episodic memory impairments following intraventricular administration of ethylcholine aziridinium ion (AF64A)

BEHAVIORALAND NEURALBIOLOGY56, 200-212 (1991)

Dose- and Delay-Dependent Working/Episodic Memory Impairments following IntraventricularAdministration of Ethylcholine Aziridinium Ion (AF64A) JAMES J. CHROBAK AND THOMAS J. WALSH *'1

Department of Pharmacology and Experimental Therapeutics, Loyola University School of Medicine, Maywood, Illinois 60153; and *Department of Psychology, Rutgers University, New Brunswick, New Jersey 08903 The present study examined the effects of intraventricular administration of the cholinergic neurotoxin ethylcholine aziridinium ion (AF64A) on performance of a radial arm maze task. Male Sprague-Dawley rats were trained to perform a delayed-nonmatch to sample radial arm maze task in which a 1-h delay was imposed between the fourth and fifth arm selections. Following acquisition, animals were injected bilaterally with AF64A (1.5 or 0.75 nmol/side) or artificial cerebrospinal fluid into the lateral cerebral ventricles and allowed 7 days to recover before behavioral testing resumed. Significant dose- and delay-dependent impairments in the radial maze performance were observed in AF64A-treated rats as evidenced by fewer correct choices following the delay and by more errors to complete the task. Long-term testing in this task revealed significant recovery of memory performance. These findings indicate dose-dependent impairments in memory following intraventricular administration of AF64A and spontaneous behavioral recovery following such insult. © 1991 AcademicPress, Inc.

Interest in the neurobiology of dementia has focused attention on the septohippocampal cholinergic system (SHC). This system plays a critical role in the neurophysiological activity of the hippocampus (HPC) and the cognitive processes it subserves. Given the pronounced disruption of hippocampal cholinergic indices observed in Alzheimer's disease (AD), the degeneration of SHC afferents may be a critical focus for the deficit in recent memory observed early in the course of this disorder (Davies & Maloney, 1976; Henke & Lang, 1983; Rylett, Ball, & Colhoun, 1983; Whitehouse, Price, Struble, Clark, Coyle, & DeLong, 1982). Experimental animal studies demonstrating that manipulation of cholinergic neurotransmission within the HPC is sufficient to produce a disruption of The authors thank Theola Demetriolou for her assistance in training and testing animals. This study was funded by a BRSG grant (PHS 07058) to T.J.W. Address correspondence and reprint requests to J. J. Chrobak at Department of Pharmacology and Experimental Therapeutics, Loyola University School of Medicine, Maywood, IL 60153. 2OO 0163-1047/91 $3.00 Copyright© 1991 by AcademicPress, Inc. All rightsof reproductionin any form reserved.

