protective effects of maternal buspirone treatment on serotonin reuptake sites in ethanol-exposed...

DEVELOPMENTAL BRAIN

RESEARCH

ELSEVIER Developmental Brain Research 92 (1996) 190-198

Research report

Protective effects of maternal buspirone treatment on serotonin reuptake sites in ethanol-exposed offspring 1

Jung-Ae Kim, Mary J. Druse * The Neuroscience Program and The Department of Molecular and Celhdar Biochemistry Loyola University Chicago, Stritch School of Medicine, 2160 S /

First AL,e., Maywood, IL 60153, USA

Accepted 27 December 1995

Abstract

Previous work in this laboratory demonstrated that in utero ethanol exposure is associated with abnormal development of the serotonergic system. Specific abnormalities included deficiencies of serotonin (5-HT) and its metabolites, and cortical 5-HT reuptake sites. The concentration of 5-HTIA receptors was also altered. The serotonin deficit was detected in the fetal ethanol-exposed brain, at an age when 5-HT would normally function as an essential trophic factor. Thus, it was hypothesized that the early 5-HT ethanol-associated deficit of an essential trophic factor (e.g. 5-HT) could contribute to subsequent developmental abnormalities in serotonergic neurons. In the present investigation we used quantitative autoradiography (QAR) to more fully characterize the developmental abnormalities in 5-HT reuptake sites in developing offspring of ethanol-fed rats. In addition, we attempted to overcome the potential negative impact of the ethanol-associated deficit of fetal 5-HT, by administering a 5-HTIa agonist, buspirone, to pregnant rats. These investigations demonstrated that postnatal (PN) 19 and/or 35 day ethanol-exposed offspring had a significant decrease in [3H]citalopram binding to 5-HT reuptake sites in the frontal cortex, parietal cortex, lateral hypothalamus, substantia nigra, medial septum, and striatum. In contrast, [3H]citalopram binding was increased in the dorsal raphe on PN5 and in the median raphe on PN19. No significant ethanol-associated changes were detected in the hippocampus CA3 region or in the amygdala. When [3H]citalopram binding was compared in the offspring of saline- and buspirone-treated dams, it appeared that maternal treatment with buspirone prevented or reversed most of the ethanol-associated developmental abnormalities in 5-HT reuptake sites. Buspirone prevented the decline in binding of [3H]citalopram in the frontal cortex, lateral hypotbalamus, substantia nigra and medial septum. Similarly, buspirone treatment prevented the ethanol-associated increase in binding in the dorsal and median raphe. Additional experiments are needed to elucidate the impact of maternal buspirone treatment on the development of other neurotransmitter systems in offspring.

Keywords: Serotonin; Fetal alcohol syndrome: Ethanol; Buspirone; Reuptake; 5-HT~

I . I n t r o d u c t i o n

Fetal atcohol syndrome (FAS) is one of the leading causes of mental retardation in the western world. In the United States alone, more than 1200 children are born with FAS each year [1]. Neuroanatomical and neurochemical studies in animal models have attempted to elucidate the underlying causes of the CNS abnormalities found in FAS. Such studies demonstrated altered development of several brain s t ructures and neu ro t r ansmi t t e r sys t ems [13,21,22,35,41]. The systems which are affected by in utero ethanol exposure include those which use the neuro-

* Corresponding author. Fax: (1) (708) 216-8523. This research was performed in partial fulfillment of the requirements

for the degree of Doctor of Philosophy (J.-A.K).

0165-3806/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII S0165-3806(96)00015-6

transmitters serotonin, dopamine, norepinephrine, gluta- mate, histamine and acetylcholine (reviewed in [21,22]).

The development of the serotonergic system, one of the most expansive neurotransmitter systems in brain, is markedly impaired by in utero ethanol exposure. Previous investigations from this laboratory demonstrated that the offspring of ethanol-fed rats have a decreased concentration of serotonin (5-HT) and its metabolite 5-hydroxyin- doleacetic acid (5-HIAA) in the brain stem and cortical regions [20,38,46]. A limited analysis of 5-HT reuptake sites found a deficiency in the cortex and cortical regions [19]. The concentration of 5-HT~ and 5-HTIA receptors was also altered in the brain stem and cortical regions of developing ethanol-exposed rats [20,45].

Of particular interest is this laboratory's observation that a marked 5-HT deficit was found on embryonic day

J.-A. Kim, M.J. Druse / Developmental Brain Research 92 (1996) 190-198 191

15 (ED15) in the brain area containing the developing raphe neurons [20]. Fetal brain 5-HT is known to act as a trophic factor, necessary for the development of serotonergic and other neurons [11,17,29,30,42,50,51]. Many of the effects of 5-HT on developing serotonergic neurons are mediated by its actions on raphe neuron 5-HT1A receptors [50,51]. Additional actions of fetal 5-HT appear to be facilitated by astroglial 5-HTIA receptors [52]. In fact, stimulation of astroglial 5-HTIA receptors results in increased synthesis and release of a serotonergic growth factor, S100fl [4,52].

