phase (W, SWS, and DS); SWS latency (defined as the interval between the second microinjection and the appearance of the first SWS episode); and DS latency (defined as the interval between the first SWS episode and the appearance of the first DS episode). Finally, because the modifications in the percentage of time spent in DS may only be due to a general modification of the total sleep time, the percentage of time spent in DS versus total sleep (DS/total sleep) was also taken into consideration as this parameter can highlight a specific effect on DS.

On completion of the experiments, an overdose of chloral hydrate was administered, and a dye microinjection (50 nl Pontamine sky blue 1%) was performed in the site of the microinjections; the brains were then removed and fixed. The position of the dye spot was reconstructed on 40-µm thick, frozen, neutral red- or cresyl violet-stained sections.

Drugs. The drugs used included NMDA, specific NMDA-receptor antagonist 2-amino-5-phosphonopentanoic acid (AP5), AMPA hydrobromide, and specific non-NMDA-receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX). All the drugs were purchased from Research Biochemicals International (Natick, MA). The substances were dissolved in 0.9% saline, and the resulting solutions were adjusted to pH 7 with NaOH.

Experimental protocol. A group of 32 rats was used. Four rats were discarded owing to the inappropriate placement of the cannulas. Six animals were used for pilot experiments and 22 for the experimental studies. The experiments were performed at 10:00 AM, during the light phase of the light-dark cycle, when rats are most asleep. A double injection protocol was adopted, each experiment consisting of two local microinjections spaced 10 min apart. This protocol required four conditions: 1) vehicle + vehicle; 2) antagonist + vehicle; 3) vehicle + agonist; and 4) antagonist + agonist. The order of the manipulations varied among animals, with an interval of at least 3 days between them, and none of the animals received the same treatment twice. Polygraphic recordings began immediately after the second injection and continued for 5 h. All the substances were injected unilaterally in a constant volume of 100 nl over a 1-min period. After the microinjections, the needle was left in place for 1 min. During the 5-h experiment, the behavior of the animals was studied with the help of a closed-circuit videocamera.

Statistical analysis. The values obtained for all the parameters considered (the duration of each vigilance state: SWS, DS, and W; the DS and SWS latencies; the DS-to-total sleep ratio) throughout the 5-h recording period were analyzed by two-way ANOVA with condition (vehicle + vehicle, antagonist + vehicle, vehicle + agonist, antagonist + agonist) as the fixed (main) effect and rat as the random effect. If statistically significant differences were detected between conditions, Tukey's post hoc multiple comparison procedure was used to determine which condition contributed to the effect. Values are given as means ± SE.

	RESULTS

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The effects on sleep and wakefulness due to the manipulation of the NMDA receptors within the NBM are described in Fig. 1. The effects of 20 nmol AP5, a specific antagonist for the NMDA receptor subtype, on the sleep-wake parameters were not different from control. NMDA (5 nmol) induced an increase in W: from 23 ± 0.9 to 36.2 ± 1.4 (ANOVA F = 16.9; P = 0.000; Fig. 1A). SWS was decreased by NMDA from 67.6 ± 1.3 to 54.3 ± 1.1 (ANOVA F = 13.6; P = 0.000; Fig.1A). There was no effect on the percentage of time spent in DS: from 9.4 ± 1.2 to 9.5 ± 0.6 (Fig. 1A). NMDA increased SWS latency from 13.3 ± 1.5 to 55 ± 6.2 (ANOVA F = 35.8; P = 0.000; Fig. 1B) but it did not significantly modify DS latency (from 27.7 ± 5.9 to 30.9 ± 4.5; Fig. 1B) as well as the DS-to-total sleep ratio (from 13.8 ± 1.4 to 14.8 ± 0.8; Fig. 1C). The pretreatment with AP5 completely antagonized the effects caused by NMDA on all the parameters considered.

View larger version (36K): [in this window] [in a new window]   Fig. 1.   Histograms showing effects of microinjections of vehicle (n = 9), 2-amino-5-phosphonopentanoic acid (AP5, 20 nmol; n = 9), N-methyl-D-aspartic acid (NMDA, 5 nmol; n = 9), and AP5 + NMDA (n = 8). A: effects on wakefulness (W), slow-wave sleep (SWS), and desynchronized sleep (DS) in percentage amount in 5 h recording. B: effects on latency to onset of first SWS episode from time of injection and on latency to onset of first DS episode from first SWS episode. C: effects on DS percentage amount vs. total sleep. Post hoc comparisons revealed significant effects of NMDA vs. vehicle. ** P < 0.01.

