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Failure of a chlordiazepoxide to reproduce the behavioral effects of muscimol administered into the basal forebrain
Behavioural Brain Research, 47 (1992) 202-205 Elsevier Science Publishers B.V.
202 BBR 01267
Failure of a chlordiazepoxide to reproduce the behavioral effects of muscimol administered into the basal forebrain Paul Dudchenko and Martin Sarter Department of Psycholog3', The Ohio State University, Columbus, OH 43210 (USA) (Received 17 July 1991) (Revised version received 6 January 1992) (Accepted 15 January 1992)
Key words: 7-Aminobutyric acid; Chlordiazepoxide; Muscimol; Basal forebrain; Intracranial injection; Conditional discrimination
Bilateral infusion of the GABA~x-receptor agonist muscimol into the basal forebrain was previously found to impair visual conditional discrimination performance in rats. In order to examine whether the GABAergie input into the basal forebrain is active during performance of this task, the benzodiazepine receptor agonist chlordiazepoxide (15, 25, 40 #g/0.5 #l/hemisphere) was bilaterally infused. Surprisingly, chlordiazepoxide did not affect performance. The impact of this result for the understanding of basal forebrain GABAergie functions is discussed.
Though the cholinergic cells of the nucleus basalis (NbM) and substantia innominata (SI) region have been implicated in a range of behavioral functions '~ 1, relatively little attention has been paid to the influence of their afferent control. In exploring the impact of this input, we have recently demonstrated that bilateral administration of the GABAA-receptor agonist muscimol directly into the NbM/SI region impairs the rat's performance of a conditional visual discrimination task 3. Furthermore, physostigmine was found to interact with the effects of muscimol on correct responding. These results have suggested that the interactions between GABAergic and cholinergic neurons in the basal forebrain may indeed be of behavioral significance. In an effort to further assess the functions of GABAergic manipulations in the NbM/SI region, the effects of administration of the benzodiazepine receptor agonist chlordiazepoxide (CDP) into the basal forebrain were tested using the same task and identical procedures as previously described 3. According to conventional models of the GABA-benzodiazepine receptor complex, the benzodiazepine receptor is allosterically coupled with the GABAa-receptor, and benzo-
Correspondence: M. Sarter, The Ohio State University, Department of Psychology, 27 Townshend Hall, Columbus, OH 43210, USA.
diazepine receptor agonists increase the gain of the GABA-gated chloride channel openings 4"5. In the current study, the intracranial administration of chlordiazepoxide (CDP) was assumed to allow the examination of the role of endogenously released GABA in the basal forebrain 's in animals performing a visual conditional discrimination task. It was anticipated that if the GABAergic input to the basal forebrain is either tonically active or activated by performance of the task, C D P would be expected to reproduce the effects of GABAA-receptor stimulation 3. Alternatively, a lack of effect of C D P infused into the basal forebrain would suggest that the GABAergic afferents to the region are not significantly active during the performance of this specific task (alternative possibilities are discussed below). Seven male Sprague-Dawley rats weighing 250350 g at the beginning of the experiment were used. Animals were housed individually in a temperature- and humidity-controlled room, food-deprived to approximately 90~o of their free-feeding weight, and maintained on a 12 h light/dark cycle. Animal training was essentially identical to that previously reported 3. Briefly, animals were initially shaped to bar press for food reinforcement, and then placed in a conditional visual discrimination task. In this task, either a constant or a flickering light (5 Hz) appeared at the central panel light for 10 s, and the animal was
203 required to respond to the left lever if the stimulus was a constant light, or the right lever if the stimulus was a flickering light. Response to the correct lever resulted in delivery of a food pellet and the onset of an inter-trial interval (panel light off) of 5 _+ 2 s, whereas response to the incorrect lever was not reinforced and initiated an inter-trial interval (panel light off) of 15 _+ 5 s. Each daily session consisted of 102 stimulus presentations (51 constant light, 51 flicker light, randomly presented) and lasted maximally 45 min. From each daily session the relative number ofcorrect responses, errors ofomission (number of stimulus presentations where no response was made), response latency, and side bias (Sahgai's ~3 Index Y) were calculated. After the animals acquired the conditional discrimination