In light of the evidence that etl,anol-exposed offspring are deficient in an essential trophic factor (e.g. 5-HT), it was hypothesized that this early deficit could contribute to subsequent developmental abnormalities in serotonergic neurons. Previous work in this laboratory used homogenate binding techniques to show that in utero ethanol exposure is associated with a significant reduction in 5-HT reuptake sites in cortical regions [19]. The present investigation used quantitative autoradiography (QAR) to more completely characterize [3 H]citalopram binding to 5-HT reuptake sites in ethanol-exposed offspring. In addition, we attempted to overcome the potential negative impact of the ethanol-associated deficit of fetal 5-HT, by administering a 5-HT1A agonist, buspirone, to pregnant rats. Specifically, we exam- ined the influence of maternal ethanol consumption on the development of 5-HT reuptake sites in multiple serotonergic brain areas of offspring, and the potential of maternal buspirone treatment to prevent or reverse the adverse effects of ethanol. Buspirone is a non-benzodiazepine anxiolytic [23], which exerts most of its effects by actions on 5-HT1A receptors [48].

2. Materials and methods

Virgin female Sprague-Dawley rats (Harlan), weighing 180-200 g, were housed in individual cages and main- tained at 23-25°C and on a constant (12/12 h) light-dark cycle. Following a 2 day period of adjustment, all rats were given a control liquid diet. The caloric composition of the diet was 21% protein, 29% fat, and 50% carbo- hydrate [33]. Water was given ad libitum. After 3 days on the control diet, rats were divided into three weight- matched groups which consumed either a control or 6.6% (v /v ) ethanol-containing liquid diet. The three groups of female rats included pair-fed control and ethanol-fed rats, and a group that consumed the control liquid diet ad libitum. The diets were administered for 6 weeks prior to breeding and during gestation. Blood alcohol levels (BALs) were determined on tail vein samples obtained 2 h after the introduction of a fresh ration of diet. An enzymatic kit (Sigma, St. Louis, MO.) was used to determine BALs.

Buspirone (RBI, Natick, MA) was administered from ED13 to ED20. The drug administration corresponded to a period when 5-HT neuronal growth and differentiation are

active. Buspirone was dissolved in a 0.9% (w/v ) sterile saline solution at a concentration of 3 mg/ml. Pregnant rats were given either a subcutaneous injection of buspirone, at a dose of 4.5 mg/kg, or an equivalent volume of saline. As noted previously [46], this dose is in the midrange of those used to assess the drug's effects on hormone release, stress-related behaviors, and alcohol consumption. Daily injections were made between 2 p.m. and 3 p.m.

2.1. Dissection and tissue sectioning

Rat offspring were killed by decapitation between 7 a.m. and 9 a.m. The brains were quickly removed and frozen on dry ice. To obtain brains from ED19 rats, mothers were decapitated, and the uterine horn was removed and placed on an ice-cold glass plate. The brains from the fetuses were quickly removed and frozen. Frozen brains were stored at -80°C until use.

Coronal brain sections of 20 /~m were cut at -18°C using a cryostat (Wild Leitz GmbH, Wetzlar, Germany). Sections were thaw-mounted onto positively charged mi- croscope slides (Fisher, Pittsburgh, PA). Four consecutive sections were placed on each slide; every fifth section was stained with 0.5% (w/v ) cresyl violet solution [34] and used to identify anatomical structures.

2.2. 5-HT reuptake sites

Serotonin reuptake sites were measured according to an established method [15]. Slide-mounted tissue sections were warmed to room temperature and then preincubated for 15 min in 50 mM Tris buffer (pH 7.4) containing 120 mM NaCI, 5 mM KC1 and 0.001% (w/v) bovine serum albu- min, in order to remove endogenous ligands. Tissue sections were then incubated for 60 rain at room temperature in the incubation buffer containing 0.8 nM [3H]citalopram (NEN, Boston, MA). (The reported K D for [3H]citalopram is approximately 0.8 nM [15]; preliminary homogenate binding studies determined a similar value.) Non-specific binding was determined in the presence of 1 /xM paroxetine (gift from Smith Kline Beecham Pharmaceuticals). Sections were then dipped (1-2 s) and washed twice for 10 min in ice-cold preincubation buffer, dipped in ice-cold distilled water for a few seconds, and dried in a cool air stream. Radiolabeled sections and a 3H-radiolabeled reference microscale were exposed to 3H-sensitive Hyperfilm (Amersham, Arlington Heights, IL) at 4°C for 4 weeks (for PN19 and PN35 brain sections) or 60 days (for ED19 and PN5 brain sections).

2.3. Analyses of autoradiograms

The illuminated image of each autoradiogram was col- lected by a solid state camera (Sony CCD Video Camera, Japan) and quantitated using a Macintosh-based image analysis system, NIH Image. The optical density of the

192 J.-A. Kim, M.J. Dru.~e / Det'elopmental Brain Research 92 (1996) 190-198

reference microscale was compared with that of ~H-labeled brain mash standards. The radioactivity of tissue sections was then converted to pmol /mg protein depending on the specific activity of the radioligand. Brain structures were identified with reference to an atlas [34]. 5-HT reuptake sites were analyzed in the regions of the raphe nuclei (dorsal raphe and median raphe), amygdala, lateral hypo-

~h

I

thalamus, substantia nigra, medial septal area, CA3 region of the hippocampus, frontal cortex and parietal cortex.

