Differential effects of prenatal cocaine exposure on selected subunit mRNAs of the GABAA receptor in rabbit anterior cingulate cortex

Journal of Chemical Neuroanatomy 24 (2002) 243 /255 www.elsevier.com/locate/jchemneu

Differential effects of prenatal cocaine exposure on selected subunit mRNAs of the GABAA receptor in rabbit anterior cingulate cortex Jed S. Shumsky a,*, Yunxing Wu b, E. Hazel Murphy a, Jonathan Nissanov a,c, Ann O’Brien-Jenkins a, Dennis R. Grayson d a

Department of Neurobiology and Anatomy, MCP Hahnemann University, 2900 Queen Lane, Philadelphia, PA 19129, USA b Johns Hopkins University School of Medicine, CMSC 9-120, 600 N. Wolfe St., Baltimore, MD 21287, USA c Computer Vision Laboratory for Vertebrate Brain Mapping, MCP Hahnemann University, 2900 Queen Lane, Philadelphia, PA 19129, USA d Department of Psychiatry, The Psychiatric Institute, University of Illinois at Chicago, 1601 West Taylor Street, Chicago, IL 60612, USA Received 14 August 2000; received in revised form 25 April 2001; accepted 4 July 2002

Abstract We have previously shown that in the dopamine-rich anterior cingulate cortex (ACC), significant changes in g-aminobutyric acid (GABA) immunoreactivity occur in the offspring of rabbits given intravenous injections of cocaine (3 mg/kg) twice daily during pregnancy. In the present study, the effects of prenatal cocaine exposure on the developmental expression of specific GABAA receptor subunit mRNAs were investigated. We compared the distribution of the a1, b2, and g2 subunit mRNAs in cocaine- and saline-treated offspring aged postnatal days 20 and 60 (P20, P60). At P20, prenatal cocaine exposure resulted in a significant increase in a1 subunit mRNA in ACC lamina III and a significant reduction in the amounts of the b2 subunit mRNA in ACC lamina II. No differences between cocaine- and saline-treated controls were detected for g2 subunit mRNA levels in ACC. Although the pattern of labeling was altered in cocaine-exposed animals, Nissl sections revealed no differences in lamination, indicating that the changes in GABAA subunit mRNAs could not be attributed to abnormal cytoarchitectonics. In P60 brains, no significant differences were observed between cocaine- and saline-treated material, indicating that the observed differences were transient. Collectively, our data show that prenatal cocaine exposure elicits differential, lamina-specific changes in mRNA levels encoding selected subunits of the GABAA receptor. Since these changes occur during a critical period when fine tuning of synaptic organization is achieved by processes of selective elimination or stabilization of synapses, we suggest that specific subunit mRNAs of the GABAA receptor play a role in cortical development. # 2002 Published by Elsevier Science B.V. Keywords: Anterior cingulate cortex; Development; In situ hybridization; Rabbit

1. Introduction Cocaine binds to the presynaptic transporters of several monoamines, including dopamine, and blocks their reuptake (reviewed in Levitt et al., 1997). If taken during pregnancy, cocaine blocks the re-uptake of monoamines not only in the mother but also in the fetus. Since monoamines play an important regulatory role in the developing central nervous system (CNS),

* Corresponding author. Tel.:
/1-215-991-8736; fax:
/1-215-8439082 E-mail address: [email protected] (J.S. Shumsky).

perturbations of these systems in the fetus are likely to have major effects on brain structure and function. For example, the dopamine (DA) D1 receptor system plays an important role in the regulation of dendritic growth. In a rabbit animal model, it has been shown that the offspring of rabbits exposed to cocaine during pregnancy are characterized both by a dysfunction of DA D1 receptors (Wang et al., 1995a,b; Friedman et al., 1996; Friedman, 1998) and by excessive growth of the dendrites of pyramidal neurons in DA-rich areas of the cortex (Jones et al., 1996). Perturbations of the monoaminergic system may also result in compensatory changes in other neurotransmitter systems, since CNS function depends on complex interactions between a variety of neurotransmitters and neuromodulators. Our