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working/episodic memory processes (Brito, Davis, Stopp, & Stanton, 1983; Chrobak, Spates, Stackman, & Walsh, 1989; Messer, Thomas, & Hoss, 1987) would seem to support this hypothesis. Ethylcholine azirinium ion (AF64A) is a nitrogen mustard analog of choline that, within restrictive dose ranges, can be used to induce selective insult to cholinergic neurons by virtue of its interaction with high-affinity choline transport (HAChT) sites (see Hanin, Fisher, Hortnagl, Leventer, Potter, & Walsh, 1987). Administration of this compound (<3 nmol) into the lateral cerebroventricles (icv) can produce a decrease in SHC indices (Chrobak, Hanin, Schmechel, & Walsh, 1988; Gaal, Potter, Hanin, Kakucska, & Vizi, 1986; Hortnagl, Potter, & Hanin, 1987; Vickroy, Watson, Leventer, Roeske, Hanin, & Yamamura, 1986) without concomitant changes in a number of other neurochemical markers (for review see Hanin et al., 1987; Hanin, 1990) nor cholinergic indices within the cortex or striatum. The regional specificity of this treatment would appear to be a consequence of a limited distribution to periventricular targets and/or the select vulnerability of SHC neurons to this insult. Intraventricular administration of AF64A produces dose-dependent behavioral impairments in cognitive performance that are associated with persistent decreases in hippocampal cholinergic indices (Chrobak, Hanin, & Walsh, 1987; Chrobak et al., 1988, 1989; Gower, Rousseau, Jamsin, Gobert, Hanin, & Wulfert, 1989; Jarrard, Kant, Meyerhoff, & Levy, 1984; Walsh, Tilson, DeHaven, Mailman, Fisher, & Hanin, 1984). The cholinospecificity of these neurochemical and behavioral alterations has been supported by findings demonstrating that they can be attenuated or blocked by preadministration of the potent HAChT inhibitor hemicholinium-3 (Chrobak et al., 1989; Potter, Tedford, Kindel, & Hanin, 1987), which prevents the interaction between AF64A and its target, the HAChT site. We have reported that a marked and long-term (>90 days) impairment in radial arm maze (RAM) performance occurs following icv administration of 3 nmol/side of AF64A (Chrobak et al., 1988). The performance of AF64A-treated rats in that study remained at chance levels, despite repeated daily testing, without any evidence of behavioral recovery over the course of 10 weeks of postoperative testing. The present study was designed to examine whether dose-related impairments in RAM performance would be observed following the administration of lower doses of AF64A. In particular, more subtle memory impairments, which might result from a more moderate insult, may produce a more suitable baseline for evaluating therapeutic pharmacological strategies designed to reverse or attenuate the AF64A-induced memory impairment. MATERIALS AND METHODS

Subjects Thirty male Sprague-Dawley rats (Charles River Breeders, Wilmington, MA) 120 days old and weighing between 250 and 300 g were used

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in the following experiment. The animals were individually housed and maintained in a colony room with a 12-h light/dark cycle. Water was freely available at all times. Beginning 5 days prior to behavioral training, daily food rations were limited in order to reduce the animals to 85% of their free-feeding weight. During preoperative training animals were allowed to gain 5 g of body weight per week. Food deprivation was maintained throughout testing, except for 7 days immediately prior to and following surgical procedures.

Preparation of Ethylcholine Aziridinium Ion AF64A was prepared from 10 mM acetylethylcholine mustard hydrochloride (RBI, Natick, MA) by dissolving the starting material in distilled water and adjusting the pH to 11.3-11.5 with concentrated NaOH. After the solution was stirred for 20 min at room temperature, the pH was lowered to 7.3-7.5 using HC1. For intraventricular administration of AF64A, solutions were diluted with artificial CSF and administered at a concentration of 1 nmol//xl. Solutions of AF64A were always prepared immediately prior to use (within 5 h) and kept on ice at all times.

Intraventricular Injection of AF64A Six days following preoperative behavioral training, animals were anesthetized with sodium pentobarbital (50 mg/kg) and positioned in a stereotaxic instrument. Blunt ear bars were used to minimize damage to the auditory meatus. A sagittal incision was made in the scalp, and two holes were drilled through the skull for placement of the injection cannula into the lateral ventricles. Rats received bilateral infusions into the lateral ventricles of either artificial CSF (3 /xl/side; N's = 10) or AF64A (1.5 or 0.75 nmol in a total of 3/xl/side; N's = 10) at the following coordinates: AP - 0 . 6 mm from bregma, ML 1.8 mm from the sagittal suture, and 2.7 mm vertical from dura (Pellegrino, Pellegrino, & Cushman, 1967). Artificial CSF preparation was as previously described (Chrobak et al., 1989). Solutions were infused unilaterally (1.0 /xl/min) into both sites using a 10-/xl Hamilton syringe positioned in a Kopf microinjector unit mounted on the stereotaxic frame. To promote the local diffusion of the perfusate, the injection cannula was left in place for a period of 2 min following each site injection. One animal in the AF64A (0.75 nmol) group died during the surgical procedure as a result of anesthetic overdose. Thus this group consisted of nine animals. Sixteen additional animals that received no behavioral training were used for biochemical analysis at a 21-day time point.