2.4. Statistical analysis

Results were analyzed using a two-way analysis of variance (ANOVA) with a randomized block design, and a

I

Fig. 1. Autoradiographic images of serotonin reuptake sites in rat brain sections, radiolabeled with [3H]citalopram. Images of 20 ~ m coronal sections, radiolabeled with 0.8 nM [3H]citalopram, are presented. A - D were taken at the level of the forebrain (A,B), midbrain (C), and brainstem (D) of PN35 rats, while E and F were sectioned at the level of the forebrain (E) and brainstem (F) of PN5 rats. G represents a coronal section of brain stem from EDI9 fetuses. Nonspecific binding in the presence of I /.zM paroxetine is depicted in tissue l¥om a 35 day rat in H. Abbreviations: FCX, frontal cortex: PCX, parietal cortex; MS, medial septum- STR, striatum: LH, lateral hypothalamus: BLA, basolateral nucleus of amygdala; CA3, hippocampus cornu ammonis 3: SN, substantia nigra; DR, dorsal raphe: MNR. median raphe.


post-hoe Tukey's protected t-test. This analysis was useful in detecting significant differences when consistent observations were made from experiment to experiment (e.g. the specific binding in the lateral hypothalamus of ethanol saline rats was less than that in age-matched control saline rats in each experimen0, despite variability in specific binding between experiments. P < 0.05 was considered statistically significant.

saline- and buspirone-treated rats were also comparable (data not shown). Consistent with a previous report, maternal treatment with 4.5 mg/kg buspirone did not alter diet consumption [24]. In addition, the mean litter size of buspirone-treated dams was comparable to that of saline- treated dams.

3.2. Specific binding of 0.8 nM [ 3H]citalopram in multiple brain regions of control rats

3. Results

3.1. Maternal weight gain and blood alcohol levels

The blood alcohol levels of chronically fed female rats ranged from 75 to 120 mg/dl . Although the female rats consumed an increasing quantity of diet as pregnancy progressed, the amount of ethanol consumed (g /kg body weight) was constant because maternal weight gain paral- leled the increased diet consumption. During pregnancy (ED0 to ED21) the maternal weight gain by pair-fed and ad lib controls, and ethanol-fed rats was not significantly different (data not shown). The weights of offspring of

Since 5-HT reuptake sites are located on nerve termi- nals, their concentration has been used as an index of serotonergic nerve fiber innervation of discrete brain areas. Fig. 1 depicts autoradiograms of brain regions from control rats, which were radiolabeled with [3H]citalopram, a specific blocker of 5-HT reuptake sites. Table 1 summarizes the specific binding of 0.8 nM [3H]citalopram to brain regions from control rats. In this table, the mean and standard error of the mean (S.E.M.) of all control values are provided. Specific binding was readily detected in the dorsal raphe by ED19 and in the parietal cortex by PN5. On PN19 and PN35 the highest binding was found in the dorsal raphe and median raphe. The rank order of binding

__• 1110.

11 140,

120, 0 o 100,

80, c

c ° - e~

40, U

~I = o

~ 0

A DORSAL RAPHE

ED 19

B _'~ 120-

PN 5 PN 19 PN 35

MEDIAN RAPHE

F"-] CS m CB m ES

EB m

100.

60.

40.

t 20.

(t) 0 . ~ PN 19 PN 35

Fig. 2. In utero ethanol exposure causes a transient increase in [3H]citalopram binding in the dorsal (A) and median raphe (B). Buspirone-treated prevented this increase. Results are expressed as the percent of the specific binding from the pair-fed control group at each age. Statistical analysis was performed on specific binding values. There was a significant main effect of ethanol in the dorsal raphe on PN5 (F(1,15) = 17.8, P = 0.02) and median raphe on PN19 (F(1,23) = 30.2, P = 0.03). The * denotes values which are significantly different from age-matched, pair-fed controls. Values from ethanol + buspirone- exposed offspring were not significantly different from those of control-saline offspring.

194 J. -A. Kim, M.J. Druse / Det,elopmental Brain Research 92 (1996) 190-198

was dorsal raphe > median raphe > lateral hypothalamus ~ substantia nigra ~ amygdala > medial septal area ~ CA3 region of the hippocampus > striatum ~ parietal cortex ~ frontal cortex.

Samples from ad lib control rats were included in initial studies of all brain areas (n = 7). These investigations found comparable specific binding in brain regions from age-matched pair-fed and ad lib control rats (99.8 + 4.5%).

Consequently, ad lib controls were not included in subsequent studies.

3.3. Effects of in utero ethanol exposure on the development of 5-HT reuptake sites

Data from the four groups of offspring are presented in Figs. 2 - 4 . For ease of visual comparison these figures

120-

100-

on mO-

60-

.~ 4o.

20-

0

A LATERAL HYPOTHALAMUS

120-

Hlj " Hbll 80-

40-

20-

0 PN 19 PN ,35

[3 MEDIAL SEPTUM

Hill 1HII PN 19 PN 35

I I CS CB

I ES EB

c e 120" o

- - 1 0 0 .

