BRAIN RESEARCH

E L S E V I E R Brain Research 702 (1995) 233-245

Research report

Behavioral and neurobiological alterations induced by the immunotoxin 192-IgG-saporin: cholinergic and non-cholinergic effects following i.c.v.

injection

T.J. Walsh a, *, R.M. Kelly a K.D. Dougherty a, R.W. Stackman a, R.G. Wiley b, C.L. Kutscher c a Department of Psychology, Rutgers University, New Brunswick, NJ 08903, USA

b VA Medical Center, Nashville, TN, USA c Department of Psychology, Syracuse University, Syracuse, NE, USA

Accepted 8 August 1995

Abstract

192-IgG-Saporin is an anti-neuronal immunotoxin that combines the 192 monoclonal antibody to the p75 neurotrophin receptor found on terminals and cell bodies of neurons in the cholinergic basal forebrain with the ribosome-inactivating protein saporin. Bilateral intraventricular injection of the 192-saporin produced a variety of dose-related behavioral, neurochemical, and histological alterations in adult male rats. While both the 2 /xg and 4 /zg dose produced comparable cholinergic hypofunction only the high dose produced behavioral changes. Behavioral deficits induced by the 4 /xg dose of 192-saporin included alterations in rotorod performance and reactivity on the hot-plate which recovered over 8 weeks. In addition, the 4/xg dose produced a persistent impairment in the acquisition and performance of standard Morris water maze task as well as a cued version of the task. The neurobiological alterations induced by 192-saporin involved both cholinergic and non-cholinergic systems. Both doses of 192-saporin produced a 60-80% decrease in high affinity choline transport in the hippocampus and cortex without altering this parameter in the striatum. In addition, there was a significant dose-related decrease of norepinephrine in the hippocampus in the high dose group. 192-saporin did not alter the content of dopamine, serotonin, or their metabolites in any region examined. 192-saporin also produced a loss of Purkinje cells in the cerebellum. This cell type also expresses the p75 receptor and appears to be a target for intraventricular 192-saporin. This complex interplay of factors makes the i.c.v, model of 192-saporin very problematic for studying the functional properties of the cholinergic basal forebrain. However, recent data suggest that injection of 192-saporin directly into components of the cholinergic basal forebrain can be used to further elaborate the function of this brain system and to model disorders of cholinergic hypofunction such as Alzheimer's disease.

Keywords: 192-Saporin; Immunotoxin; Alzheimer's disease; Cholinergic basal forebrain; Spatial memory; Animal model

1. Introduction

The cholinergic basal forebrain (CBF) provides the primary cholinergic innervation of the neocortex, hip- pocampus (HPC), and amygdala [26]. Cholinergic neurons in the nucleus basalis of Meynert (nBM) project to the neocortex and the amygdala, while those in the medial septum/vertical limb of the diagonal band ( M S / V L ) pro- ject to the HPC as well as the entorhinal and cingulate cortices. Evidence derived from behavioral, pharmacologi- cal, neurochemical, and lesion studies supports a critical

* Corresponding author. Fax: (1) (908) 445-2263; E-mail: tomw@gandalf.rutgers.edu

0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993(95)01050-5

involvement of the CBF in certain types of learning and memory (see [43,44] for review). For example, a number of laboratories have observed training-induced increases in high-affinity choline transport (HAChT) in the HPC, and to a lesser extent in cortex, of rats following acquisition of both aversively and appetitively motivated tasks [2,9,42,47]. In addition, manipulation of the integrity of the CBF with cholinergic antagonists, lesions of the medial septum or nBM, and severing the fimbria-fornix produces impairments in a variety of spatial memory tasks in ro- dents (reviewed in [29,43]).

The CBF has also been a major focus in the study of Alzheimer's disease (AD) because of the notable correla- tion between (i) the degree of cholinergic hypofunction, (ii) the number of plaques and tangles, and (iii) the extent

234 T.J. Walsh et al. /Brain Research 702 (1995) 233-245

of the dementia [1,31,49]. Furthermore, the only drug to date that has significantly alleviated the cognitive deficits in AD is the cholinesterase inhibitor THA, which has produced a modest improvement in patients in early stages of the disease [34,40]. Therefore, a logical strategy for developing an animal model of AD is to focus on the 'target' symptoms of memory deficits and cholinergic hypofunction [44].

Neurotoxins used to lesion the CBF lack neurochemical specificity (e.g. excitotoxins) or have proved to be of limited use in creating models of cholinergic degeneration. Intraventricular injection of the cholinergic toxin AF64A selectively lesions cholinergic input to the HPC [6]. How- ever, it produces an incomplete lesion (less than 50% cholinergic loss), it cannot be injected directly into tissue due to a small window of specificity [25], and there has been considerable variability in results between different laboratories [12]. While AF64A can produce a selective degeneration of the cholinergic septohippocampal pathway with a specific profile of memory impairments it cannot be used to selectively damage other components of the CBF or to produce a more complete lesion. This limits its utility as a model of the extensive CBF degeneration observed in AD.

The immunotoxin IgG-192-saporin might provide a more useful model of cholinergic hypofunction since it is easy to use, and its effects are selective and reproducible [50-52]. Immunotoxins are conjugates of a monoclonal antibody, which targets a specific antigen, combined with a ribosome-inactivating protein (RIP). These toxins irre- versibly halt protein synthesis which results in cell death [4,50]. Since immunotoxins recognize and destroy only antibody-targeted cells it is possible to create highly selec- tive lesions which can mimic neurodegenerative disorders and/or address fundamental neurobiological questions.

