Behavioral Neuroscience 2013, Vol. 127, No. 5, 619-627

© 2013 American Psychological Association 0735-7O44/13/$12.O0 DOI: 10.1037/a0033939

BEHAVIORAL NEUROSCIENCE AT 30: REPRINTED ARTICLE

Selective Immunotoxic Lesions of Basal Forebrain Cholinergic Cells: Effects on Learning and Memory in Rats Mark G. Baxter and David J. Bucci

Linda K. Gorman

University of North Carolina at Chapel Hill

Johns Hopkins University

Ronald G. Wiley

Michela Gallagher

Department of Veterans Affairs Medical Center and Vanderbilt University

University of North Carolina at Chapel Hill

Male Long-Evans rats were given injections of either 192 IgG-saporin, an apparently selective toxin for basal forebrain cholinergic neurons (LES), or vehicle (CON) into either the medial septum and vertical limb of the diagonal band (MS/VDB) or bilaterally into the nucleus basalis magnocellularis and substantia innominata (nBM/SI). Place discrimination in the Morris water maze assessed spatial learning, and a trial-unique matching-to-place task in the water maze assessed memory for place information over varying delays. MS/VDB-LES and nBM/SI-LES rats were not impaired relative to CON rats in acquisition of the place discrimination, but were mildly impaired relative to CON rats in performance of the memory task even at the shortest delay, suggesting a nonmnemonic deficit. These results contrast with effects of less selective lesions, which have been taken to support a role for basal forebrain cholinergic neurons in learning and memory.

The rat basal forebrain contains a continuum of magnocellular cholinergic neurons that provide the major source of cholinergic innervation of the cerebral cortex, hippocampus, and amygdala (Bigl, Woolf, & Butcher, 1982; Mesulam, Mufson, Wainer, & Levey, 1983). Although the specific nomenclature and subdivision of the basal forebrain cholinergic system remain a matter of debate, several populations of neurons are distinguished by their efferent targets. The medial septum (MS) and vertical limb of the

diagonal band (VDB) contain cholinergic neurons that innervate the hippocampus and cingulate/infralimbic cortices (Amaral & Kurz, 1985; Wainer & Mesulam, 1990). The nucleus basalis magnocellularis (nBM) and substantia innominata (SI) contain cholinergic cells that provide widespread innervation of the neocortical mantle and amygdala (Heckers & Mesulam, 1994; Wainer & Mesulam, 1990). The loss of cholinergic neurons in the basal forebrain of patients with Alzheimer's disease (Davies & Maloney, 1976) has informed nearly 20 years of research on the "cholinergic hypothesis" of dementia and learning and memory (Bartus, Dean, Beer, & Lippa, 1982; Olton, Givens, Markowska, Shapiro, & Golski, 1991). This hypothesis specifies that the basal forebrain cholinergic system (BFCS) plays a central role in normal learning and memory and that pathological states involving mnemonic dysfunction are due to loss or dysfunction of neurons in the BFCS. Support for this hypothesis comes from a variety of studies showing consistent loss of cholinergic markers in Alzheimer's disease (CoUerton, 1986; Coyle, Price, & DeLong, 1983; Quirion et al., 1986), as well as deficits in learning and memory induced by administration of anticholinergic drugs, or by lesions of the basal forebrain (Caine, Weingartner, Ludlow, Cudahy, & Wehry, 1981; Drachman & Leavitt, 1974; Hepler, Olton, Wenk, & Coyle, 1985; Kesner, 1988; Stevens, 1981; Wenk, Markowska, & Olton, 1989). Recently, this hypothesis has been criticized on the grounds that loss of cholinergic neurons may not result in dementia, and basal forebrain lesions, which remove noncholinergic cells as well as cholinergic netirons, produce behavioral deficits that do not necessadly cor-

Mark G. Baxter and David J. Bucci, Curriculum in Neurobiology, University of North Carolina at Chapel Hill; Linda K. Gorman, Department of Anesthesiology and Critical Care Medicine. Johns Hopkins University; Ronald G. Wiley, Neurology Service-127, Department of Veterans Affairs Medical Center, and Vanderbilt University; Michela Gallagher, Department of Psychology, University of North Carolina at Chapel Hill. This study was supported by National Institute on Aging Grant POlAG09973 and by a National Science Foundation Predoctoral Fellowship. Portions of the research reported in this article have appeared elsewhere in preliminary form (Baxter et al., 1994; Gallagher, Gill, Baxter, & Bucci, 1994). We thank W. Bass, C. Cahill, and L. Watterson for assistance with water maze testing and data management; A. Chiba and L. Thai for assistance with surgery; and M. Conley for assistance with immunocytochemistry. Correspondence concerning this article should be addressed to Mark G. Baxter, Curriculum in Neurobiology, Campus Box 7320, University of North Carolina, Chapel Hill, NC 27599. Electronic mail may be sent via Internet to [email protected] This article is reprinted from Behavioral Neurocience, 1995, Vol. 109, No. 4, 714-722.