0891-0618/02/$ - see front matter # 2002 Published by Elsevier Science B.V. PII: S 0 8 9 1 - 0 6 1 8 ( 0 2 ) 0 0 0 6 7 - 4

244

J.S. Shumsky et al. / Journal of Chemical Neuroanatomy 24 (2002) 243 /255

studies have focused on the g-aminobutyric acidA (GABA)-ergic system. While there is little evidence that cocaine acts directly on the GABAergic system, there is increasing evidence of interactions between the DA system and the GABA system, and DA D1 receptors may play a role in modulating GABA release (Beauregard and Ferron, 1991; Aceves et al., 1992; Campbell et al., 1993; Cameron and Williams, 1994). In previous studies, we have reported that prenatal cocaine exposure significantly alters the development of the GABA system in cocaine-exposed offspring (Wang et al., 1995a,b, 1996; Levitt et al., 1997; Murphy et al., 1997b; Stanwood et al., 2001). These alterations occur over a range of prenatal doses of cocaine (Murphy et al., 1997b) and appear to be restricted to DA-rich regions (Stanwood et al., 2001). For example, chronic administration of cocaine to pregnant dams results in increased GABA immunoreactivity in the anterior cingulate cortex (ACC) of the offspring (Wang et al., 1995a,b) which receives substantial DA innervation. It also results in increased parvalbumin immunoreactivity in ACC (Wang et al., 1996), which is important because parvalbumin is a calcium binding protein associated with a subset of cortical GABAergic neurons. We have interpreted our results as suggesting a dysfunction of the GABAergic system, possibly related to decreased D1 receptor-mediated release of GABA. This increased GABA immunoreactivity is present at birth and is maintained into maturity, suggesting a long-lasting or permanent effect on GABA system function following prenatal cocaine exposure. To better understand the involvement of the GABAergic system and the functional consequences of these changes in animals exposed prenatally to cocaine, we are now studying the plasticity of the g-aminobutyric acidA (GABAA) receptor subunit mRNAs in response to chronic cocaine exposure in utero. GABAA receptors are hetero-oligomeric complexes which include Cl  channels and recognition sites for multiple allosteric modulators such as barbiturates, benzodiazepines (BZDs), steroids and ethanol (for reviews see MacDonald and Olsen, 1994; Rabow et al., 1995; Mehta and Ticku, 1999). Molecular cloning of GABAA receptor subunits has shown the existence of at least eighteen different subunits in mammalian CNS, including six a subunits, three b subunits, three g subunits, one d subunit, one o subunit, one p subunit, and three r subunits (for reviews, see MacDonald and Olsen, 1994; Rabow et al., 1995; Mehta and Ticku, 1999). Each GABAA receptor subunit exhibits a distinct regional distribution in CNS indicating that different combinations of subunits are present in different neuronal populations both during development (Laurie et al., 1992a; Fritschy et al., 1994) and in the adult (Laurie et al., 1992b; Wisden et al., 1988; Fritschy and Mohler, 1995). Studies have shown that the mRNA

