Laboratory of Neuropsychopharmacology, Department of Psychiatry and
Behavioral Sciences, Emory University School of Medicine, Atlanta,
Georgia
The effects of a single injection of haloperidol (2.0 mg/kg), a typical
antipsychotic drug, on neurotensin (NT) concentrations and
NT/neuromedin N (NT/NN) mRNA expression in adult female and male rats
were examined. There were significant estrous cycle stage-related
differences in both NT concentrations and NT/NN mRNA expression in
female control rats. Although acute administration of haloperidol
increased NT concentrations and NT/NN mRNA expression in the
caudate/putamen and nucleus accumbens of both male and female rats,
haloperidol did not increase NT/NN mRNA expression during diestrus 2 or
NT concentrations during proestrus in the nucleus accumbens of female
rats. These results indicate the presence of both sex- and estrous
cycle-related differences in the regulation of NT-containing neurons
and in the effects of antipsychotic drug administration on the NT
system of the rat brain.
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Introduction |
A
burgeoning literature supports the view that sex differences in the
course and treatment response of patients with schizophrenia exist.
These include the following: 1) a delayed age of onset in women,
possibly attributable to the number of women becoming schizophrenia
during their 40s and 50s; 2) better treatment response in women; 3)
less severe course of illness; and 4) better outcome in women (Castle
et al., 1995
). It is unclear, however, whether the sex differences
observed in schizophrenia are due to actual sex differences in the
manifestation of the disorder, or whether women are more susceptible to
distinct subtypes of schizophrenia. Because of the increased incidence
of psychotic episodes at times of low estrogen (menopause and the
puerperal period), however, estrogen has been hypothesized to have a
protective, possibly antidopaminergic, effect in schizophrenia.
The neurotensin (NT) system is one neurotransmitter system regulated by
estrogen. NT is a tridecapeptide that was first structurally characterized from extracts of bovine hypothalamus by Carraway and
Leeman (1973). Estrogen has been repeatedly demonstrated to exert
effects on NT-containing neurons in the rat brain (Alexander et al.,
1989a; Herbison and Theodosis, 1991; Watters and Dorsa, 1998).
Moreover, numerous studies have demonstrated interactions between
dopamine (DA) neurons and estrogen (Becker, 1999), and the
former has long been known to be intimately related to NT circuits.
NT was first hypothesized to be an endogenous antipsychotic due to the
numerous similarities between the effects of centrally administered NT
and peripherally administered antipsychotic drugs (Nemeroff, 1980;
Bissette and Nemeroff, 1995). Clinical studies of drug-free
schizophrenic patients have repeatedly demonstrated that there is a
subset of such patients with decreased cerebrospinal fluid (CSF)
concentrations of NT. After antipsychotic drug treatment, these CSF NT
concentrations are increased toward control concentrations (Lindström et al., 1988; Garver et al., 1991; Sharma et al., 1997). There also appears to be a correlation between NT concentrations in the CSF and the magnitude of psychopathology, including negative symptoms (Garver et al., 1991; Breslin et al., 1994). Schizophrenic patients with low CSF concentrations of NT are lithium nonresponders, and have a greater degree of thought disorder, negative symptoms, delusions-hallucinations, behavioral disorganization, and impaired functioning.
One of the most consistent findings implicating the NT circuits to the
mechanism of action of antipsychotic drugs is the effect of
antipsychotic drug administration on regional NT concentrations in the
rat brain. Govoni et al. (1980) first reported that the clinically
efficacious antipsychotic drugs haloperidol, chlorpromazine, trifluoroperazine, and pimozide specifically increased NT
concentrations in the nucleus accumbens and caudate/putamen of rats,
whereas clinically ineffective phenothiazines (e.g., promazine and
promethazine), as well as other classes of psychotherapeutic agents
such as tricyclic antidepressants, anxiolytics, and antihistamines
failed to alter NT concentrations in any brain region examined (Govoni
et al., 1980; Myers et al., 1992). In contrast to the effects of
typical antipsychotic drugs, antipsychotic drugs classified as
"atypical" due to their lack of extrapyramidal side effects and
superior efficacy in treatment-resistant schizophrenia (e.g.,
clozapine) increase NT concentrations only in the nucleus accumbens
(Kilts et al., 1988).