Most cholinergic neurons in the CBF express p75 low- affinity neurotrophin receptors that are involved in mediat- ing the effects of nerve growth factor [39]. 192-IgG is a monoclonal antibody to the rat p75 receptor. Intraventricu- lar injection of radiolabelled 192-IgG results in the accu- mulation of the antibody in the cholinergic neurons of the basal forebrain [35,36]. Other cell groups of the basal forebrain, and the p75-negative cholinergic interneurons of the striatum are left unlabelled [53]. 192-Saporin combines the 192-IgG monoclonal antibody to saporin, a potent RIP derived from Saponaria officinalis. The immunotoxin tar- gets the p75 receptor localized on cholinergic nerve termi- nals in neocortex and HPC and on cholinergic cell bodies in the CBF but not on those cholinergic cell groups found in the upper brainstem [53]. This regional selectivity is important since the upper brainstem complex of choliner- gic neurons is spared in AD [54]. Following injection into the CBF, 192-saporin (i) is taken up by cholinergic neu- rons, (ii) is transported to the cell body by retrograde transport, and (iii) disrupts ribosome function and protein synthesis resulting in the death of the cell [4,14]. Site-

specific injection of 192-saporin produces a selective loss of cholinergic neurons and neurochemical markers to- gether with reduced theta activity within the HPC [20,48]. The p75 receptor is also found on a limited population of noncholinergic cell bodies primarily located in the brain stem, cerebellum and spinal cord [32], and there is evi- dence that 192-saporin acts upon these cells if injected i.c.v. [14]. 192-Saporin appears to be a superior tool with which to model the cognitive and neurochemical sequelae of chronic cholinergic hypofunction. The following experi- ments characterized the dose-related behavioral and neuro- chemical effects induced by i.c.v, injection of 192-saporin. The essential questions were (1) how selective is i.c.v. injection of 192-saporin?, (2) how can 192-saporin be used?, and (3) how should it be used?

2. Materials and methods

2.1. Subjects

Adult male Sprague-Dawley rats were used in the fol- lowing studies. Rats were singly housed in a temperature- and humidity controlled colony room and maintained on a 12:12 h light/dark cycle, with lights on at 07.00 h. All behavioral testing was conducted during the light phase. Food and tap water were freely available for the duration of the experiment. Daily water intake was monitored for 2 weeks prior to, and 4 weeks following, surgery and body weight was recorded every other day.

2.2. Immunotoxin preparation

192-saporin was made as previously described [51]. Briefly, purified 192 IgG from ascites and purified saporin from Saponaria officinalis were derivatized with SPDP followed by reduction of saporin-SPDP, mixing the two components and purification of the hetero disulfide conju- gate by column chromatography.

2.3. Surgery

On the day of surgery rats were divided into 4 groups. Rats in the 4 /xg group (n = 17) received bilateral i.c.v. injections of 2 /xg 192-saporin (4 /zg total), rats in the 2 /xg group (n = 17) received bilateral injections of 1.0 /xg 192-saporin (2/zg total), one group of control rats (n = 10) received bilateral injections of artificial CSF and a second group of control rats (n = 10) did not undergo any surgery. Rats were anesthetized with bilateral intramuscular injec- tions of a solution containing ketamine (100.0 mg/ml), xylazine (20.0 mg/ml), and acepromazine (10.0 mg/ml). Subjects were positioned in a Kopf stereotaxic instrument, and a midline sagittal incision was made in the scalp. Holes were drilled in the skull over the lateral ventricles 0.6 mm posterior to bregma and 1.8 mm lateral to the

T.J. Walsh et al. /Brain Research 702 (1995) 233-245 235

sagittal suture. Using a Kopf microinjection unit and a Hamilton microliter syringe positioned 2.7 mm ventral to the surface of the brain, rats were injected with either artificial CSF, 1.0 /xg 192-saporin in saline or 2 /xg 192-saporin in saline into each lateral ventricle (8.0 ~1/3.5 rain).

2.4. Locomotor activity

Locomotor activity, rotorod performance, and hot-plate reactivity were assessed 1 week prior to surgery and again 1, 2, and 8 weeks after surgery.

Four photocell chambers were used to measure horizon- tal locomotor activity. The chambers were constructed of black plexiglas and measured 40 cm long by 23 cm wide by 19 cm high. A row of 3 photocells was positioned along each side wall 5.5 cm above the chamber floor such that photobeams intersected the chamber at 7.0 cm intervals. Each photobeam interruption was recorded as a single activity count by an IBM personal computer.

2.5. Motor coordination

A rotorod apparatus was used to assess motor coordina- tion. The rod was constructed of a plastic cylinder (21 cm circumference) covered with cloth. Two metal disks ex- tending in 10 cm arcs around the rod prevented rats from ambulating along the length of the rod. The disks were positioned on the rod such that a 9.0 cm space existed between them, and rats were placed within this space for trials. The rod was connected at one end to a peristaltic pump (Masterflex-Cole Parmer) with the speed adjusted to 10 rpm. The rod was positioned 142 cm above the floor, and a large carton filled with 17 cm of bedding and cotton served to cushion the rat if it fell from the rod. On the first day of rotorod testing rats received 3 trials with an inter- trial interval (ITI) of 30 rain. Rats were placed one at a time on the rotating rod, and the latency to fall was recorded to a maximum of 180 s. Rats had 3 chances to remain on the rod for at least 10 s. At subsequent testing times, each rat had only 1 trial, with 3 chances to remain on the rod for at least 10 s. If rats did not remain on for at least 10 s, a score of 0.0 was recorded for latency to fall.

2.6. Behavioral reactivity

A hot plate apparatus (Omnitech) was used to assess reactivity to a thermal stimulus. The hot plate surface temperature was maintained at 60°C. Rats were placed one at a time onto the plexiglass-enclosed hot plate surface where each rat remained until it licked one of its 4 paws or until 30 s passed.

2.7. Standard Morris water maze task

was 120 cm in diameter and 56 cm deep. Four equally spaced points around the edge of the pool were designated as start positions and served to divide the pool into 4 imaginary quadrants. The removable escape platform was 35 cm high and consisted of a steel base column topped with a grooved, circular plexiglas disk 15.4 cm in diame- ter. The pool was filled with tap water made opaque by the addition of nontoxic white paint (Pearl Tempera), and the level of the water was maintained at 2 cm above the escape platform's surface. The temperature of the water remained at 21-23°C.

2. 7.1. Habituation

Twenty-four hours prior to training, rats were habitu- ated to the water maze. Rats were placed in the pool at start position 3 and allowed to swim for 60 s. Following the 60 s swim, the escape platform was placed in the center of the pool and the rat was placed on the platform for 30 s.

2. 7.2. Training trials

For each training trial the rat was placed into the pool at one of the 4 start points with its snout facing the center of the pool. The rat was released to swim until it located the escape platform or until 60 s passed. When a rat escaped onto the platform, it remained there for 30 s. If a rat did not escape onto the platform within 60 s, it was placed there by the experimenter and remained there for 30 s. After 30 s the rat was placed into a holding bin (stainless steel mesh cage 23 cm by 20 cm by 17 cm) with a metal cover for a 30 s ITI. Each rat underwent 4 trials a day for 10 consecutive days of training. Throughout water maze training the escape platform was situated in the middle of quadrant 1. During each day of training, each of the 4 start points was used once in a pseudo-random order.