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relate with the degree of cholinergic loss (Fibiger, 1991; Wenk, Harrington, Tucker, Ranee, & Walker, 1992). A more direct investigation of the role of the BFCS in leaming and memory has been made possible by the development of an apparently selective lesioning technique that appears to remove only cholinergic neurons in the basal forebrain. 192 IgG-saporin, an immunotoxin composed of a monoclonal antibody to the "lowaffinity" p75 nerve growth factor (NGF) receptor coupled to a ribosome-inactivating cytotoxin, saporin (Wiley, Oeltmann, & Lappi, 1991), produces apparently selective lesions of basal forebrain cholinergic neurons when infused directly into basal forebrain nuclei (Heckers et al, 1994). Of the few studies that have examined the behavioral effects of this toxin, the first one found a deficit in a place discrimination task using the Morris (1981) water maze after intraventricular administration of 192 IgG-saporin (Nilsson et al., 1992). However, the interpretation of these results as specific to spatial leaming and memory is questionable; a subsequent study noted disruption of both place leaming and a nonspatial cue-guided task following intracerebroventricular infusions of the immunotoxin (Berger-Sweeney et al., 1994). Nonspecific behavioral impairment may be due to the cerebellar damage that occurs following intracerebroventricular infusions of 192 IgGsaporin (Heckers et al., 1994). However, direct infusions of 192 IgG-saporin into basal forebrain nuclei do not result in cerebellar damage (Heckers et al, 1994). Basal forebrain lesions produced by these direct infusions do not affect acquisition or retention of a passive avoidance response or spatial altemation in a T maze (Wenk, Stoehr, Quintana, Mobley, & Wiley, 1994) and produce only mild performance deficits in spatial leaming in the Morris water maze (Berger-Sweeney et al, 1994). As a further extension of these findings, the present study examined the effects of these lesions on place leaming in the Morris water maze, in a testing protocol sensitive to the effects of aging in rats (Gallagher, Burwell, & Burchinal, 1993), thus permitting direct comparison of the effects of these lesions in young animals to the effects of aging on spatial cognition. The BFCS, particularly the medial septal area, is also involved in working memory function (Givens & Olton, 1990, 1994). Therefore, we also examined the effects of these lesions on performance of a matching-to-place task in the Morris water maze, in which rats were tested for memory of novel locations over varying delays.

Method

sterile phosphate-buffered saline was microinjected into control rats, whereas 192 IgG-saporin (provided by Ronald Wiley) was microinjected into lesion rats at a concentration of 0.375 |J-g/|Jil of sterile phosphate-buffered saline. The general surgical procedure was identical for all groups. All surgical procedures were conducted under pentobarbital (Nembutal, 55 mg/kg ip) anesthesia, supplemented with additional injections of Nembutal or administration of methoxyfiurane (Metofane) inhalation anesthetic as needed during surgery. The anesthetized rat was placed in a stereotaxic apparatus (Kopf Instruments, Tujunga, CA), an incision was made to expose the skull, and the skin was retracted. After surgery, the wound was cleaned and closed with sterile wound clips. Sham surgery. After the skull was exposed, the skin was closed with sterile wound clips. No other surgical procedures were performed. MS/VDB surgeries. Two holes were drilled in the skull at stereotaxic coordinates AP = 4-0.45 mm and ML = ± 0.6 mm from bregma according to the Paxinos and Watson (1986) brain atlas. Injections were made at two depths for each site: one at DV = -7.8 mm and one at DV = -6.2 mm from the skull surface measured at bregma. A 28-gauge Hamilton syringe filled with either sterile phosphate-buffered saline (control surgeries) or 192 IgG-saporin (lesion surgeries) was lowered into the desired location and left in place 30 s before beginning the infusion. Saline or immunotoxin was slowly infused (0.05 ixl/min) into the injection site. A total volume of 0.3 |JL1 was infused into the sites at DV = -7.8 mm, and a total volume of 0.2 |jil was infused into the sites at DV = —6.2 mm. The syringe was left in place for 9 min after each 0.3-JJL1 injection and 6 min after each 0.2-(xl injection to limit diffusion up the needle track. nBM/SI surgeries. Four holes were drilled in the skull at stereotaxic coordinates AP = —0.75 mm and ML = ± 2.3 mm (medial sites) or ML = ± 3.3 mm (lateral sites) from bregma. Injections were made at DV = —7.8 mm (medial sites) or DV = -8.1 mm (lateral sites) from the skull surface measured at bregma. A 28-gauge Hamilton syringe filled with either sterile phosphatebuffered saline (control surgeries) or 192 IgG-saporin (lesion surgeries) was lowered into the desired location and left in place for 30 s before beginning the infusion. Saline or immunotoxin was slowly infused (0.10 |jLl/min) into the injection site. A total volume of 0.2 (xl was infused into each site. The syringe was left in place for 3 min after each injection to limit diffusion up the needle track.

