JOURNALOF

PSYCHIATRIC

Journal of Psychiatric Research 38 (2004) 521–529RESEARCH

www.elsevier.com/locate/jpsychires

Differential effects of typical and atypical antipsychotics onnerve growth factor and choline acetyltransferase expression in

the cortex and nucleus basalis of rats

Vinay Parikh, Mohammad M. Khan, Alvin Terry, Sahebarao P. Mahadik*

Department of Psychiatry and Health Behavior, Medical College of Georgia, Georgia, USA

Medical Research Service Line, Veterans Affairs Medical Center, 1 Freedom Way, Augusta, GA 30904, USA

Program in Clinical and Experimental Therapeutics, University of Georgia College of Pharmacy (Augusta Campus), Augusta, GA, USA

Received 15 January 2003; received in revised form 8 March 2004; accepted 10 March 2004

Abstract

Previously we reported that chronic exposure to haloperidol (HAL), but not the atypical antipsychotics risperidone (RISP) or

clozapine (CLOZ), resulted in reductions in brain choline acetyltransferase (ChAT) immunoreactivity and impaired water maze task

performance in rats. In the present study, we compared the effects of these antipsychotic drugs on the expression of nerve growth

factor (NGF) as well ChAT the in the rat cortex and nucleus basalis of Meynert (NBM) in an effort to determine the underlying

mechanism for the differential drug effects observed previously. We also evaluated the effects of these compounds in a crossover

design to evaluate specific neurochemical consequences of switching between typical and atypical antipsychotics, a common practice

observed in the clinical setting. Male Wistar rats (250–300 g) were exposed to HAL (2.0 mg/kg/day), RISP (2.5 mg/kg/day), or

CLOZ (20 mg/kg/day) for 45 days or a pre-treatment regimen consisting of administering either RISP/HAL (i.e., RISP followed by

HAL) or CLOZ/HAL, or a post-treatment regimen consisting of administering: HAL/RISP or HAL/CLOZ. The duration of each

treatment in the crossover study was also 45 days. NGF and ChAT immunoreactivity were measured by quantitative immuno-

histochemistry in some sub-cerebral cortical regions and NBM after drug exposures. NGF protein was also measured by an enzyme-

linked ImmunoSorbent assay (ELISA) in rat sensorimotor cortex. The results indicated that HAL (but not RISP or CLOZ)

significantly reduced NGF levels in some sub-cortical regions and ChAT immunoreactivity in both cortex and NBM. However, pre-

treatment with CLOZ prevented the HAL-associated decreases in NGF and ChAT, while post-treatment with either RISP or CLOZ

(i.e., after the administration of HAL) appeared to restore NGF and ChAT to control levels. These data indicate that antipsychotic

drugs exert dissimilar effects on the levels of NGF and ChAT in the brain, which may contribute to their differential effects on

cognitive function. The crossover data further suggest that certain atypical antipsychotic drugs (e.g., clozapine) may have the

potential to prevent or reverse the deleterious effects of HAL on important neurochemical substrates of cognitive function.

� 2004 Elsevier Ltd. All rights reserved.

Keywords: Antipsychotics; Nerve growth factor; Choline acetyltransferase; Haloperidol; Risperidone; Clozapine

1. Introduction

Atypical antipsychotic drugs such as clozapine

(CLOZ), risperidone (RISP), and olanzapine (OLZ),

when compared to typical antipsychotics such as halo-

peridol (HAL), are associated with fewer extrapyrami-

dal symptoms (Buckley, 2001) and are reported to be

* Corresponding author. Tel.: +1-706-733-0188x2490; fax: +1-706-

823-3977/3949.

E-mail address: mahadik@psychnts4.mcg.edu (S.P. Mahadik).

0022-3956/$ - see front matter � 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.jpsychires.2004.03.008

more effective in reducing negative symptoms as well as

to improve cognitive performance in schizophrenic pa-

tients (Sharma and Mockler, 1998; Kern et al., 1999;

Cuesta et al., 2001; Keefe et al., 1999). The latter find-

ings are important, because a wide range of cognitive

deficits (i.e., attention, learning, memory, and executive

function) are observed in schizophrenic patients evenduring the early stages of the illness (Tollefson, 1996;

Friedman et al., 1999). Accordingly, the management of

these symptoms is essential to improving the disease

outcome.

