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. 2002 Jul 1;22(13):5597-605.
doi: 10.1523/JNEUROSCI.22-13-05597.2002.

The nitric oxide synthase inhibitor NG-Nitro-L-arginine increases basal forebrain acetylcholine release during sleep and wakefulness

Jacqueline Vazquez  1 , Ralph Lydic, Helen A Baghdoyan
Affiliations

The nitric oxide synthase inhibitor NG-Nitro-L-arginine increases basal forebrain acetylcholine release during sleep and wakefulness

Jacqueline Vazquez et al. J Neurosci. .

Abstract

Cholinergic neurotransmission in the basal forebrain changes across the sleep/wake cycle, and considerable data show cortical activation by ACh originating from basal forebrain neurons. These findings have stimulated efforts to elucidate molecular modulators of ACh release within the basal forebrain. Basal forebrain cholinergic neurons contain nitric oxide synthase (NOS), the enzyme that produces the gaseous neuromodulator nitric oxide. This study tested the hypothesis that administration of an NOS inhibitor to the basal forebrain would alter basal forebrain ACh release, sleep, and respiratory rate. Seven cats were instrumented for recording sleep and wakefulness and for in vivo microdialysis and microinjection. Compared with Ringer's solution (control), microdialysis delivery of the NOS inhibitor N(G)-nitro-l-arginine (NLA; 10 mm) increased ACh release during wakefulness (33%), non-rapid eye movement (NREM) sleep (70%), and rapid eye movement (REM) sleep (16%). Mean +/- SEM ACh levels (pmol/10 min) during control and NLA dialysis, respectively, were 0.58 +/- 0.03 and 0.77 +/- 0.06 in wakefulness, 0.36 +/- 0.01 and 0.61 +/- 0.06 in NREM sleep, and 0.68 +/- 0.06 and 0.79 +/- 0.09 in REM sleep. Increases in ACh release were not evoked by dialysis delivery of the less active enantiomer N(G)-nitro-d-arginine. Dialysis administration of NLA did not alter respiratory rate. Sleep-dependent changes in basal forebrain ACh release were localized specifically to lateral basal forebrain regions and did not occur in medial basal forebrain sites. Microinjection of NLA into the lateral basal forebrain did not significantly alter the sleep/wake cycle. In contrast to NLA-induced depression of REM sleep and ACh release in the cat pons, the present results demonstrate that NLA increased ACh release in the cat basal forebrain and had no effect on sleep. The different effects of NLA on ACh release in the cat pons and cat basal forebrain may prove relevant for developing compounds that differentially alter cholinergic neurotransmission in specific brain regions.