Radial Arm Maze Training and Testing Training and testing of animals in the radial arm maze were as previously described (Chrobak et al., 1989). Briefly, animals were habituated to the

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maze apparatus and food pellets (94 mg, Noyes) for 5 days. Following habituation, rats were trained on a standard RAM task. In this task the rat was placed in the center of an eight-arm radial maze and was allowed to visit all eight arms, which were baited with a single food pellet. Reentry into an arm previously visited within any daily trial was scored as an error. Following acquisition (15 standard trials), rats were trained to perform this task with a 1-h delay imposed between the fourth and fifth arm choices (15 additional trials). Plexiglas barriers were used to prevent access to four arms during a predelay session. The set of arms chosen for each daily session varied quasirandomly. The arrangement of four adjacent arms and four alternate arms (a plus configuration) was not utilized. Typically, animals chose the four available arms in four choices; the occurrence of an error during the predelay session was less than one error in five trials. Following the delay, the animals were returned to the maze and allowed to choose among all eight arms. Only those arms not visited during the predelay session contained food pellets; thus, the animal is reinforced for nonmatching to the predelay sample set. Rats were allowed to choose until the four remaining baited arms were visited or until a total of 10 postdelay choices were made. Entry into an arm visited during the predelay session (retroactive error, retroactive to the delay) or reentry into a postdelay chosen arm (proactive error) constituted a postdelay error. The number of correct choices (CC, 0-4), postdelay errors (PDE, 0-10), retroactive errors (0-4), proactive errors (0-9), and latency per arm choice served as dependent measures. Following acquisition of the delay task, rats were allowed 1 week before surgery to regain body weight. At this point animals were treated with AF64A or artificial CSF as described. One week following surgery, behavioral testing resumed. Animals were initially tested for 16 postoperative sessions with delay intervals of 0 (standard RAM task, 4 trials), 1 (6 trials), or 2 (6 trials) h imposed between the fourth and fifth arm choice; the sequence of delay intervals chosen was determined quasirandomly. Following this initial testing, these animals were used to examine the effects of a proprietary experimental pharmacological agent (BurroughsWelcome) on reversing the memory impairments associated with an AF64A-induced deficit. Over the course of this testing period, a substantial improvement in the performance of the AF64A-treated rats that was not related to the drug manipulation was observed; i.e., it occurred in drugand saline-treated animals. Following the completion of drug testing (4 weeks; 50 days postsurgery), animals were tested to examine whether the imposition of longer delay intervals would affect their performance. Beginning at 60 days postsurgery, animals were tested for an additional 14 trials with delay intervals of 1, 2, 4, 8, or 12 h imposed between the fourth and fifth maze selection.

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High-Affinity Choline Transport Assay Animals were sacrificed by decapitation 21 days following surgery. Brains were rapidly removed over ice, and the brain was then blocked at the level of the posterior septum. Visual inspection of the fimbriafornix was made, and the anterior section was saved-for standard histological examination of the region surrounding the injection site. The hippocampus was dissected from the posterior region and assayed for HAChT. Briefly, samples were homogenized on ice in 20 vol of 0.32 M sucrose using hand-held Potter-Elvehjem grinders. Homogenates were then centrifuged at 1000g for 10 min (4°C). The supernatant was then recentrifuged at 20,000g for 20 rain and the resulting P2 pellet was resuspended in the original volume of sucrose. Triplicate aliquots (50/zl) of this suspension were then added to 450 pJ of ice-cold buffer containing 0.2 /~M choline, 0.25 /zCi [3H]choline (NEN, 80.0 Ci/mmol), 126 mM NaC1, 9.6 mM KC1, 4.2 mM MgSO4, 2.4 mM CaC12-H20, 252 mM dextrose, and 40.0 mM Tris base. After incubation for 4 min at 37°C, tubes were placed on ice and quenched immediately with the addition of 4 ml of ice-cold buffer. Tissue was collected on glass fiber filter (Gelman type A / E ) by vacuum filtration. After washing twice with 4 ml of icecold buffer, the filters were placed in vials and 10 ml of scintillation fluid was added. Twenty-four hours following the addition of scintillation fluid, vials were assayed for radioactivity in a Packard Tri-Carb (4530) counter. HAChT was defined as that occurring at 37°C, minus that at 4°C, and is expressed as picomol/4 min/mg tissue. Tissue protein content was determined according to the procedure of Bradford (1976), using bovine serum albumin as the reference standard.