8 e0-

.o

20-

C SUBSTANTIA NIGRA

120"

llll IHll 0

PN 19 PN ,.35

D STRIATUM

1111 1111 PN 19 PN 35

120

,oo

8 so

~o

4o

20

E

FRONTAL CORTEX ~40

20

C PN 19 PN ,:35

F

PARIETAL CORTEX

llJl INii llll PN 5 PN 19 PN 35

Fig. 3. In utero ethanol exposure is associated with a decrease in [3H]citalopram binding at PNI9 a n d / o r PN35 in the lateral hypothalamus, medial

septum, substantia nigra, striatum, frontal cortex and parietal cortex. Buspirone prevented this decrease in the lateral hypothalamus, medial septum. substantia nigra and frontal cortex. Results are expressed as the percent of the specific binding from the pair-fed control group at each age. Statistical

analysis was performed on specific binding values. There were significant main effects of alcohol in the lateral hypothalamus on PNI9 ( F ( 1 , 2 7 ) = 13.7, P = 0 . 0 1 ) , in the medial septum on PNI9 ( F ( 1 , 2 7 ) = 8.0, P = 0.03), in the substantia nigra on PNI9 ( F ( 1 , 2 7 ) = 27.0, P = 0 . 0 0 2 ) and PN35 (F(1,27) = 5.9, P = 0.05), in the striatum on PN19 (F(1,27) = 8.7, P = 0.03), in the frontal cortex on PNI9 (F(1,27) = 4.2, P = 0.09) and in the parietal cortex on PN19 (F(1,23) = 8.7, P = 0.03). eThere was also a significant ethanol × buspirone interaction on P N I 9 in the lateral hypothalamus ( F = 9.8,

P = 0.02) and in the medial septum ( F = 6.4, P = 0.04). The * denotes values which are significantly different from age-matched, pair-fed controls. Values from ethanol + buspirone-exposed offspring were not significantly different from those of control-saline offspring in the lateral hypothalamus, medial septum, substantia nigra and frontal cortex.


Table 1

Specific binding of [3H]citalopram to 5-HT reuptake sites in the offspring of control, saline-treated dams

Brain region Age Specific binding (pmol /mg protein)

Frontal cortex PN19 0.19 + 0.04 PN35 0.24 + 0.05

Parietal cortex PN5 0.28 + 0.06 PN19 0.33 + 0.07 PN35 0.29 + 0.06

Striatum PN19 0.32 ± 0.05 PN35 0.34 _+ 0.06

CA3 PN 19 0.44 ± 0.08 PN35 0.53 4-0.10

Medial septum PN19 0.62 5:0.09 PN35 0.57 ± 0.11

Lateral hypothalamus PN 19 1.09 + 0.15 PN35 0.98 _+0.10

Amygdala PN19 0.78 + 0.t 1 PN35 1.01 ±0.12

Substantia nigra PN19 1.13 ± 0.12 PN35 1.07 ± 0.09

Median raphe PN 19 1.59 ± 0.10 PN35 1.34±0.11

Dorsal raphe ED 19 0.39 ± 0.08 PN5 0.36 + 0.04 PN19 1.77_+ 0.08 PN35 1.76 _+ 0.08

Data are presented as the mean __+ S.E.M. ED and PN are abbreviations for embryonic and postnatal days, respectively. The sample size was 6 -8 with the exception of the parietal cortex (PN5, n = 5), and the dorsal raphe (PN5, n = 4).

depict the binding in saline- and buspirone-treated ethanol-exposed offspring and in buspirone-treated control offspring relative to that of age-matched saline-treated control offspring (% of the age-matched control-saline rat (CS)). Multiple (6-8) individual experiments were performed in which one sample of each age, dietary and drug treatment group was included in each binding assay and on

a single film. The results were analyzed by a two-way ANOVA, with each experiment evaluated as a block. Statistical findings are detailed in the legend to each figure.

When the offspring of saline-treated, pair-fed CS and ethanol-saline dams (ES) were compared, we detected a transient increase in [3H]citalopram binding to 5-HT reuptake sites in the dorsal and median raphe (Fig. 2A,B). This increase was found in each experiment in the dorsal raphe on PN5 and in the median raphe on PN19. Although [3H]citalopram binding in EDI9 and PN5 control buspirone-treated rats appears less than that in CS rats (Fig. 2A), the values were not significantly different. In contrast, to the transient increase in the dorsal and median raphe of ethanol-treated rats, [3H]citalopram binding to 5-HT reuptake sites was decreased in the lateral hypothalamus and substantia nigra of ES offspring at both PN19 and PN35 (Fig. 3A,C). In fact, the specific binding of 0.8 nM [3H]citalopram was lower in the lateral hypothalamus and substantia nigra on PN19 in all 7 experiments; decreased binding was found in the lateral hypothalamus in 6 of 7 experiments on PN35. In addition, a transient decrease in [3H]citalopram binding was consistently found in the frontal and parietal cortex, medial septum, and striatum of ES offspring (Fig. 3B,C-F). The concentration of 5-HT reuptake sites was unchanged in two brain regions, the hippocampal CA3 region and the amygdala (Fig. 4A,B).

3.4. Effects of maternal buspirone treatment of 5-HT reuptake sites in control and ethanol-exposed rat offspring

[3H]Citalopram binding to 5-HT reuptake sites in control rats was not significantly affected by maternal buspirone treatment (Figs. 2-4). In contrast, buspirone prevented some of the ethanol-associated developmental abnormalities in 5-HT reuptake sites. Buspirone prevented the decline in binding of [3H]citalopram in the frontal

120.

100.

8

Ot ,.F.¢ e0. "10 c ,,o 40. o

.rE

A B CA ,3 AMYGDALA

12o..

r---1 CS

0 I

PN lg PN35 PN 19 PN35

Fig. 4. Neither in utero ethanol or buspirone exposure affected [3H]citalopram binding in the CA3 region of the hippocampus or in the amygdala. Results are expressed as the percent of the specific binding from the pair-fed control group at each age. Statistical analysis was performed on specific binding values. There were no significant ethanol- or buspirone-related effects.

