Central cholinergic depletion induced by 192 IgG-Saporin alleviates the sedative effects of propofol in rats

L. Pain*,1, H. Jeltsch2, O. Lehmann2, C. Lazarus2, F. Z. Laalou1 and J. C. Cassel2

1GRERCA, U405 INSERM et Service d’Anesthésie, CHU Hautepierre, 1 Avenue Molière, F-67000 Strasbourg, France. 2LN2C, UMR 7521 CNRS/Université Louis Pasteur, 12 rue Goethe, F-67000 Strasbourg, France*Corresponding author

Accepted for publication: July 18, 2000


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We examined the effect of central cholinergic depletion on the sedative potency of propofol in rats. Depletion was produced by intracerebroventricular administration of an immunotoxin specific to cholinergic neurones (192 IgG-Saporin; 2 µg). As a result of this lesion, acetylcholine concentration was reduced by about 40% in the frontoparietal cortex and in the hippocampus but was essentially normal in the striatum and cerebellum. Sedation in rats was assessed as the decrease in locomotor activity. Sedative potency of propofol (30 mg kg–1 i.p.) was reduced by about 50% in rats who received the injection of 192 IgG-Saporin as compared to controls. These results show that a central cholinergic depletion alleviates the sedative effect of propofol, and indicates that basal forebrain cholinergic neurones might mediate part of the sedative/hypnotic effects of propofol.

Br J Anaesth 2000; 85: 869–73

Keywords: anaesthetics i.v., propofol, acetylcholine; age factors; rat


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The cholinergic system is one of the most important modulatory neurotransmitter systems in the brain, and has long been recognized as playing a key role in the regulation of consciousness.1 2 Briefly, the cholinergic system is distributed in a variety of different nuclei of which two groups (basal-forebrain and pedunculopontine) have extensive projections to the cortex and thalamus. Pedunculopontine cholinergic neurones are considered to control rapid eye movement sleep or dreaming, while basal-forebrain cholinergic pathways appear to generate and integrate conscious awareness.

Cellular and molecular studies suggest that cholinergic neurotransmission is a potential mediator of general anaesthetic actions.35 It has been largely suggested that some anaesthetics produce their effects via actions on both muscarinic and nicotinic receptors. In this respect, propofol (2,6-diisopropylphenol), an intravenous general anaesthetic, has been shown to directly act on nicotinic acetylcholine receptors, more precisely on the subtype of nicotinic receptors found in the central nervous system ({alpha}4ß2).68 Propofol also has dose-dependent inhibitory effects on acetylcholine release in the frontal cortex and hippocampus, as shown by in vivo microdialysis in the rat brain.9 10 Though there are some reports of the effects of propofol on cholinergic neurotransmission, to date there is no clinical and/or physiological evidence that an inhibitory effect on cholinergic function may be involved in the sedative/hypnotic effects of propofol.

To what extent cholinergic neurons mediate the hypnotic effects of propofol, is of particular relevance to the effects of anaesthetics in aged patients. There is evidence for an impairment of cholinergic function in normal as well as in neuropathological ageing,11–14 but little is known about any age-related modification of anaesthetic effects in the central nervous system.10 Thus an investigation of the interactions between anaesthetics and cholinergic lesions seems worthwhile, particularly if one considers the effects of anaesthetics may depend upon age.

We therefore wondered to what extent the cholinergic system may be involved in propofol-induced sedative/hypnotic effects. To address this question, we assessed the effect of central cholinergic depletion on the sedative effect of propofol in rats. Cholinergic depletion was performed by intracerebroventricular (ICV) administration of 192 IgG-Saporin, a monoclonal antibody to the p75NGF receptor coupled to the ribosomal toxin saporin. This neurotoxin is selective for cholinergic neurons of the basal forebrain.1519 Using a locomotor activity test, we measured the sedative potency of propofol in lesioned rats as compared to controls. The extent of the cholinergic damage was assessed by measuring acetylcholine concentrations in various regions of the brain.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
All procedures involving animals and their care were conducted in accordance with the institutional guidelines that are in compliance with national (concil directive #87848, October 19, 1987, Ministère de l’Agriculture et de la Forêt, Service Vétérinaire de la Santé et de la Protection Animales; permission #6212 to J-C. Cassel) and international (NIH publication, N°86–23, revised 1985) laws and policies.