522 V. Parikh et al. / Journal of Psychiatric Research 38 (2004) 521–529

Recently, we demonstrated that chronic exposure by

rats to HAL impaired spatial learning performance and

that this impairment was concomitant with a loss of

choline acetyltransferase (ChAT) immunoreactivity in

certain brain regions such as the cortex and hippocam-pus (Terry et al., 2002, 2003). This finding is important,

since cholinergic activity in these brain regions influences

or modulates a number of cognitive processes (reviewed,

Perry et al., 1999). Interestingly, other reports have in-

dicated that HAL exposure in rats reduces the levels of

the endogenous neurotrophin, nerve growth factor

(NGF) in the brain (Alleva et al., 1996; Angelucci et al.,

2000). Since the survival and function of adult mam-malian cholinergic neurons (particularly those projecting

from the basal forebrain to the cortex and hippocampus)

is dependent on NGF (Rylett and Williams, 1994), we

further investigated the effects of antipsychotics on this

specific neurotrophin. Furthermore the basal forebrain

has long been suggested to play a crucial role in the

psychopathology of schizophrenia, particularly in at-

tentional deficits and altered information processing(Stevens, 1973; Heimer, 2000). Most of the cells in the

nucleus basalis of Meynert (NBM) project to all cortical

regions (Zaborszky et al., 1999). These projections are

important facilitators of synaptic plasticity (Sachdev

et al., 1998), and damage to these projections is probably

involved in the attentional abnormalities of several ad-

ditional neuropsychiatric disorders as well (Sarter and

Bruno, 1998). We were, therefore, particularly interestedin possible molecular mechanisms underlying the differ-

ential effects of the aforementioned antipsychotic drugs

on the NBM-cortical pathway. The effects of chronic

exposure to HAL on NGF and ChAT in the NBM-

cortical pathway in rats were compared to the effects

following exposure to RISP and CLOZ. Three cortical

areas (cingulate, secondary motor, and sensorimotor

representing limbic, neocortical, sensory, and motor ar-eas, respectively) and the NBM (the locus of a dense

group of NGF dependent cholinergic neurons that have

projections to cortex) were examined. We also evaluated

these compounds in a crossover design to evaluate spe-

cific neurochemical consequences of switching between

typical and atypical neuroleptics, a common practice

observed in the clinical setting. The switching of neuro-

leptics is often necessary from a therapeutic standpointto address challenges associated with adverse drug effects

or inadequate efficacy in individual patients.

2. Materials and methods

2.1. Animals

Male albino Wistar rats (225–250 g) were obtained

from Harlan Sprague–Dawley, Inc. (Indianapolis, IN)

and housed in a temperature-controlled room (25 �C)

with a 12-h light/dark cycle. Upon arrival, each animal

was provided with tap water and food (Purina Rat

Chow�) ad libitum for one week. Thereafter, tap water

was replaced with the solutions described below. All

procedures employed during this study were reviewedand approved by the Medical College of Georgia

Committee on Animal Use for Research (CAURE) and

the Veterans Affairs Medical Center Subcommittee on

Animal Use. These procedures were consistent with

AAALAC guidelines as per the Public Health Service

(PHS) Policy on Humane Care and Use of Laboratory

Animals (2000, reprint).

2.2. Drug treatments

Forty-five days were chosen as the duration for each

drug treatment based on several of our earlier studies

that showed significantly different effects from exposure

to HAL vs. exposure to atypical antipsychotics (Maha-

dik et al., 1988; Korenovsky et al., 1990; Mahadik and

Mukherjee, 1995; Parikh et al., 2004; Terry et al., 2002,2003). All the drugs were prepared daily and adminis-

tered in solutions that replaced drinking water. HAL

(Sigma Chemicals, St. Louis, MO), RISP (Janssen

Pharmaceutica, Trenton, NJ) and CLOZ (ICN Bio-

medicals Inc., OH) were each dissolved in 0.1 M solu-

tion of acetic acid and subsequently diluted with tap

water (1:100) to administer the daily dose of drug. The

amount of drug intake was estimated daily and adjust-ments were made depending on the amount of the fluid

consumed and the weight of the animal. Rats

(N ¼ 10–12/group) were exposed to HAL (2 mg/kg/day),

RISP (2.5 mg/kg/day), or CLOZ (20 mg/kg/day) for 45

days. The HAL dose was chosen based on previous

studies (Mahadik et al., 1988; Terry et al., 2002, 2003).