Subjects

Behavioral Testing

Forty-five male Long-Evans rats (Charles River, Raleigh, NC), 2-3 months old, were housed singly in the psychology department vivarium beginning 1 week prior to surgery. Food and water were available ad libitum, and the colony was maintained on a 12-h light-dark cycle (lights on at 7 a.m.). All surgical procedures and behavioral testing took place during the light phase.

The behavioral testing protocol began after 14 days of postoperative recovery. Leaming and memory were assessed in the Morris (1981) water maze. The maze was a round tank, 1.81 m in diameter and 58 cm deep, filled to a depth of 35.5 cm with tepid (27° C) water made opaque by the addition of white tempera paint. A retractable circular platform, 12 cm in diameter, was located 1 cm below the surface of the water. When retracted, the platform was at the bottom of the pool, 30 cm below the surface of the water, unavailable for escape. The maze was surrounded by white curtains, on which black cloth visual stimuli of various shapes and sizes were placed. A camera was located above the center of the maze, which relayed images to a vidéocassette recorder and an HVS Image Analysis VP-112 tracking system. Data from trials in

Surgery Each rat was randomly assigned to one of five groups: sham (n = 5), MS/VDB-control (n = 8), nBM/SI-control (n = 8), MS/VDB-lesioned (n = 12), or nBM/SI-lesioned (n = 12). Lesion and control surgeries were identical in every respect, except that

MNEMONIC EFFECTS OF BASAL FOREBRAIN IMMUNOLESIONS the water maze were analyzed with software written by Richard Baker (HVS, Hampton, United Kingdom). Behavioral testing consisted of four phases, which took place in the following order: habituation, place training, cue training, and delayed match-to-place memory testing. Animals were handled daily for 3 days before beginning behavioral testing. Habituation. One habituation trial took place on the first day. The platform was retracted and the rat was allowed to swim in the maze for 60 s, after which time the rat was removed from the pool. Place training. One session took place each day for 8 consecutive days, with three trials in each session, beginning tiie day after habituation. The platform was in a constant location in the soutiieast quadrant of the pool, 43 cm from the wall of the tank. On each training trial, the rat was lowered by hand into the pool, facing the inside wall of the tank, at one of four pseudorandomly varied start points (such that the same start point was not used twice in one session) spaced equally around the rim of the tank. Ninety seconds were allowed for the rat to reach tiie platform; if the rat did not reach the platform in this time, it was led there by the experimenter. The rat was allowed to remain on the platform for 15 s, at which time it was removed from the pool and returned to a holding cage lined with paper towels for a 30-s intertriai interval. Every sixth trial was a probe trial, during which the platform was reti-acted and unavailable for escape for the first 30 s of the trial; after this time it was raised and made available for escape. The probe tiials were in all other ways identical to training trials. These interpolated probe trials were used to assess the development of an accurate search strategy. Cue traiuing. One session with six trials took place after the last day of place training. The submerged platform was replaced with a visible platform, with a black surface extending 2 cm above the surface of the water. The platform was moved to different locations in the pool between trials, in order to test visual acuity and swim ability independent of the ability to process spatial information. Each rat was given 30 s to reach the platform, and was allowed to remain there briefiy before being returned to a holding cage for 5 s before the next trial. Match-to-place memory task. This protocol began after a 4-day period with no behavioral testing following the completion of cue training. The place ti-aining platform was retracted, and a moveable platform was placed in the maze, its surface 1 cm below the surface of the water. The moveable platform was positioned in a different location in the maze for each session of testing; each location was in a different quadrant of the maze and at a different distance from the wall of the tank from the previous day ' s position. The platform never returned to the original training quadrant. Sixteen sessions took place, one per day. Two trials were given per session. The trials were otherwise identical to the training trials given during place training, except that a variable delay was imposed between the first tiial (the information trial) and the second ti-ial (the retention tiial). Four delays were used: 30 s, 5 min, 20 min, and 3 hr. The 30-s delay was used only in the first four sessions to acclimate rats to the new procedure. The subsequent 12 sessions used all four delays in a repeating cycle, moving from the shortest delay to the longest delay in successive sessions, resulting in three blocks of testing with each delay occurring once in each block.