2. 7.3. Probe trials Probe trials were introduced following the second train-

ing trial on days 1, 4, 7, and 10. During probe trials the escape platform was removed from the pool. Each rat was placed into the pool at start position 3 and allowed to swim for 30 s. After 30 s the rat was removed from the pool and placed into the holding bin for the 30 s ITI before training trial 3. During the ITI, the escape platform was reposi- tioned into the middle of quadrant 1. Probe trials were recorded using a Chromotrack V video tracking system (San Diego Instruments) interfaced to an personal com- puter; the swim paths of the rats were recorded and the following indices were derived: (1) the average distance the rat swam from the site in which the escape platform was located during training (ADT), and (2) the percent of time the rat spent in the quadrant in which the escape platform was located during training trials.

2.8. Cued Morris water maze task

Twenty-two days after surgery rats were trained on a standard Morris water maze task (MWM). The maze pool

After 10 days of training on the standard MWM task, subjects were trained in a cued version of the task. During

236 1],1. Walsh et al, / Brain Research 702 (1995) 233-245

the cued task the escape platform protruded 1.0 cm above water level, and black tape around the periphery of the platform served to enhance its visibility from any start point at the edge of the pool. Rats underwent 16 training trials (4 days of 4 trials). Trials were conducted in the same way as during the standard task, except that each day the visible platform was located in a different quadrant of the pool. As in the standard task rats experienced a trial from all of the 4 start points in a pseudo-random order on each day of testing.

2.9. Neurochemical analysis

Following the 8-week behavioral assessment rats were sacrificed by decapitation. Their brains were removed immediately and tissue samples were dissected out and processed for various analyses.

Samples were homogenized in 0.5 ml ice cold 0.4 N perchloric acid. Homogenates were then centrifuged at 14,000 rpm for 20 min at 4°C and 20 /zl of the supernatant was injected for the determination of 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), ho- movanillic acid (HVA), and norepinephrine (NE). External standard samples were obtained from Sigma. The mobile phase consisted of 0.15 M monochloroacetate, 0.86 mM sodium octylsulfate, 3.5% acetonitrile, 1.9% tetrohydrofu- ran and was adjusted to pH 3.0. The HPLC system con- sisted of a Waters 510 HPLC pump, Waters 460 electro- chemical detector with applied potential set at 0.80 V, and a Biophase ODS 5 /xm column (BAS, West Laffayette, IN).

2.10. Cerebellar histology

2.9.1. HAChT Sodium-dependent HAChT was assessed in synapto-

somes prepared from homogenates of HPC, frontal cortex, and striatum, as described in detail by Opello et al. [30]. Tissue samples were homogenized in 20 volumes of 0.32 M sucrose solution using hand-held Potter-Elvehjem grinders. Homogenate was centrifuged at 3000 rmp at 4°C for 10 rain, and the resulting supernatant was centrifuged at 13,000 rpm for 20 min. The pellet produced by centrif- ugation was resuspended in half of the original volume of sucrose solution. Fifty /zl aliquots of this suspension were added to two tubes containing 450.0 ~1 ice-cold buffer which contained 0.2 /zM choline, 0.25 /zCi [3H]choline (specific activity 80.0 Ci /mmol , DuPont, New England), 126.0 mM NaC1, 9.60 mM KC1, 4.20 mM MgSO 4, 2.4 mM CaCI 2 - H 2 0 , 252.0 mM dextrose, and 40.0 mM Tris base; and a second set of two tubes containing 450.0 /xl of the same buffer with the exception that NaCI was replaced by 252.0 m M sucrose. These suspension-buffer solutions were incubated for 4 min at 37°C and immediately quenched with 3.0 ml ice cold Na+-free buffer. Tissue was rinsed several times with Na+-free buffer and collected using a Brandel Cell Harvester onto Type G F / B (What- man) glass fiber filters. Filters were placed in scintillation vials with 6.0 ml of Scintiverse BD scintillation fluid. Approximately 12 h after the addition of scintillation fluid, the vials were assayed for radioactivity in a Packard TriCarb(4530) scintillation counter with a 66% counting efficiency for 3H. The protein content of the samples was determined according to the procedure of Bradford [5] using bovine serum albumin (Sigma Chemical, St. Louis, MO) as a reference standard. Data were analyzed as picomoles of choline taken u p / m g protein/4 min.

2.9.2. HPLC Frozen homogenates of HPC, frontal cortex, and stria-

turn were analyzed by high pressure liquid chromatog- raphy with electrochemical detection (HPLC-EC), [24,30].

At the time of sacrifice, the cerebella of a subset of rats (4 /.Lg n = 5; 2 /~g n = 5, Con n = 5) were removed and drop fixed in a solution of 0.1 M phosphate buffer, 10% formalin, and 30% sucrose. Matching paravermal sections (50 /.tm) were stained with hematoxylin and eosin. Purk- inje cells were counted throughout the anterior-posterior extent (lobules I -X) of the sagittal plane. In each sample, all of the visible Purkinje cells were counted from the inferior regions of a folium, up along the lateral aspect, across the dorsal surface, and down the posterior aspect of the folium. The total number of Purkinje cells in both the superior and inferior regions of lobules I - X was deter- mined.

2.11. Cholinergic fiber density following i.c.u. 192-saporin

A separate group of naive rats received bilateral i.c.v. injections of either CSF, or 1 or 2 /zg of 192-saporin. They were sacrificed 60 days later with an overdose of sodium pentobarbital and perfused through the heart with 150 ml 0.05 M phosphate-buffered saline (0.9% PBS) followed by 300-400 ml of ice cold 4.0% paraformal- dehyde/0.1 M PB (pH 7.0) over 30 min. Brains were immediately removed and incubated overnight at 4°C in a 1:1 solution of 4% paraformaldehyde/0.1 M PBS then stored at 4°C in a 10.0% sucrose solution for at least 24 h. Coronal sections (50 /zm) were cut on a freezing micro- tome. Cholinergic (acetylcholinesterase-positive) fibers were stained using the technique described by Tago and colleagues [41]. Slices were incubated in 0.003% hydrogen peroxide/0.1 M maleate buffer (pH 6.0) solution for 30 min, washed in 0.1 M maleate buffer, then placed in a dilute (1:20) Karnovsky-Roots stain solution for 45 rain. Slices were then rinsed in 0.1 M Tris buffer (pH 7.6) followed by further staining in a 0.05% DAB/0.003% hydrogen peroxide solution for 30 rain. Slices were rinsed in 0.05 M Tris buffer to stop the reactions. After process- ing the sections were mounted onto gelatin coated slides.