Materials
Animals
We used 16 female Long Evans rats (Janvier, France) that weighed 200–210 g. They were housed in a colony room maintained on a 12:12 h dark-light cycle (lights on at 7:00 AM), with food and water provided ad libitum. The colony and testing rooms were temperature controlled (21°C).

Drugs
Propofol (10 mg ml–1, Diprivan; Zeneca, Paris, France) was dissolved in 10% intralipid. Drugs were prepared immediately before use and injected intraperitoneally (i.p.) in a volume of 3 ml kg–1.

Apparatus
Eight identical activity cages (size, 45 x 30 x30 cm) were used. Each cage had an infrared detector (IPR124, Talco, Paris, France) placed behind a Fresnel lens and located at the roof of the cage, which allows monitoring of animal movement in different sections of the cage. The signal was fed into a computer that summed all horizontal movements (one unit representing one crossing from one section to the other) during three consecutive periods of 5 min.

Study design
Rats were randomly allocated to two groups as follows: rats with saporin lesions (SAPO; n=8) and sham-operated controls (SHAM; n=8). During surgery, the rats received ICV infusions of 192 IgG-Saporin to induce a cholinergic depletion (SAPO), or vehicle (SHAM). The sedative effect of propofol (30 mg kg–1, intraperitoneally) was measured using a crossover behavioural experiment that measured locomotor activity. Finally the extent of the cholinergic depletion was assessed neurochemically by determining the concentration of acetylcholine in the frontoparietal cortex, hippocampus, striatum and cerebellum.

Surgery
All surgical procedures19 were conducted under aseptic conditions, using equitesin (composed of pentobarbital, chloral hydrate and alcohols) anaesthesia (3.0 ml kg–1, i.p.). Following a mini-craniotomy, injections into the lateral ventricles were performed stereotaxically using a 2-µl Hamilton syringe at the following co-ordinates (in mm): A: –0.8 (from Bregma), L: ±1.4 (from midline); V: –4.3 (from Bregma), with the incisor bar set at the level of the interaural line. Following injection, the needle was left in situ for 5 min, retracted over 2 mm, and maintained there for another 4 min before complete retraction. Rats with saporin lesions received an ICV injection of 2 µg of the immunotoxin 192 IgG-Saporin (2 µg per rat; 1 µl per lateral ventricle, concentration 1 µg µl–1 of phosphate-buffered saline). In the control group, rats were treated in a similar manner, except that phosphate-buffered saline was injected in place of 192 IgG-Saporin.

Behavioural experiment
Propofol-induced sedation was assessed as follows:20 21 the rat was placed in an activity cage with its horizontal movements being recorded using a detector located at the top of the cage. When placed in such a cage, undrugged rats exhibited a locomotor activity aimed at exploring which decreased progressively over time. Activity counts expressed as metric units explored per 5 min were used as activity scores. The experiment was performed over 2 days. The first day, half the rats of each group were injected intraperitoneally with propofol (30 mg kg–1), the remainder injected with vehicle. All rats were then returned to their home cage. Ten minutes later, each rat was placed in one of the eight experimental cages and tested for 15 min. During the test, locomotor activity was recorded during three consecutive periods of 5 min, abbreviated T1, T2 and T3 hereafter. The second day, the procedure was reversed (vehicle instead of propofol, and vice versa).

Comparison of the activity scores found after the injection of vehicle in rats with saporin lesions with those of sham-operated controls allowed assessment of the effect of the cholinergic lesion. Comparison of the activity scores found after propofol injection with those after vehicle allowed determination ofthe effects of the cholinergic lesion on the sedative potency of propofol. Sedative potency was calculated for each rat as follows: (activity score after vehicle injection minus activity score after propofol injection)/(activity score after vehicle injection plus activity score after propofol injection).

Neurochemical control
To control for the efficiency of the cholinergic lesions, six rats of each group (SHAM or SAPO) were arbitrarily chosen by an experimenter who was unaware of the rats’ activity scores. These were killed by microwave irradiation in order to inactivate brain enzymes such as acetylcholinesterase. Following decapitation, the brain was extracted and dissected on a cold plate in order to separate the striatum, frontoparietal cortex, and hippocampus. The hippocampus was further split into its dorsal and ventral areas. Left and right structures from each rat were pooled, weighed and maintained at –80°C until neurochemical determination.