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Neurobiological Analysis Several different neurobiological assessments were performed after the completion of behavioral testing to verify the lesions. Most rats (conti-ol, n = 17; MS/VDB-lesioned, n = 9; nBM/SIlesioned, n = 9) were decapitated and the brains microdissected and frozen. Cortex (frontal, sensorimotor, and parietal cortex pooled), hippocampus, and stiiatum were later processed for determination of choline acetyltransferase (ChAT) activity to provide a quantitative assessment of the degree to which cholinergic denervation had been achieved. The remaining rats were transcardially perfused with saline, followed by 8% formalin with 2% sucrose in 0.1 M sodium phosphate buffer (pH 7.4), followed 30 min later by 0.1 M sodium phosphate buffer (pH 7.4) with 10% sucrose and 1% dimethyl sulfoxide (DMSO). The brain was then removed from the skull, placed in 0.1 M sodium phosphate buffer (pH 7.4) witii 20% sucrose and 1% DMSO, and was then allowed to sink before slices were taken on a sliding microtome. Adjacent series of sections from this tissue were processed for acetylcholinesterase (AChE) histochemistry to map the removal of hippocampal-cortical cholinergic innervation, and immunocytochcmistry for ChAT and parvalbumin were determined at the lesion sites. Parvalbumin immunostaining is found in GABAergic neurons of the basal forebrain (Freund, 1989), permitting an assessment of potential damage to noncholinergic neurons at the lesion site. This analysis was performed on these few rats primarily to confirm the selectivity of neuron loss at the lesion site as reported by others (e.g., Heckers et al., 1994) and to provide some qualitative assessment of the neuroanatomical extent of the lesion. Radioenzymatic determination of ChAT activity. ChAT activity was determined in the left hippocampus, left cortex, and pooled left and right striata in the rats that were not processed for immunocytochemistiy and AChE histochemistry. ChAT activity was measured by formation of ["*C] acetylcholine formed from ["'CJacetylcoenzyme-A and choline (Fonnum, 1969). A 40-jj,l aliquot of homogenized tissue was combined with 10 |jil of a solution containing 2% Triton X-100 and 50 mM ethylenediaminetetiraacetic acid (pH 7.4). Incubation was carried out for 15 min at 37° C. The incubation mixture (final volume, 100 \ú) contained sodium chloride, 200 mM; sodium phosphate (pH 7.0), 50 mM; eserine, 0.075 mM; choline chloride, 6 mM; ['^'CJacetylcoenzymeA, 0.1 mM; and bovine serum albumin, 0.5 mg/ml. The newly formed ['*C]acetylcholine was exti-acted into a hydrophobic mixture containing sodium tetraphenylboron. Protein content of the homogenates was assayed by the method of Bradford (1976), with bovine serum albumin as a protein standard. AChE histochemistry. AChE histochemistry was performed essentially as described elsewhere (Kamovsky & Roots, 1964). Immunocytochemistry. Immunocytochemistry for ChAT and parvalbumin was performed by means of the avidin-biotin complex technique according to methods described elsewhere (Fitzpatiick, Conley, Luppino, Matelli, & Diamond, 1988), using monoclonal antibodies to ChAT (Boehringer Mannheim, Indianapolis, IN) or parvalbumin (Sigma Chemical, St. Louis, MD).

