Intraseptal Flumazenil Enhances, while Diazepam Binding Inhibitor Impairs, Performance in a Working Memory Task

NEUROBIOLOGY OF LEARNING AND MEMORY ARTICLE NO.

66, 341–352 (1996)

0074

Intraseptal Flumazenil Enhances, while Diazepam Binding Inhibitor Impairs, Performance in a Working Memory Task CHRISTOPHER D. HERZOG, ROBERT W. STACKMAN,1

AND

THOMAS J. WALSH2

Department of Psychology, Rutgers University, New Brunswick, New Jersey, 08903

tics (File & Pellow, 1987). In addition, BDZ’s produce a well-documented amnestic effect in humans and laboratory animals across a variety of cognitive paradigms (for reviews see Lister, 1985; and Thie´bot, 1985). The behavioral effects of BDZs result from an interaction at central BDZ receptors. The BDZ receptor is an integral component of a macromolecular GABAA/BDZ/Cl0 receptor complex. Agonists of the BDZ receptor enhance GABAA-mediated postsynaptic inhibition via an enhancement of the affinity of the GABAA receptor and an enhancement of GABAA-mediated Cl0 influx. In contrast, the BDZ inverse agonists decrease receptor affinity and decrease Cl0 channel opening frequency (Duman, Sweetnam, Gallombardo, & Tallman, 1987; Polc, Bonetti, Schaffner, & Haefely, 1982). The BDZ receptor ligand flumazenil acts as a true pharmacological antagonist in that it blocks the effects of BDZ agonists and inverse agonists. Flumazenil does not modulate receptor affinity or influence Cl0 influx (Hunkeler, Mohler, Pieri, Polc, Bonetti, Cumin, Schaffner, & Haefely, 1981). Despite the distinct pharmacological nature of the BDZ receptor antagonist and inverse agonist, these ligands can produce similar behavioral effects. That is, like the inverse agonists, flumazenil: (i) can enhance performance in aversively motivated tasks (Lal, Kumar, & Forster, 1988; Lal & Forster, 1990; Brioni, Arolfo, Jerusalinsky, Medina, & Izquierdo, 1991; Raffalli-Sebille & Chapouthier, 1991; McNamara & Skelton, 1993a) and (ii) can induce anxiogenic effects in some paradigms (Lee & Rodgers, 1991; File, Lister, & Nutt, 1982). There has been debate as to whether these effects of flumazenil are the result of intrinsic actions of the compound or the result of an antagonism of endogenous BDZ receptor ligands. Wolfman and co-workers (1991) demonstrated that inhibitory avoidance training caused a region-

GABAA/benzodiazepine receptors in the medial septum modulate the activity of cholinergic neurons that innervate the hippocampus. Injection of benzodiazepine (BDZ) agonists into the medial septum impairs working memory performance and decreases high-affinity choline transport (HAChT) in the hippocampus. In contrast, intraseptal injection of the BDZ antagonist flumazenil increases HAChT and prevents the memory deficits induced by systemic BDZs. The present studies attempted to further characterize the behavioral effects of medial septal injections of flumazenil to an endogenous negative modulator of the GABAA/BDZ receptor complex, diazepam binding inhibitor (DBI). Male Sprague–Dawley rats were cannulated to study the effects of intraseptal injections of these BDZ ligands on spatial working memory, anxiety-related behaviors in the elevated plus maze, and on general locomotor activity. Intraseptal flumazenil (10 nmol/0.5 ml) produced a delay-dependent enhancement of DNMTS performance after an 8-h, but not a 4-h, delay interval. This promnestic dose of flumazenil had no effect on locomotor activity and did not produce changes in measures of anxiety on the plus maze. Intraseptal injection of DBI had no effect (8 nmol/0.5 ml) or slightly impaired (4 nmol/0.5 ml) DNMTS radial maze performance following an 8-h delay, without producing changes in locomotion or plus maze behavior. These data demonstrate that flumazenil has a unique profile of activity in enhancing working memory following intraseptal injection. q 1996 Academic Press, Inc.

INTRODUCTION Benzodiazepines (BDZ) are widely prescribed as anxiolytics, anticonvulsants, and sedative–hypno1 Current address: Department of Psychology, Dartmouth College, Hanover, NH, 03755. 2 The authors thank Papia Bhattacharya for help with the intracranial injections and behavioral testing. This work was supported by NSF Grant IBN 9514557 to T. J. W. Address correspondence and reprint requests to Thomas Walsh, Department of Psychology, Rutgers University, New Brunswick, NJ 08903. Telephone: (908)445-2907. E-mail: [email protected].