T.J. Walsh et al. / Brain Research 702 (1995) 233-245 237

The slides were then processed in a graded series of alcohols (dehydration), N-butyl alcohols (transition stage), and Histosol (clearing). Slides were coverslipped with a synthetic mounting medium.

Direct visual inspection was used to qualitatively assess cholinergic fiber density in most cortical and subcortical regions. In addition, quantitative evaluation of fiber loss was performed in frontal, parietal, and pyriform cortices using a counting grid. The grid consisted on an array of 5 parallel lines. At 400 X each line covered 0.09 mm of tissue and lines were 0.07 mm apart, therefore a 0.09 mm by 0.28 mm core of cortex was sampled. The grid was centered on a radial line through the cortex which was perpendicular to a tangent drawn on the cortical surface. Cortex was viewed at 400 X and each intersection of fibers with horizontal lines of the grid was counted. Fiber density of a given area was expressed as the sum of intersections on all 5 lines. A coronal section of one side of the brain was used at the level of the anterior commis- sure ( - 0 . 3 mm from bregma). In the pyriform cortex the grid was placed immediately below the rhinal fissure. For the parietal cortex the grid was placed at the point where the arc of the external capsule fibers reaches the most lateral extent. For the frontal cortex, the grid was placed immediately above the cingulum dorsal to the corpus callosum.

3. Results

The data derived from the CSF-injected group and the non-injected group were comparable for all behavioral and neurochemical measures, and their data has been combined to form a single group designated as Control.

3.1. Body weight

A two-way repeated measures ANOVA (treatment X time) revealed a non-significant treatment effect (F2,51 = 0.489, n.s.), but significant effects of time (/73, 6 = 1.06, P = 0.0001) and treatment by time interactions (F6.~53 = 2.86, P = 0.011). All rats increased body weight over time

but the 4 /zg dose of 192-saporin produced a significant 5% decrease in body weight compared to the Control group which was only evident one week following surgery. Body weights were comparable at all other times.

3.2. Water intake

192-saporin had no effect on water intake following surgery. A two-way repeated measures ANOVA revealed a significant time effect for water intake (F3. 6 = 6.25, P = 0.0005) but non-significant treatment (F2,5~ = 0.837, n.s) and treatment by time (F3.153 = 0.967, n.s.) effects.

3.3. Locomotor activity

192-saporin did not affect locomotor activity following surgery. A two-way repeated measures ANOVA revealed a significant time effect (F2, 4 = 4.40, P = 0.015) for activ- ity, but non-significant treatment (F2,51 = 0.251, n.s.) and interaction (F2,102 = 2.26, n.s.), effects. Motor activity de- creased with repeated testing and this was comparable across treatment groups.

3.4. Rotorod and hot plate

192-saporin did produce a dose- and time-dependent alteration in both rotorod and hot-plate performance. For rotorod, a two-way repeated measures ANOVA revealed significant time (/72, 4 = 3.17, P = 0.04) and treatment by time interactions (F4.102 = 5.86, P = 0.0003) but a non- significant overall treatment effect (F2,51 = 1.97, n.s.). Post-hoc analyses revealed that the 4 /~g group exhibited significantly shorter latencies than (i) the Control group at week 1, and (ii) the 2 /xg group at week 2 (Fig. 1, Panel A).

For hot-plate, a two-way repeated measures ANOVA revealed significant t r e a t m e n t ( F 2 , 4 9 = 7.78, P = 0.001), time (F2. 4 = 7.61, P = 0.0008) and treatment by time in- teraction (F4,98 = 4.09, P = 0.004) effects. Post-hoc analy- ses revealed that the 4 /zg group exhibited significantly longer latencies than both the Control and 2 /xg groups at 1 and 2, but not 8, weeks following surgery (Fig. 1, Panel B).

250 -

200"

150 "

1 O0 -

50

R O T O R O D B.

500 ql l +

~ 3oo

200

W E E K P O S T S U R G E R Y

H O T P L A T E

=

W E E K P O S T S U R G E R Y

• 4 # g

2#g • CON

Fig. 1. Dose-related effects of 192-saporin on performance on the rotorod (Panel A) and the hot-plate test (Panel B). Data are presented as mean ( 4- S.E.M.) percent of the pre-operative baseline recorded 7 days prior to surgery. Rats were tested again 1 (7), 2 (14), and 8 (56) weeks following surgery. * P < 0.05, 4 p.g group vs. Controls, a p < 0.05, 4 /xg group vs. 2 p.g group, Fisher's LSD test.

238 T.J. Walsh et al. / Brain Research 702 (1995) 233-245

50

STANDARD MORRIS WATER MAZE TASK

40 ~,,~T "_ T * * a a , • 4 pg x ~ q t , , ~ T I a a + 2 pg ~ , * • CON

>- 30 a * * O

20 l-,,- g

]0

0 t I i i J r t J i i 1 2 3 4 5 6 7 8 9 10

DAY

Fig. 2. Dose-related effects of 192-saporin on acquisition of the standard M W M task. Rats were g iven four trials per day. Data are presented as

mean ( + S .E.M.) latency to find the escape platform during each 4 trials. Groups arc designated as Con (Controls) , 2 /xg (saporin) , 4 / x g (saporin).

Rats were tested 32-42 days following surgery in this task. * P < 0 . 0 5 . 4

,ag g roup vs. Controls , and ~' P < 0 . 0 5 , 4 # g group vs. 2 # g group,

Fisher's LSD test.

3.5. Standard water-maze task

3.5.1. Acquisition Latencies for all groups were similar on the first acqui-

sition trial in the water maze (F2,5~ = 0.68, n.s.); Controls = 4 5 . 2 7 + 4 . 5 ; 4 ~g dose=43 .95+_4 .93 ; 2 /,~g d o s e = 38.06 _+ 4.42 (mean seconds 4- S.E.M.)