task to a criterion of 3 days at >75~o correct response animals were anesthetized with pentobarbital (50 mg]kg; i.p.), placed in a stereotaxic instrument (David Kopf, Tijuna, CA), and surgically implanted with bilateral cannulae directed 1 mm above the NbM/SI region of the basal forebrain. These guide cannulae were placed according to the coordinates of Paxinos and Watson~: AP: + 8.2 mm (from interaural line); L: + 3.0 mm; V: 2.8 mm (from interaural zero) and fixed to the skull with dental cement. Dummy cannulae were inserted into the guide cannulae to prevent blockage. Animals were allowed free access to food and water for a postoperative recovery time of 1 week. After postoperative return to baseline (>75~'o correct responding for 3 consecutive days), animals received a series of intracranial injections via a Hamilton mierosyringe attached by polyethylene tubing to an injection needle. The injection needle projected 1 mm beyond the end ofthe guide cannulae. The benzodiazepine receptor agonist chlordiazepoxide (Sigma; 15, 25, 40 llg]0.5 ld]hemisphere dissolved in 0.9~o sterile s a l i n e ) o r the vehicle was administered 15 min before testing in the behavioral task. Subsequent drug administrations were separated by at least one criterion day. Upon completion of the drug regimen, animals were sacrificed and cannulae placement was verified histologically. Data were analyzed with a single factor repeated ANOVA (SPSS Int. B.V., AC Gorinchem, The Netherlands). Infusions of chlordiazepoxide into the region of the substantia innominata did not appear to affect the animal's behavior in the conditional visual discrimination task. Statistically, there were no significant effects on correct responding (FL4; df3,9; P = 0.31), errors of omission (Fo.59; df 3,12; P = 0.632), or response time (Fo.32 ; df 3,9; P = 0.808; see Fig. 1). Index Y, the measure of side bias, was not examined statistically as the high percentage of correct responses in each treatment
condition precluded the possibility of ally significant side bias. The current data demonstrate that infusions of the benzodiazepine receptor agonist ehlordiazepoxide into the basal forebrain region does not affect visual conditional discrimination performance in rats. This finding suggests that the GABAergic input into this area 16 is not tonically active and is not activated by performance in this task. The lack of effect of CDP appears unlikely a result ofpotential technical problems related to the infusion of the drug into the brain. Previous studies that utilized intracranial infusions of benzodiazepine receptor ligands suggest that a relevant range of concentrations was tested in the present experiment, and that a dose of 40 fig/0.5/~l/hemisphere (the highest dose tested herein) may even be a relatively high concentration 6'9. Furthermore, the excellent solubility of CDP does not suggest that the drug diffuses in a way that was radically different from the diffusion of muscimol in our previous study. IF CDP differed from muscimol only in this respect though, one would have expected that the relatively high concentrations of CDP used here would at least be distinguishable from saline and perhaps comparable to lower doses of muscimol. Alternative explanations for the current results are also possible. For example, it may be speculated that the GABAA-receptor in the NbM/SI region are of a population not associated with a benzodiazepine receptor site. However, this appears unlikely as studies using in vitro autoradiography have shown that the NbM/SI region shows a relatively high density ofbenzodiazepine binding sites 16. The current results suggest that the activity of cells of the NbM/SI region is not controlled by GABAergic inhibition. Indeed, other studies have suggested that the GABAergic input to the NbM/SI region is not tonic. For example, Blaker I showed that injection of the GABAA-receptor antagonist bicuculline into the NbM/SI region did not affect cortical acetylcholine (ACh) turnover. Likewise, injection ofpirotoxin (which reduces the gating functions ofthe GABAA-receptor via the associated barbiturate receptor 5) into the NbM/SI region failed to affect cortical ACh turnover ~7. Taken together with the current results, these findings may suggest that the GABAergic input to the NbM/SI region is either not tonically active, or not active during performance of the tasks used in this experiment. In summary, although recent studies have suggested the GABAergic input to the basal forebrain may have a tonically active component 2, the current results appear most consistent with the interpretation that, during performance of a conditional visual discrimi-
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This work was supported in part by Grants from the Ohio Department of Aging and the U.S. Public Health Service (MH 46869-01AI).