341 1074-7427/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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ally selective release of an endogenous BDZ-like molecule with agonist activity. Training induced a substantial release of ‘‘endozepines’’ in the medial septum (MS), hippocampus (HPC), amygdala, and frontal cortex. Pretraining infusions of flumazenil enhanced retention of the avoidance behavior, possibly via a blockade of the pharmacological activity of the endogenous compound (Wolfman, Da Cunha, Jerusalinsky, Levi de Stein, Viola, Izquierdo, & Medina, 1991). Such data support the notion that the BDZ antagonist has no intrinsic activity; the behavioral effects of flumazenil result from blocking the actions of endogenous BDZ receptor ligands. The present series of experiments compared the effects of flumazenil and the endogenous BDZ inverse agonist, diazepam binding inhibitor (DBI), in several behavioral paradigms that assess working memory, anxiety, and locomotor behavior. Discrete intraseptal injections were used to specifically examine the role of medial septum BDZ receptors in mediating the behavioral effects of flumazenil and DBI. The BDZ chlordiazepoxide impairs spatial working memory in rats when injected into the MS but not the amygdala, lateral septum, cingulate gyrus, or nucleus basalis (Stackman & Walsh, 1992; 1995). These data suggest that the MS is the primary neural site mediating the amnestic effect of this BDZ. Direct administration of BDZ receptor agonists into the amygdaloid complex produces an anxiolytic effect that is similar in magnitude to that produced by systemic administration, suggesting that this behavioral property of BDZs is mediated by receptors within the amygdaloid complex (Tomaz, DickinsonAnson, & McGaugh, 1991; Shibata, Yamashita, Yamamoto, Ozaki, & Ueki, 1989). However, administration of BDZs to amygdala-lesioned rats produces an anxiolytic effect (Yadin, Thomas, Strickland, & Grishkat, 1991). Therefore, it is possible that additional central regions, such as the MS, contribute to the expression of BDZ-induced anxiolysis. The following experiments examined the influence of the infusion of flumazenil and DBI into the MS upon (i) appetitive spatial memory; (ii) anxiety as assessed in the elevated plus maze; and (iii) gross locomotor activity. GENERAL METHOD Subjects Male Sprague–Dawley rats (Harlan Sprague– Dawley, Indianapolis, IN) weighing 250–275 g at the onset of each experiment served as subjects. All animals were individually housed in a temperature-

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controlled colony room with a 12-h light/dark cycle with lights on at 0700 h. Food and tap water were freely available except during behavioral testing. Animals in radial arm maze (RAM) experiments were reduced to 85% or their free feeding body weight 5 days prior to the beginning of behavioral training. Thereafter, subjects were provided with sufficient food to allow a 5-g weight gain per week for the duration of testing. Apparatus and Procedure Radial arm maze. An elevated RAM constructed of black plexiglas with eight equally spaced arms (8 1 50 cm) that extended outward from an octagonal central arena (20 cm across) was used. At the end of each arm was a 4-mm-deep food well in which the food pellets were placed. Food pellets (94 mg, standard formula plus chocolate, P. J. Noyes Inc., Lancaster, NH) were used as reinforcers for correct arm choices. Standard RAM procedure. Prior to training subjects received 1 habituation trial per day for 5 days. During these trials chocolate pellets were scattered throughout the maze and each animal was allowed to freely explore for 5 min per day. Following habituation trials the animals were trained for 25 trials on the standard RAM task. In this task, a trial consisted of an animal being placed in the center of the maze and allowed to visit each of the eight arms that were baited with a single chocolate pellet. Each trial (1/day) continued until the rat had visited all eight arms or until 5 min had elapsed. Reentry into an arm previously visited within any single trial was recorded as an error. Delayed-non-match-to-sample (DNMTS) RAM procedure. Following completion of standard RAM trials the animals were trained, for 20 trials (1/day), to perform a working memory version of the task, in which a 1-h delay was imposed between the fourth and fifth arm choices. Removable barriers (12 cm wide 1 25 cm high) made of clear Plexiglas were used to block four arms during each predelay training session. The remaining four arms were open and baited with a food pellet. The configuration of blocked and baited arms varied in a random manner for each daily session. Therefore, rats were presented with trial-dependent information each day (working memory). During the predelay training session each rat was placed on the maze and allowed to enter and retrieve pellets from the open arms. After entering all four open arms the rat was returned to its home cage for a 1-h delay. During the

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postdelay test session rats were returned to the maze and allowed access to all eight arms; however, only those previously blocked arms were now baited (delayed non-match to sample). To successfully perform the task, animals had to retain information about the configuration of open and closed arms for that given day and retrieve/utilize that information in order to perform accurately during the postdelay test session. DNMTS RAM task: Dependent measures. Dependent measures during the postdelay test session were: (i) the number of correct choices in the first four arm selections (CC) and (ii) the total number of errors which were scored as either retroactive (RE) (reentry into an arm baited during the predelay training session) or proactive (PE) (perseverative reentries into any postdelay choice). By definition, RE are first-time reentries into arms entered during the predelay session. These arms should be avoided during the test session since pellets were retrieved from these arms during the predelay training session. PE are reentries into arms selected during the postdelay test session. Therefore, PE can be either repeated entries into correct postdelay choices or repeated entries into predelay choices. By this criteria, a limit of only four RE can be committed during the postdelay session, as a second repeated entry into a predelay choice would be scored as a PE. Intracranial cannulation and drug administration. Following standard and DNMTS RAM acquisition trials, rats were given food ad libitum for 48 h prior to intraseptal cannulation. Rats were anesthetized with an intramuscular injection of an anesthetic mixture (ketamine, 50 mg/kg; xylazine, 2.61 mg/kg; and acepromazine 0.65 mg/kg) and secured in a stereotaxic frame. Blunt tip ear bars were used to prevent damage to the auditory meatus. Rats were implanted with a single stainless steel guide cannula (22 gauge, Plastics One Inc., Roanoke, VA) aimed dorsal to the MS (AP Å / 2.0 mm from bregma, ML 0.0 mm, DV 4.0 mm below the cortical surface) (Pellegrino, Pellegrino, & Cushman, 1979). These placements allowed for the respective injection cannulae (28 gauge) to extend an additional 1.5 mm to permit intraseptal infusions. Stainless steel mounting screws and cranioplastic cement were used to anchor the guide cannula to the skull. Following a 7-day recovery from surgery animals were returned to the delay RAM task and tested for 5 days in order to demonstrate that the intraseptal implantation of cannulae did not affect baseline performance. The implantation of intraseptal cannulae