Administration of the typical antipsychotic drug haloperidol also
increases the number of NT/neuromedin N (NT/NN) mRNA-expressing neurons
in the neostriatum of the rat brain. Merchant et al. (1992), using in
situ hybridization histochemistry, demonstrated that a single dose of
haloperidol leads to a nearly 3-fold increase in the number of cells
expressing NT/NN mRNA in the dorsolateral striatum, whereas the
atypical antipsychotic drug clozapine had no effect in this brain
region. Both clozapine and haloperidol were found to increase NT/NN
mRNA levels in the shell section of the nucleus accumbens.
To date, all of the studies examining the effects of antipsychotic
drugs on the NT system have been conducted in adult male rats. For all
of the above-cited reasons it was of interest to scrutinize the effects
of antipsychotic drug administration in female as well as male rats.
These studies examined basal levels of NT/NN mRNA expression and NT
concentrations in both male and female rats, as well as the effects of
an acute injection of the typical antipsychotic drug haloperidol on
NT/NN mRNA expression, as well as NT tissue concentrations.
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Materials and Methods |
Animals and Housing.
Sexually mature male and female
Sprague-Dawley rats (150-200 g; Harlan Sprague-Dawley, Inc.,
Indianapolis, IN) were housed in same sex groups of four, under 24-h
light/dark cycle (lights on 7:00 AM; lights off 7:00 PM) in an
environmentally controlled animal facility. Food and water were
available ad libitum. All rats were handled daily for 1 week before
treatment. The Emory Institutional Animal Care and Use Committee
approved all animal protocols.
Treatment and Tissue Preparation.
Stage of estrous was
assessed in female rats by vaginal lavage. Based on the number and type
of cells present in the daily swab, females were divided into four
groups: diestrus 1 (D1), diestrus 2 (D2), proestrus (P), and estrus
(E). Only those rats showing at least two regular 4-day cycles were
included in the study. Male (n = 5-10) and female rats
(n = 5-10/group) received a single s.c. injection of
either haloperidol (2.0 mg/kg) or vehicle (1.0 ml/kg, 0.3% tartaric acid).
For measurement of NT concentrations by radioimmunoassay (RIA), vaginal
lavages were obtained from all female rats 1 h before drug
administration (4:00 PM) and 1 h before they were sacrificed (10:00 AM, 18 h after the single injection). Based on the final swab and the results of the previous day's swabs, the females were
categorized as being either in D1, D2, P, or E at the time of death.
For measurement of NT/NN mRNA expression by RNase protection assay
(RPA) and in situ hybridization, vaginal lavages were obtained 1 h
before the rats were injected (9:00 AM) and at the time of kill (4:00
PM, 7 h after the single injection).
Rats used for examination of NT concentrations or NT/NN mRNA expression
by RPA were sacrificed by decapitation, and the brains were rapidly
removed and frozen. The brains were dissected freehand on ice based on
the method of Glowinski and Iversen (1966). The brain regions dissected
included the prefrontal cortex, nucleus accumbens, anterior and
posterior caudate/putamen, hippocampus, substantia nigra (SN), and
ventral tegmental area (VTA). Individual brain regions were stored at
70°C in polypropylene microcentrifuge tubes until assay.
Animals used for in situ hybridization analysis of NT/NN mRNA
expression were anesthetized using Euthanasia-5 solution (Henry Schein,
Inc., Port Washington, NY) and transcardially perfused via the
ascending aorta with cold 0.9% NaCl (200 ml) followed by cold 4%
paraformaldehyde in 0.1 M phosphate buffer (pH 7.6) for 8 min (250 ml).
The brains were then removed and postfixed in 4% paraformaldehyde for
24 h at 4°C and then transferred to 20% sucrose for 48 h.