192-saporin produced a dose-related impairment in the acquisition and performance of the standard MWM task. A two-way repeated measures A N O V A revealed significant treatment (Fz5 t = 10.79, P = 0 . 0 0 0 1 ) and time effects (Fg, t8 = 51.22, P = 0.0001) and a non-significant interac- tion ( F I s , 4 5 9 = 1.03, n.s.) for acquisition (latency) in the water-maze task. All groups exhibited shorter latencies with continued testing but the 4 /xg group never exhibited latencies that were comparable to those of the Control group (Fig. 2). Post-hoc analyses revealed that the 4 /~g group displayed significantly longer latencies than the Control group on days 2 through 10. The latencies of the 2 /zg group were not different from those of the Controls at any time point.

12

CUED WATER MAZE TASK

I0 a

>- 8 ~ 2pg 0 Z • CON uJ 6 - I,-

5 4 -

2 J | , i l 2 3 4

DAY

Fig. 4. Dose-related effects of 192-saporin on acquisition of the cued M W M task. Rats were given four trials per day. Data are presented as

mean ( ± S .E .M.) latency to find the visible escape platform during each 4

trials. Rats were tested 52-55 days following surgery in the cued task.

P < 0.05, 4 /zg g roup vs. Controls , a p < 0.05, 4 p,g g roup vs. 2 ,ttg

group, F isher ' s P L S D test.

3.5.2. Probe trials

During probe trials there was a significant effect of treatment (F2,3t =3 .86 , P = 0 . 0 3 2 ) and time (F3,93 = 111.19, P = 0.0001) on ADT, and a non-significant inter- action (F~,,~ 3 = 1.43, n.s.). Post-hoc analyses revealed that during the probe trials on days 7 and 10, the 4 /zg rats swam significantly further from the platform site relative to rats in the Control group (Fig. 3, Panel A). Two-way repeated measures ANOVAs comparing the percent of time rats spent in the platform quadrant of the pool during probe trials revealed a main effect of t r e a t m e n t ( F 2 , 3 1 =

11.58, P = 0.0002) and trial (F3.93 = 45.47, P = 0.0001). There was no interactive effect of these two f a c t o r s (F6,93 = 1.86, n.s.). On days 4, 7, and 10, rats in the 4 / z g group spent significantly less time swimming in the platform quadrant than Control rats (Fig. 3, Panel B). Rats in the Control and 2 /xg groups did not differ on any probe measure during any probe trial.

.7.6. Cued water maze task

Latencies for the 192-saporin-treated groups were 2 3 - 90% longer than those of the Control group on the first

A B !JO hO *

40 • 4/Jg 2#g

<0 3o ~ 30 • CON

20 20 •

PROBE DAY PROBE DAY

Fig. 3. Dose-related effects of 192-saporin on performance during the Probe trials. These trials, in which the escape platform was removed, were introduced following the second training trial on days l, 4, 7, and 10 in the standard M W M task. Panel A presents the the effects of 192-saporin on average distance to target (mean ± S.E.M. A D T in cm) . Panel B presents the effects of 192-saporin on the percent time spent in the target quadrant during the probe trials (mean ± S.E.M.). * P < 0.05, 4 # g group vs. Controls , " P < 0.05, 4 p~g g roup vs. 2 /.~g group, F i she r ' s L S D test.

T.J. Walsh et al. ~Brain Research 702 (1995) 233-245 239

Table 1 Regional sodium-dependent high-affinity choline uptake

Hippocampus Frontal cortex Striatum

Con 100.00 + 11.64 100.00 + 14.21 100.00 + 13.72 2.0/xg 39.415:8.96 * 35.59-1- 8.14 * 117.965:23.30 4.0/zg 19.76+ 2.81 * 34.68+ 6.82 * 141.41 5:19.17

Rats were sacrificed 58-62 days following surgery. Data are presented as mean (5: S.E.M.) percent of control values. Control values are: hippocam- pus 11.90 + 1.56, frontal cortex 6.40 + 0.99, striatum 33.03 5:7.17. * P(0.05 vs. Con, Fisher's LSD test.

acquisi t ion trial in the cued water maze task. This group difference approached but did not reach statistical signifi-

cance (F2,st = 2.94, P = 0.069); Control~ = 7.95 + 1.06; 4 /xg dose = 15.77 ___ 3.95; 2 /.~g dose = 9.78 + 1.46 (mean seconds + S.E.M.).

192-saporin produced a dose-related impai rment in per- formance of the cued water maze task. A two-way re- peated measures A N O V A revealed s ignif icant t reatment

(F2,51 = 7.33, P = 0 . 0 0 1 ) and t ime (F3, 6 = 13.32, P = 0.0001) effects, but a non-s igni f icant interact ion (F6,153 = 0.613, n.s.) effect (Fig. 4, Panel A). All groups acquired the task, exhibi t ing shorter latencies as t raining cont inued; but the 4 / z g group had s ignif icant ly longer latencies than

the Controls at each of the 4 days of testing.

3. 7. Hippocampus

3. 7.1. High affinity choline transport Both doses of 192-saporin s ignif icant ly decreased

H A C h T in the HPC. A one way A N O V A revealed a s ignif icant t reatment effect (F2,5o = 22.44, P = 0.0001). Post-hoc analyses revealed that 192-saporin s ignif icant ly decreased transport by 60% in the 2 /~g group and 80% in the 4 /~g group, relative to the Control group. There was

no signif icant difference be tween the decrease observed in the 2 /xg and 4 / z g groups (see Table 1).

3.7.2. Monoamine neurotransmitters 192-saporin also s ignif icant ly affected NE in the HPC.

A one-way A N O V A revealed a s ignif icant t reatment effect

(F2,50 = 9.01, P -- 0.0005). Post-hoc analyses revealed that the 4 ~ g dose of 192-saporin decreased the concentra t ion

Table 2 Regional catecholamines, indoleamines and metabolites

Hippocampus Frontal cortex Striatum

NE Con 100.02___ 3.84 99.98 + 3.94 99.94 ___ 12.32 2.0 88.67+6.45 87.155:6.92 114.535:20.39 4.0 70.59:1:4.45 * 84.295:6.93 112.265:24.77

DA Con N.D. 99.97 5:21.40 100.01 + 8.82 2.0 N.D. 79.675:14.58 97.065:5.13 4.0 N.D. 120.38 5:22.97 93.60 + 5.85

DOPAC Con N.D. 99.69 5:22.85 99.99 5:5.35 2.0 N.D. 55.82+ 8.46 100.305:7.63 4.0 N.D. 62.825:12.30 86.735:5.52

5-HT Con 100.085:6.30 100.035:4.87 100.06+ 4.96 2.0 92.165:7.64 100.905:4.38 90.175:6.31 4.0 97.79+5.43 101.90+ 4.94 89.825:4.41