Figure 2 shows the effects on sleep and wakefulness due to the manipulation of AMPA receptors within the NBM. Similar to the results obtained with AP5, DNQX (2 nmol), a specific antagonist for the non-NMDA-receptor subtypes, had no effects per se on any of the parameters considered. AMPA (0.4 nmol) increased W: from 23.3 ± 1.8 to 45.1 ± 3.3 (ANOVA F = 5.4; P = 0.025; Fig. 2A) and decreased SWS: from 65.2 ± 1.7 to 49.2 ± 2.7 (ANOVA F = 3.3; P = 0.075; Fig. 2A). Moreover, different from NMDA, AMPA decreased DS: from 11.5 ± 0.6 to 5.7 ± 0.9 (ANOVA F = 4.8; P = 0.032; Fig. 2A). As regards the latencies, the effect of AMPA on SWS latency is very similar to the effect of NMDA: AMPA induced an increase from 15.8 ± 2.1 to 61.9 ± 8 (ANOVA F = 12.3; P = 0.001; Fig. 2B). Furthermore, different from NMDA, AMPA was also able to cause an increase in DS latency: from 23.8 ± 2.8 to 46.1 ± 7.4 (ANOVA F = 2.65; P = 0.110; Fig. 2B) and a decrease in DS-to-total sleep ratio: from 14.9 ± 0.8 to 10.1 ± 1.2 (ANOVA F = 3.19; P = 0.081; Fig. 2C). The pretreatment with DNQX counteracted the effects of AMPA, except for the DS latency.

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Histograms showing effects of microinjections of vehicle (n = 9), 6,7-dinitroquinoxaline-2,3-dione (DNQX, 2 nmol; n = 7), -amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA, 0.4 nmol; n = 9) and DNQX + AMPA (n = 7). A: effects on wakefulness (W), slow-wave sleep (SWS), and desynchronized sleep (DS) in percentage amount in 5 h recording. B: effects on latency to onset of first SWS episode from time of injection and on latency to onset of first DS episode from first SWS episode. C: effects on DS percentage amount vs. total sleep. Post hoc comparisons revealed significant effects of AMPA vs. vehicle. * P < 0.05; ** P < 0.01.

	DISCUSSION

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The results presented in this paper show the effects of the stimulation of NBM excitatory amino acid (EAA) receptors on sleep and wakefulness. Local microinjections of EAA agonists NMDA and AMPA within the NBM increase W and decrease SWS. Moreover, our findings demonstrate that the NBM stimulation of AMPA-subtype receptors can modify DS, which significantly decreases. The sleep and wakefulness modifications induced by NMDA and AMPA are antagonized by the specific NMDA antagonist AP5 and by the specific non-NMDA antagonist DNQX, respectively.

The present study suggests that both NMDA and AMPA receptors are involved in the NBM modulation of sleep and wakefulness. As a matter of fact, NMDA and AMPA cause similar effects on W and SWS, supporting previous conclusions that both NMDA and non-NMDA receptors are involved in the activation of cortically projecting cholinergic neurons (21, 25).

Interestingly enough, the results reported in this paper demonstrate that AMPA (but not NMDA) injections within the NBM cause a decrease in DS. In addition to the well-known pivotal role played by the NBM in SWS modulation, recent data indicate that NBM cholinergic and cholinoceptive mechanisms can exert an influence on DS (3, 16, 19). In a previous study we showed that the decrease in DS produced by carbachol (a cholinergic agonist) microinjections into the rat NBM is blocked by an EAA antagonist preinjection (16). That finding indicates that the effect on DS due to carbachol injections into NBM is mediated by glutamatergic receptors. It is possible to hypothesize that carbachol causes a release from local glutamatergic elements and the subsequent excitation of glutamatergic receptors induces a DS decrease. Moreover, it has already been suggested that cholinergic terminals could directly affect glutamate transmission on presynaptic glutamatergic elements (26). The present results demonstrate that the AMPA-receptor subtype is responsible for the DS decrease.