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alone was previously shown to produce no impairment in delay RAM performance (Stackman & Walsh, 1992). Animals were then habituated to the intraseptal injection procedure during the second week of postoperative testing. During the two injection habituation trials, rats were removed from the RAM following completion of the predelay training session and held gently, and injection cannulae were inserted into the guide cannulae. These habituation injection cannulae were not longer than the respective guide cannulae; thus, in this way the animals could be exposed to all aspects of the injection procedure without inducing injection-related tissue damage. During this initial procedure no solutions were infused through the injection cannulae. During a third injection habituation trial, rats received a single infusion of 0.5 ml of artificial cerebrospinal fluid (CSF in mM: NaCl 150, KCl 2.9, MgCl2 1.6, HCO3 35.9, CaCl2 –2H2O 1.7, and dextrose 2.2) into the respective region. We have found that this injection habituation protocol facilitates intracranial infusions and greatly reduces the inherent stress that may preclude observation of the more subtle drug effects. Subjects in experiments assessing anxiety and locomotor activity underwent intraseptal cannulation surgery as described above and were allowed 7 days of postoperative recovery. In these studies rats received one intraseptal injection 5 min prior to behavioral testing. The injection procedure used in all experiments involved the infusion of drug or vehicle into the MS via the injection cannulae connected to a 10-ml Hamilton syringe by polyethylene tubing. All infusions were delivered in a volume of 0.5 ml, at an injection rate of 0.167 ml/min maintained by a microinjection syringe pump (CMA 100, Carnegie-Medicin, Stockholm, Sweden). The injection cannula remained in place for an additional 2 min following infusions to allow for adequate diffusion of the drug. Elevated plus maze. The plus maze was constructed of black plexiglas and designed so that each of its four arms was elevated 50 cm above the testing room floor. Two open arms (45 1 10 cm) and two closed arms (45 1 10 1 40 cm) extended in opposing directions away from the center of the maze. A video camera suspended above the maze recorded behavior for later analysis. Immediately following intraseptal injection and prior to plus maze testing, rats were placed individually into an enclosed arena constructed of cardboard (48 1 41 1 66 cm) that was located in a separate testing room. Preliminary studies conducted in our laboratory, and by others (Pel-

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low & File, 1986), indicate that this procedure increases exploratory behavior on the plus maze. After a 5-min exposure to the arena, each rat was placed onto the center of the plus maze, initiating a 5-min interval during which exploratory activity was recorded. Placement of rats toward an open or closed arm was randomly assigned across all treatment groups. The floor of the maze was cleaned with 70% ethanol after each session. An experimenter blind to the treatment conditions scored video tapes recording the number of entries and time spent on each arm. Locomotor activity. Locomotor activity was measured with the use of six activity chambers. Each chamber was constructed of black plexiglas (40 1 23 1 19 cm) and contained six pairs of photocell beam sensors mounted 5.5 cm above the floor of the chamber. Photocell beam interruptions were automatically recorded by an IBM PC XT computer interfaced with the chambers. Cumulative photocell interruptions over 5-min bins were calculated. Baseline motor activity was evaluated for 30 min prior to intraseptal injections. Upon completion of the intraseptal infusion, rats were returned to the activity boxes and motor activity was evaluated for 60 mins. Data were computed as the percentage of baseline activity 15, 30, and 60 mins postinfusion. Histological analysis. After behavioral testing was completed each animal received a final intraseptal infusion of 0.5 ml of cresyl violet dye through the guide cannulae. Brains were then removed 10 min following intraseptal infusion, frozen, and sectioned to determine gross localization of dye. Injection locations were characterized by comparison of tissue to coronal sections of the atlas of Pellegrino et al. (1979). EXPERIMENT 1A: INTRASEPTAL FLUMAZENIL ENHANCES SPATIAL WORKING MEMORY Posttraining intraseptal injection of the GABAA agonist, muscimol, or BDZ agonists, such as CDP, impairs spatial memory in rats (Chrobak, Stackman, & Walsh, 1989; Brioni, Decker, Gamboa, Izquierdo, & McGaugh, 1990; Stackman & Walsh, 1992; 1995; McNamara & Skelton, 1993b) and decreases hippocampal high-affinity choline transport (HAChT) and acetylcholine turnover and release. (Blaker, Cheney, Gandolfi, & Costa, 1983; Walsh, Stackman, Emerich, & Taylor, 1993; Imperato, Dazzi, Obinu, Gessa, & Biggio, 1993). In contrast, administration of GABA and BDZ antagonists enhances retention of inhibitory avoidance in mice and