RISP and CLOZ doses were chosen based on several

published studies that reported the effect of these drugson a variety of behavioral and pharmacological pa-

rameters in rats (e.g., Arnt, 1996; Didriksen, 1995;

Skarsfeldt, 1996). In the crossover study, the animals in

the pre-treatment group (N ¼ 10–12/group) were ex-

posed to RISP (2.5 mg/kg/day) or CLOZ (20 mg/kg/day)

prior to exposure to HAL (2 mg/kg/day) (RISP/HAL or

CLOZ/HAL). Animals in the post-treatment group were

administered either RISP (2.5 mg/kg/day) or CLOZ (20mg/kg/day) after exposure to HAL (2 mg/kg/day). The

duration of each treatment in the crossover study was 45

days. Tap water containing 0.1 M acetic acid (1:100) was

provided for rats in the control groups (N ¼ 10–12) for

45 and 90 days, respectively, to assure that any unan-

ticipated effects did not result from the vehicle used.

2.3. Immunohistochemistry

Rats (N ¼ 4–6/group) were deeply anaesthetized with

ketamine/xylazine and perfused with a cold 0.01 M

V. Parikh et al. / Journal of Psychiatric Research 38 (2004) 521–529 523

phosphate buffer saline (PBS) injected into the left

ventricle to remove circulating blood elements. Their

brains were quickly removed and cryoprotected in an

embedding media. Coronal sections (20 lm in thickness)

were cut at a specific anatomical landmark; interaural9.48 mm, bregma 0.48 mm (Paxinos and Watson, 1998)

to obtain sections with subfields of cerebral cortex

(cingulate, secondary motor, and sensorimotor), and

interaural 7.70 mm, bregma )1.30 mm for NBM using a

cryostat microtome (Leica CM 3050S, Leica Microsys-

tems Inc., Chantilly, VA) at )20� 2 �C. Fresh frozen

sections were fixed in ice-cold acetone for 30 min and

air-dried. Then the sections were rinsed in 0.01 M PBScontaining Tween 20 (PBST). After blocking with 10%

donkey serum for 1 h, sections were washed and incu-

bated overnight at 4 �C with rabbit anti-mouse poly-

clonal NGF antibody (1:100) or monoclonal ChAT

antibody (10 lg/ml) (Chemicon International Inc.,

Temecula, CA). After three or four washes, secondary

antibodies (Jackson ImmunoResearch Laboratories

Inc., West Grove, PA), specifically donkey anti-rabbitcy3 (1:100) for NGF or donkey anti-mouse cy2 (1:200)

for ChAT, were applied.

In order to determine the co-localization of NGF and

ChAT in the cortex and NBM, double immunostaining

for NGF and ChAT was performed. Fixed sections were

washed in PBST and blocked with 10% normal donkey

serum for 1 h. Sections were incubated overnight at 4 �Cwith anti-mouse polyclonal NGF antibody (1:100) andmonoclonal ChAT antibody (Chemicon International

Inc., Temecula, CA). Then sections were washed and

incubated for 1 h with donkey anti-rabbit cy3 antibody

(1:100) and donkey anti-mouse cy2 (1:200). Sections

were washed several times with PBST and visualized.

Controls for double-labeling included reversing the or-

der of primary antibodies as well as omitting the first or

second primary antibody.For ChAT immunohistochemistry in NBM, the

method used was modified from that used by Angelucci

et al., 2000). Fixed sections were incubated with 0.3%

H2O2 in 0.3% horse serum for 5 min to block endoge-

nous peroxidase activity. Sections were washed with

PBST and blocked with 10% normal horse serum for 1 h

and then incubated with 10 lg/ml mouse monoclonal

anti-ChAT antibody (Chemicon International Inc.,Temecula, CA) overnight at 4 �C. The sections were

washed three times with PBST and then incubated with

a diluted solution (1:20) of biotinylated horse anti-

mouse IgG for 2 h. After washing, the sections were

incubated with avidin-HRP for 1 h and staining was

developed with DAB. The sections were washed with

water, dehydrated using graded concentrations of eth-

anol and xylene, and coverslipped. As controls, thisimmunohistochemical procedure was carried out on

representative sections with the omission of primary or

secondary antibody.

2.4. Quantitative image analysis of immunohistograms

Immunohistograms (two sections from each of 4–6

animals) from each treatment group were analyzed with

a Zeiss Axioplan-2 microscope equipped with CCDcamera, PC computer, and Zeiss KS-300 image analy-

ses software by an experimenter blinded to the study

code. To quantify the amount of NGF and ChAT

immunoreactivity, each section of the cortical area was

examined for total area of red and green fluorescence

intensity. In NBM, each section was examined for total

staining intensity of DAB stained elements. Digital

images were imported to Zeiss KS-300 image analysessoftware and quantified by measuring the total intensity

of immunoreactivity via a relative densitometric mea-

surement. Total staining intensity is expressed in arbi-

trary intensity units. Values for each animal represent

the image intensity of the cell density to count profile

per mm2 for each section. Only intact neurons were

included and the mean values from all sections of each

animal in a group were calculated and used in statisticalanalyses.