It is well-known that mesopontine cholinergic nuclei play a key role in DS generation (10, 15). Therefore, it is possible to speculate that if local NBM-injected AMPA activates a projection system inhibiting the mesopontine cholinergic neurons, this inhibition will probably cause a reduction in DS. It has been demonstrated that in rodents the non-NMDA glutamate receptor subtypes are localized on noncholinergic (GABAergic) NBM neurons (17). This finding suggests that GABAergic neurons within the NBM can be affected by AMPA. Therefore, the hypothetical inhibitory projection could be represented by GABAergic cells. GABAergic neurons are numerous in the NBM of the rat (7), and although many of these GABAergic cells are interneurons, some of them are the source of ascending and descending projections (1, 8). The descending GABAergic projections have already been proposed as candidates for the NBM-mediated inhibitory effects on DS (3). However, it has also been shown that the cholinergic neurons in the NBM are more sensitive to a glutamatergic non-NMDA component, whereas the noncholinergic neurons are more sensitive to an NMDA component (21, 28, 35). Furthermore, cholinergic projections from the NBM to the mesopontine cholinergic nuclei have been described (22). Consistent with these data, the hypothetical inhibitory projection may also be represented by a subpopulation of cholinergic neurons.

Nevertheless, the situation is complicated by the presence of other noncholinergic and non-GABAergic neuronal populations. Thanks to in vitro electrophysiological experiments, Fort et al. (6) described at least three different types of noncholinergic neurons in the NBM, and noncholinergic non-GABAergic neurons in the NBM give rise to ascending and descending projections (8, 30).

It is interesting to note that some common drugs that are able to affect sleep and wakefulness, such as ketamine, a dissociative anesthetic, and dextromethorphan, an antitussive drug, have been shown to interfere with EAA transmission (32, 36). This is consistent with the present results and with data recently published by other authors on the role of the glutamatergic system on sleep and wakefulness modulation (2, 9, 19, 20).

We can exclude that the effects on sleep and wakefulness described in this paper are the result of motor activation: the increase in wakefulness observed after EAA agonist administration was not associated with an increase in motor locomotion or with the appearance of behavioral syndromes. Moreover, it has been shown that the areas anterior to the NBM have a role in motor activity (4), but there is no evidence suggesting that the NBM plays a role in motor activity.

EAA agonists are widely used to cause neurodegeneration or neuronal excitation. The doses of AMPA and NMDA used in this study were similar to the doses administered by other authors to cause neuronal excitation without neurodegeneration. Page et al. (21) showed that doses of NMDA and AMPA equivalent to the ones microinjected in the present study exert excitatory, but not neurotoxic, effects in the NBM of the rat. Moreover, another group used doses of NMDA and AMPA similar to ours to excite neurons in the globus pallidus (29), and the amount of NMDA used in our study is also equivalent to the one microinjected in the septum of rats by Puma et al. (23) to cause an increase in theta waves. It has been shown that doses ten times higher than the ones used in these experiments are necessary to cause neuronal degeneration in the NBM (21, 35).

The effects of the very low doses of NMDA and AMPA used in our experiments are receptor mediated, as revealed by the fact that they are counteracted using the specific antagonists AP5 and DNQX, respectively. Furthermore, it should be emphasized that it has been possible to reproduce the effects induced on sleep and wakefulness by the glutamatergic agonist doses used in this study with a second injection performed on the same site of the same animal 1 wk later (pilot experiments not shown), indicating that the glutamate agonist-induced excitation of NBM neurons does not cause a desensitization or degeneration of neuronal cells surrounding the tip of the injection needle.

In conclusion, the present findings indicate that, in addition to already known modulations of sleep and wakefulness by NBM cholinergic or cholinoceptive mechanisms (3, 16, 19), the NBM is a sensitive action site for exogenously administered glutamatergic drugs and the activity of both NMDA and non-NMDA receptors in the NBM may exert a modulatory influence on the projection neurons involved in sleep and wakefulness. The present results represent the first evidence linking two different classes of EAA receptors localized in the NBM to sleep and wakefulness. Furthermore, the different effects of NMDA and non-NMDA agonists on DS parameters strongly suggest a different role for NBM NMDA and non-NMDA receptors in sleep modulation.