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Figures

Fig. 1.
Fig. 1.
A, Polygraph recordings used to identify and quantify states of sleep and wakefulness. Eachpanel shows a 60 sec recording obtained from the same cat. B, ACh chromatograph peaks obtained using microdialysis and HPLC/EC. Representative ACh samples collected during Ringer's solution dialysis (Control) and during dialysis with Ringer's solution containing NLA are shown for wakefulness (left), NREM sleep (middle), and REM sleep (right). Note the increase in peak height, indicating an increase in amount of ACh (in picomoles) caused by dialysis administration of NLA. Resp, Respiration;LGB, lateral geniculate body.
Fig. 2.
Fig. 2.
ACh release in the lateral basal forebrain region during one experiment. Each histogram represents the amount of ACh in one 10 min dialysis sample. Six samples (sample numbers 1–6) were collected during dialysis with Ringer's solution to obtain control levels of ACh release during wakefulness (1, 2), NREM sleep (3–5), and REM sleep (6). The dialysis probe then was perfused with Ringer's solution containing NLA and 10 dialysis samples (7–16) were collected during wakefulness (7–9, 13, 16), NREM sleep (10–12, 14), and REM sleep (15).
Fig. 3.
Fig. 3.
Dialysis delivery of NLA to the lateral basal forebrain region significantly increased ACh release. ACh release is plotted as a function of arousal state and drug administration. Data were obtained from 18 experiments using five cats. Two-way ANOVA revealed significant (p < 0.0001) drug and state main effects on ACh release and no significant drug-by-state interaction. The Tukey–Kramer test showed that NLA significantly (*p < 0.05) increased ACh over control levels during wakefulness (n = 73 control dialysis samples and 73 NLA samples) and NREM sleep (n = 109 control and 80 NLA samples). During REM sleep (n = 18 control and 25 NLA samples), the NLA-induced increase in ACh release did not reach statistical significance. Portions of the control microdialysis data have been reported previously (Vazquez and Baghdoyan, 2001).
Fig. 4.
Fig. 4.
Dialysis delivery of NDA to the lateral basal forebrain region had no effect on ACh release. ACh release is plotted as a function of arousal state and drug administration. Data were obtained from 930 min of dialysis during five experiments in four cats. Two-way ANOVA showed no significant drug main effect on ACh release, a significant (p = 0.0145) state main effect, and no significant drug-by-state interaction. ACh release was quantified during wakefulness (n = 19 control dialysis samples and 19 NDA samples), NREM sleep (n= 26 control and 17 NDA samples), and REM sleep (n= 7 control and 6 NDA samples).
Fig. 5.
Fig. 5.
Histological localization of basal forebrain microdialysis sites. A, Line drawing of a cat forebrain adapted from coronal atlas plate A15.6 of Berman and Jones (1982). (Theboxed area is enlarged in B.)B, Coronal section processed for GFAP shows a lesion from one representative dialysis site in the lateral basal forebrain region (right arrowhead) and one representative dialysis site in the medial basal forebrain region (left arrowhead). The lateral dialysis site is located in the SI ∼4 mm from the midline. The medial dialysis site is localized within the DBV ∼1 mm from the midline. AC, Anterior commissure;CA, caudate; IC, internal capsule;LV, lateral ventricle.
Fig. 6.
Fig. 6.
Schematic localization of basal forebrain dialysis sites. Dialysis sites are represented as 2-mm-long, 0.5-mm-widecylinders to indicate the length and diameter of the dialysis probe membrane. Gray cylinders localize 31 lateral basal forebrain dialysis sites in six cats; white cylinders localize seven medial basal forebrain dialysis sites in four cats. Cylinders are shown on coronal atlas plates at 14.5 and 15.6 mm anterior to stereotaxic zero [modified fromBerman and Jones (1982)]. Tick marks on axes indicate 0.5 mm increments. AC, Anterior commissure;CA, caudate; IC, internal capsule;LV, lateral ventricle; OC, optic chiasm.
Fig. 7.
Fig. 7.
State-dependent changes in ACh release are localized to lateral regions of the basal forebrain. ACh release is plotted as a function of the sleep/wake state for lateral and medial basal forebrain regions. There was no significant effect of arousal state on ACh release in medial basal forebrain regions. One-way ANOVA showed that ACh release in lateral sites was state-dependent (p < 0.0001). Lateral site data are based on 3530 min of dialysis, including 133 dialysis samples collected during wakefulness, 187 NREM sleep samples, and 33 REM sleep samples obtained from 31 experiments in six cats. Medial site data are based on 940 min of dialysis, including 37 dialysis samples collected during wakefulness, 53 NREM sleep samples, and four REM sleep samples obtained from seven experiments in four cats. The asteriskindicates a significant difference in ACh release between the lateral and medial basal forebrain regions.
Fig. 8.
Fig. 8.
Histological localization of basal forebrain microinjection sites. A, Cat forebrain, adapted from coronal atlas plate A14.5 of Berman and Jones (1982). (The boxed area is enlarged in B.) B, Coronal section stained with cresyl violet shows a representative microinjection-induced lesion (arrow) in the lateral basal forebrain region. The microinjection site is located in the SI ∼4 mm from the midline. Comparison with Figure 6 confirms that NLA was microinjected into the same lateral basal forebrain region where NLA was administered by microdialysis. AC, Anterior commissure; CA, caudate; IC, internal capsule; LV, lateral ventricle; OC, optic chiasm.
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