After postfixing, the brains were rinsed in double-distilled
H2O, dried, and stored at 70°C until use. Tissue sections (30 µm) were cut on a microtome and collected in
24-well series. The level of the appearance of the anterior commissure
was marked for future reference. Tissue sections were stored in
cryoprotectant solution (30% ethylene glycol and 20% glycerol in 25 mM phosphate buffer, pH 7.4) at 20°C. Before slide mounting the
tissue, the tissue slices were rinsed in 50 mM phosphate buffer (pH
7.6) to remove cryoprotectant. The slides were then allowed to dry at
room temperature and stored at 20°C until use.
RPA.
Total RNA was extracted from individual brain
regions using a phenol/guanidinium isothiocyanate/chloroform method.
The tissue was homogenized in TRI Reagent (phenol and guanidine
isothiocyanate in a monophase solution; Molecular Research Center,
Inc., Cincinnati, OH), chloroform was added, and the sample was
centrifuged at 12,000g for 15 min at 4°C. The homogenate
then separates into three phases, with the top aqueous phase containing
the RNA. The RNA was then precipitated with isopropanol, washed with
75% ethanol, and resuspended in distilled DEPC-treated water.
Total RNA content was determined by optical densitometry at 260 nm.
Riboprobes were prepared as described above for the in situ
hybridization with the exception that [32P]UTP
was incorporated as the radiolabel. The probe was then gel purified and
used in conjunction with the Ambion RPA II RNase protection kit.
Nonlabeled sense strand RNA (for determination of a specific signal)
was generated using SP6 RNA polymerase and XbaI linearized
plasmid using a similar method.
The RNase protection assay was performed using the RPA II ribonuclease
protection assay kit (Ambion, Austin, TX). Briefly, extracted RNA was
combined with 32P-labeled probe and allowed to
hybridize overnight at 45°C. 32P-Labeled
antisense probe to rat actin RNA was also added to serve as an internal
RNA-loading control for each sample. An RNase cocktail (RNase A/RNase
T1) was added, and the samples were incubated for 30 min. The RNases
were then deactivated using an ethanol mixture (solution Dx, RPA II
kit; Ambion), the protected fragments ethanol precipitated, and
resuspended in gel-loading buffer (solution E, RPA II kit; Ambion). The
protected fragments were separated on a denaturing polyacrylamide gel.
To determine the amount of nonspecific probe binding, nonlabeled sense
strand RNA (a different length than the native sense strand RNA) was
added to separate samples. The gel was then exposed to an
autoradiographic film, and the resulting bands were quantified by
densitometry on a Nikon microphot with a charge-coupled device video
system using an Apple Quadra 950 computer with NIH IMAGE software.
Hybridization times and exact buffer concentrations were adjusted to
maximize sensitivity and specificity.
In Situ Hybridization.
Template plasmid consisting of a
336-base pair EcoRv/BglII fragment (nucleotides
626-961) of the rat NT/NN gene (Kislauskis et al., 1988) in a
BamHI/SmaI-digested pGEM4 (Promega, Madison, WI)
was generously provided by P. Dobner (University of Massachusetts Medical Center, Worcester, MA).
35S-Labeled antisense riboprobes were generated
using EcoRV linearized plasmid, nucleotides,
[35S]-UTP, and T7 RNA polymerase (protocol
adapted from the T7/T3 MAXIscript kit; Ambion).
35S-Labeled sense strand RNA was generated using
SP6 RNA polymerase, and XbaI linearized plasmid by the same method.
Unincorporated nucleotides were removed from the reactions using Quick
Spin Columns (Boehringer Mannheim, Indianapolis, IN). The
35S-labeled riboprobes were then diluted to
1 × 107 cpm/µl in hybridization buffer
[62.5% formamide, 12.5% dextran sulfate, 0.375 M NaCl, 2.5%
Denhardt's solution, 12.5 mM Tris (pH 8.0), 1.25 mM EDTA, pH 8.0] and
stored at 20°C until use.
The protocol for in situ hybridization was adapted from the methods of
Simmons et al. (1989). Briefly, the slide-mounted tissue was digested
with proteinase K followed by acetylation in acetic anhydride. The
slides were then rinsed in 2× standard saline citrate buffer
(NaCl/sodium citrate) and then quickly dehydrated in ascending concentrations of fresh ethanol. After dehydration, slides were dried
at room temperature, and then 100 µl (1 × 106 cpm) of riboprobe mixture (riboprobe in
hybridization solution with tRNA and dithiothreitol) was added to each
slide. The slides were then covered with parafilm, and stored overnight
at 60°C in covered trays.