2.5. NGF ELISA

The animals (n ¼ 6/group) were sacrificed after 45

days of treatment. Their brains were removed and the

dorsal cortical tissue (mainly consisting of frontal and

parietal cortexes) dissected out, and then snaps frozen inliquid nitrogen and stored at )70 �C. The frozen tissues

were homogenized in an ice-cold buffer solution con-

sisting of 100 mM Tris/HCl, pH 7.0, containing 2%

bovine serum albumin (BSA), 1 M NaCl, 4 mM EDTA,

2% Triton X-100, 0.1% sodium azide, 5 lg/ml aprotinin,

0.5 lg/ml antipain, 157 lg/ml benzamidine, 0.1 lg/ml

pepstatin A, and 17 lg/ml phenylmethyl-sulphonyl

fluoride using polytron homogenizer (Brinkmann In-struments, New York, USA). The homogenate was

centrifuged at 15,000g for 30 min. The supernatants

were collected and used for ELISA. Quantification of

endogenous NGF was done using a two-site enzyme

immunoassay kit (Chemicon International Inc., Teme-

cula, CA). The sensitivity of ELISA was 10–15 pg/ml

and no significant crossreactivity was observed with

other neurotrophins like BDNF, NT3, or NT4/5. Dataare represented as pg/g wet weight and all assays were

performed in duplicate.

2.6. Statistical analyses

The data are expressed as means� SEM. One-way

analysis of variance (ANOVA) was applied to compare

the difference between control and treatment groups.When significant differences were observed, pairwise

multiple comparisons using the Student–Newmann–

Keuls (SNK) test were performed.

524 V. Parikh et al. / Journal of Psychiatric Research 38 (2004) 521–529

3. Results

3.1. Effect of chronic antipsychotic exposure on NGF

Expression in cortex

NGF was predominantly localized within the neuro-

nal cell bodies, including within the nuclear region. The

data from quantitative image analysis of NGF immu-

noreactivity in three cortical areas: cingulate, secondary

motor, and sensorimotor cortex, are presented in Table

1. Antipsychotic treatment produced significant effects

in all examined cortical areas (cingulate, F ð7; 25Þ ¼ 5:63,P < 0:01; secondary motor, F ð7; 26Þ ¼ 3:25, P < 0:05and sensorimotor F ð7; 25Þ ¼ 18:16, P < 0:001). The

largest differences in staining intensity associated with

antipsychotic exposure were observed in the sensori-

motor cortex. NGF immunoreactivity was markedly

lower in cingulate and sensorimotor cortex in HAL-

treated animals as compared to control animals (cin-

gulate, 7132� 311 vs. 9298� 431 units, P < 0:01, SNK

test and sensorimotor cortex, 5034� 363 vs. 9487� 534units, P < 0:001, SNK test). There was a trend that the

secondary motor area had lower NGF immunoreactiv-

ity in HAL treated animals (P ¼ 0:1 vs. control,

P < 0:05 vs. CLOZ). There were also significant differ-

ences between the controls, RISP, and CLOZ groups

(specifically, the CLOZ group was associated with the

highest immunoreactivity, P < 0:001 vs. control and

RISP, SNK test) in the sensorimotor cortex (Table 1).Quantitative immunohistochemical analysis also re-

vealed that post-treatments with RISP or CLOZ after

chronic exposure to HAL significantly increased NGF

Table 1

NGF levels in rat cortexA

Treatment Cingulate Secondary

motor

Sensorimotor

CON45 9798� 431 9546� 367 9487� 534

CON90 9581� 349 9266� 236 9392� 395

HAL 7132� 311�� 7655� 371a 5034� 363���

RISP 9014� 541 8911� 295 8845� 526

CLOZ 10815� 589 10355� 445 11814� 902b;c

RISP/HAL 8092� 390 7856� 635 6095� 362

HAL/RISP 8846� 408 9179� 813 7188� 370d

CLOZ/HAL 8382� 583 8392� 571 6792� 431d

HAL/CLOZ 9494� 410 9466� 509 7854� 149e

CON45, 45 days vehicle control; CON90, 90 days vehicle control;