Analysis of Behavioral Data Several measures were used to assess accuracy of performance in the water maze. The primary measures were cumulative proximity and cumulative search error on training trials, and a learning

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index computed from the probe trials given over the course of learning. These measures rely on a computation of average distance from the platform during the trial and are described more fully elsewhere (Gallagher et al., 1993). Briefly, the rat's distance from the platform is sampled 10 times per second during the trial; these distances are averaged in 1-s bins. Cumulative search error is the sutnmed 1-s averages of the proximity measure corrected for the particular platform location and start location by subtracting the proximity score that would be produced by perfect performance on the trial; cumulative proximity is this value uncorrected for perfect performance (used only in the delayed match-to-place task). The learning index is a derived measure from average proximity (cumulative search error divided by the length of the probe trial) on the second, third, and fourth interpolated probe trials during place training; scores from these trials are weighted and summed to provide a unitary measure of spatial learning. Lower scores on the index indicate accurate search in the vicinity of the target location and hence good learning; higher scores indicate a more random search and poor learning. Two additional training trial measures that have been routinely used in this task were also evaluated. Swim time refers to the total time taken by the rat to reach the platform. Pathlength is the total distance taken by the rat to reach the platform. Swim speed, time spent in the training quadrant, and time spent in concentric zones in the maze on the habituation trial were analyzed by a one-way analysis of variance (ANOVA) to test for potential differences in swimming ability or pattern that predated spatial learning. For place training, the means of performance measures (cumulative search error, swim time, and pathlength) on blocks of training trials (Trials 1-5, 7-11, 13-17, 19-23) were analyzed by repeated-measures ANOVA, in addition to the performance during probe trials (Trials 6, 12, 18, 24) as assessed by cumulative search error (repeated-measures ANOVA), as well as the learning index calculated from average proximity on Probe Trials 2-4 (one-way ANOVA). For cue training, swim time averaged over the block of six cue training trials was analyzed (one-way ANOVA). Cumulative proximity and pathlength for the three blocks of testing at all delays were analyzed for the matching-to-place task in an overall four-way ANOVA (Lesion X Delay X Block X Trial), followed by more focused analyses (individual ANOVAs on smaller components of the overall ANOVA) to examine any significant interaction effects. Statistical analysis was performed with SuperANOVA software (Abacus Concepts, Berkeley, CA) and with SYSTAT version 5.1 (SYSTAT, Evanston, IL).

Results No differences were observed in any of the behavioral measures analyzed for sham, MS/VDB-control, or nBM/SI-control rats; therefore these groups were pooled into a single control group for statistical analysis.

Neurobiological Analysis ChAT activity. ChAT activity (nmol/hr/mg protein) for the control group and two lesion groups is presented in Table 1 (excluding 3 rats that were eliminated from analysis for reasons described later). There were significant effects of lesion on hip-

Table 1 Choline Acetyltransferase Activity (nmol/hr/mg Protein) in Control, MS/VDB-Lesioned, and nBM/Sl-Lesioned Rats Anatomical region Group

Hippocampus

Cerebral cortex

Stdatum

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74.6 ± 1.6 7.5 ± 0.4* 70.3 ± 2.8

48.0 ± 3.9 35.0 ± 4 . 1 18.0 ± 1.3*

174.6 d-.5.1 174.3 dt 13.8 179.6 it 10.1

Note. Data are means ± standard errors. MS/VDB = medial septumvertical limb of the diagonal band; nBM/SI = nucleus basalis magnocellulads-substantia innominata. * /? < .05 versus control group.