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rats (Brioni & McGaugh, 1988; Lal et al., 1988) and enhances retention of place learning in the Morris water maze (Brioni et al., 1991; McNamara & Skelton, 1993a). Intraseptal infusion of flumazenil (i) increases hippocampal HAChT (Walsh et al., 1993) and (ii) increases acetylcholine release in HPC (Imperato, Dazzi, Obinu, Gessa, & Biggio, 1994). Therefore, the amnestic effects of GABA and BDZ agonists may be a consequence of a disruption of hippocampal cholinergic activity during a critical phase of memory formation (see Walsh & Stackman, 1992). Since spatial memory can be disrupted by a septal BDZ mechanism, it may be possible to enhance such performance by antagonizing this mechanism. Subjects Sixteen male Sprague–Dawley rats served as subjects. Rats were food deprived and trained on the standard and delay versions of the RAM prior to cannulation surgery (see General Methods). DNMTS testing resumed 1 week following surgery where a 1-h delay was imposed between pre- and postdelay sessions. Rats were then exposed to longer delay intervals (8 h followed by 4 h). Intraseptal Infusions and DNMTS Behavioral Testing On Tuesdays and Fridays, immediately after the predelay training session, rats received intraseptal injections of flumazenil [10 nmol (1.5 mg) or 20 nmol (3.0 mg)] (generously provided by Hoffmann–La Roche, Nutley, NJ) or drug vehicle (5.0% polyethylene glycol). The influence of flumazenil on the retention of working memory was determined during the subsequent postdelay test session. The dose range of flumazenil was chosen based upon preliminary behavioral and neurochemical investigations and previous studies (Stackman & Walsh, 1992). During the first phase of treatment, a within-subjects crossover design was utilized to examine the effects of intraseptal flumazenil upon performance of the DNMTS-RAM task with an 8-h delay. Upon completion of the flumazenil dose response at the 8-h delay, the effect of intraseptal flumazenil upon performance on the DNMTS-RAM task with a 4-h delay was determined in eight of the rats. Following completion of the experiment, all animals were tested for 5 additional drug-free trials to determine whether baseline performance had been affected by the intracranial injections. Histological analyses were performed as described above to determine cannulae placement. All rats exhibited correct cannula placements within the MS, and dye did

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FIG. 1. (A) Dose-related effect of intraseptal flumazenil (10 or 20 nmol) on the DNMTS RAM task following an 8-h delay interval. Data are presented as the mean number of correct choices and the mean number of postdelay errors ({ SEM). Posttraining intraseptal injections of flumazenil (10 nmol) significantly increased the number of correct choices and decreased the number of postdelay retroactive errors (* p õ .05 vs Veh group, Fisher’s LSD test). (B) Dose-related effect of intraseptal flumazenil (10 or 20 nmol) on measures of correct choice and postdelay errors following a 4-h delay interval in the DNMTS RAM task. Data are presented as mean number of correct choices and mean number of postdelay errors ({ SEM).

not appear to diffuse beyond the lateral extent of the MS. Results Implantation of MS cannulae did not disrupt performance, as there were no differences on any measure of performance when the first 5 trials following surgery were compared with the last 5 preoperative trials: [CC t(15) Å 0.89, n.s.; RE t(15) Å 01.26, n.s.; PE t(15) Å 01.10, n.s.]; all paired two-tailed t tests. Posttraining intraseptal flumazenil significantly enhanced retention (see Fig. 1A) of working memory in the DNMTS task with an 8-h delay. One-factor repeated measures ANOVAs revealed that flumazenil significantly increased CC, F(2,30) Å 3.39; p Å .047; and decreased RE, F(2,30) Å 3.96; p Å .029, without affecting PE, F(2,30) Å 1.79; n.s. The results of post-hoc analyses are indicated in Fig. 1A. A randomly chosen subset (N Å 8) of the original group of rats was used to evaluate the efficacy of intraseptal flumazenil upon retention with a 4-h delay imposed between predelay training and the postdelay test session. One-factor (intraseptal treatment) repeated measures ANOVAs revealed nonsig-

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nificant effects of treatment: CC, F(2,14) Å 0.25; n.s.; RE, F(2,14) Å 1.14; n.s., PE, F(2,14) Å 0.14; n.s. Repeated infusions into the MS did not influence baseline performance, as there was no difference in DNMTS performance when the last 5 drug-free trials were compared to the first 5 postoperative trials. [CCt(15) Å 01.16, n.s.; RE t(15) Å 01.40, n.s.; PE t(15) Å 0.58, n.s.], all paired two-tailed t tests. These results demonstrate the ability of posttraining intraseptal flumazenil to enhance retention of spatial memory at long delays. Systemic administration of flumazenil enhances retention in aversive paradigms such as the standard Morris water maze (Brioni et al., 1991; McNamara & Skelton, 1993b) and passive avoidance (Lal, Kumar, & Forster, 1988). The flumazenil-induced enhancement of retention in the DNMTS paradigm is indicated by a greater number of CC and the commission of fewer RE, compared to vehicle-treated rats. The cognitive demand of this task can be manipulated simply by increasing the delay interval. Thus, rats performing accurately at a 1-h delay (mean CC of ú 3.0) will exhibit delay-dependent impairments at 4- and 8-h retention intervals. In a previous report (Stackman & Walsh, 1992), we demonstrated the lack of

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influence of intraseptal flumazenil upon performance in the DNMTS task with a 1-h delay. Given the present data, it appears that performance is at a ceiling level at the 1-h delay, which limits the assessment of the performance-enhancing potential of flumazenil. However, the present data suggest that intraseptal flumazenil appears to render spatial information that is to be maintained for the 8-h delay interval less susceptible to decay over time. Performance of flumazenil-treated rats in this paradigm following an 8-h retention interval is comparable to that exhibited by control rats following a 1-h delay. EXPERIMENT 1B: INTRASEPTAL FLUMAZENIL HAD NO EFFECT ON BEHAVIOR IN THE ELEVATED PLUS MAZE Flumazenil has been shown to have a mild anxiogenic effect at low to moderate doses when delivered systemically to mice and rats in some, but not all, paradigms that assess anxiety (File, Lister, & Nutt, 1982; Pellow & File, 1986; Treit, 1987; Lee & Rodgers, 1991; McNamara & Skelton, 1993a; Pesold & Treit, 1994). Therefore, it was the aim of the present experiment to determine whether intraseptal flumazenil produces an anxiogenic effect in a task commonly used to assess the pharmacological activity of anxiolytics and anxiogenics, the elevated plus maze.