HAL, haloperidol treated; RISP, risperidone treated; CLOZ, clozapine

treated; RISP/HAL and HAL/RISP, pre- and post-treated with ris-

peridone; CLOZ/HAL and HAL/CLOZ, pre- and post-treated with

clozapine Statistical analysis was carried out using one-way analysis of

variance for main effect followed by SNK test for post hoc group

comparisons. ��,���P < 0:01, 0.001 HAL vs. CON45; aP < 0:05 HAL

vs. CLOZ; bP < 0:001 CLOZ vs. CON45; cP < 0:001 CLOZ vs. RISP;dP < 0:05 HAL/RISP, CLOZ/HAL vs. HAL; and eP < 0:01 HAL/

CLOZ vs. HAL.AData are represented as means�SEM. Levels indicate fluorescence

intensity units quantified by image analysis of NGF immunoreactivity.

immunoreactivity compared to that observed in animals

exposed only to HAL (P < 0:05 HAL/RISP vs. HAL,

P < 0:01 HAL/CLOZ vs. HAL). Furthermore, pre-

treatment with CLOZ also prevented the reduction in

NGF immunoreactivity observed in animals adminis-tered HAL alone (P < 0:05 CLOZ/HAL vs. HAL). The

effect of chronic exposure to antipsychotics on NGF

protein levels in the cortex (predominantly sensorimotor

cortex) of rat as measured by ELISA is shown in Fig. 1.

These data parallel the immunohistochemical differ-

ences, i.e., a significant reduction in NGF protein oc-

curred with exposure to HAL compared to controls

(1310� 73 vs. 750� 66 pg/gwt, P < 0:01). No change inNGF protein levels was seen after exposure to RISP,

and a significant increase (P < 0:05 vs. control, RISP)

occurred following exposure to CLOZ. Post-treatment

with either RISP or CLOZ partially restored (P < 0:05HAL/RISP, CLOZ/HAL, HAL/CLOZ vs. HAL) and

pre-treatment with CLOZ prevented (P < 0:05 CLOZ/

HAL vs. HAL) the HAL induced decline in NGF

expression.

3.2. Effect of antipsychotic treatment on ChAT Expres-

sion in cortex and NBM

Quantitative data from ChAT immunoreactivity in

the sensorimotor cortex and NBM are shown in Table 2.

ChAT immunoreactivity was measured and quantified

in sensorimotor cortex only since the largest differencesin NGF immunoreactivity were observed in this cortical

region. Significant treatment effects were observed on

ChAT immunoreactivity, both in sensorimotor cortex

(F ð7; 28Þ ¼ 12:54, P < 0:001) and NBM (F ð7; 39Þ ¼19:55, P < 0:001). Post hoc comparisons indicated a

Fig. 1. Effect of NGF protein levels in rat cortex (sensorimotor area)

after chronic exposure with antipsychotics. NGF was measured in

dorsal cortical tissue, which mainly consisted of frontal and parietal

cortexes. Bar graphs show means� SEM (n ¼ 6 animals/group).

CON45, vehicle control (45 days); CON90, vehicle control (90 days);

HAL, haloperidol treated; RISP, risperidone treated; CLOZ, clozapine

treated; RISP/HAL and HAL/RISP, pre- and post-treated with ris-

peridone; CLOZ/HAL and HAL/CLOZ, pre- and post-treated with

clozapine. ��, P < 0:01, HAL vs. CON45; (a) P < 0:05, CLOZ vs.

CON45; (b) P < 0:05 CLOZ vs. RISP, and (c) P < 0:05 HAL/RISP,

CLOZ/HAL, HAL/CLOZ vs. HAL. Values represent pg/gwet weight.

Table 2

ChAT levels in sensorimotor cortex and NBMA

Treatments Sensorimotor cortex NBM

CON 5111� 272 6988� 368

CON90 5082� 347 6853� 411

HAL 2896� 157��� 4267� 178���

RISP 4519� 383 6375� 210

CLOZ 6089� 416a;b 8612� 375a1;b1

RISP/HAL 4014� 178c 5813� 355c1

HAL/RISP 3350� 177 4983� 282

CLOZ/HAL 4534� 360d 7334� 215d1

HAL/CLOZ 3710� 222 5596� 418e

CON45, vehicle control (45 days); CON90, vehicle control (90

days), HAL, haloperidol treated; RISP, risperidone treated; CLOZ,

clozapine treated; RISP/HAL and HAL/RISP, pre- and post-treated

with risperidone; CLOZ/HAL and HAL/CLOZ, pre- and post-treated

with clozapine. Statistical analysis was carried out using one-way

analysis of variance for main effect followed by SNK test for post hoc

group comparisons. ���P < 0:001 HAL vs. CON45; a;a1P < 0:05, 0.01

CLOZ vs. CON45; b;b1P < 0:01, 0.001 CLOZ vs. RISP; c;c1P < 0:05,

0.01 RISP/HAL vs. HAL; d;d1P < 0:01, 0.001CLOZ/HAL vs. HAL;

and eP < 0:05 HAL/CLOZ vs. HAL.AData are represented as means�SEM. Levels indicate fluorescence

(sensorimotor cortex) and DAB (NBM) intensity units quantified by

image analysis of ChAT immunoreactivity.