196 J.-A. Kim, M.J. Druse / Del,elopmental Brain Research 92 (1996) 190-198

cortex on PN19, in the lateral hypothalamus on PN 19 and PN35, and in the substantia nigra on PN35; binding in the medial septal area of EB rats was greater than that of ES offspring. Similarly, buspirone treatment reversed the ethanol-associated increase in binding in the dorsal raphe (PN5) and in the median raphe (PN19). In contrast, maternal buspirone treatment had no effect on the decreased number of reuptake sites in the parietal cortex and striatum.

4. Discussion

The concentration of 5-HT reuptake sites in the dorsal raphe appears to peak at about 3 weeks of age; at this age the dorsal raphe contained the highest concentration of 5-HT reuptake sites in brain [36]. A dense patch of 5-HT reuptake sites was found in the parietal cortex at PN5 and PN19. The distribution of 5-HT reuptake sites resembles the innervation pattern of 5-HT immunopositive fibers [161.

In the offspring of ethanol-fed rats 5-HT reuptake sites were increased in the dorsal raphe at PN5, and in the median raphe at PNI9. The increased number of 5-HT reuptake sites could indicate 5-HT hyperinnervation (sprouting) in response to an ethanol-associated 5-HT deficit. A similar hyperinnervation of 5-HT fibers was observed in the brainstem of neonatal rats in which 5-HT had been depleted by the 5-HT neurotoxin, 5,7-dihydroxy- tryptamine [36].

In contrast to the transient increase in [3H]citalopram binding in the cell body regions, reuptake sites were decreased at PNI9 in several projection areas including the frontal cortex, parietal cortex, lateral hypothalamus, substantia nigra, medial septal area, and the striatum. At PN35 5-HT reuptake sites were also reduced in the lateral hypothalamus and substantia nigra. The observations in the frontal cortex confirm a prior analysis of a limited number of brain areas [19]. A deficit of reuptake sites is likely indicative of abnormal development of serotonergic fibers in ethanol-exposed offspring. Development may be impaired if there is a lower number of raphe 5-HT neurons and/or fewer projections from raphe neurons to target areas. In support of the first possibility, there is evidence that in utero ethanol exposure results in decreased cell number in regions such as somatosensory cortex, the hippocampal CA3 region and the cerebellum [5,6,32].

Alternatively, 5-HT projections to target brain areas could be diminished if ethanol affected one or more factors required for guidance of these projections - cell adhesion molecules, trophic factors, or chemotactic factors. One trophic factor known to be affected by ethanol is 5-HT [20,38]. In utero ethanol exposure also appears to alter neuronal development by affecting either the concentration of certain trophic factors or the responsiveness of developing neurons to these trophic factors [7-9,27,28,37,44,47].

Treatment of pregnant rats with the 5-HTIA agonist, buspirone, prevented the abnormal development of 5-HT reuptake sites in the dorsal raphe (PN5), medial septal area (PN 19), frontal cortex (PN 19), substantia nigra (PN35) and lateral hypothalamus (PN19 and PN35) of the offspring of ethanol-exposed rats. This suggests that at least part of the ethanol-associated abnormal development of serotonergic neurons was due to the low fetal concentration of 5-HT and hence to the low level of stimulation of 5-HTIA receptors. The present and prior studies emphasize the important role of fetal 5-HT as a developmental signal [31] and essential trophic factor. 5-HT is known to influence growth cone elongation and neurite outgrowth to target areas [25,26], as well as the maturation of fetal serotonergic neurons [4,10,17,50,51]. In fact, depletion of the 5-HT signal, via use of a tryptophan hydroxylase inhibitor (p- chlorophenylalanine (PCPA)), resulted in somewhat similar abnormalities as those which accompany in utero ethanol exposure. For example, PCPA treatment led to 5-HT depletion in the cell bodies and axons of embryonic raphe nuclei of the B4-B9 complex, as well as delayed differentiation of the postsynaptic target areas of 5-HT neurons [29,30] and long-term alterations in the density of 5-HT uptake sites [42].

The observed protective effects of the 5-HTIA agonist, buspirone, are consistent with a previous report from this laboratory which demonstrated that a similar treatment prevented the cortical deficit of 5-HT and its metabolite, 5-HIAA in ethanol-exposed offspring [46]. Although we cannot definitively prove that 5-HTtA receptors were involved in the protective effects of buspirone, it is well known that many of the trophic actions of 5-HT on fetal serotonergic neurons are mediated by neuronal as well as astroglial 5-HTIA receptors [4,50,51]. The astroglial effects may involve 5-HTLA agonist-stimulated release of the astroglial neurotrophic factor, S100¢1 [52]. In addition, ipsapirone, another 5-HTIA agonist, has been shown to have protective effects against the damaging effects of prenatal cocaine exposure [2].

The results of the present experiments also indicate that in utero ethanol exposure has region- and age-specific effects on the development of 5-HT fibers. Similar region- specific effects of in utero ethanol exposure have been observed in the hippocampus. Hippocampal pyramidal cells in the CA1 region are decreased by prenatal ethanol exposure, whereas the cells in the CA3 region were unaf- fected [5]. The present studies also indicate that many of the detected developmental abnormalities are transient. Transient, age-specific ethanol effects have also been demonstrated in the cerebellum [49].