Results Statistical analysis revealed no significant differences between vehicle- and CSF-treated animals on any behavioral measure (e.g., number of entries or time spent on each type of arm; all p’s ú .05). Data from these two treatment groups were therefore combined to form one group designated Control. Flumazenil-treated rats did not exhibit an anxiogenic profile in the elevated plus maze (see Table 1). Statistical analyses (unpaired two-tailed t tests) revealed a nonsignificant treatment effect for all behavioral measures assessed: number of open entries t(28) Å 1.315, n.s.; closed entries t(28) Å 1.038, n.s.; percent open entries t(28) Å .586, n.s.; ratio of open/ total entries, t(28) Å 0.592, n.s.; percent time on open arms, t(28) Å 0.810, n.s.; percent time on closed arms t(28) Å 00.655, n.s.; ratio of time on open/total t(28) Å .758, n.s. These findings demonstrate that infusions of a promnestic dose of a BDZ antagonist into the MS did not alter the expression of anxiety-related behaviors as measured by the elevated plus maze. EXPERIMENT 1C: INTRASEPTAL FLUMAZENIL DID NOT INFLUENCE LOCOMOTOR ACTIVITY

Thirty-two naive, male Sprague–Dawley rats as described previously were used. Animals were individually housed in a temperature-controlled colony room with free access to food and water except during behavioral testing.

Performance on the RAM task may be influenced by alterations in locomotion (e.g., increased locomotor activity lending to enhanced performance). In order to determine if the behavioral effects of flumazenil could be attributed to alterations in locomotor activity, a final group of rats was implanted with guide cannula aimed dorsal to the MS and the doserelated influence of intraseptal injections of flumazenil upon locomotor activity was assessed as described in General Methods.

Behavioral Testing and Drug Administration

Results

Following intraseptal cannulation and 7 days of postsurgical recovery, each rat received an intraseptal injection of flumazenil (10 nmol, N Å 16), vehicle (5% polyethylene glycol, N Å 8), or artificial CSF (N Å 8). Immediately following injection, rats were placed individually into the enclosed arena constructed of cardboard that was located in a separate testing room. After a 5-min exposure to the arena, each rat was placed onto the center of the plus maze initiating a 5-min interval during which exploratory activity was recorded as described above. Following histological analysis, correct placement of cannula was observed in all but two of the rats which were subsequently excluded from all behavioral data.

Intraseptal infusion of flumazenil did not significantly influence locomotor activity (see Table 2). A two-factor (flumazenil dose, postinfusion time) repeated measures ANOVA revealed a nonsignificant effect of treatment, F(3, 18) Å 0.94; n.s.; a significant effect of time, F(2, 36) Å 29.75 p õ .0001; and a nonsignificant treatment 1 time interaction, F(6, 36) Å 0.158; n.s. Therefore, infusion of 5, 10, or 20 nmol of flumazenil into the MS did not alter locomotor activity. Additionally, based upon the significant effect of time but nonsignificant treatment 1 time interaction, flumazenil did not alter normal habituation to the activity chamber across the 60-min test interval.

Subjects

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TABLE 1 Intraseptal Injections of Flumazenil or DBI Did Not Dignificantly Alter Measures of Anxiety on the Elevated Plus Maze Time (seconds) Intraseptal injection Experiment 1B Control Flu 10 nmol Experiment 2B Control DBI 4 nmol DBI 8 nmol

Open arms

Closed arms

17.9 { 3.8 23.1 { 5.1

259.9 { 7.8 251.7 { 8.5

15.4 { 4.9 30.8 { 9.9 18.2 { 10.8

275.9 { 16.5 234.0 { 12.8 246.6 { 18.2

Entries Percent open

Open arms

Closed arms

Percent open

6.0 { 1.3 7.7 { 1.7

1.4 { .31 2.1 { .47

10.6 { 1.2 11.7 { 1.0

12.6 { 2.9 15.2 { 3.3

5.3 { 1.6 11.7 { 3.8 7.3 { 4.5

.78 { .15 1.2 { .40 1.1 { .66

7.6 { 1.9 8.2 { 1.2 8.9 { 1.6

15.7 { 5.7 13.5 { 4.9 7.2 { 3.7

Note. Data are presented as group mean values ({SEM).

(Venault et al., 1986; also see Raffalli-Sebille & Chapouthier, 1991) the degree to which such behavioral facilitation may be observed in tasks which involve processing and utilization of discrete trial-specific information is limited by the anxiogenic and proconvulsant effects of b-CCM (see Sarter et al., 1988). Nevertheless, the selective BDZ inverse agonists ZK 93,426 and MDL 26,479 enhance radial maze performance in basal forebrain-lesioned rats (Hodges et al., 1989; Holley et al., 1993) and attenuate scopolamine-induced impairments in spatial delayed alteration tasks (Holley et al., 1992). DBI, an endogenous negative modulator of the BDZ receptor complex, appears to exhibit a similar pharmacological profile to that of the anxiogenic bcarboline esters (see Barbaccia et al., 1990). Intracerebroventricular injections of DBI produce proconflict effects which are reversed by flumazenil as well as an attenuation of the anti-conflict effects of diazepam (Ferrero et al., 1986). An abundance of DBI and two of its breakdown products (ODN and TTN) have