5-HIAA Con 99.935:3.49 100.09+ 7.15 99.99+ 4.10 2.0 94.415:3.65 104.35+ 4.16 91.225:6.55 4.0 93.885:3.04 101.075:4.92 94.295:4.74

Rats were sacrificed 58-62 days following surgery and the regional monoamine content was determined by HPLC with electrochemical de- tection. Data are expressed as mean ( + S.E.M.) percent of control values. N.D., not determined. * P(0.05 4.0 ~g vs. Con.

of NE in the HPC by 30%, relative to the Control group. The 2 /.tg dose of 192-saporin produced a non-s igni f icant

12% decrease in NE (Table 2). 192-Saporin did not alter the concentra t ion of 5 -HT

(F2,50 = 0.392, n.s.) or 5 - H I A A (F2,50 = 0.995, n.s.) in the HPC.

3.8. Frontal cortex

3.8.1. High affinity choline transport 192-Saporin s ignif icant ly decreased H A C h T in the

frontal cortex (F2,48 = 13.02, P = 0.0001). Both the 4 /xg and the 2 ~ g dose decreased uptake by approximately 64% relative to the Control values (Table 1).

3.8.2. Monoamine neurotransmitters 192-Saporin did not s ignif icant ly alter the concentra-

t ions of NE, DA, DOPAC, 5 -HT or 5 - H I A A in the frontal

cortex (all F2,46_50's = 0 .04 -2 .08 , all P ' s n.s.). However , 4 /.tg of 192-saporin did produce a non-s igni f icant 16% decrease in NE in the frontal cortex (Table 2).

Table 3 Number of Purkinje cells in sagittal sections of cerebellum

Superior folia Inferior folia Total

Con 157.40 + 18.40 669.40 5:74.80 826.80 + 89.70 2.0 p,g 72.40 5:12.40 + (46) 615.60 + 81.50 (92) 688.00 5:87.00 (83) 4.0/zg 63.80 5:17.10 * (40) 438.20 + 41.90 (65) 502.00 5:51.90 * (61)

Data are expressed as the mean ( + S.E.M.) total number of Purkinje cells counted in matching paravermal sections of cerebellum as well as number of cells occurring in superior and inferior regions of folia from lobules I-IX (numbers in parentheses represent percent of control values). * P(0.05 4.0 p.g vs. Con. ÷ P(0.05 2.0 /xg vs. Con.

241) T.J. Walsh et al. / Brain Research 702 (1995) 233-245

0

0

[ .......

.?-. X

~z ~ <

..,...,

"~t~

..~ x

.= -~

. ~ & x ~

.,.4

. ~ u

"~ d

~ .~"

._~ ~ .~ ~ [ '- o

T.J. Walsh et aL /Bra in Research 702 (1995) 233-245 241

3.9. Striatum

3.9.1. High affinity choline transport and monoamine neu- rotransmitters

192-Saporin produced no alterations in choline uptake, or in the regional concentrations of NE, DA, DOPAC, HVA, 5-HT or 5-HIAA in the striatum (all F2,49'S 0.23- 1.52, all P ' s n.s) ( Tables 1 and 2).

3.10. Cerebellar histology

192-Saporin produced a dose-related decrease in the number of Purkinje cells in the cerebellum (Table 3). Two-way ANOVAs revealed significant effects of treat- ment (F2,12 = 4.35, P = 0.038) and region (F], 2 = 181.96, P = 0.0001) but no interaction (F2,12 = 2.15, n.s.) on the number of Purkinje cells in matched sagittal sections of cerebellum. Post-hoc analyses revealed that rats in the 2 /.tg and 4/.~g groups had significantly fewer Purkinje cells in the superior regions of cerebellar lobules than controls; and 4 /.~g rats had significantly fewer Purkinje cells in ventral regions of cerebellar lobules compared to Controls.

3.11. Cholinergic fiber density

In naive rats i.c.v. 192-saporin produced a selective profile of cholinergic fiber loss in both subcortical and cortical structures. The fiber loss was only evident in the terminal fields of the CBF, most notably olfactory bulb, HPC, and neocortex. These areas represent the major targets of cholinergic projections from the horizontal limb of the diagonal band, the MS/VL, and the nBM, respec- tively. In the HPC, both doses eliminated all fibers throughout the structure including the pyramidal cell fields, the dentate gyrus, and the subiculum (Fig. 5). In contrast, the cholinergic innervation of the basolateral and lateral nuclei of the amygdala was intact. This observation is in agreement with prior reports [14].

The decrease in fiber density in neocortex was depen- dent on dose and region. The parcellation of different cortical areas by Lysakowski and coworkers [22] is fol- lowed below. The low dose removed virtually all fibers in the cingulate, retrosplenial, and frontal cortices in all ani- mals and in the parietal cortex in most animals. When fibers remained in the neocortex they were evident along the lateral extent of the cortex in parietal and insular areas. The high dose removed virtually all cholinergic fibers with the exception of sparse enclaves in the insular area or the amygdalar cortex.

Quantification of 192-saporin-induced changes in fiber densities in frontal, parietal, and pyriform cortices is shown in Fig. 6. A split plot ANOVA revealed that fiber density differed as a function of dose (F4,20 = 165.55, P = 0.001), and cortical area (F2,20 = 9.80, P = 0.001). The dose by area interaction was also marginally significant (F4,2~ = 2.80, P = 0.053). Analyses of simple main effects revealed

C H O L I N E R G I C F I B E R D E N S I T Y

1 O0 -

N so

~ 40 +

2 0 , + a *

0

[ ] 4pg [ ] 2pg [] CON

FRONTAL PYRIFORM PARIETAL

Fig. 6. Dose-related effects of 192-saporin on cholinergic (ACHE + ) fiber density in frontal, pyriform, and parietal, cortices. Non-behaviorally tested rats were sacrificed 60 days following surgery and AChE + fibers were examined according to the Tago procedure described in Section 2. Data are presented as mean ( + S.E.M.) total number of cholinergic fibers counted in a defined area of cortex (see Section 2). * P < 0.05, 4 ~g group vs. Controls, a p < 0.05, 4/.~g group vs. 2 /xg group, ÷ P < 0.05, 2 /.tg group vs. Controls, Tukey's HSD test.

that 192-saporin significantly reduced cholinergic fiber density in each cortical area ( P ' s = 0.001). Post hoc pair- wise comparisons (one-tailed) revealed that both doses of 192-saporin produced comparable decreases in fiber den- sity in frontal cortex (85-92%). However, dose-related effects were observed in both the pyriform and parietal cortices with the high dose decreasing fiber density signifi- cantly more than the low dose (90% vs. ~ 65% decrease in both areas).