In some regions (e.g. parietal cortex and striatum) buspirone treatment did not prevent or reverse the abnormalities of 5-HT reuptake sites. This may be due to differences in the timing of treatment relative to that of key developmental processes in the unresponsive regions or to a requirement for a different concentration of a 5-HT

J. -A. Kim, M.J. Druse / Developmental Brain Research 92 (1996) 190-198 197

agonist. For example, in cultured cells and in vivo, 5-HT's effects were dose dependent. That is, low agonist concentrations inhibited growth of cultured 5-HT neurons, whereas high concentrations stimulated growth [50]. Similar dual dose effects were demonstra ted in whole animal studies [43]. Alternat ively, the persistent e thanol-associated 5 -HT terminal abnormali t ies may be caused by ethanol-associ-

ated changes, such as a def ic iency of addit ional neu-

rotrophic factors, by alterations in other neurotransmit ter systems, which impact the 5 -HT system, or by changes in hormonal function. As noted previously, in utero ethanol

exposure also affects the product ion of other essential

trophic factors or the response of neurons to these factors. Buspi rone is a non-benzodiazep ine anxiolyt ic drug [48],

whose actions appear to be mediated pr imari ly through 5-HTIA receptors. At doses comparable to those used in the present study buspirone acts on the 5-HTtA receptor of

serotonergic dorsal raphe neurons [48]. Buspi rone acts on h ippocampal pyramidal cells of the CA1 region, as a

partial 5-HT1A agonist; these actions are postsynaptic [3]. Busp i rone ' s effects on body temperature and corticotropin occur as a consequence of s t imulat ion of presynaptic and

postsynaptic 5-HTIA receptors, respectively [14]. The present study provides important informat ion re-

garding one mechan i sm by which in utero ethanol expo-

sure adversely affects the deve lopment of the serotonergic

system, and a potential therapeutic agent which may prevent or reverse the effects of ethanol. One must be cau- tious in extending these observat ions unt i l addit ional informat ion has been gathered regarding busp i rone ' s effects on other neurotransmit ter systems. In addition, it will be

impor tant to clearly establish how buspirone exerted its protective effect. Al though it was presumed that bus-

p i rone ' s effects were media ted by its actions on 5-HTIA receptors, we cannot e l iminate the possibi l i ty that other systems were involved. For example, at concentra t ions much higher than those used in this study, buspi rone

displaces [3H]spiroperidol f rom striatal dopamine D 2 bind-

ing sites [12], and appears to have D 2 antagonis t properties. It may also have weak a-adrenoreceptor activity [39], and can st imulate norepinephr ine release [18], through its

actions on D 2 receptors [40].

Acknowledgements

This research was supported by a grant from the USPHS, AA03490.

References

[1] Abel, E.L. and Sokol, R.J., A revised conservative estimate of the incidence of FAS and its economic impact, Alcohol. Clin. Exp. Res., 15 (1991) 514-524.

[2] Akbari, J.M., Whitaker-Azmitia, P.M. and Azmitia, E.C., Prenatal

cocaine decreases the trophic factor S-100 beta and induced micro- cephaly: reversal by postnatal 5-HT1A receptor agonist, Neurosci. Lett., 170 (1994) 141-144.

[3] Andrade, R. and Nicoll, R.A., Novel anxiolytics discriminate between postsynaptic serotonin receptors mediating different physio- logical responses on single neurons of the rat hippocampus, Naunyn-Schmiedeberg 's Arch. Pharmacol., 336 (1987) 5-10.

[4] Azmitia, E.C., Dolan, K. and Whitaker-Azmitia, P.M., S-100 B but not NGF, EGF, insulin or calmodulin is a CNS serotonergic growth factor, Brain Res., 516 (1990) 354-356.

[5] Barnes, D.E. and Walker, D.W., Prenatal ethanol exposure perma- nently reduces the number of pyramidal neurons in rat hippocampus, Dev. Brain Res., 1 (1981) 333-340.

[6] Bonthius, D.J. and West, J.R., Alcohol-induced neuronal loss in developing rats: increased brain damage with binge exposure, Alco- hol. Clin. Exp. Res., 14 (1990) 107-118.

[7] Breese, C.R., D'Costa, A., Ingram, R.L., Lenham, J. and Sonntag, W.E., Long-term suppression of insulin-like growth factor-1 in rats after in utero ethanol exposure: relationship to somatic growth, Z Pharmacol. Exp. Ther., 264 (1993) 448-456.

[8] Brodie, C., Kentroti, S. and Vernadakis, A., Growth factors attenu- ate the cholinotoxic effects of ethanol during early neuroembryogen- esis in the chick embryo, Int. J. Dev. Neurosci., 9 (1991) 203-213.

[9] Brodie, C. and Vemadakis, A., Ethanol increases cholinergic and decreases GABAergic neuronal expression in cultures derived from 8-day-old chick embryo cerebral hemispheres: interaction of ethanol and growth factors, Dev. Brain Res., 65 (1992) 253-257.

[10] Budnik, V., Wu, C.F. and White, K., Altered branching of serotonin-containing neurons in Drosophila mutants unable to synthesize serotonin and dopamine, J. Neurosci., 9 (1989) 2866-2877.