levels of individual GABAA receptor subunits are also modulated by different neurotransmitter/receptor systems. For example, excitatory synaptic transmission increases the expression of selected GABAA receptor subunits. The levels of the a1, b1, b2, and g2 subunit mRNAs and their corresponding protein products are reduced after treatment of primary cerebellar granule cell cultures with the N -methyl-D-aspartate (NMDA) selective glutamate receptor antagonist, MK-801, while a persistent stimulation of NMDA receptors increases GABAA receptor subunit expression (Memo et al., 1991; Harris et al., 1994; Zhu et al., 1995; Wang et al., 1998). Moreover, these changes are accompanied by alterations in the sensitivities of the receptors to GABA and their responsiveness to BZDs. The data suggest that excitatory afferent synaptic signaling may modulate the expression of GABAA receptors and hence modify the quality of inhibitory tone. Evidence is also accumulating that suggests an interaction between the DA, GABA, and glutamate neurotransmitter receptor systems. For example, DA D1 receptor activation stimulates the accumulation of GABA in slices of substantia nigra pars reticulata of 6-hydroxydopamine-lesioned rats (Aceves et al., 1992). DA afferents terminate on GABA-containing neurons in rat anteromedial cerebral cortex (Verney et al., 1990). In addition, chronic treatment with haloperidol, a DA receptor blocker, significantly increases GABA immunoreactive terminals in rat frontal cortex, suggesting that blockade of DA receptors may increase the levels of GABA (Vincent et al., 1991). Finally, in cortical pyramidal neurons, DA D1 receptors tend to be localized on dendritic spines in close apposition to large asymmetric (presumed glutamatergic) synapses, suggesting a role for DA D1 receptors in modulating glutamate input. These neurons are densely innervated by GABAergic terminals which indicates that an interplay between DA and GABA may modulate the extent of excitatory activity (Vincent et al., 1991). GABA may also modulate the expression of GABAA receptors. The expression of certain subunits of the GABAA receptor complex is promoted by GABA (for review, see Schousboe and Redburn, 1995). Thus, if GABA release is reduced in rabbits exposed prenatally to cocaine, then the developmental patterns of expression of specific GABAA receptor subunits may, in turn, be altered. As a first step towards examining regional changes in receptor subunit expression in response to in utero cocaine exposure, we selected the a1, b2 and g2 mRNAs because they are the most abundantly expressed GABAA receptor subunits throughout the mammalian CNS (Benke et al., 1991). We also provide an improved method for laminar analysis of the distribution of these subunit mRNAs in the ACC, and describe the effects of prenatal cocaine exposure on their distribution at two significant developmental ages.

J.S. Shumsky et al. / Journal of Chemical Neuroanatomy 24 (2002) 243 /255

2. Methods Dutch Belted rabbits (Myrtle’s Rabbitry, Thompson Station, TN) were bred in our animal facility as previously described (Murphy et al., 1995), and females were administered cocaine (3 mg/kg, IV) twice daily from gestation days 8/29. Equivalent volume saline injections were given to control animals. Pair-feeding was not used since cocaine administration does not induce anorexia under our rabbit model (Murphy et al., 1997a). During and following cocaine exposure, maternal weight gain and the birth weight of the kits do not differ from those of saline-treated control animals (Murphy et al., 1997a). Kits were born on days 30 or 31. Cross-fostering was not used since rabbits interact with their offspring for only an average of 2/3 min daily for feeding, and postnatal weight gain of cocaineexposed offspring does not differ from postnatal weight gain of saline-exposed offspring (Murphy et al., 1997a). For comparison between cocaine-exposed animals and controls, four offspring at the postnatal ages of 20 days (P20) or 60 days (P60) were sacrificed. All animals were sacrificed by a lethal dose of sodium pentobarbital (100 mg/kg IP), and brains were rapidly removed and frozen in isopentane cooled to /35 8C with dry ice. Tissues were stored at /70 8C until use. Parasagittal and coronal sections (14 mm) were cut using a cryostat (Hacker Instruments) and mounted onto silanated slides (Digene). 2.1. RNA isolation from rabbit cerebellum Cerebella were dissected from adult Dutch Belted rabbits (Myrtle’s Rabbitry, Thompson Station, TN) and immediately frozen in dry ice. RNA was isolated from the frozen cerebellum with the commercially available Tri-reagent kit as described by the manufacturer (Mol. Res. Ctr., Cincinnati, OH). The concentration of total RNA was determined spectrophotometrically at 260 and 280 nm. 2.2. Primers, cDNA synthesis, PCR amplification and subcloning of cDNA fragments Receptor subunit cDNA amplification primers were designed based on the published rat cDNA sequence of the a1 (Khrestchatisky et al., 1989), b2 (Ymer et al., 1989) and g2 subunits (Shivers et al., 1989). The target cDNAs correspond to the intracellular loop, since this region contains the most unique nucleotide sequences amongst subunits. The primer sequences and locations within the corresponding rodent sequences are indicated in Table 1. Each primer contained a 3 bp clamp and a hexanucleotide sequence for a specific restriction enzyme (i.e. 5?-Eco RI and 3?-Hind III) to facilitate directional cloning of the amplified cDNA fragment.