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nation task, the GABAergic input to the NbM/SI region is not active. However, support for this hypothesis requires the determination of a behavioral function that can be modulated by infusions of CDP into the basal forebrain, i.e., that is dependent on an increase in endogenously released GABA in the basal forebrain. As we have assumed that performance of well-trained animals of a conditional visual discrimination task might be based on the uncontrolled processing of procedural information8, and as the basal forebrain does not appear as a prime substrate involved in the mediation of habits 7, it may be speculated that tasks that address the controlled processing of more declarative information may correlate with changes in basal forebrain GABAergic activity.
1
0
sal
15
25
40
ehlordiazepoxide ( u g / 0 . 5 u l / h s )
Fig. 1. Effects of bilateral infusions of the bcnzodiazepine receptor agonist chlordiazepoxide on performance of rats in a visual conditional discrimination task. hs, hemisphere; sal, saline.
1 Blakcr, W.D., GABAergic control ofthe cholinergic projections to the frontal cortex is not tonic, Brain Res., 325 (1985) 389-390. 2 Bourdelais, A. and Kalivas, P.W., Amphetamine lowers extracellular GABA concentration in the ventral pallidum, Brain Res., 516 (1990) 132-136. 3 Dudchenko, P. and Sarter, M., GABAergic control of basal forebrain cholinergic neurons and memory, Behav. Braha Res., 42 (1991) 33-41. 4 ttaefely, W.E., Pharmacology of allosteric modulation of GABAA receptors by benzodiazepine receptor ligands. In E.A. Barnard and E. Costa (Eds.), AIlosteric Mo&dation of Amino Acid Receptors: Therapeutic hnplications, Raven Press, New York, 1989, pp. 47-69. 5 tlaefely, W.E., Structure and function of the benzodiazepine receptor, Chhnia, ! i (1987) 389-396. 6 Kataoka, Y., Shibata, K., Gomita, Y. and Ueki, S., The mammillary body is a potential site of antianxiety action of benzodiazepines, Brain Res., 241 (1982) 374-377. 7 Mishkin, M., Malamut, B. and Bachevalier, J., Memories and habits: two neural systems. In G. Lynch, J.L. McGaugh and N.M. Weinberger (Eds.), Neurobiology of Learning and Memory, Guilford Press, New York, 1984, pp. 65-77. 8 Morris, R.G.M., Is the distinction between procedural and declarative memory useful with respect to animal models of amnesia? In G. Lynch, J.L. McGaugh and N.M. Weinberger (Eds.), Neurobiology of Leanthzg and 2~lemory, Guilford Press, New York, 1984, pp. 119-124. 9 Nagy, J., Zambo, K. and Decsi, L., Anti-anxiety action of diazepam after intra-amygdaloid application in the rat, Neurophannacology, 18 (I 979) 573-576. 10 Olton, D.S., Markowska, A., Voytko, M.L, Givens, B., Gorman, L. and Wenk, G., Basal forebrain cholinergic system: a functional analysis. In T.C. Napier, P.W. Kalivas and I. Itanin (Eds.), The Basal Forebrab~:Anatomy to Function, Plenum Press, New York, in press. 11 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1982.
205 12 Pirch, J.H., Ridgon, G.C., Rucker, H. and Turco, K., Basal forebrain modulation of cortical cell activity during conditioning. In T.C. Napier, P.W. Kalivas and I. Hanin (Eds.), The Basal Forebrahz: Anatomy to Function, Plenum Press, New York, in press. 13 Sahgal, A., Some limitations of indices derived from signal detection theory: evaluation of an alternative index for measuring bias in memory tasks, Psychopharmacology, 91 (1987) 517-520. 14 Sarter, M., Retrieval of procedural information: insensitive to changes in activity ofindividual neurotransmitter systems? Psychobiology, 18 (1990) 451-459.
15 Sarter, M., Bruno, J.P. and Dudchenko, P., Activating the damaged basal forebrain cholinergic system: tonic stimulation versus signal amplification, Psychopharmacology, 101 (1990) 1-17. 16 Sarter, M. and Schneider, H.H., High density ofbenzodiazepine binding sites in the substantia innominata of the rat, PhannacoL Biochem. Behav., 30 (1988) 679-682. 17 Wood, P.L. hnd Richard, J., GABAergic regulation of the substantia innominata-cortical cholinergie pathway. Neuropharmacology, 21 (1982) 962-972.