The parafilm was removed after incubation for 24 h, and the
slides were rinsed in 4× standard saline citrate before RNase digestion (1:500 dilution of 10.0 mg/ml RNase A). The slides were then
rinsed, gradually desalted, and incubated for 30 min at 60°C. Slides
were then quickly dehydrated in ethanol (containing salt and
dithiothreitol), drained well, allowed to dry, and exposed to Kodak
Biomax MR film. After exposure to film, the slides were emulsion coated
and counterstained.
Film autoradiographs were digitized and quantified using NIH IMAGE. A
14C standard curve (Amersham, Piscataway,
NJ) was included in each film cassette for use in standardizing
quantification between films. NT/NN mRNA expression was measured in the
core and shell subdivisions of the nucleus accumbens at 2.4 mm anterior
to bregma according to the atlas of Palkovits and Brownstein (1988).
RIA.
NT concentrations were determined using a highly
specific and sensitive NT RIA. Brain regions were extracted in ice-cold
1.0 M HCl by ultrasonic dismembranation, and the homogenates
centrifuged at 10,000g for 15 min at 4°C. The supernatant
was then transferred to a fresh microcentrifuge tube, vortexed, and
duplicate 100-µl aliquots were transferred to borosilicate glass
tubes and stored at 70°C. On the day of the assay the frozen
aliquots were lyophilized, reconstituted in assay buffer, and then
assayed by a single equilibrium RIA according to methods previously
described (Bissette et al., 1984). The assay buffer consisted of 10 mM
NaH2PO4, 0.15 M NaCl, 0.01% NaN3, 0.1% gelatin, 2.5 mM EDTA, and
0.05% Triton X-100 adjusted to pH 7.6 with NaOH. The antiserum used
(Peninsula Laboratories, Inc., Belmont, CA) is directed toward
the middle portion of the NT molecule and was used at a final dilution
that provides 30% binding of the labeled NT (normally 1:8500).
Synthetic NT1
13 (Bachem Inc., Torrance, CA) was
used as a standard, and monoiodinated [Tyr3]-NT
was obtained from DuPont/NEN (Wilmington, DE). Goat anti-rabbit antiserum (Arnel Products, New York, NY) was used as second antibody. The assay has a sensitivity of 1.25 pg/tube and an
IC50 of 80 pg/tube. The pellets from the
extraction were resuspended in 1.0 M NaOH by sonication and assayed for
protein concentration by the method of Lowry et al. (1951) with BSA
used as standard. NT concentrations are expressed as picograms NT per
milligram protein.
Data from the studies examining sex- and estrous cycle-related
differences in the effects of antipsychotic drug administration were
first analyzed by two-way ANOVA (sex × treatment) or (cycle stage × treatment). After a significant effect of sex, basal
female and male NT/NN mRNA expression or NT concentrations were
compared by t test. After a significant effect of cycle,
female and male NT/NN mRNA expression or NT concentrations were
examined for basal cycle differences by one-way ANOVA. After
significant cycle × treatment interactions, sex × treatment
interactions, or significant differences by one-way ANOVA,
Student-Newman-Keuls multiple comparisons test was applied to identify
significantly differing groups.
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Results |
Sex- and Estrous Cycle-Related Differences in Basal NT/NN mRNA
Expression and Response to Acute Antipsychotic Drug
Administration.
Comparison of basal NT/NN mRNA expression
(determined by RPA) in female versus male rats showed that female rats
had significantly higher levels of basal NT/NN mRNA expression in the
nucleus accumbens (P < .05) and the SN
(P < .05) (Table 1).
Furthermore, basal NT/NN mRNA expression in the anterior
caudate/putamen was significantly lower in female rats compared with
male rats (P < .05) (Table 1).