Contrary to the cholinergic denervation of the CBF, fiber density in the target areas of the pedunculopontine nucleus and the laterodorsal tegmental nucleus,the brain- stem cholinergic nuclei, was intact (see [26,37]). There was no loss of fibers in thalamic nuclei, superior colliculus, periaqueductal gray, or the lateral septum.

4. Discussion

i.c.v, injection of 192-saporin produced a variety of behavioral, neurochemical, and histological alterations. Be- havioral deficits induced by the 4/.Lg dose of 192-saporin included transient alterations in (i) motor coordination and (ii) reactivity on the hot-plate, and (iii) a persistent impair- ment in the acquisition and performance of a MWM task. The neurobiological alterations induced by 192-saporin involved both cholinergic and non-cholinergic systems.

Both doses of 192-saporin produced a 60-80% decrease in HAChT in the HPC and cortex without affecting uptake in the striatum. While both doses produced comparable decreases in cholinergic parameters only the 4 g g group exhibited any behavioral alterations. Therefore, it is diffi- cult to attribute the behavioral deficits to the loss of cholinergic function in cortex and HPC. Regional concen- tration of 5-HT, DA, and their metabolites were not af- fected by 192-saporin. However, 4 /~g 192-saporin did

242 T.J. Walsh et al. / Brain Research 702 (1995) 233-245

significantly decrease NE in the HPC compared to the 2 /~g 192-saporin group and the controls. 192-Saporin also significantly decreased the number of Purkinje cells in the cerebellum in a dose-dependent manner consistent with a previous report [14]. The distribution of Purkinje cell loss reflects the localization of the p75-positive Purkinje cells in the cerebellum [10,32]. Based upon the profile of neuro- biological alterations it is parsimonious to attribute the behavioral deficits induced by 192-saporin to the com- bined influence of cholinergic hypofunction, Purkinje cell loss, and changes in forebrain NE content (described be- low). The loss of cholinergic parameters cannot be the sole or primary cause of the behavioral deficit since the cholin- ergic deficits were comparable for both 192-saporin groups while only the 4 ~g group exhibited behavioral deficits. However, there were dose-related decreases in cholinergic fiber density in both pyriform and parietal cortices which might contribute to the differential behavioral effects ob- served in the 2 and 4/~g groups (see below).

The 4 /~g dose of 192-saporin produced a significant decrease of NE in HPC. It is unclear whether this reflects a loss of NE neurons in the locus coeruleus (LC) or a secondary change in NE dynamics subsequent to the cholinergic hypofunction. If 192-saporin damaged LC cells, there should have been a decrease in NE in all of its targets including the striatum and frontal cortex, which was not observed in this study. Cholinergic hypofunction induced by AF64A is also associated with a decrease in NE in the HPC [16] which is thought to reflect increased NE release. Therefore, the decreased NE in the HPC observed in the present study might also indicate adaptive changes secondary to the cholinergic hypofunction. How- ever, Nilsson and coworkers [28] reported a small, but significant, increase in hippocampal NE following i.c.v. 192-saporin (4.62 /xg). A morphological assessment of LC neurons or an in vivo microdialysis study of NE release might further reveal the effects of 192-saporin on nora- drenergic neurons.

I.c.v. 192-saporin produced a dose and region-depen- dent loss of cerebellar Purkinje cells. Significant degenera- tion was observed following both 2 /zg and 4 p,g of 192-saporin in the superior regions of cerebellar folia but only the higher dose damaged the ventral folia. The contri- bution of this prominent Purkinje cell loss to the 192- saporin-induced alterations in motor behavior, reactivity, and spatial learning needs to be further explored. While forebrain cholinergic systems have been implicated in motor behavior and stimulus reactivity [33,46], the equiva- lent decreases in HAChT in both dose groups argue against the hypothesis that cholinergic hypofunction mediates these effect.

Spatial learning and memory in the standard MWM task was also impaired in the 4/xg group. This group exhibited longer escape latencies throughout training and impaired performance during the probe trials (time spent in previ- ously correct quadrant, average distance to target). Again,

this deficit might reflect a combined alteration in neuro- transmitters in the forebrain and Purkinje cell loss. There is a growing appreciation that the cerebellum is involved in a variety of non-motor behaviors including cognitive func- tion [19]. In the rat and primate brain the deep nuclei of the cerebellum project to regions of the mesencephalic tegmentum and thalamus which in turn project to limbic and cortical areas which have been implicated in cognitive processes [27,38]. Cerebellar lesions can impair both asso- ciative and nonassociative learning processes [19,21]. In fact, in preliminary studies we have found that i.c.v. injection of OX7-saporin, an immunotoxin that targets Purkinje cells [7], does produce alterations in spatial learn- ing and memory (Kelly and Walsh, in preparation). This is consistent with documented spatial learning and memory deficits in the pcd (Purkinje cerebellar degenerate) mouse strain [11].

Other reports have also observed deficits in a MWM task following i.c.v. 192-saporin. Both Nilsson et al. [28] and Berger-Sweeney et al. [3] reported that i.c.v, injection of 3.6-5.0 /~g 192-saporin impaired acquisition or perfor- mance of this task. The water maze deficit appeared to relate to a loss of cortical cholinergic innervation since an intrabasalis, but not an intraseptal, injection of 192-saporin produced comparable deficits to the i.c.v, injection [3]. Other reports also suggest a dissociation in the involve- ment of MS-hippocampal and nBM-cortical projections in different behavioral tasks. Radiofrequency lesions of the MS produce deficits in the acquisition of an appetitive radial-arm maze task without affecting performance in the MWM despite a 70% decrease in ChAT activity in the HPC [8]. Therefore, spatial tasks other than the MWM might be more sensitive for detecting subtle effects of cholinergic hypofunction on cognitive behaviors.