202 BBR 01267
Failure of a chlordiazepoxide to reproduce the behavioral effects of muscimol administered into the basal forebrain Paul Dudchenko and Martin Sarter Department of Psycholog3', The Ohio State University, Columbus, OH 43210 (USA) (Received 17 July 1991) (Revised version received 6 January 1992) (Accepted 15 January 1992)
Key words: 7-Aminobutyric acid; Chlordiazepoxide; Muscimol; Basal forebrain; Intracranial injection; Conditional discrimination
Bilateral infusion of the GABA~x-receptor agonist muscimol into the basal forebrain was previously found to impair visual conditional discrimination performance in rats. In order to examine whether the GABAergie input into the basal forebrain is active during performance of this task, the benzodiazepine receptor agonist chlordiazepoxide (15, 25, 40 #g/0.5 #l/hemisphere) was bilaterally infused. Surprisingly, chlordiazepoxide did not affect performance. The impact of this result for the understanding of basal forebrain GABAergie functions is discussed.
Though the cholinergic cells of the nucleus basalis (NbM) and substantia innominata (SI) region have been implicated in a range of behavioral functions '~ 1, relatively little attention has been paid to the influence of their afferent control. In exploring the impact of this input, we have recently demonstrated that bilateral administration of the GABAA-receptor agonist muscimol directly into the NbM/SI region impairs the rat's performance of a conditional visual discrimination task 3. Furthermore, physostigmine was found to interact with the effects of muscimol on correct responding. These results have suggested that the interactions between GABAergic and cholinergic neurons in the basal forebrain may indeed be of behavioral significance. In an effort to further assess the functions of GABAergic manipulations in the NbM/SI region, the effects of administration of the benzodiazepine receptor agonist chlordiazepoxide (CDP) into the basal forebrain were tested using the same task and identical procedures as previously described 3. According to conventional models of the GABA-benzodiazepine receptor complex, the benzodiazepine receptor is allosterically coupled with the GABAa-receptor, and benzo-
Correspondence: M. Sarter, The Ohio State University, Department of Psychology, 27 Townshend Hall, Columbus, OH 43210, USA.
diazepine receptor agonists increase the gain of the GABA-gated chloride channel openings 4"5. In the current study, the intracranial administration of chlordiazepoxide (CDP) was assumed to allow the examination of the role of endogenously released GABA in the basal forebrain 's in animals performing a visual conditional discrimination task. It was anticipated that if the GABAergic input to the basal forebrain is either tonically active or activated by performance of the task, C D P would be expected to reproduce the effects of GABAA-receptor stimulation 3. Alternatively, a lack of effect of C D P infused into the basal forebrain would suggest that the GABAergic afferents to the region are not significantly active during the performance of this specific task (alternative possibilities are discussed below). Seven male Sprague-Dawley rats weighing 250350 g at the beginning of the experiment were used. Animals were housed individually in a temperature- and humidity-controlled room, food-deprived to approximately 90~o of their free-feeding weight, and maintained on a 12 h light/dark cycle. Animal training was essentially identical to that previously reported 3. Briefly, animals were initially shaped to bar press for food reinforcement, and then placed in a conditional visual discrimination task. In this task, either a constant or a flickering light (5 Hz) appeared at the central panel light for 10 s, and the animal was
203 required to respond to the left lever if the stimulus was a constant light, or the right lever if the stimulus was a flickering light. Response to the correct lever resulted in delivery of a food pellet and the onset of an inter-trial interval (panel light off) of 5 _+ 2 s, whereas response to the incorrect lever was not reinforced and initiated an inter-trial interval (panel light off) of 15 _+ 5 s. Each daily session consisted of 102 stimulus presentations (51 constant light, 51 flicker light, randomly presented) and lasted maximally 45 min. From each daily session the relative number ofcorrect responses, errors ofomission (number of stimulus presentations where no response was made), response latency, and side bias (Sahgai's ~3 Index Y) were calculated. After the animals acquired the conditional discrimination task to a criterion of 3 days at >75~o correct response animals were anesthetized with pentobarbital (50 mg]kg; i.p.), placed in a stereotaxic instrument (David Kopf, Tijuna, CA), and surgically