pocampal, F(2, 29) = 352.1, p < .0001, and cortical, F(2, 29) = 15.2, p < .0001, ChAT activity; no significant effect of lesion was evident for striatal ChAT activity, F(2, 28) < 1, p > .05. MS/ VDB-lesioned rats had significantly decreased hippocampal ChAT activity (p < .001, Tukey HSD) compared with control rats; decreased cortical ChAT activity approached significance (p = .06, Tukey HSD). nBM/SI-lesioned rats had normal hippocampal ChAT activity but significantly decreased cortical ChAT activity (p < .001, Tukey HSD) compared with control rats. Immunocytochemistry. Photomicrographs of ChAT immunostaining at the level of the injection sites in the septal area and substantia innominata are shown in Figure 1 for control, MS/VDB-lesioned, and nBM/SI-lesioned rats. ChAT-positive (cholinergic) neurons are apparent in both areas in control brains. A loss of ChAT-positive neurons in the septal area is seen in the MS/VDB-lesioned brain, and a loss of ChATpositive neurons in the substantia innominata is apparent in the nBM/SI-lesioned brain. Photomicrographs of parvalbumin immunocytochemistry at the level of the septal area and the substantia innominata are also shown in Figure 1 for MS/VDBlesioned and nBM/SI-lesioned rats, respectively. No loss of parvalbumin-immunoreactive (GABAergic) cells at the injection site is apparent in either lesion group. AChE histochemistry. The pattern of AChE histochemistry matched the regional depletion of cholinergic activity shown by the radioenzymatic ChAT assay: loss of staining throughout the dorsal-ventral extent of the hippocampus in the MS/VDB-lesioned rats and loss of cortical staining in the nBM/SI-lesioned rats. The MS/VDB cholinergic neurons project to cingulate cortex as well as the hippocampus (Amaral & Kurz, 1985). Consistent with this topography of MS/VDB projections, some loss of AChE staining in the cingulate cortex was seen in MS/VDB-lesioned rats. This finding is likely to account for the trend of reduced ChAT acdvity in cortex from MS/VDB-lesioned rats shown in Table 1 (as the dissection of cortex included cingulate and infralimbic regions). Similarly, some preservation of AChE staining in these areas was noted in nBM/SI-lesioned rats (as lesions of the nBM/SI would spare the VDB neurons that give rise to these cholinergic projections). Four rats were excluded from the behavioral analysis on the basis of the neurobiological analysis: 1 MS/VDB-lesioned rat with normal hippocampal ChAT activity, 1 nBM/SI-control rat with diminished cortical ChAT activity (in the range of the nBM/SIlesioned rats), 1 nBM/SI-lesioned rat with substantially preserved

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Figure 7. a-c: Photomicrographs taken at the level of the septal area, a: Choline acetyltransferase (ChAT) immunostaining in a control brain, b: Absence of ChAT-immunopositive neurons in a MS/VDB-Iesioned brain, c: Presence of parvalbumin-immunopositive neurons at the lesion site in a MS/VDB-lesioned brain, d-f: Photomicrographs taken at the level of the substantia innominata (SI), d: ChAT immunostaining in the SI in a control brain, e: Absence of ChAT-immunopositive neurons in a nBM/SI-lesioned brain, f: Presence of parvalbumin-immunopositive neurons at the lesion site in an nBM/SI-lesioned brain. All photomicrographs are at lOx magnification. MS/VDB = medial septum-vertical Umb ofthe diagonal band; nBM/SI = nucleus basalis magnocellularis-substantia innominata.

numbers of ChAT neurons in the nBM/SI (as seen by immunocytochemistry), and 1 MS/VDB-control rat that died before neurobiological analysis could be performed.

Behavioral Testing Habituation. There were no significant differences in swim speed, F{2, 37) < l,p > .05, or time spent in the training quadrant, f (2, 37) < 1, p > .05, on the habituation trial between control rats and either of the lesion groups. nBM/SI-lesioned rats spent a greater percentage of time (94.9 ± 1.6) in the outer concentric zone ofthe maze than either the MS/VDB-lesioned (86.3 ± 3.3) or control (81.9 ± 2.1) rats, F{2, 37) = 7.5, p = .002 (followed by Tukey HSD post hoc tests). This small difference in time spent close to the wall of the tank suggests that nBM/SI-lesioned rats were slightly more thigmotaxic on the habituation trial than either of the other groups. Place training. As shown in Figure 2A, all rats quickly became proficient at locating the submerged platform during the training trials by the cumulative search error measure. In addition, there were no apparent differences in performance on

the probe trials, as assessed by proximity of the search to the target location across the four probe trials (Figure 2B) or by the composite learning index (Figure 2C). There was no significant effect of lesion on the proximity measure across the four probe trials, F{2, 37) < 1, ;> > .05, or on the learning index, F(2, 37) < l,p > .05. There was a main effect of lesion on training trial performance (Figure 2, top left), F(2, 37) = 5.9,p = .0059, due to the poor performance of the nBM/SI-lesioned rats on the first block of trials relative to the MS/VDB-lesioned and control rats. Trial X Lesion interaction: F(6, 111) = 2.3, p = .0410. The pattern of results for the other two training trial measures (swim time and pathlength, data not shown) were similar (main effects of lesion were due to slightly worse performance of nBM/SI-lesioned rats on Block 1). Cue training. Time to reach the platform (seconds) during the block of cue-training trials (mean ± standard error) for the three groups was: control, 10.3 ± 1.2; MS/VDB-lesioned, 8.0 ± 0.9; nBM/SI-lesioned, 10.6 ± 1.1. There was no significant difference between the control group and either lesion group, F(2, 37) < 1, p > .05.