Statistics RAM indices were evaluated with multifactor (treatment × delay) analyses of variance for split-plot factorial designs. Significant treatment x delay interactions were further evaluated for simple main effects followed by Spjotvall-Stoline t' tests (Kirk, 1982). Biochemical measures were evaluated with independent t tests. RESULTS Performance on the RAM task prior to surgery was comparable in both the CSF-treated and the AF64A-treated groups. Statistical analyses on the number of correct choices in the first four postdelay choices (CC) and the number of postdelay errors (PDE) for the last five preoperative trials revealed no significant group effect, trials effect, or group by trials interaction (p's < 0.50), indicating that all groups were performing with comparable proficiency prior to surgery. Upon the resumption of testing, CSF-treated rats performed as accurately as prior to surgery, making 3.3-

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M E M O R Y I M P A I R M E N T IN A F 6 4 A - T R E A T E D RATS [] [] []

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FIG. l. The effects of AF64A (1.5 or 0.75 nmol/side/icv) on the performance of a R A M task with varying delays (hours) imposed between the fourth and fifth arm selections. Data presented are for the first 16 postoperative trials. This figure presents the number of correct choices (CC, left) made in the first four postdelay choices and the number of postdelay errors (PDE, right). Data are presented as means m SEM. (*p < .05, **p < .01 vs CSF controls at the same delay interval, Spjotvall-Stoline t test.)

3.5 CC and 0.6-1.2 PDE. Further, their performance was unaffected by the imposition of delay intervals less than 8 h (see Figs. 1 and 2). We have observed that groups trained to perform this D N M T S - R A M task with the present protocol are typically unaffected by the imposition of delay intervals of less than 8 to 12 h, although individual animals vary considerably. Systematic training at longer delays can result in improved ability to perform at much longer delays (see Beatty and Shavalia, 1980). Postoperatively, AF64A-treated rats demonstrated a significant ira4.0-

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FIG. 2. The effects of AF64A (1.5 or 0.75 nmol/side/icv) on the performance of a R A M task with varying delay intervals (hours) imposed between the fourth and fifth maze selections. Data presented are for the last 14 postoperative trials, which began 60 days postsurgery. This figure presents the number of correct choices (CC). Data are presented as means + SEM. No significant differences were observed.

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pairment in the performance of this task compared to CSF-treated animals during the initial 3-week testing period. A two-way R M A N O V A on the number of CC and PDE indicated significant treatment effects [(CC), F(2, 26) = 5.35, p > .0114; (PDE) F(2, 26) = 5.1, p > .038], delay effects [(CC), F(2, 52) = 14.53, p < .0001; (PDE) F(2, 52) = 15.4, p < .0001], and treatment x delay interactions [(CC) F(4, 52) = 3.26, p -- .018; (PDE) F(4, 52) = 2.87, p = .032; Fig. 1]. Tests for significant simple main effects followed by post hoc comparisons indicated a significant impairment in the number of CC in the 1.5-nmol group at the 1-h (q'T'(2, 78) = 4.05, p < .05) and 2-h (q'T'(2, 78) = 4.58, p < .01) delay interval, as well as in the 0.75-nmol group at a 2-h delay (q'T'(2, 78) = 3.87, p < .05) compared to the CSF-treated group at the same delay interval. Similarly, significant increases in the number of PDE in the 1.5-nmol group at the 1-h (q'T'(2, 78) = 3.62, p < .05) and 2-h (q'T'(2, 78) = 5.13, p < .01) delay interval, and at a 2-h delay in the 0.75-nmol group (q'T'(2, 78) = 4.46, p < .01), were observed. It should be noted that during the drug testing both AF64A-treated groups demonstrated significant decreases in the number of CC and decreases in PDE when a 4-h delay was imposed between the fourth and the fifth arm selections (data not shown). Thus, both groups of AF64A-treated rats were moderately impaired on both measures of R A M performance, and these impairments varied as a function of the delay interval imposed between the fourth and fifth arm choice. Analysis of the postdelay errors indicated that the impairment in postdelay performance was attributable to an increase in retroactive errors (entry into arms chosen during the predelay session) as opposed to proacrive errors (entry into arms chosen during the postdelay session). Twoway A N O V A on the number of retroactive errors indicated a significant treatment [F(2, 26) = 4.46, p = .021], delay [F(2, 52) = 18.41, p < .0001], and treatment x delay interaction [F(4, 52) = 4.41, p = .0042]. Tests for simple main effects followed by post hoc comparisons indicated a significant increase in the number of retroactive errors in both groups of AF64A-treated rats at the 2-h delay interval [(q'T'(2, 78) > 4.79, p < .01); Table 1]. No significant treatment [F(2, 26) = 2.51, p = .10] or delay effects [F(2, 52) = 2.67, p = .08) were observed in the number of proactive errors (Table 1). No significant difference in the AF64A-treated animal's latency per choice to perform the R A M task was observed (Table 1). A two-way A N O V A on this measure indicated no treatment effect [F(2, 26) = 0.5], a significant delay effect [F(2, 26) = 15.22, p < .0001], and no significant delay × treatment interaction [F(4, 52) = 1.6, p = .19]. Thus, while rats performed the maze task more rapidly as the retention interval increased, this effect did not vary as a function of treatment.