[11] Chubakov, A.R., Gromova, E.A., Konovalov, G.V., Sarkisova, E.F. and Chumasov, E.I., The effects of serotonin on the morpho-func- tional development of rat cerebral neocortex in tissue culture, Brain Res., 369 (1986) 285-297.

[12] Cimino, M., Ponzio, F., Achilli, G., Vantini, G., Perego, C., Algeri, S., and Garrattini, S., Dopaminergic effects of buspirone, a novel anxiolytic agent, Biochem. Pharmacol., 32 (1983) 1069-1074.

[13] Clarren, S.K., Alvord, E.C. Jr., Sumi, S.M., Streissguth, A.P. and Smith, D.W., Brain malformations related to prenatal exposure to ethanol, J. Pediatr., 92 (1978) 64-67.

[14] Cowan, P.J., Anderson, I.M. and Grahame-Smith, D.G., Neuroen- docrine effects of azapirones, J. Clin. Psychopharmacol., 10 (1990) 21S-25S.

[15] D'Amato, R.J., Larget, B.L., Snowman, A.M. and Snyder, S.H., Selective labeling of serotonin uptake sites in rat brain by [3H]citalopram contrasted to labeling of multiple sites by [3H]imipramine, J. Pharmacol. Exp. Ther., 242 (1987) 364-371.

[16] D'Amato, R.J., Blue, M.E., Largent, B.L., Lynch, D.R., Ledbetter, D.J., Molliver, M.E. and Snyder, S.H., Ontogeny of the serotonergic projection to rat neocortex: Transient expression of a dense innervation to primary sensory areas, Proc. Natl. Acad. Sci. USA, 84 (1987) 4322-4326.

[17] DeVitry, F., Hamon, M., Catelon, J., Bubois, M. and Thibault, J., Serotonin initiates and autoamplifies its own synthesis during mouse central nervous system development, Proc. Natl. Acad. Sci. USA, 83 (1986) 8629-8633.

[18] Done, C.J. and Sharpt, T., Biochemical evidence for the regulation of central noradrenergic activity by 5-HT1A receptors: microdialysis studies in the awake and anesthetized rat. Neuropharmacology, 33 (1994) 411-421.

[19] Druse, M.J. and Paul, L.H., Effects of in utero ethanol exposure on serotonin uptake in cortical regions, Alcohol, 5 (1989) 455-459.

[20] Druse, M.J., Kuo, A. and Tajuddin, N., Effects of in utero ethanol exposure on the developing serotonergic system, Alcohol. Clin. Exp. Res., 15 (1991) 678-684.

[21] Druse, M.J., Effects of in utero ethanol exposure on the development of CNS neurotransmitter systems. In: M.W. Miller (Ed.), Develop-

198 J.-A. Kim, M.J. Druse / Det,elopmental Brain Research 92 (1996) 190-198

merit of the Central Nert,ous System. Effects oJ AIcohol and Opiates. Wiley-Liss, New York, 1992, pp. 139-167.

[22] Druse, M.J., Effects of maternal alcohol consumption on the devel oping nervous system. In: R.R. Watson (Ed.), Alcohol and Neurobi- ology. Brain De~elopment and Hotvnone Regulation, CRC Press. New York, 1992, pp. 1-30.

[23] Feighner, J.P., Merideth, C.H. and G.A. Hendrickson, A double-blind comparison of buspirone and diazepam in outpatients with general- ized anxiety disotder, J. Clin. Psychiatry, 43 (1982) 103-108.

[24] Fletcher, P.J. and Davies, M., The involvement of 5-hydroxytryp- taminergic and dopaminergic mechanisms in the eating induced by buspirone, gepirone and ipsapirone, Br. J. Pharmacol., 99 (1990) 519-525.

[25] Haydon, P.G., McCobb, D.P. and Kater, S.B., Serotonin selectively inhibits growth cone motility and synaptogenesis of specific identified neurons, Science, 236 (1984) 561-564.

[26] Haydon, P.G., McCobb, D.P. and Kater. S.B., The regulation of neurite outgrowth, growth cone motility, and electrical synaptogenesis by serotonin, J. NeurobioL, 18 (1987) 197-215.

[27] Heaton, M.B., Swanson. D.J., Paiva, M. and Walker, D.W., Ethanol exposure affects trophic factor activity and responsiveness in chick embryo, Alcohol, 9 (1992) 161-166.

[28] Heaton, M.B., Paiva, M., Swanson, D.J. and Walker. D.W., Respon- siveness of cultured septal and hippocampal neurons to ethanol and neurotrophic substances, J. Neurosci. Res., 39 (1994) 305-318.

[29] Lauder, J.M. and Krebs, H., Serotonin as a differentiation signal ira early neurogenesis, Det~. Neurosci., I (1978) 15-30.

[30] Lauder, J.M., Towle, A.C., Patrick, K., Henderson, P. and Krebs, H., Decreased serotonin content of embryonic raphe neurons following maternal administration of p-chlorophenylalanine: a quantitative immunocytochemical study, Det,. Brain Res., 20 (1985) 107 114.

[31] Lauder, J.M., Ontogeny of the serotonergic system in the rat: serotonin as a developmental signal, Ann. NY Acad. Sci,, 600 (1990) 297-313.