245

One microgram of total RNA from rabbit cerebella was reverse transcribed using 200 units of MMLV reverse transcriptase (BRL) in the presence of 2.5 mM random hexamers (Pharmacia) for 1 h at 37 8C using buffers and conditions recommended by the manufacturer (BRL). The resultant cDNA was amplified using a DNA thermal cycler (Perkin /Elmer Cetus) and the thermal stable Hot Tub polymerase (Amersham) as previously described (Bovolin et al., 1992; Grayson et al., 1993). Amplification was performed for 35 cycles using reduced stringency annealing conditions. The cycling parameters included incubation at 94 8C, 45 s (denaturation), 55 8C, (annealing), and 72 8C, 1 min, for extension. Following the last cycle, the reaction was incubated at 72 8C for an additional 15 min to allow more complete extension. Aliquots (1/20) of the amplification products were analyzed on 1.6% agarose gels (w/v, agarose/0.5 /Tris / HCl, borate, EDTA), which showed the presence of single bands of approximately 231 bp for the a1 subunit, 221 bp for the b2 subunit and two bands for the g2 subunit with sizes of 235 and 259 bp, respectively. The remainder of each product was purified using disposable PCR purification columns (Qiagen) and cloned into the Eco RI and Hind III sites of pGEM-1 as described previously (Bovolin et al., 1992; Grayson et al., 1993). Automated sequencing of both strands of each of the cloned amplification products was performed using a Model 373 DNA Sequencer (ABI) with fluorescent dideoxynucleotides and the 373A Sequence Navigator Software (ABI). 2.3. cRNA probe preparation The GABAA receptor subunit mRNA plasmids were linearized using either the Hind III (sense) or Eco RI (antisense) restriction enzymes. [35S]-labeled sense and antisense cRNA probes were prepared by in vitro transcription of the linearized templates using the Riboprobe in vitro Transcription System (Promega) according to the manufacturer’s protocols. Following in vitro transcription, residual plasmid templates were digested with RQ1 RNase-free DNase (Progema). Probes were purified using NucTrap Probe Purification Columns (Stratagene). Concentrations of the purified probe solutions were estimated by counting a small aliquot of probe. Specific activities on the order of 2/ 2.5 /106 cpm/ng cRNA were routinely obtained. 2.4. In situ hybridization In situ hybridization was performed according to a previously described protocol (Marlier et al., 1993). Briefly, sections were postfixed in freshly prepared 4% para -formaldehyde (Fisher) in 1 /phosphate-buffered saline, pH 7.4 (PBS, Gibco) for 5 min. Free amino acid

J.S. Shumsky et al. / Journal of Chemical Neuroanatomy 24 (2002) 243 /255

246

Table 1 A list of primers used for amplification of partial cDNAs Subunit

Location of primers corresponding to rat brain

Predicted length of products

References

a1

5? 3? 5? 3? 5? 3?

D  231 bp

(Khrestchatisky et al., 1989)

D  221 bp

(Ymer et al., 1989)

D  259 and 235 bp

(Shivers et al., 1989)

b2 g2

primer primer primer primer primer primer

(1091 /1114 (1298 /1321 (1091 /1114 (1298 /1321 (1091 /1114 (1298 /1321

bp) bp) bp) bp) bp) bp)