BAXTER, BUCCI, GORMAN, WILEY, AND GALLAGHER

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Figure 2. Place training performance for control (CON), MS/VDB-lesioned (MS/VDB), and nBM/SI-lesioned (nBM/SI) rats. A: cumulative search error during blocks of training trials. B: cumulative search error during probe trials. The cumulative search error for the first 30 s of the habituation (Hab.) trial is included for comparison to indicate the learning that takes place before the first probe trial. C: spatial learning index computed from probe trial performance during place training. Data are the mean from each block of training trials; error bars are one standard error of the mean. MS/VDB = medial septum-vertical limb of the diagonal band; nBM/SI = nucleus basalis magnocellularis-substantia innominata.

Match-to-place memory tests. Performance during memory testing (cumulative proximity) is shown in Figure 3. It is evident that performance in the information trial was similar for all three groups and that the control rats exhibited substantial and relatively stable retention across all four delays. Both lesion groups were impaired relative to the control group across delays; this effect was not obviously delay dependent. The overall ANOVAs revealed highly significant Trial X Lesion interactions; pathlength, F(2, 37) = 9.9, p = .004; cumulative proximity, F(2, 37) = 9.7, p = .004. Focused analysis revealed no effect of lesion on measures of performance on Trial 1, both measures, F(2, 37) < 1, p > .05, but significant main effects of lesion on measures of performance on Trial 2, pathlength, F(2, 37) = 8.1, p = .0012; cumulative proximity, F(2, 37) = 8.3, p = .001. Post hoc analysis on this between-groups effect on Trial 2 performance (Newman-Keuls tests) revealed no differences in performance between MS/VDBlesioned and nBM/SI-lesioned rats but significant (p < .05) differences between each of the lesion groups and control rats. The lesion effect on Trial 2 performance did not interact with delay for either measure, pathlength, F(6, U l ) = 1.6, ;? > .05; cumulative proximity, F(6, 111) = 1.8, p > .05.

Discussion The present study examined the effects of apparently selective removal of cholinergic input to either hippocampus (MS/VDBlesioned) or cortex (nBM/SI-lesioned) on spatial learning and memory assessed in the water maze. Acquisition of the spatial learning task was largely unaffected by either lesion; both lesions produced minor deficits in the match-to-place memory task, but these effects were not delay dependent. Many other studies have examined the effects of medial septal lesions on learning and memory. For example, ibotenic acid lesions of the medial septal area, which decrease hippocampal ChAT activity by 54%-66%, produce deficits in spatial learning (Hagan, Salamone, Simpson, Iversen, & Morris, 1988). The medial septal lesions in this study produced a 90% reduction in hippocampal ChAT activity but no deficit in spatial learning. This result strongly suggests that selective removal of the cholinergic innervation of the hippocampus is not sufficient to impair spatial learning in the water maze. It is possible that learning and memory deficits observed after other MS/VDB lesions are the result of damage to noncholinergic neurons either alone or in combination with removal of cholinergic inputs.

MNEMONIC EFFECTS OF BASAL FOREBRAIN IMMUNOLESIONS

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Trial 1 30 sec 5min 20min 3hr Trial 2 - Delay Figure 3. Match-to-place memory-testing performance (cumulative proximity) for control (CON), MS/VDB-lesioned (MS/VDB), and nBM/ Sl-lesioned (nBM/SI) rats. Data shown are the means of Trial 1 performance across all 12 sessions of testing and the means of Trial 2 performance across the three sessions at each delay; error bars are one standard error of the mean. MS/VDB = medial septum-vertical limb of the diagonal band; nBM/SI = nucleus basalis magnocellularis-substantia innominata.