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TABLE 1 T h e Effect of Bilateral Intraventricular Administration of A F 6 4 A (1.5 or 0.75 n m o l / s i d e / i c v on Postdelay Indices of Radial A r m Maze Performance at 0-, 1-, and 2-h Retention Intervals No delay

1-h delay

2-h delay

Retroactive errors CSF 0.75 1.5

0.73 + 0.14 0.61 + 0.17 1.00 + 0.22

1.03 + 0.17 0.96 + 0.12 1.62 + 0.16 ns

0.87 + 0.13 1.83 + 0.29* 1.90 + 0.28*

Proactive errors CSF 0.75 1.5

0.05 + 0.03 0.19 + 0.11 0.25 + 0.11

0.12 + 0.04 0.11 + 0.04 0.38 + 0.07

0.26 + 0.12 0.22 + 0.07 0.42 + 0.16

Latency per choice (s) CSF 0.75 1.5

10.7 10.1 12.5

+ 0.8 + 0.4 + 1.4

10.8 8.5 8.1

+ 2.3 + 0.6 + 0.9

7.5 6.6 6.9

+ 1.3 + 0.5 + 0.7

* p < .05 compared to CSF group at the same delay interval.

The AF64A-treated groups demonstrated significant improvement in their ability to perform this task with repeated testing (see Fig. 2). Although the performance of the CSF-treated group was superior to that of either AF64A-treated group on both measures at all delay intervals during this testing period, a two-way R M A N O V A on the final postoperative trials indicated no significant treatment effect on the number of CC and PDE [F's(2, 26) < 1.0, p's = .4], a significant delay effect [F 's(4, 104) > 14.0, p's < .001], and no treatment by delay interaction IF's(8, 104) < 1.0, p's > .4]. Thus, all groups were equally affected by the longer retention intervals. Histological examination of the posterior septal region indicated that the fimbria-fornix was intact in all animals. Several animals in the 1.5nmol group exhibited ventricular dilatation, typically unilaterally, in the dorsal aspect of the ventricle. As opposed to our previous observations at a 3-nmol dose (Chrobak et al., 1988, 1989), no apparent tissue necrosis was observed in areas adjacent to the ventricular space. Neurochemical analysis of separate animals receiving bilateral administration of AF64A and sacrificed 21 days postoperatively demonstrated a significant dose-related decrease in HAChT (see Table 2, compared to CSF controls) in both groups: 26% in the 1.5-nmoi group IT(9) - 2.96, p --- .008] and 10% in the 0.75-nmol group [T(10) = 2.56, p = .014]. HAChT in the 1.5-nmol group was also significantly different from that in the 0.75-nmol group [T(9) = 1.87, p = .05].