groups were acetylated with 0.25% acetic anhydride (Sigma) in 0.1 M triethanolamine (Sigma), pH 8.0 for 10 min. Slides were then dehydrated and allowed to air dry. Hybridization was performed at 55 8C overnight in a solution which contained 50% formamide, 10% Dextran sulfate, 0.5 M NaCl, 1/Denhardt’s solution, 1 mM Tris, pH 8.0, 10 mM EDTA, pH 8.0, 500 mg/ml salmon sperm DNA, 100 mM dithiothreitol (DTT, Sigma) with 4 /106 cpm receptor subunit cRNA in a volume of 50 ml per slide. Both sense and antisense probes for each subunit were applied to control and experimental tissue. Following hybridization, excess solution was removed by immersing slides in 4 /SSC. Single stranded RNAs were digested with 20 mg/ml RNase A (Boerhinger) for 30 min at 37 8C. Subsequently, sections were washed successively in 2 /SSC and 1/SSC at room temperature for 10 min each, and in 0.1 /SSC at 55 8C for 30 min, dehydrated and air dried. Sections were exposed to bMax-Hyperfilm (Amersham) for 2/7 days. All sections using a given subunit were developed together on the same film, and background measurements confirmed that background readings remained uniform across the film. The OD range measured for each subunit is much less than that of film saturation. The developed autoradiographs were analyzed using the imaging system described below. After exposure to film, sections were also coated with nuclear track emulsion (Kodak NTB3, diluted 1:1 with water) and exposed for 10 /21 days to reveal silver grain localization of the hybridized probe. After developing, sections were counter-stained for Nissl substance with cresyl violet. 2.5. Data analysis Each animal used for analysis was taken from a separate litter of kits. In situ signals were visualized using film. Matched sections of ACC from four pairs of cocaine and saline-treated rabbit brains were quantitatively analyzed. Additionally, four coronal sections were collected for each subject and sequential sections were used for each of the a1, b2 and g2 GABAA receptor subunits in order to reduce within-subject variability. Nissl stained sections were used for the identification of specific laminae.

Histologically counterstained sections and their corresponding autoradiographic films were captured for image analysis using a Sony XC-77 camera mounted on a lightbox for macroautoradiographic analysis (acquisition pitch /18.4 mm/pixel). Image capture and analysis utilized BRAIN 2.0 (Computer Vision Laboratory for Vertebrate Brain Mapping, http://www.neuroterrain. org) and NIH IMAGE 1.62 both operating on a Macintosh. Autoradiograms were calibrated using optical density (OD) standards (Eastman Kodak). The use of OD standards permits quantification in absolute OD units. Histologic and autoradiographic images were aligned by the principal axis method (Nissanov and McEachron, 1991). Equivalent regions of interest (ROIs) for the laminae I, II, III, V and VI of ACC were delineated on the histologic image and their optical densities were measured from the corresponding autoradiographic images. The ROIs employed were each 368 by 37 mm oriented along the laminar plane and situated at the center of each ACC layer. At each age (P20, P60) we collected data from four sequential sections from four pairs of cocaine- or saline-exposed animals, with each pair matched for cortical level, and the data were processed together. Two-way analysis of variance (ANOVA) was performed between prenatal treatment and ACC layer, with ACC layer taken as a repeated measure. Post hoc analysis was performed using unpaired t-tests. Secondary analysis of our results was performed using grain counting in order to determine whether the OD changes observed were restricted to the soma. To do so, a systematic unbiased sampling protocol was employed (Howard and Reed, 1998). Briefly, using StereoInvestigator (Microbrightfield) the five ACC layers were delineated at low resolution, an overlaying grid was defined, and cells within count boxes were examined. On average 25 cells were examined for layer I, 27 cells for layer II, 14 cells for layer III, 38 cells for layer V, and 30 cells for layer VI. Both the number of grains overlaying the cell body and the area of each counted cell were determined. The ratio of grains to cell area was determined for each ACC layer and compared between prenatal treatments using unpaired t-tests. The advantage of grain count analysis is the greater resolution offered by this method over OD measure-

J.S. Shumsky et al. / Journal of Chemical Neuroanatomy 24 (2002) 243 /255

247

ment. However, OD analysis is well recognized for the greater dynamic range that it supports and the reduced variance that it yields (Rogers, 1979). Thus, our primary mode of analysis in the present study was OD.