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TABLE 1
Basal NT/NN mRNA expression and NT-like immunoreactivity in adult male
and female rats
NT/NN mRNA expression was determined by RPA. NT-like immunoreactivity
was determined by RIA. There was a significant sex effect on NT/NN mRNA
expression in the nucleus accumbens (F1,72 = 5.8, P < .05) and SN (F1,67 = 7, P < .01). There was a significant effect of sex on
NT-like immunoreactivity in the prefrontal cortex
(F1,98 = 6.1, P < .05), posterior
caudate/putamen (F1,90 = 4.6, P < .05), SN (F1,91 = 8.4, P < .005),
and VTA (F1,94 = 4.0, P < .05).
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There was a significant effect of estrous cycle stage in the
nucleus accumbens, anterior caudate/putamen, and VTA. Analysis by
one-way ANOVA (Fig. 1a) demonstrated that
these estrous cycle-related differences were due to differences in
basal NT/NN mRNA expression in all three brain regions; NT/NN mRNA
expression was highest in female rats during D2.
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Fig. 1.
Estrous cycle-related differences in NT/NN mRNA
expression (a) and NT concentrations (b) in the nucleus accumbens,
anterior caudate/putamen, and VTA of the rat brain. NT/NN mRNA
expression was determined by RNase protection assay in adult female and
male rats (n = 5-10/group). There was a significant
effect of estrous cycle stage on NT/NN mRNA expression in the nucleus
accumbens (F4,66 = 3.7, P < .005), anterior caudate/putamen (F4,61 = 8.1, P < .001), and VTA
(F4,820 = 8.6, P < .001).
Further analysis by one-way ANOVA demonstrated that these estrous
cycle-related differences were due to differences in basal NT/NN mRNA
expression in all three brain regions (nucleus accumbens:
F4,3121=4.3, P < .001;
anterior caudate/putamen: F4,142 = 6, P < .001; VTA: F4,428 = 3.9, P < .05). There was a significant effect of
estrous cycle stage on NT-like immunoreactivity in the prefrontal
cortex (F4,90=2.7, P < .05), posterior caudate/putamen (F4,87 = 2.9, P < .05), SN (F4,83 = 4.9, P < .001), and VTA
(F4,86 = 3.3, P < .05).
Analysis by one-way ANOVA demonstrated that these estrous cycle-related
differences were due to differences in basal NT-like immunoreactivity
in the VTA (F4,39 = 2.7, P < .05). *P < .05 compared with NT/NN mRNA expression
during P and E. **P < .05 compared with NT/NN mRNA
expression during D1, P, and E. ***P < .05 compared
with NT/NN mRNA expression during P. +P < .05 compared with NT
concentrations during D1 and D2. ++P < .05 compared with NT concentrations during all other estrous cycle
stages and in males.
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Two-way ANOVA of NT/NN mRNA expression (sex × treatment) and
(cycle × treatment) indicated a significant treatment effect in
the nucleus accumbens (Fig. 2), anterior
caudate/putamen (Fig. 3), and the
posterior caudate/putamen (Fig. 4).
Administration of haloperidol significantly increased NT/NN mRNA
expression in the anterior and posterior caudate/putamen in male and
female rats in all stages of the estrous cycle. In the nucleus
accumbens, haloperidol did not increase NT/NN mRNA expression in female
rats during D2. There were no significant (sex × treatment) or
(cycle × treatment) interactions on NT/NN mRNA expression in any
brain region examined.
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Fig. 2.
Estrous cycle-related differences in the effects of
acute haloperidol administration on NT/NN mRNA expression and NT
concentrations in female and males rats in the nucleus accumbens. Male
(n = 10) and female rats (n = 5-10/each estrous cycle stage) received a single s.c. injection of
either haloperidol (2.0 mg/kg) or vehicle. NT/NN mRNA expression was
measured by RNase protection assay. NT-like immunoreactivity was
determined by RIA. Two-way ANOVA (cycle × treatment) indicated a
significant treatment effect (F1,66 = 27.6, P < .001) and (F1,86 = 84.8, P < .001) on NT/NN mRNA expression and NT-like
immunoreactivity, respectively. There was a significant (cycle
stage × treatment) interaction in the nucleus accumbens
(F4,86 = 4.6, P < .001).
*P < .05 compared with corresponding control.