implanted with bilateral cannulae directed 1 mm above the NbM/SI region of the basal forebrain. These guide cannulae were placed according to the coordinates of Paxinos and Watson~: AP: + 8.2 mm (from interaural line); L: + 3.0 mm; V: 2.8 mm (from interaural zero) and fixed to the skull with dental cement. Dummy cannulae were inserted into the guide cannulae to prevent blockage. Animals were allowed free access to food and water for a postoperative recovery time of 1 week. After postoperative return to baseline (>75~'o correct responding for 3 consecutive days), animals received a series of intracranial injections via a Hamilton mierosyringe attached by polyethylene tubing to an injection needle. The injection needle projected 1 mm beyond the end ofthe guide cannulae. The benzodiazepine receptor agonist chlordiazepoxide (Sigma; 15, 25, 40 llg]0.5 ld]hemisphere dissolved in 0.9~o sterile s a l i n e ) o r the vehicle was administered 15 min before testing in the behavioral task. Subsequent drug administrations were separated by at least one criterion day. Upon completion of the drug regimen, animals were sacrificed and cannulae placement was verified histologically. Data were analyzed with a single factor repeated ANOVA (SPSS Int. B.V., AC Gorinchem, The Netherlands). Infusions of chlordiazepoxide into the region of the substantia innominata did not appear to affect the animal's behavior in the conditional visual discrimination task. Statistically, there were no significant effects on correct responding (FL4; df3,9; P = 0.31), errors of omission (Fo.59; df 3,12; P = 0.632), or response time (Fo.32 ; df 3,9; P = 0.808; see Fig. 1). Index Y, the measure of side bias, was not examined statistically as the high percentage of correct responses in each treatment
condition precluded the possibility of ally significant side bias. The current data demonstrate that infusions of the benzodiazepine receptor agonist ehlordiazepoxide into the basal forebrain region does not affect visual conditional discrimination performance in rats. This finding suggests that the GABAergic input into this area 16 is not tonically active and is not activated by performance in this task. The lack of effect of CDP appears unlikely a result ofpotential technical problems related to the infusion of the drug into the brain. Previous studies that utilized intracranial infusions of benzodiazepine receptor ligands suggest that a relevant range of concentrations was tested in the present experiment, and that a dose of 40 fig/0.5/~l/hemisphere (the highest dose tested herein) may even be a relatively high concentration 6'9. Furthermore, the excellent solubility of CDP does not suggest that the drug diffuses in a way that was radically different from the diffusion of muscimol in our previous study. IF CDP differed from muscimol only in this respect though, one would have expected that the relatively high concentrations of CDP used here would at least be distinguishable from saline and perhaps comparable to lower doses of muscimol. Alternative explanations for the current results are also possible. For example, it may be speculated that the GABAA-receptor in the NbM/SI region are of a population not associated with a benzodiazepine receptor site. However, this appears unlikely as studies using in vitro autoradiography have shown that the NbM/SI region shows a relatively high density ofbenzodiazepine binding sites 16. The current results suggest that the activity of cells of the NbM/SI region is not controlled by GABAergic inhibition. Indeed, other studies have suggested that the GABAergic input to the NbM/SI region is not tonic. For example, Blaker I showed that injection of the GABAA-receptor antagonist bicuculline into the NbM/SI region did not affect cortical acetylcholine (ACh) turnover. Likewise, injection ofpirotoxin (which reduces the gating functions ofthe GABAA-receptor via the associated barbiturate receptor 5) into the NbM/SI region failed to affect cortical ACh turnover ~7. Taken together with the current results, these findings may suggest that the GABAergic input to the NbM/SI region is either not tonically active, or not active during performance of the tasks used in this experiment. In summary, although recent studies have suggested the GABAergic input to the basal forebrain may have a tonically active component 2, the current results appear most consistent with the interpretation that, during performance of a conditional visual discrimi-
204 100
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T
80
1
-7--
60
v
e. o
r
P.
,t0 20
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sal
15
25
40
chlordiazepoxide (ug/0.5ul/hs)
This work was supported in part by Grants from the Ohio Department of Aging and the U.S. Public Health Service (MH 46869-01AI).