The role of the cortical cholinergic projections from the nBM/SI in leaming and memory has been questioned, based on the lack of correspondence between cortical ChAT depletion and behavioral impairment. For example, nBM lesions produced by quisqualic acid and ibotenic acid result in similar cortical ChAT depletion, but only ibotenic acid lesions produce substantial deficits in leaming and memory (Wenk et al., 1992). Hagan et al. (1988) noted a lack of impairment in water maze performance after ibotenic acid lesions of the nBM, which produced less than 40% depletion of cortical ChAT activity. The present study found a similar absence of leaming impairment after nBM/SI lesions that depleted cortical ChAT activity by 63%. These data are consistent with the hypothesis that damage to other types of cells may underlie the leaming and memory deficits consequent to nBM/SI damage. The present results are largely in agreement with other studies examining effects of 192 IgG-saporin lesions in the basal forebrain on water maze performance. Similar to the results of the present study, spatial leaming and memory were substantially preserved following 192 IgG-saporin lesions of the MS/VDB; however, performance was impaired following 192 IgG-saporin lesions of the nBM (Berger-Sweeney et al., 1994). More circumscribed damage to the corticopetal cholinergic system was produced in the present study, perhaps accounting for this discrepancy. We found no reduction in ChAT activity in either the striatum or hippocampus of rats that received immunotoxin into the nBM/SI region. In contrast, Berger-Sweeney et al. reported some loss of striatal ChAT-positive intemeurons and also observed some loss of cho-

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linergic input to the hippocampus in their nBM/SI-lesioned rats (Berger-Sweeney et al., 1994; Heckers et al, 1994). Striatal damage produces deficits in the spatially guided version of the water maze task (Whishaw, Mittleman, Bunch, & Dunnett, 1987); it is also possible that the combined loss of hippocampal and cortical cholinergic afférents resulted in a more severe deficit than the loss of either one alone. Our examination of performance in the delayed matching-toplace task revealed deficits in both lesion groups that did not depend on the delay interval. Similar delay-independent deficits following 192 IgG-saporin basal forebrain lesions have been observed in an operant-delayed spatial nonmatch-to-position task (Robinson, Wiley, Lappi, & Crawley, 1994). This type of deficit is difficult to interpret as a memory impairment, which would be expected to emerge with increasing delays. For example, a previous assessment of rats with entorhinal-perirhinal cortex damage in the same task used in the present study revealed normal retention at a 30-s delay interval but impaired retention at a 5-min delay interval (Nagahara, Otto, & Gallagher, 1995). However, we did not observe a delay-related decrement in performance in the control rats in this study, raising the possibility that delay-dependent effects of the lesions would have emerged at longer delays. It is possible that delay-related declines in performance in normal rats would have been exacerbated in the lesioned rats, resulting in an delay-dependent effect in addition to the delay-independent effect we observed. Our current data cannot address this possibility. The presence of a deficit even at the briefest (30 s) delay in this study suggests that an altemative interpretation is necessary for the effects of cholinergic denervation of hippocampus or cortex. For example, recent evidence has implicated the basal forebrain system in attentional processing rather than modulation of leaming and memory per se (Muir, Everitt, & Robbins, 1994; Pang, Williams, Egeth, & Olton, 1993; Robbins et al., 1989; Voytko et al., 1994). By this view, the deficit in the delayed match-to-placc task might be interpreted as the result of a mild impairment in attentional processing in the information trial rather than impaired memory for the location on the retention trial. It would be of interest, therefore, to examine animals with these selective cholinergic lesions on tasks specifically designed to assess attention. Age-related deficits in leaming and memory are often linked to deficits in cholinergic function, by analogy to the cholinergic deficits seen in Alzheimer's disease. Indeed, decreases in cholinergic markers for the basal forebrain system are most pronounced in aged rats with greater spatial leaming impairment (Dunbar, Rylett, Schmidt, Sinclair, & Williams, 1993; Fischer, Chen, Gage, & Björklund, 1991; Fischer, Gage, & Björklund, 1989; Gallagher et al., 1990; Ingram, London, & Goodrick, 1981). To the extent that loss of basal forebrain cholinergic neurons may play a role in agerelated cognitive dysfunction, basal forebrain immunolesions should provide a useful model of the contribution of this cholinergic loss to learning and memory deficits. As we have noted elsewhere (Gallagher, Gill, Baxter, & Bucci, 1994), the absence of spatial leaming impairment following basal forebrain immunolesions suggests that this is not a useful model for age-related impairments in leaming and memory and that changes in the cholinergic system cannot uniquely account for these impairments.

BAXTER, BUCCI, GORMAN, WILEY, AND GALLAGHER

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Received October 28, 1994 Revision received December 20. 1994 Accepted January 19, 1995

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