Cross-hatched bar is percentage of control of vehicle-treated
animals ± S.E.M. S.E.M. shown is the largest S.E.M. from a
control group.
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Fig. 3.
Estrous cycle-related differences in the effects of
acute haloperidol administration on NT/NN mRNA expression and NT
concentrations in female and males rats in the anterior
caudate/putamen. Male (n = 10) and female rats
(n = 5-10/each estrous cycle stage) received a single
s.c. injection of either haloperidol (2.0 mg/kg) or vehicle. NT/NN mRNA
expression was measured by RNase protection assay. NT-like
immunoreactivity was determined by RIA. Two-way ANOVA (cycle × treatment) indicated a significant treatment effect
(F1,61 = 81, P < .001) and
(F1,88 = 154.6, P < .001)
on NT/NN mRNA expression and NT-like immunoreactivity, respectively.
*P < .05 and **P < .001 compared with
corresponding control. Cross-hatched bar is percentage of control of
vehicle-treated animals ± S.E.M. S.E.M. shown is the largest
S.E.M. from a control group.
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Fig. 4.
Estrous cycle-related differences in the effects of
acute haloperidol administration on NT/NN mRNA expression and NT
concentrations in female and males rats in the posterior
caudate/putamen. Male (n = 10) and female rats
(n = 5-10/each estrous cycle stage) received a single
s.c. injection of either haloperidol (2.0 mg/kg) or vehicle. NT/NN mRNA
expression was measured by RNase protection assay. NT-like
immunoreactivity was determined by RIA. Two-way ANOVA (cycle × treatment) indicated a significant treatment effect
(F1,44 = 51.9, P < .001)
and (F1,87 = 212, P < .001) on NT/NN mRNA expression and NT-like immunoreactivity,
respectively. *P < .05 and **P < .01 compared with corresponding control. Cross-hatched bar is percentage of
control of vehicle-treated animals ± S.E.M. S.E.M. shown is
the largest S.E.M. from a control group.
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In situ hybridization was used to examine NT/NN mRNA expression in the
shell and core subdivisions of the nucleus accumbens (Fig.
5). Haloperidol significantly increased
NT/NN mRNA expression in both the core and shell subdivisions of the
nucleus accumbens. There was a significant (cycle stage × treatment) interaction in the shell subdivision of the nucleus
accumbens. Haloperidol did not significantly increase NT/NN mRNA
expression in the shell subdivision of the nucleus accumbens during D2.
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Fig. 5.
Estrous cycle-related differences in basal and
haloperidol-induced NT/NN mRNA expression in the shell (a) and core (b)
subdivisions of the nucleus accumbens. Adult male and female rats
(n = 5-10/group) received a single s.c. injection of
either haloperidol (2.0 mg/kg) or vehicle and were sacrificed by
pentobarbital overdose 7 h after the single injection. NT/NN mRNA
expression was measured by in situ hybridization. Two-way ANOVA (cycle
stage × treatment) demonstrated a significant effect of cycle in
both the core (F3,101 = 7.1, P < .001) and shell (F3,98 = 36, P < .001) subdivisions of the nucleus accumbens.
There was also a significant treatment effect in both the core
(F1,101 = 105, P < .001)
and shell (F1,98 = 36, P < .001) subdivisions. There was a significant (cycle stage × treatment) interaction (F3,98 = 3.2, P < .05) in the shell subdivision of the nucleus
accumbens. *P < .001 compared with control in the same
estrous cycle stage. +P < .001 compared with controls in D1 and E.
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Sex- and Estrous Cycle-Related Differences in Basal NT
Concentrations and Response to Acute Antipsychotic Drug
Administration.
There was a significant effect of sex in the
prefrontal cortex, posterior caudate/putamen, SN, and VTA. Further
comparison of basal NT tissue concentrations in female versus male rats
demonstrated that female rats had significantly higher levels of basal
NT-like immunoreactivity in the prefrontal cortex (P < .05) and SN (P < .05) (Table 1).