V. Parikh et al. / Journal of Psychiatric Research 38 (2004) 521–529 525

significant reduction in ChAT intensity with HAL as

compared to that seen in the sensorimotor cortex of

control animals (P < 0:001, SNK test). No such reduc-

tion in ChAT immunoreactivity was observed in animals

after exposure to RISP, but a marked increase in ChAT

immunoreactivity was detected after administration of

CLOZ (P < 0:05 CLOZ vs. CON45, P < 0:01 CLOZ vs.RISP). Interestingly, ChAT immunoreactivity in the

sensorimotor cortex was found to be higher after pre-

treatment with RISP or CLOZ (P < 0:05 RISP/HAL

and P < 0:01 CLOZ/HAL, respectively) when compared

to HAL (Table 2). The changes in ChAT immunoreac-

tivity observed in sensorimotor cortex paralleled the

changes seen in cholinergic cell bodies of NBM. Forty-

five days of exposure to HAL resulted in a significantreduction in the area of ChAT staining (P < 0:01 vs.

control) (Table 2). Morphological changes included a

reduction in the cytoplasmic volume in the cells of ani-

mals administered HAL, whereas normal cellular mor-

Fig. 2. Photomicrographs illustrating the double immunostaining of NGF an

the neuronal cell bodies, ChAT (green) on the synaptic terminals and proj

Bar¼ 10 lm.

phology with a marked increase in the area of ChAT

immunostaining was observed in those animals treated

with CLOZ (P < 0:01 vs. control). Pretreatment with

RISP or CLOZ partially, but significantly, prevented the

decline in ChAT staining (P < 0:01 RISP/HAL vs.HAL, CLOZ/HAL vs. HAL, P < 0:001) and post-

treatment with CLOZ partially restored ChAT immu-

noreactivity (P < 0:05 HAL/CLOZ vs. HAL) (Table 2).

There were no differences in either the immunoreactivi-

ties of NGF and ChAT, or NGF protein levels between

the control animals treated with the vehicle alone for 45

days and those controls treated for 90 days (Tables 1

and 2). Hence, for statistical comparisons in the cross-over study, only data from the control animals treated

for 45 days were used.

3.3. Co-localization of NGF and ChAT in cortex and

NBM

Double immunostaining of NGF and ChAT in the

cortex is shown in Fig. 2(a)–(c). For clarity, ChAT(green), NGF (red), and ChAT and NGF together

(yellow indicates co-localization) are shown in (a), (b),

and (c), respectively, in the same section. ChAT immu-

noreactivity (green) was mainly observed in projections

and in the synaptic terminals surrounding the neuronal

cell bodies containing NGF (red). Some of the synaptic

terminals surrounding the cortical neurons stained po-

sitive for both ChAT and NGF (yellow), indicatinguptake of NGF. A significant positive correlation was

observed between NGF and ChAT immunoreactivities

in sensorimotor cortex (r2 ¼ 0:9; P < 0:01). Double

immunostaining of ChAT (green) and NGF (red) in

cholinergic neuron of NBM is shown in Fig. 3(a)–(c).

Fig. 3(c) shows co-localization of NGF and ChAT

(yellow) in neuronal cell suggesting retrograde transport

of NGF from cortex to NBM.

4. Discussion

In the present study, 45 days of exposure to HAL

markedly reduced NGF levels in various cortical areas

d ChAT in the sensorimotor cortex. (a–c) Localization of NGF (red) in

ections surrounding the cell body (marked with arrows) respectively.

Fig. 3. Photomicrographs illustrating the double immunostaining of NGF and ChAT in NBM. (a–c) Localization of NGF (red) and ChAT (green)

and co-localization (yellow) in the neuronal cell bodies. Bar¼ 30 lm.