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nation task, the GABAergic input to the NbM/SI region is not active. However, support for this hypothesis requires the determination of a behavioral function that can be modulated by infusions of CDP into the basal forebrain, i.e., that is dependent on an increase in endogenously released GABA in the basal forebrain. As we have assumed that performance of well-trained animals of a conditional visual discrimination task might be based on the uncontrolled processing of procedural information8, and as the basal forebrain does not appear as a prime substrate involved in the mediation of habits 7, it may be speculated that tasks that address the controlled processing of more declarative information may correlate with changes in basal forebrain GABAergic activity.
1
0
sal
15
25
40
ehlordiazepoxide ( u g / 0 . 5 u l / h s )
Fig. 1. Effects of bilateral infusions of the bcnzodiazepine receptor agonist chlordiazepoxide on performance of rats in a visual conditional discrimination task. hs, hemisphere; sal, saline.
1 Blakcr, W.D., GABAergic control ofthe cholinergic projections to the frontal cortex is not tonic, Brain Res., 325 (1985) 389-390. 2 Bourdelais, A. and Kalivas, P.W., Amphetamine lowers extracellular GABA concentration in the ventral pallidum, Brain Res., 516 (1990) 132-136. 3 Dudchenko, P. and Sarter, M., GABAergic control of basal forebrain cholinergic neurons and memory, Behav. Braha Res., 42 (1991) 33-41. 4 ttaefely, W.E., Pharmacology of allosteric modulation of GABAA receptors by benzodiazepine receptor ligands. In E.A. Barnard and E. Costa (Eds.), AIlosteric Mo&dation of Amino Acid Receptors: Therapeutic hnplications, Raven Press, New York, 1989, pp. 47-69. 5 tlaefely, W.E., Structure and function of the benzodiazepine receptor, Chhnia, ! i (1987) 389-396. 6 Kataoka, Y., Shibata, K., Gomita, Y. and Ueki, S., The mammillary body is a potential site of antianxiety action of benzodiazepines, Brain Res., 241 (1982) 374-377. 7 Mishkin, M., Malamut, B. and Bachevalier, J., Memories and habits: two neural systems. In G. Lynch, J.L. McGaugh and N.M. Weinberger (Eds.), Neurobiology of Learning and Memory, Guilford Press, New York, 1984, pp. 65-77. 8 Morris, R.G.M., Is the distinction between procedural and declarative memory useful with respect to animal models of amnesia? In G. Lynch, J.L. McGaugh and N.M. Weinberger (Eds.), Neurobiology of Leanthzg and 2~lemory, Guilford Press, New York, 1984, pp. 119-124. 9 Nagy, J., Zambo, K. and Decsi, L., Anti-anxiety action of diazepam after intra-amygdaloid application in the rat, Neurophannacology, 18 (I 979) 573-576. 10 Olton, D.S., Markowska, A., Voytko, M.L, Givens, B., Gorman, L. and Wenk, G., Basal forebrain cholinergic system: a functional analysis. In T.C. Napier, P.W. Kalivas and I. Itanin (Eds.), The Basal Forebrab~:Anatomy to Function, Plenum Press, New York, in press. 11 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1982.
205 12 Pirch, J.H., Ridgon, G.C., Rucker, H. and Turco, K., Basal forebrain modulation of cortical cell activity during conditioning. In T.C. Napier, P.W. Kalivas and I. Hanin (Eds.), The Basal Forebrahz: Anatomy to Function, Plenum Press, New York, in press. 13 Sahgal, A., Some limitations of indices derived from signal detection theory: evaluation of an alternative index for measuring bias in memory tasks, Psychopharmacology, 91 (1987) 517-520. 14 Sarter, M., Retrieval of procedural information: insensitive to changes in activity ofindividual neurotransmitter systems? Psychobiology, 18 (1990) 451-459.
15 Sarter, M., Bruno, J.P. and Dudchenko, P., Activating the damaged basal forebrain cholinergic system: tonic stimulation versus signal amplification, Psychopharmacology, 101 (1990) 1-17. 16 Sarter, M. and Schneider, H.H., High density ofbenzodiazepine binding sites in the substantia innominata of the rat, PhannacoL Biochem. Behav., 30 (1988) 679-682. 17 Wood, P.L. hnd Richard, J., GABAergic regulation of the substantia innominata-cortical cholinergie pathway. Neuropharmacology, 21 (1982) 962-972.