EXPERIMENT 2 The results of Experiment 1 highlight the ability of intraseptal flumazenil to enhance retention in an appetitive spatial working memory task. Intraseptal flumazenil-induced retention enhancement does not appear to involve an influence upon anxiety or locomotor activity. A number of endogenous BDZ ligands, endozepines, have been isolated from the mammalian brain, with highest concentrations in limbic system structures such as the MS and HPC. Thus, it is of interest to determine if such an endogenous BDZ receptor ligand could also modulate memory processes when injected into the MS. It has been previously demonstrated that HAChT in the HPC can be elevated by approximately 25– 30% following intraseptal administration of flumazenil or the BDZ inverse agonist b-CCM (Walsh et al., 1993). Although b-CCM has been shown to enhance both habituation to a novel environment and memory for a simple passive avoidance task in mice

TABLE 2 The Dose-Related Effects of Flumazenil and DBI on Horizontal Locomoter Activity Were Assessed Preinjection baseline

Intraseptal Experimental 1C Vehicle Flu 5 nmol Flu 10 nmol Flu 20 nmol Experiment 2C Control DBI 4 nmol DBI 8 nmol

Post 15 min % baseline

245.1 { 26.2 267.3 { 12.1 251.8 { 13.2 245.1 { 18.0

95.2 108.4 96.3 84.1

284.5 { 15.6 285.2 { 19.9 307.2 { 16.8

102.5 { 6.7 122.7 { 12.7 99.6 { 7.1

{ { { {

30.2 28.4 23.1 16.7

Post 30 min % baseline

Post 60 min % baseline

46.2 59.9 58.4 44.6

21.5 21.9 19.4 20.8

{ { { {

21.6 27.5 12.3 10.2

51.6 { 7.7 52.8 { 11.6 46.9 { 4.3

{ { { {

10.1 13.2 7.8 7.3

37.7 { 9.8 32.9 { 11.6 44.7 { 16.5

Note. Intraseptal injection of these BDZ ligands did not alter motor activity. Data are presented as group mean activity counts ({SEM).

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been found in brain regions containing BDZ receptors, and it has been demonstrated that these peptides bind preferentially to BDZ receptors located on the GABAA/BDZ/Cl0 complex (Guidotti et al., 1989; Alho et al., 1990). Experiment 2 examined the behavioral effects of intraseptal injections of DBI on DNMTS RAM performance, elevated plus maze behavior, and locomotor activity. EXPERIMENT 2A: INTRASEPTAL DBI DID NOT INFLUENCE SPATIAL WORKING MEMORY Eight male Sprague–Dawley rats as described in Experiment 1 were trained preoperatively on the standard and the DNMTS RAM task (1-h delay). Rats were implanted with guide cannulae to assess the effect of intraseptal injection of DBI on DNMTS working memory performance following 8- and 4-h retention intervals. An elevated RAM as described for Experiment 1 was used. On Wednesdays and Saturdays rats received intraseptal injections of one of the doses of DBI [4 nmol (15 mg) or 8 nmol (30 mg) (obtained from Bachem California)] or artificial CSF immediately after the predelay training session. The influence of DBI on behavioral performance during the postdelay test session following an 8-h delay interval was determined using a within-subjects crossover design. The effect of intraseptal DBI on postdelay performance was then similarly determined following a 4h delay interval in the DNMTS RAM task. Upon completion of behavioral testing, histological analyses were performed. Behavioral data collected from one animal was not included in the statistical analyses due to improper cannula placement. Results Implantation of MS cannulae did not alter postsurgical DNMTS RAM performance. There were no significant differences on any measure of behavioral performance when the first 5 trials following surgery were compared with the last 5 preoperative trials: CC t(6) Å 1.353, n.s.; RE t(6) Å .89, n.s.; PE t(6) Å 0.655, n.s., all paired two-tailed t tests. Figure 2A presents the effects of intraseptal infusions of DBI on measures of DNMTS performance during the postdelay test session following a delay interval of 8 h. One-factor repeated measures ANOVAs revealed that neither dose of DBI significantly altered performance on RE, F(2, 12) Å 0.33, n.s. or PE, F(2, 12) Å 0.78, n.s. However, there was a significant trend for DBI to impair CC at the 8-h delay

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F(2, 12) Å 3.50, p Å .063. Post-hoc analyses (Fisher’s LSD test) revealed that 4 nmol of DBI significantly decreased the number of CC by 27%. The 8 nmol dose decreased CC by 23% but this was not significant. The results of intraseptal infusions of DBI on working memory performance following a delay interval of 4 h are presented in Fig. 2B. Posttraining infusion of DBI into the MS did not affect working memory performance. One-factor repeated measures ANOVAs revealed nonsignificant effects of treatment: CC, F(2, 10) Å 0.63, n.s.; RE, F(2, 10) Å 0.60, n.s.; PE, F(2, 10) Å 2.25, n.s. EXPERIMENT 2B: INTRASEPTAL DBI HAD NO EFFECT ON BEHAVIOR IN THE ELEVATED PLUS MAZE Twenty-seven naive male Sprague–Dawley rats were cannulated and randomly assigned to one of three treatment groups. Behavior in the elevated plus maze was assessed following intraseptal injection of 4 or 8 nmol DBI, or artificial CSF, according to the procedure detailed in Experiment 1. Results Intraseptal DBI had no significant effect on any measure in the plus maze (see Table 1). One-factor ANOVAs were performed on a number of open arm entries F(2, 24) Å 0.26, n.s.; closed entries F(2, 24) Å 0.17, n.s.; percent open entries F(2, 24) Å 0.83; time on open arms F(2, 24) Å 0.83 n.s.; time on closed arms F(2, 24) Å 1.81 n.s.; percent time spent on open arms F(2, 24) Å 0.89, n.s. Intraseptal injections of DBI did not significantly alter elevated plus maze behavior. EXPERIMENT 2C: INTRASEPTAL DBI HAD NO EFFECT ON LOCOMOTOR ACTIVITY Eighteen naive male Sprague–Dawley rats served as subjects for the final experiment designed to assess the dose-related effects of intraseptal DBI on locomotor activity. The injection protocol and activity chambers were as described in General Methods. Intraseptal infusions of either dose of DBI did not significantly influence locomotor activity (see Table 2). A two-factor (DBI dose, postinfusion time) repeated measures ANOVA revealed a nonsignificant effect of treatment, F(2, 15) Å 0.83, n.s.; a significant effect of time, F(2, 30) Å 56.71, p õ .0001, reflecting within-trial habituation; and a nonsignificant treatment 1 time interaction F(4, 30) Å 1.17, n.s.