The 2 /.~g dose of 192-saporin produced an extensive loss of HAChT in the HPC and cortex but did not impair the acquisition or performance of the water maze task. The lack of a cognitive deficit might indirectly support the involvement of cholinergic systems that are not affected by 192-saporin in spatial memory. Cholinergic neurons in the nBM have extensive neocortical projections but also inner- vate limbic targets, in particular the amygdala [26]. Mallet and coworkers [23] reported that nBM injections of ph- thalic acid produced working memory deficits in a spatial learning task, and these deficits were associated with a large decrease in ChAT activity in amygdala but not in cortex. In addition, injection of the muscarinic antagonist, scopolamine, into the basolateral amygdala impaired work- ing memory in an appetitive Y-maze task [17]. Therefore, the cholinergic innervation of the amygdala might also participate in memory processes mediated by limbic and cortical regions. Heckers and colleagues reported that i.c.v. 192-saporin spared a population of ChAT-positive neurons in the nBM as well as AChE-positive fibers in the amyg- dala, despite a nearly complete loss of cholinergic innerva- tion of cortex [14]. There is a subgroup of cholinergic cells

T.J. Walsh et al. / Brain Research 702 (1995) 233-245 243

in the nBM that project to the amygdala and do not express p75 [13]. In the present study, naive rats that were sacrificed 60 days after i.c.v. 192-saporin exhibited intact cholinergic innervation of the amygdala evidenced by nor- mal density of AChE-positive fibers in both lateral and basolateral nuclei. Since the nBM-amygdala pathway is intact in 192-saporin-treated rats it is possible that it can support some cognitive processes despite the loss of corti- cal and hippocampal cholinergic innervation. This could account for the intact performance of the 2 p~g 192-saporin group. While this pathway would also be intact in the 4/xg group, the other changes such as Purkinje cell loss and decreased NE in the HPC might have exacerbated any behavioral deficit.

The cued version of the MWM task assesses both sensorimotor ability and the acquisition of a visual dis- crimination. In this cued task the 4/~g 192-saporin group took significantly longer than controls to escape onto a visible platform throughout training. While these impair- ments are consistent with those reported by Berger-Swee- ney et al. [3] following i.c.v, injections of 192-saporin, they are contrary to other reports of intact cued water maze performance following compromise of the CBF. Choliner- gic deafferentation of HPC produced by i.c.v, injections of AF64A [30], intraseptal infusion of 192-saporin [3] or quisqualic acid [8] did not impair rats' performance on a cued or visual discrimination water maze task. The ability of the 4/xg group to swim in the pool at speeds similar to controls and to escape onto the visible platform more rapidly than onto the submerged platform, suggests that their primary motor and visual systems were not impaired.

Histological analysis of naive rats revealed that i.c.v. 192-saporin produced an extensive loss of cholinergic fibers in the primary targets of the CBF; neocortex, HPC, and olfactory bulb. In the cortex, quantitative assessment revealed a dose-related loss of cholinergic fibers in the parietal and pyriform cortices with the 4 /zg dose decreas- ing density ( ~ 90%) significantly more than the 2 /zg dose ( ~ 65%). Assuming a similar profile of cholinergic fiber loss in the behaviorally-tested rats, the residual cholinergic activity in parietal and pyriform cortices in the 2 /xg group might have been sufficient to maintain spatial learning in the MWM task. Deficits in spatial memory have been observed following parietal cortex lesions in rats [18].

As previously reported, the cholinergic innervation of the lateral and basolateral nuclei of the amygdala was not affected (see above). In addition, there was no apparent reduction in fiber density in the thalamus, colliculi, or lateral septum; the primary terminal fields of the upper brainstem cholinergic complex [26,37]. These cholinergic fibers do not express p75 and are not viable targets for 192-saporin [53].

Both doses of 192-saporin produced extensive choliner- gic denervation of isocortex and allocortex. There was widespread loss of cholinergic fibers throughout areas of motor, sensory, and association cortex. The main motor

cortex (area 4), the frontal eye fields (area 8) and the other motor areas (area 10, 11) were almost totally devoid of cholinergic input. The parietal cortex, which comprises primary sensory, sensorimotor, secondary sensory and as- sociation cortices in the rat,[22] was also deprived of cholinergic fibers (areas 3a, 3b, 2, 7, 5, 39, 40 and 43) following 192-saporin. Area 3a contains a motor cortex for front and hind limbs, vestibular cortex and primary so- matosensory cortex receiving fibers from all over the body. The primary visual cortex (area 17) and the secondary visual cortex (area 18) were also totally deprived of cholin- ergic fibers. To the extent that these sensory/motor func- tions are modulated by subcortical cholinergic input 192- saporin could produce, at least subtle, deficits in sensory or motor ability. The paralimbic cortical areas (pyriform, entorhinal, perirhinal and insular-areas 51, 28, 35, 13, respectively) show enclaves of greatly reduced fibers in cortices least affected and almost no fibers whatever in cortices more strongly affected by 192-saporin. This stands in contrast to a previous study where paralimbic choliner- gic fibers were reduced only partially by a larger dose of i.c.v. 192-saporin than we used [14].

5. Conclusions

Bilateral i.c.v, injection of 192-saporin produced a ro- bust cholinergic hypofunction in the targets of the CBF. This immunotoxin can be used to study mechanisms of cholinergic degeneration, neuroplasticity following such an insult, and the impact of cholinergic hypofunction on other systems. However, the behavioral deficit induced by i.c.v. 192-saporin reflects an apparent combined influence of (i) the decrease in HAChT, (ii) the decrease in norepinephrine in the HPC, and (iii) the loss of Purkinje cells in the cerebellum. This complex interplay of factors makes the i.c.v, model of 192-saporin problematic for studying the role of the CBF in normative behavior and in disease states. However, a number of reports have demonstrated that injection of 192-saporin directly into components of the CBF can produce a selective loss of cholinergic neu- rons, and decreases in cholinergic parameters in the targets of these neurons [15,20,45,48]. Therefore, site-specific in- jection of 192-saporin might provide a viable approach to further delineate the biology and function of the CBF and to model disorders of cholinergic hypofunction such as Alzheimer's disease.

Acknowledgements

The authors would like to thank Linda King for her expertise in performing the HPLC analyses. This work was supported in part by an NSF Grant (IBN 9222097) and a gift in memory of Colonel Norman C. Kalmar to T.J.W.

244 T.J. Walsh et al. /Brain Research 702 (1995) 233-245

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