To determine acetylcholine concentrations by high performance liquid chromatographic analysis, tissue samples were homogenized in 1 N formic acid/acetone (18/8.5, vol./vol.). Formic extracts were then purified by a tetraphenylboron exchange of amines in 3-heptanone, followed by 0.1 N HCl extraction. High performance liquid chromatographic analysis was performed on a C(18)Spherisorb ODS2 reverse phase column (3 µm pore size, 7 mm in diameter, 10 cm long). The mobile phase consisted of 0.05 M KH2PO4, pH=7, containing 600 mg litre–1 of tetramethylammonium chloride and 25 mg litre–1 sodium octane sulphate. The flow rate was 0.8 ml min–1. Acetylcholine was converted into betaine in a post-column reactor with covalently bound acetylcholinesterase and choline oxidase. The resulting hydrogen peroxide was detected using a 50740 ESA cell working electrode at 0.3 V.

Statistical analysis
For behavioural experiments, two dependent variables were used: activity score after vehicle injection and sedative potency of propofol. Statistical analyses of these variables were performed using analysis of variance with repeated measures (between factor: SHAM or SAPO, within factor: time). For neurochemical control, one way analyses of variance as performed on acetylcholine concentration in the different brain structures, namely hippocampus (dorsal and ventral), frontoparietal cortex, striatum and cerebellum. P<0.05 was considered indicative of statistical significance.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Following injection of vehicle alone, the absolute activity scores decreased progressively in both groups during the test (activity scores, mean (SEM); T1: 306 (20) vs. 278 (12); T2: 240 (21) vs. 249 (24); T3: 189 (15) vs. 193 (24), for groups SHAM and SAPO, respectively). Analysis of variance showed a significant effect of the Time (P<0.05), but no effect of Group, SHAM or SAPO (P=0.83).

The sedative potency of propofol (30 mg kg–1 i.p.) was significantly decreased in SAPO, as compared to SHAM rats (Figure 1). The time course of the sedative effect appeared similar in both groups. Analysis of variance showed a significant effect of Group, SAPO or SHAM, but no statistical significant effect of Time nor interaction between Group and Time (Group, P<0.05; Time, P=0.07; GroupxTime, P=0.67).



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Fig 1 Sedative potency of propofol 30 mg kg–1 i.p. (T1: 10–15 min, T2: 15–20 min, T3: 20–25 min, after the injection) in rats who received intraventricular injections of 2 µg of 192 IgG-Saporin (SAPO) or of 2 µl of phosphate-buffered saline solution (SHAM). Sedative potency was expressed as the ratio: (activity score after vehicle injection minus activity score after propofol injection)/(activity score after vehicle injection plus activity score after propofol injection). Results are expressed as mean±SEM.

 
Acetylcholine concentrations were significantly reduced in the frontoparietal cortex and hippocampus for SAPO, as compared to SHAM rats (all P<0.05) (Table 1). Acetylcholine concentrations in the striatum and cerebellum were not significantly altered by 192 IgG-Saporin (striatum, P=0.62; cerebellum, P=0.68).


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Table 1 Mean concentrations of acetylcholine in various brain structures of rats who received intraventricular injections of 2 µg of 192 IgG-Saporin (SAPO) or of 2 µl of a phosphate-buffered saline solution (SHAM). All data are given in ng/mg microwaved tissue. Values in parentheses correspond to SEM. *P<0.05 vs. SHAM
 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In the present study we report for the first time that cholinergic neurotransmission may be involved in the sedative effects of propofol.