There was a significant effect of estrous cycle stage on NT-like
immunoreactivity in the prefrontal cortex, posterior caudate/putamen, SN, and VTA (Fig. 1b). Analysis by one-way ANOVA demonstrated that
these estrous cycle-related differences were due to differences in
basal NT-like immunoreactivity in the VTA only. In the VTA, NT-like
immunoreactivity was significantly higher during D1 (P < .05) compared with NT-like immunoreactivity in all other estrous cycle stages.
Two-way ANOVA of NT-like immunoreactivity (sex × treatment) and
(cycle × treatment) indicated a significant treatment effect in
the nucleus accumbens (Fig. 2), anterior caudate/putamen (Fig. 3), and
the posterior caudate/putamen (Fig. 4). Haloperidol significantly increased NT-like immunoreactivity in male and female rats in the
anterior and posterior caudate/putamen. There were no significant (sex × treatment) interactions in any other brain region
examined. In the nucleus accumbens, there was a significant (cycle
stage × treatment) interaction. In this brain region, haloperidol
increased NT-like immunoreactivity in males and in females in all
stages of the estrous cycle except for P.
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Discussion |
In the same year that NT was hypothesized to be an endogenous
neuroleptic (Nemeroff, 1980), the first report appeared concerning the
effects of antipsychotic drug administration on NT-like
immunoreactivity was published (Govoni et al., 1980). To date, all
clinically effective antipsychotic drugs examined have specific effects
on the NT system of the rat brain (for review, see Kinkead et al.,
1999). Previously, acute administration of haloperidol has been shown
to increase NT/NN mRNA expression (Merchant et al., 1992) and NT-like
immunoreactivity (Frey et al., 1986; Eggerman and Zahm, 1988; Zahm,
1992; Zahm et al., 1996) in both the dorsal and ventral striatum of
adult male rats. Within the ventral striatum, the effects of
haloperidol have been reported to be limited to the shell subdivision
(Merchant et al., 1992; Zahm, 1992; Zahm et al., 1996).
The results of these current studies demonstrate the necessity of
considering both sex and the estrous cycle stage of female rats when
examining the regulation of NT-containing neurons. Significant differences in basal NT/NN mRNA expression and NT concentrations between male and female rats were found in the prefrontal cortex, nucleus accumbens, hippocampus, and SN. In addition to differences between the sexes, both NT/NN mRNA expression and NT concentrations varied significantly across the estrous cycle of female rats in the
VTA, nucleus accumbens, and anterior caudate/putamen. Analysis of NT/NN
mRNA expression by in situ hybridization demonstrated that the estrous
cycle-related differences in basal NT/NN mRNA expression were limited
to the shell subdivision of the nucleus accumbens, and not to the core.
The highest levels of NT/NN mRNA expression are seen in D2. It is
possible from the time course of the increase in NT/NN mRNA expression
during D2, to increases in NT concentrations during P that the
increases in peptide concentrations may be due to the increase in gene
expression. NT neurons in the VTA project to the nucleus accumbens
(specifically the shell subdivision of the nucleus accumbens) and NT
neurons within the nucleus accumbens have many local axon collaterals
(Kalivas and Miller, 1984). It is unclear whether the estrous
cycle-related increases in NT peptide are associated with changes in
peptide release. Microdialysis studies are needed to specifically
answer this question.
After examination of the effects of acute administration of the typical
antipsychotic drug haloperidol, striking results were found in the
nucleus accumbens. Haloperidol administration increased NT/NN mRNA
expression and NT concentrations in both male and female rats. In
contrast, haloperidol administration did not increase NT/NN
mRNA expression in female rats in D2, or NT concentrations in female
rats in P. In both cases, it appears that haloperidol did not have a
significant effect due to the significantly higher basal NT/NN mRNA
expression and NT concentrations during D2 and P, respectively. In situ
hybridization analysis of NT/NN mRNA expression in the nucleus
accumbens demonstrated that the lack of effect of haloperidol
administration during D2 was confined to the shell subdivision of the
nucleus accumbens.