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FIG. 2. (A) Dose-related effect of intraseptal DBI (4 or 8 nmol) on the number of correct choices and postdelay errors following an 8-h delay interval in the DNMTS RAM task. Intraseptal injection of 4 nmol of DBI significantly decreased the number of correct choices at the 8-h delay. Neither dose affected the number of postdelay errors. (B) Dose-related effect of DBI (4 or 8 nmol) on the number of correct choices and postdelay errors following a 4-h delay interval in the DNMTS RAM task. Posttraining injections of DBI had no effect on measures of cognitive performance during the postdelay session.

GENERAL DISCUSSION Intraseptal infusion of flumazenil (10 nmol) enhanced performance in an appetitively motivated working memory task. This cognitive enhancing dose did not alter anxiety-related behaviors on an elevated plus maze, and it did not affect locomotor activity or normal habituation in the activity chamber. Experiment 1A demonstrated the ability of a single posttraining injection of flumazenil (10 nmol) to enhance retention of spatial information over an 8h delay. A similar but nonsignificant trend following a 4-h delay also suggests that this BDZ antagonist may alter normal delay-dependent decay of information in this spatial working memory paradigm. Although similar results have been observed following systemic administration of flumazenil in an aversively motivated task (McNamara & Skelton, 1993a), the site-specific manipulation in Experiment 1 strongly suggests that the promnestic effect of GABA and BDZ antagonists are a consequence of interactions with GABA/BDZ receptors in the MS. The lack of a significant enhancement following infusions of 20 nmol may reflect a nonlinear dose–response curve. Previous dose–response studies have demonstrated an inverted U-shaped function for the

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expression of flumazenil-induced anxiogenesis when injected systemically (File et al., 1982). There is evidence that GABA/BDZergic mechanisms in the amygdala can modulate memory processes (Brioni, Nagahara, & McGaugh, 1989). However, infusions of flumazenil (10 nmol) into the MS, but not the amygdala, can prevent systemic chlordiazepoxide-induced amnesia (Stackman & Walsh, 1992). Such data suggest that septal BDZ receptors are responsible for mediating the memory-disruptive effects of the drug. In contrast to the present intraseptal flumazenil-induced enhancement of working memory retention, bilateral intra-amygdaloid injections of 10 nmol flumazenil failed to influence DNMTS performance (Stackman & Walsh, 1992). Together, these data suggest a dissociation between the contribution of amygdaloid and medial septal BDZ receptors in supporting accurate performance in this spatial memory task. However, a role for the MS in aversive memory processes is supported by the observation that intraseptal injection of GABAergic drugs can modulate retention of inhibitory avoidance and water maze tasks (Brioni et al., 1990; Nagahara, Brioni, & McGaugh, 1992). Further, the present results demonstrate that intraseptal flumazenil does not produce an anxiogenic effect that could complicate the analysis of performance

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changes. The degree to which anxiolysis contributes to the amnestic effects of intra-amygdaloid-administered GABA and BDZ compounds remains to be determined. Activation of hippocampal and cortical cholinergic systems has been previously demonstrated by measuring HAChT following training on spatial RAM tasks (Toumane, Durkin, Marighetto, & Jaffard, 1989; Durkin, 1994). Infusion of BDZ receptor antagonists and inverse agonists into the MS potentiates presynaptic cholinergic activity in the HPC, as measured by HAChT (Walsh et al., 1993), and by acetylcholine release (Imperato et al., 1994). Furthermore, the memory-enhancing dose of flumazenil in the present experiment significantly augments both dentate granule cell field responses and the induction of long-term potentiation evoked by angular bundle stimulation (Stackman, Walsh, Brucato, & Swartzwelder, 1996). Therefore, rather than enhancing spatial working memory by an exclusive interaction with GABAergic systems, flumazenil seems to facilitate normal mnemonic processes by its ability to interact with the septohippocampal cholinergic system during a time when this system is actively engaged in processing information acquired during the predelay training session. Given that flumazenil was administered posttraining, it is unlikely that the compound enhanced the acquisition of spatial information by altering attentional/perceptual processes during the predelay training session. The possibility of an action of flumazenil at the time of the test session (i.e., Ç1 h following injection) is unlikely given that the halflife of flumazenil in brain is approximately 16 min (Lister, Breenblatt, Abernethy, & File, 1984). It seems more likely that the BDZ antagonist augmented the neural processes induced by the training experience. Intraseptal injection of CDP immediately following but not 30 or 45 min following the predelay session impairs postdelay performance (Stackman & Walsh, 1995). The behavioral consequence of intraseptal infusions of flumazenil may therefore be due to antagonism of BDZ receptors during a time period when processing of the predelay configuration contributes to retention over the delay interval. Experiment 1B demonstrated that intraseptal infusions of flumazenil do not produce behavioral changes on an elevated plus maze. No differences were observed between control and flumazeniltreated animals for arm entries or time spent on each type of arm. Although systemic injections of flumazenil have been found to have an anxiogenic effect in some behavioral paradigms (File, Lister, &