To our knowledge, we show for the first time that cholinergic depletion in the basal forebrain alleviates the sedative effect of propofol in rats. Indeed, the activity found after propofol treatment was significantly less in rats which had sustained ICV injection of 192 IgG-Saporin than in controls. Sedative potency of propofol was decreased by about 50% but the time-course of the sedative effect appeared unchanged (Fig. 1). This finding cannot be attributed to the pre-existence of impaired locomotion due to 192 IgG-Saporin, as the activity scores following injection of vehicle were comparable in the experimental and control groups. Evidence that 192 IgG-Saporin induced cholinergic depletion in the basal forebrain is substantiated by reduced acetylcholine concentration in the hippocampus and frontoparietal cortex (Table 1). From previous experiments using intraventricular administration of 192 IgG-Saporin, it is known that the extent of cholinergic depletion depends on the amount of 192 IgG-Saporin injected. Close-to-maximum effects are generally obtained with about 5 µg.22 In our study, the reduction of acetylcholine concentration was about 40%. Importantly for locomotion and movement, the concentration of acetylcholine in the striatum was unchanged, an observation which is in line with previous reports.22 23 Similarly, the concentration of acetylcholine in the cerebellum was not significantly affected by 192 IgG-Saporin.

How might a depletion of basal forebrain cholinergic neurons alleviate the depressant effect of propofol on the central nervous system? Physiological studies have highlighted the role of cholinergic neurotransmission in the maintenance of consciousness.1 2 Depending on the brain area, cholinergic neurotransmission exerts either an excitatory or an inhibitory role on consciousness. For example, small amounts of acetylcholine injected into the tegmental reticular formation cause sleep whereas infusions of acetylcholine in the region of the amygdala, septum or hippocampus stimulate arousal behaviour.24 From our results, it is clear that the basal forebrain cholinergic system participates in propofol-induced sedation although its alteration has no major effect on exploratory activity in our experimental conditions. We suggest that propofol depresses central nervous system excitability by a mechanism that probably involves, at least in part, inhibition of the excitatory effect of the basal forebrain cholinergic system associated with the maintenance of consciousness. However, it is generally accepted that propofol acts as a potent gamma-aminobutyric acid (GABA) agonist. Propofol potentiates the inhibitory actions of the GABAA receptor and its pharmacological mechanisms have been investigated in some detail.2527 Using in vivo microdialysis, Kikuchi et al.9 have shown that the inhibitory effects of propofol on acetylcholine release is mediated by GABAA receptors in the frontal cortex and in the hippocampus in rats. Because propofol acts directly on ligand-gated ion channels of GABAA and neuronal nicotinic receptors, the mechanisms by which the hypnotic effects of propofol occur may involve an inhibition of specific neuronal acetylcholine receptors in addition to an interaction with GABAA receptors implicated in the modulation of the activity of cholinergic neurons.

The interaction between propofol and central cholinergic function is of great interest in investigations of the mechanisms which underlie the age-dependent effects of propofol. Recent experimental studies showed that brain sensitivity to propofol can be modified by ageing.10 28 29 In particular, the anaesthetic potency of propofol appears reduced by ageing, independently of any pharmacokinetic modifications. Larsson and Wahlstrom29 demonstrated that brain concentrations of propofol need to be increased in aged as compared to young rats, in order to obtain the same depressant effect of propofol on electroencephalographic parameters. As previously mentioned, central cholinergic function is impaired by normal ageing.1114 For example, in the aged brain cholinergic markers such as the activity of choline acetyltransferase and the levels of hemicolinium-3 binding are reduced in the medial septum, the hippocampus and the cingulate cortex.13 Similarly, the biosynthesis of nicotinic receptors is reduced by ageing as shown by a 30–50% reduction of mRNA levels in the brain of 29- to 32-month-old rats.12 Because ageing alters cholinergic function and in the light of our present findings, one could hypothesise that the weaker potency of propofol to induce sedative/hypnotic effects in the aged subjects is due to the alteration of cholinergic function.

In summary, our study shows that the sedative effect of propofol is partly mediated by cholinergic mechanisms and can be attenuated by an alteration of cholinergic function in the basal forebrain. This finding points towards a key role of cholinergic mechanisms in the effects of propofol. Moreover, our results support the working hypothesis that the ageing process might reduce the brain sensitivity to propofol due to age-related changes in cholinergic function. To what extent this could apply to the anaesthetic effect of propofol or of other anaesthetics needs further investigation.


    Acknowledgements
 
The authors thank Olivier Bildstein and René Paul for technical assistance. This work was supported by MENRT/IFR37 (projet de soutien des IFR aux sciences du vivant, IFR 37, 1999/2000). F-Z Laalou was supported by Fondation pour la Recherche Médicale.


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