The unique estrous cycle regulation and effects of haloperidol
administration on NT/NN mRNA expression in the shell subdivision of the
nucleus accumbens are interesting in light of the role of this
particular brain region in the antipsychotic properties of
antipsychotic drugs. The induction of the immediate early gene c-fos in the nucleus accumbens was shown to be an excellent
predictor of antipsychotic drug potential (Robertson et al., 1994). In
addition, both typical and atypical antipsychotic drugs increase NT/NN
mRNA expression in the shell subdivision of the nucleus accumbens, whereas only typical antipsychotic drugs increase NT/NN mRNA expression in the caudate/putamen (Merchant et al., 1992). Estrous cycle-related regulation of NT/NN mRNA expression and NT tissue concentrations (and
subsequently NT release?) might be the mechanism by which ovarian
hormones exert antipsychotic-like properties.
Although it is unclear which hormone (or hormones) is responsible for
the estrous cycle regulation of the NT system, previous studies have
demonstrated that estrogen can regulate NT/NN gene expression in
hypothalamic nuclei of the rat (Alexander et al., 1989a,b; Alexander,
1993; Alexander and Leeman, 1994). Alexander et al. (1989) demonstrated
that estrogen-induced increases in NT/NN mRNA expression in the
preoptic nuclei are essential for the preovulatory surge of luteinizing
hormone. It was further demonstrated that in ovariectomized rats,
estrogen differentially regulates NT/NN mRNA expression in subdivisions
of the arcuate nucleus and the median eminence and that there are
estrous cycle-related differences in NT/NN mRNA expression in the
dorsomedial division of the arcuate nucleus (Alexander, 1993). These
experiments extend the findings of ovarian hormone regulation of
NT-containing neurons to nonhypothalamic nuclei. Recently, Watters and
Dorsa (1998) described a mechanism by which estrogen may regulate NT/NN
mRNA expression (the NT gene lacks an estrogen response element) via interactions with the cAMP cascade.
Although NT/NN mRNA expression is regulated by estrogen, it is possible
that other ovarian hormones influence NT-containing neurons in these
brain regions or that the effects of estrogen on NT-containing neurons
are secondary to the effects of estrogen on other neurotransmitter
systems. For example, extracellular DA levels, as well as DA-mediated
behaviors are both regulated by ovarian hormone levels (Castner et al.,
1993; Morissette and Di Paolo, 1993; Diaz-Veliz et al., 1994; Xiao and
Becker, 1994; Becker, 1999). The effects of estrogen on the DA system
are complex, nevertheless, it has been hypothesized that estrogen has
an antidopaminergic (possibly antipsychotic drug-like) effect.
Additionally, the fact that haloperidol administration did not further
increase the elevated NT/NN mRNA expression and NT concentrations seen
during D2 and P, respectively, indicates possible regulation of
NT-containing neurons via the same mechanisms. These findings, in
combination with the close association between NT and DA in both the
mesolimbic and nigroneostriatal pathways, indicate the possibility that
ovarian hormone effects on NT-containing neurons may be secondary to
modulation of the DA system.
These results are of interest in light of the evidence that NT may
function as an endogenous antipsychotic agent (Nemeroff, 1980) and the
evidence of sex differences in schizophrenia (Castle et al., 1995;
Canuso et al., 1998). Women have a better treatment response, less
severe symptomatology, and a better outcome compared with men, leading
to the hypothesis that estrogen may have a protective effect in
schizophrenia (Seeman, 1996). Women also have a higher incidence of
neuroleptic-induced Parkinsonism, akathisia, and tardive dyskinesia. A
recent study examining the neuroleptic-like properties of progesterone
demonstrated that progesterone also shares many of the same behavioral
effects as atypical antipsychotic drugs (Rupprecht et al., 1999).
Further studies are needed to determine the mechanism behind the
regulation of the NT-containing neurons in these brain regions, as well
as to determine whether NT release is altered in concert with the mRNA
and peptide concentration changes.
NT, neurotensin;
DA, dopamine;
CSF, cerebrospinal fluid;
NT/NN, neurotensin/neuromedin N;
D1, diestrus 1;
D2, diestrus 2;
E, estrus;
P, proestrus;
RIA, radioimmunoassay;
RPA, RNase protection assay;
SN, substantia nigra;
VTA, ventral tegmental
area.
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Abstract
Introduction