526 V. Parikh et al. / Journal of Psychiatric Research 38 (2004) 521–529

of the brain in rats. Changes in the cholinergic marker,ChAT, in the cell bodies of NBM and cholinergic pro-

jections to sensorimotor cortex paralleled the changes in

NGF levels. These finding are in general agreement with

earlier studies in which chronic treatment with HAL

reduced the levels of NGF and ChAT immunoreactivity

in the rat brain (Alleva et al., 1996; Angelucci et al.,

2000; Mahadik et al., 1988; Terry et al., 2002). The ef-

fects on ChAT and NGF following exposure to HALwere associated with altered morphology, i.e., cellular

shrinkage and reduction in dendritic fibers (Angelucci et

al., 2000), but not with cell death as observed in earlier

studies examining the effects of exposure to HAL or

fluphenazine (Mahadik et al., 1988; Jeste et al., 1992).

The deleterious effects described above were not ob-

served with RISP or CLOZ and in fact, CLOZ slightly

increased NGF immunoreactivity in the sensorimotorcortex and increased ChAT immunoreactivity in cortical

cholinergic projections and cholinergic cell bodies of

NBM. The differences in cortical NGF immunoreactiv-

ity indicate that these drugs may alter the expression of

NGF protein in different ways, which is further sup-

ported by similar differences in the ELISA data on NGF

protein in the rat cortex. The changes in ChAT immu-

noreactivity in NBM and co-localization of NGF andChAT in the cortex support the suggestion that drug

related effects on NGF in the cortex may (in turn) result

in alterations in cholinergic markers in the basal fore-

brain (i.e., reduced with HAL and increased with

CLOZ).

With regard to potential implications of data de-

scribed above, the role of NGF in the regulation of

cholinergic activity, and thereby, cognitive performance,is well established. Numerous studies in animals have

reported that NGF can prevent the metabolic or exci-

totoxic injury to hippocampal and cortical neurons in

vivo (Mattson et al., 1995; Shimohama et al., 1993;

Semkova et al., 1996) as well as in vivo (Buchan et al.,

1990; Shigeno et al., 1991; Pechan et al., 1995). NGF

also has been found to increase the survival and function

of CNS cholinergic neurons, regulate ChAT activity,and improve cognitive deficits (Mohammed et al., 1990).

These and other studies have established that NGF is animportant trophic factor particularly for the survival

and neuroplasticity of cholinergic neurons in the adult

hippocampus and cerebral cortex, which are associated

with cognitive performance in both animals and human

(Garofalo et al., 1992; Lo, 1995; Thoenen, 1995).

In an additional series of experiments, pre- or post-

treatment with RISP or CLOZ appeared to prevent and

restore, respectively, the HAL-induced decline in NGFand ChAT expression in the cortex. Moreover these

changes again paralleled the changes in ChAT immu-

noreactivity in the cholinergic cell bodies of NBM.

These findings suggest that atypical antipsychotics like

RISP or CLOZ (via effects on the plasticity of cholin-

ergic neurons mediated via NGF) exert neuroprotective

(and/or restorative) actions against the negative effects

of chronic HAL exposure. The levels of NGF weremuch higher after post-treatment (compared to pre-

treatment) with RISP or CLOZ. In contrast, the levels

of ChAT immunoreactivity were much higher after pre-

treatment as compared to post-treatment with these

compounds. These observations may suggest that the

effects of antipsychotics on NGF precede the effects on

ChAT. It is interesting to note that the crossover effects

associated with CLOZ (compared to RISP) were morerobust implying that there are differential potencies with

regard to the effects of these agents on NGF and ChAT.

Since only a single dose of each neuroleptic was evalu-

ated in this study, it may be possible that the differences

in NGF and ChAT among the drugs observed after this

length of exposure could be dose related rather than the

result of some unique property of the different drugs.

Overall, our results are suggestive of a link between thedecline in NGF and ChAT associated with chronic HAL

administration (and the positive changes in these pro-

teins observed with atypical antipsychotics in the

crossover studies), but do not demonstrate a causal

connection. Further studies are warranted to address the

issue of long-term effects of antipsychotics on the cho-

linergic system and its causal relationship with NGF.

In a previous study we found that the doses of anti-psychotics used in the present study achieved drug

V. Parikh et al. / Journal of Psychiatric Research 38 (2004) 521–529 527

plasma levels well within the therapeutic range used for

the treatment of schizophrenia (Parikh et al., 2003a).

These plasma concentrations were also within the range

needed to provide the level of D2 receptor occupancy

often associated with notable behavioral effects in ani-mals (Arnt, 1996; Didriksen, 1995; Skarsfeldt, 1996).