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CHROBAK AND WALSH TABLE 2 The Effect of Bilateral Intraventricular A d m i n istration of A F 6 4 A (1.5 or 0.75 nmol/side/icv) on High-Affinity Choline Transport in the Hippocampus 21 days Postinfusion CSF 20.4 _+ 0.6 n=5

0.75 nmol

1.5 nmo]

18.4 -+ 0.5* n=6

15.1 -+ 1.4"* n=6

Note. Values represent m e a n s + S E M expressed as pmol c h o l i n e / m g protein/4 min. * p < .05, ** p < .01 vs CSF controls, independent t tests.

DISCUSSION Rats treated with doses of AF64A (1.5 and 0.75 nmol/side/icv), which produces modest reductions in HAChT (<30%), showed a dose-dependent impairment in RAM performance. AF64A-treated animals, depending upon the length of the retention interval, made significantly fewer correct choices during the postdelay session and significantly more errors to complete the postdelay task. This impairment was associated with an inability to respond discriminantly during the postdelay session to arms chosen prior to the delay interval, as evidenced by a significant increase in retroactive errors and no change in proactive errors. Despite an initial impairment in the performance of the RAM task, AF64A-treated rats improved dramatically during the course of postoperative testing. Although the absolute performance levels in the AF64A-treated group never matched those of CSF-treated controls, these animals recovered their ability to perform this task when considerable delay intervals (4-8 h) were imposed between arm choices. Marked disruptions during the last 14 trials were not observed in AF64A-treated animals until the imposition of delay intervals that significantly impaired control performance. The performance deficit induced by these doses of AF64A could be characterized by an inability to maintain representations of previous choice events over extended delay intervals. This deficit is thus reflected in an inability to perform accurately when longer delays were inserted between the fourth and fifth choice. Analysis of animals' performance during the predelay session, during the standard RAM trials, and during the postdelay session in which no increase in proactive errors were observed indicates that the rats were capable of avoiding previously entered arms as long as the retention interval was on the order of seconds or minutes. These findings provide further evidence that disruption of hippocampal neurophysiology, following chronic insult to this structure or insult to cholinergic afferents, produces a deficit that can be specifically related to mnemonic function.

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These data also indicate that substantial behavioral recovery can occur following an AF64A-induced insult. These results differ from our previous observations that provided no evidence of behavioral recovery in AF64Atreated rats at a 3 nmol/side dose (Chrobak et al., 1987, 1988). In contrast, previous findings suggested that these animals tend to adopt alternate performance strategies that preclude accurate performance of working/episodic tasks. Evidence indicating behavioral recovery, as well as anatomical and neurochemical recovery, following electrolytic or chemical lesions of the medial septum or fimbria-fornix, which disrupt the septohippocampal projection and result in a decrease in hippocampal cholinergic parameters, has been reported (Gage, Bjorklund, Stenevi, & Dunnett, 1983; Oderfeld-Nowak, Potempska, & Oderfeld, 1977). Although time-dependent alterations in HAChT were not assessed in the present study, similar doses (1, 2, or 3 nmol) of AF64A have been shown to produce significant reductions in ACh levels with maximal decreases occurring at the latest time point sampled (28 days) (Hortnagl, Potter, & Hanin, 1988). Leventer, McKeag, Clancy, Wulfert, and Hanin (1987) have reported sustained decreases in HAChT following icv administration of 3 nmol for up to i year postadministration. Recent observations indicate that a recovery in presynaptic cholinergic indices can occur following doses less than 1 nmol within 3-6 months following treatment (El-Tamer, Corey, Wulfert, & Hanin, in press). The possibility that such changes may have functional relevance to the observed behavioral phenomenon awaits further investigation. It appears that recovery of cholinergic tone and cognitive function can occur following AF64A-induced insult to the SHC system; further studies will be required to elucidate the relationship between these phenomena. While subtle impairments in memory performance may be experimentally useful for further characterizing the nature of the mnemonic impairment, changes in baseline performance on the RAM task utilized make it difficult to evaluate the acute effects of agents to attenuate AF64Ainduced cognitive impairments at these doses of AF64A. Several studies, however, have reported that cholinomimetic agents can reverse or attenuate AF64A-induced cognitive impairments in a variety of tasks (Ogura, Yamanishi, & Yamatsu, 1987; Nakahara, Iga, Mizobe, & Kawanishi, 1988; Speiser, Reicher, Gitter, & Cohen, 1989; Brandeis, Dachir, Sapir, Levy, & Fisher, 1990). To date, we have not observed any acute or subchronic treatment with a cholinomimetic compound to significantly attenuate the AF64A-induced deficit in DNMTS-RAM performance observed following a 3-nmol dose of AF64A (unpublished observations). However, treatment with the neurotrophic agent AGF2 can induce behavioral recovery in the standard (nondelayed) version of the task (Emerich & Walsh, 1990) following a 3-nmol dose of AF64A. Our ability to evaluate such treatment in animals treated with lower doses would be confounded by the spon-