[32] Miller, M.W. and Potempa, G., Numbers of neurons and glia in mature rat somatosensory cortex: Effects of prenatal exposure t~ ethanol, J. Comp. Neurol., 293 (1990) 92-102.

[33] Noronha, A.B. and Druse, M.J., Maternal ethanol consumption and synaptic membrane glycoproteins in offspring, J. Neurosci, Res., 8 (1982) 83-97.

[34] Paxinos, G. and Watson, C.. The Rat Brain in Stereotaxic Coordi nates, Academic Press, Sydney, 1986.

[35] Pentney R.J. and Miller, M.W., Effects of ethanol on neuronal morphogenesis. In: M.W. Miller (Ed.), Dez,elopment ~(the Central Nert,ous System. Effects ~[" Alcohol and Opiates, Wiley-Liss, New York, 1992, pp. 71-108.

[36] PranzateHi, M.R. and Martens, J.M., Plasticity and ontogeny of the central 5-HT transporter: effect of neonatal 5. 7- dihydrox- ytryptamine lesions in the rat, Dec. Brain Res., 70 (1992) 191-195.

[37] Rahman, H.. Kentroti. S. and Vernadakis, A., The critical period for ethanol effects on cholinergic neuronal expression in neuroblast-en- riched cultures derived from 3-day-old chick embryo: NGF amelio- rates the cholinotoxic effects of ethanol, lnt. J. Det~. Neurosci.. 12 (1994) 397-404.

[38] Rathbun, W. and Druse, M.J., Dopamine, serotonin and acid metabolites in brain regions from the developing offspring of ethanol treated rats, J. Neurochem., 44 (1985) 57-62.

[39] Rimele, T.J., Henry, D.E., Lee, D.K.H., Geiger, G., Heaslip, R.J. and Grimes, D., Tissue-dependent alpha adrenoceptor activity of buspirone and related compounds, J. Pharmacol. Exp. Ther., 241 (1987) 771-778.

[40] Sanger, D.J. and Shoemaker, H., Discriminative stimulus properties of 8-OH-DPAT: relationship to affinity to 5-HTIA receptors, Psy- chopharmacology, 108 (1992) 85-92.

[41] Savage, D.D. and Swartzwelder, H.S., Effects of perinatal ethanol exposure on hippocampal formation. In: R.R. Watson (Ed.), Alcohol and Neurobiology. Brain DeL, elopment and Hormone Regulation, CRC Press, New York, 1992, pp. 177-200.

[42] Shemer, A., Whitaker-Azmitia, P.M. and Azmitia, E.C.. Effects of prenatal 5-methoxytryptamine and parachlorophenylalanine on serotonergic uptake and behavior in the neonatal rat, Pharmacol. Biochem. Behat,., 30 (1988) 547-851.

[43] Shemer. A.V., Azmitia, E.C. and Whitaker-Azmitia, P.M. (1991) Dose-related effects of prenatal 5-methoxytryptamine (5-MT) on development of serotonin terminal density and behavior, Det). Brain Res., 59 (1991) 59-63.

[44] Singh, S.P., Srivenugopal, K.S., Ehmann, S., Yuan, X.-H. and Snyder, A.K., Insulin-like growth factors (IGF-I and IGF-II), IGF- binding proteins and IGF gene expression in the offspring of ethanol-fed rats, J. Lab. Clin. Med., 125 (1994) 183-192.

[45] Tajuddin, N. and Druse, M.J., Chronic maternal ethanol consumption results in decreased serotonergic 5-HT I receptors, Alcohol, 5 (1989) 465-470.

[46] Tajuddin, N.F. and Drnse, M.J., Treatment of pregnant alcohol-con- suming rats with buspirone: effects of serotonin and 5-hydroxyin- doleacetic acid content in offspring, Alcohol. Clin. Exp. Res., 17 (1993) 110-114.

[47] Valles, S., Lindo, L., Montoliu, C., Renau-Piqueras, J. and Guerri, C., Prenatal exposure to ethanol induces changes in the nerve growth factor and its receptor in proliferating astrocytes in primary culture, Brain Res., 656 (1994) 281-286.

[48] VanderMaelen, C.P., Matheson, G.K., Wilderman, R.C. and Patter- son, L.A.. Inhibition of serotonergic dorsal raphe neurons by sys- temic and iontophoretic administration of buspirone, a non-benzodiazepine anxiolytic drug, Eur. J. Pharmacol., 129 (1986) 123-130.

[49] Volk, B., Delayed histogenesis in the 'embryofetal alcohol syndrome', Acta Neuropathol. (Berl.), 39 (1977) 157-163.

[50] Whitaker-Azmitia, P.M. and Azmitia, E.C., Autoregulation of fetal serotonergic neuronal development: role of high affinity serotonin receptors, Neurosci. Lett., 67 (1986) 307-312.

[51] Whitaker-Azmitia, P.M., Shemer, A.V., Caruso, J., Molino, L. and Azmitia, E.C., Role of high affinity serotonin receptors in neuronal growth, Ann. NYAcad. Sci., 600 (1990) 315-330.

[52] Whitaker-Azmitia, P.M. and Azmitia, E.C., Astroglial 5-HTIA receptors S-100 beta in development and plasticity, Per.wect. De~,. Neurobiol.. 2 (1994) 2333-2338.

protective effects of maternal buspirone treatment on serotonin reuptake sites in ethanol-exposed...

Documents