The differential effects of antipsychotics on NGF ex-

pression may provide a biochemical basis for the con-

trasting behavioral effects of various antipsychotic drugs

reported in animals (Mahadik et al., 1988; Kinon and

Lieberman, 1996; Terry et al., 2002, 2003), as well as in

patients with psychotic disorders (Green and Braff,

2001). However, the mechanisms associated with thedifferential drug effects on the expression of NGF may

be very complex. HAL clearly differs from RISP and

CLOZ in its neurotransmitter receptor reactivity profile

(Bymaster et al., 1996; Kapur et al., 1999; Seeman,

2002), as well as in receptor occupancy (Kapur et al.,

1999). HAL is known to produce potent D2 antagonistic

activity, whereas atypical antipsychotics like RISP and

CLOZ block both the D2 and 5HT2 receptors (Meltzer,1995). Theoretically, any of a number of downstream

biochemical effects initiated by dopamine receptor ac-

tivity could be involved in the differential effects of the

antipsychotics on NGF and ChAT levels. Reports of

working memory deficits in mice deficient in D2 and D3

receptors (Glickstein et al., 2002) as well as in monkeys

treated chronically with drugs that potently block D2

receptors such as haloperidol (Castner et al., 2000) fur-ther suggest that dopamine receptor activity is an im-

portant mechanistic consideration when comparing the

cognitive effects of antipsychotics.

The clinical implications of these findings may be very

important, specifically in the context of treatment-out-

come of schizophrenia. At present, there is general

agreement that cognitive performance is the key psy-

chopathology that must be improved to improve theoverall quality of life of schizophrenic patients (Tollef-

son, 1996; Green and Braff, 2001). Both learning and

memory are impaired at very early stages of schizo-

phrenia, and deficits in ChAT activity have been shown

to correlate with decrements in cognitive function in

schizophrenics (Powchik et al., 1998). We have clearly

demonstrated in previous studies in rats that HAL im-

pairs spatial learning performance and reduces the ex-pression of cholinergic markers in the hippocampus and

cerebral cortex (Terry et al., 2003). Based on the current

results, we hypothesize that these deleterious effects of

HAL may be related to adverse effects on NGF. Inter-

estingly, lower plasma NGF levels were reported in

neuroleptic free schizophrenic patients (Bersani et al.,

1999) as well as those treated with HAL (Aloe et al.,

1997). In addition, we found significantly lower plasmaNGF levels in both drug-na€ıve first-episode psychotic

patients and in patients with chronic schizophrenia who

had been treated over a long period of time with typical

antipsychotics. In contrast, NGF levels were substan-

tially higher in patients treated with atypical antipsy-

chotics (Parikh et al., 2003c). Therefore, augmentation

of NGF activity by atypical antipsychotics could rep-

resent a target for normalizing cognitive deficits early inthe illness and in preventing further deterioration.

The implications of findings of the crossover study

could be important for patients who are refractory or

intolerant to conventional antipsychotics (Fitton and

Heel, 1990; Lane et al., 2002; Lindenmayer et al., 2001;

Umbricht et al., 2002). Data from our study support the

contention that post-treatment with atypicals may be

beneficial in such patients. Similarly, findings from ourstudy of pre-treatment with atypicals may be applicable

in situations where atypicals have been discontinued

because of side effects such as weight gain, cardiovas-

cular complications, diabetes mellitus, or agranulocy-

tosis (Bechara and Goldman-Levine, 2001; Zaluska and

Gajewska, 1995; Bettinger et al., 2000; Leppig et al.,

1989), and then shifted back to typical antipsychotics.

In summary, the present data indicate that, unlikeHAL, exposure to CLOZ or RISP in rats sustained (or

in some cases even increased) levels of NGF in cortical

cholinergic receptive neurons, effects that were paral-

leled by similar positive effects on ChAT levels in the

basal forebrain cholinergic corticopetal projection sys-

tem. Thus, atypical antipsychotics like RISP or CLOZ

may have neurotrophic and/or neuroprotective actions

on cortical cholinergic function. These properties pro-vide one potential mechanism for the differential effects

of the aforementioned antipsychotics on cognitive

function. The crossover data further suggest that certain

atypical antipsychotic drugs (e.g., clozapine) may have

the potential to prevent or reverse the deleterious effects

of HAL on important neurochemical substrates cogni-

tive function.

Acknowledgements

This study was supported in part by Janssen Phar-

maceutica Research Foundation and the National In-

stitute of Mental Health (MH 066233 to A.V.T.). The

authors thank Abhishek Kalla for his excellent technical

assistance and Ms. Karen Ship for her assistance re-garding grammatical, syntax, and sentence structure

editing. The current address for Dr. Vinay Parikh is the

Department of Psychology, Ohio State University, Co-

lumbus, OH.

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