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taneous recovery reported. At present, we are examining methods for inducing a progressive insult to the SHC system using AF64A: however, the regenerative capacity of these neural circuits and the factors that influence recovery are critical areas of investigation. The present findings indicate that dose-dependent impairments in working/episodic memory can be induced by graded doses of the cholinotoxin AF64A. Such deficits may be a consequence of graded alterations in the functional activity of the SHC system. Analysis of SHC and other neurochemical systems in animals' behavior following intraventricular AF64A treatment could be utilized to further address this issue as well as to examine the relationship between cholinergic neuronal activity and mnemonic performance. REFERENCES Beatty, W. W., & Shavalia, D. A. (1980). Rat spatial memory: resistance to retroactive interference at long retention intervals. Animal Learning and Behavior, 8, 550-557. Bradford, M. M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248-254. Brandeis, R., Dachir, S., Sapir, M., Levy, A., & Fisher, A. (1990). Reversal of age-related cognitive impairments by an ml cholinergic agonist, AF102B. Pharmacology, Biochemistry, and Behavior, 36, 89-95. Brito, G. N. O., Davis, B. J., Stopp, L. C., & Stanton, M. E. (1983). Memory and the septo-hippocampal cholinergic system in the rat. Psychopharmacology, 81, 315-320. Chrobak, J. J., Hanin, I., & Walsh, T. J. (1987). AF64A (ethylcholine aziridinium ion), a cholinergic neurotoxin, selectively impairs working memory in a multiple component T-maze task. Brain Research, 414, 14-21. Chrobak, J. J., Hanin, I., Schmechel, D. E., & Walsh, T. J. (1988). AF64A-induced working memory impairment: behavioral, neurochemical and histological correlates. Brain Research, 463, 107-117. Chrobak, J. J., Spates, M., Stackman, R. W., & Walsh, T. J. (1989). Hemicholinium-3 prevents the working memory impairments and the cholinergic hypofunction induced by ethylcholine aziridinium ion (AF64A), Brain Research, 504, 269-275. Davies, P., & Maloney, A. J. F. (1976). Selective loss of central cholinergic neurons in Alzheimer's disease. Lancet, 1403. El-Tamer, A., Corey, J., Wulfert, E., & Hanin, I. (1991). Reversible cholinergic changes induced by AF64A in rat hippocampus and possible septal compensatory effect. Neuropharmacology, in press. Emerich, D. F., & Walsh, T. J. (1990). Ganglioside AGF2 promotes task-specific recovery and attenuates the cholinergic hypofunction induced by AF64A. Brain Research, 527, 299-307. Gaal, Gy., Potter, P. E., Hanin, I., Kakucska, I., & Vizi, E. S. (1986). Effects of intracerebroventricular AF64A administration on cholinergic, serotonergic and catecholaminergic circuitry in rat dorsal hippocampus. Neuroscience, 19, 1197-1205. Gage, F. H., Bjorklund, A., Stenevi, U., & Dunnett, B. (1983). Functional correlates of compensatory collateral sprouting by aminergic and cholinergic afferents in the hippocampal formation. Brain Research, 268, 39-47. Gower, A. J., Rousseau, D., Jamsin, P., Gobert, J., Hanin, I., & Wulfert, E. (1989).

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