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Nutt, 1982; Lee & Rodgers, 1991), our data parallel previous reports illustrating that systemic or central injections fail to alter animals’ normal preference for closed arms on the elevated plus maze (Pellow & File, 1986; Pesold & Treit, 1994). Furthermore, these findings are in agreement with prior experiments where intraseptal infusions of flumazenil alone failed to alter open arm activity on the elevated plus maze, but did block the anxiolytic effect of intraseptal midazolam, a BDZ agonist (Pesold & Treit, 1994). Posttraining injection of DBI, unlike flumazenil, did not enhance performance in the delay task. In fact, the low dose (4 nmol) of DBI impaired retention when an 8-h delay interval was imposed, as indicated by a significant decrease in the number of correct choices; however, this dose did not appear to alter postdelay errors. Additionally, 8 nmol of DBI nonsignificantly increased the number of both retroactive and proactive errors following the 4-h delay. It would be interesting to examine the effects of intraseptal ODN and TTN in our paradigms since these DBI breakdown products have a more potent proconflict effect following intraventricular injection (Slobodyansky et al., 1989). In addition, these peptides are localized primarily in neurons, unlike DBI which is localized in both glia and neurons, and they interact with different subcategories of GABAA receptors (Alho et al., 1985; Guidotti et al., 1989). Despite the report that central administration of DBI produced a proconflict effect in rats (Slobodyansky et al., 1989), discrete intraseptal injection of DBI did not produce behavioral changes indicative of anxiety in this task. The plus maze test of anxiety has been found to be sensitive to both anxiogenic and anxiolytic compounds which interact with the GABA/BDZ receptor complex. For example, the BDZ inverse agonists b-CCM (Belzung et al., 1991), FG 7142 and CGS 8216 (Pellow & File, 1986) reduce measures of open arm exploration, while benzodiazepines increase the number of open arm entries and relative time spent on open arms in the elevated maze (Pellow & File, 1986). In summary, the data presented here demonstrate that intraseptal injection of the BDZ antagonist flumazenil enhances performance in a spatial working memory task. In contrast, the endogenous BDZ receptor inverse agonist DBI appeared to disrupt working memory. This impairment is interesting since it differs from behavioral findings following systemic administration of other BDZ inverse agonists such as ZK 93,426 and MDL 26,479 (Holley et al., 1993). Intraseptal administration of either flumazenil or DBI did not alter motor activity or anxiety-related behaviors in the plus maze. Flumazenil

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appears to be a useful tool in further elucidating the role of BDZ receptors in different memory processes. In addition, pharmacological antagonism of central BDZ receptors may have therapeutic potential for the treatment of cognitive disorders associated with compromised septohippocamal cholinergic function (Walsh, 1993).

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ing inhibitor (DBI): Biological activity and presence in rat brain. Proceedings of the National Academy of Science USA, 83, 827–831. File, S. E., Lister, R. G., & Nutt, D. J. (1982). The anxiogenic action of benzodiazepine antagonists. Neuropharmacology, 21, 1033–1037. File, S. E., & Pellow, S. (1987). Behavioral pharmacology of minor tranquilizers. Pharmacological Therapeutics, 35, 265–290.

Alho, H., Bovolin, P., & Slobodyansky, E. (1990). Diazepam binding inhibitor (DBI) processing: Immunohistochemical studies in rat brain. Neurochemical Research, 15, 209–216.

Guidotti, A., Alho, H., Berkovich, A., Cox, D., Ferrarese, C., Slobodyansky, E., Santi, M., & Wambebe, C. (1989). DBI processing: Allosteric modulation at different GABA/Benzodiazepine receptor subtypes. In E. A. Barnard & E. Costa (Eds.), Allosteric modulation of amino acid receptors: Therapeutic implications. (pp. 100–123). New York: Raven Press.

Alho, H., Costa, E., Ferrero, Pl, Fujimoto., M. Cosenze-Murphy, D., & Guidotta, A. (1985). Diazepam binding inhibitor: A neuropeptide located in selected neuronal populations in rat brain. Science, 229, 179–182.

Hodges, H., Thrasher, S., & Gray, J. (1989). Improved radial maze performance induced by the benzodiazepine antagonist ZK 93 426 in lesioned and alcohol-treated rats. Behavioral Pharmacology, 1, 45–55.

Barbaccia, M., Berkovich, A., Guarneri, P., & Slobodyansky, E. (1990). Diazepam binding inhibitor: The precursor of a family of endogenous modulators of GABAA receptor function. History, perspectives, and clinical implications. Neurochemical Research, 15, 161–168.

Holley, L., Dudchenko, P., & Sarter, M. (1992). Attenuation of muscarinic receptor blockade-induced impairment of spatial delayed alternation performance by the traizole MDL 26,479. Psychopharmacology, 109, 223–230.

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