3. Results 3.1. Partial cDNA cloning of a1, b2, and g2 subunit cDNAs The intracellular loop portion of the a1, b2, and g2 GABAA receptor subunit cDNAs were cloned from rabbit cerebellar RNA using RT-PCR and primers corresponding to each region of the rat subunit. This region was chosen as it shows the highest degree of sequence divergence between subunits and is likely to produce clones with the lowest possible overlap and highest extent of specificity. The nucleotide sequences of these newly cloned subunit cDNAs were determined and found to be highly similar to those previously cloned from rat brain. We observed a 93% nucleotide sequence identity between the rabbit and rat a1 subunit, 90% for the b2 subunit, 92% for the g2L subunit and 91% for the g2S subunit cDNAs. All substitutions, however, were located in third base pair positions with respect to codon usage, resulting in 100% concordance in amino acid sequence between these two species. A representative comparison (rat and rabbit) of the a1 subunit nucleotide and amino acid sequences is shown in Fig. 1. Differences in cDNA sequence are highlighted with a * symbol at the site of the mismatch. 3.2. Specificity controls Numerous control experiments were performed to verify the specificity of our probes. For each receptor subunit studied, we confirmed that the corresponding sense probe yielded strictly non-specific labeling as compared with the antisense labeled probe. This is shown for the g2 receptor subunit in Fig. 2. In addition, we have performed competition experiments to verify the specificity of our cloned probes. As members within the same subfamily show a higher degree of sequence identity, we chose to examine the competition amongst subunits of the a family. For example, an excess of unlabeled a1 cDNA completely blocked the a1 in situ hybridization, whereas there was no effect of unlabeled a1 cDNA on the a2 in situ hybridization pattern (data not shown). 3.3. Effects of prenatal cocaine exposure on rabbits aged P20 We analyzed the distribution of OD corresponding to the a1, b2, and g2 GABAA receptor subunit mRNAs in

Fig. 1. Comparison of the rat and rabbit a1 GABAA receptor cDNA and amino acid sequences. The rabbit cDNA fragment was isolated by RT-PCR from cerebellar RNA isolated from adult cerebella as described in Section 2. The amplification primers are shown in bold and correspond to that portion of the a1 receptor subunit sequence located between putative transmembrane domains M3 and M4. All mismatches between the two species are indicated (*). While the extent of sequence identity was only 93% at the nucleotide level, all differences were conservative resulting in 100% concordance between amino acid sequences.

the ACC of rabbits aged P20. These films show, particularly for the a1 and b2 receptor subunit mRNAs, that the distribution of label differs across laminae (Fig. 3). The distribution is such that the signal density is lowest in lamina I, highest in lamina II and intermediate in laminae III, V and VI. In saline-exposed animals this distribution makes lamina II appear particularly prominent and results in a clear demarcation of the border between lamina II and III. In contrast, in cocaineexposed rabbits, the distribution of signal across laminae II and III appears much more homogeneous, and the II/III border is less distinct. This difference between cocaine- and saline-exposed animals is shown in histograms in Fig. 4A, in which we plot the ratio of OD in lamina II/OD of lamina III. An OD ratio greater than 1 indicates relatively higher levels in lamina II compared with lamina III. For each subunit, the mean OD ratio is higher for saline- than for cocaine-treated animals, although the difference does not quite reach statistical significance for the g2 subunit mRNAs. Analysis of mean optical densities using ANOVA revealed a significant interaction for the distribution of the a1 and b2 GABAA receptor subunit mRNAs (but not for g2) across the ACC layers: a1 [F (4,16) /3.0, P B/0.05], b2 [F (4,24) /6.9, P B/0.001], and g2 [F (4,24) /1.1, n.s.]. ANOVA confirms that differences

248

J.S. Shumsky et al. / Journal of Chemical Neuroanatomy 24 (2002) 243 /255

homogeneity of in situ signal for the a1 and b2 mRNAs in cocaine-exposed offspring compared with controls results from two changes: (i) a1 mRNA levels were significantly higher in ACC layer III of cocaine-exposed animals (P B/0.05); and (ii) b2 mRNA levels were significantly lower in ACC layer II of cocaine-exposed animals (P B/0.001). To evaluate the cellular location of the changes, grain counts were performed over the soma on a subset of slides from each animal. Interestingly, significant differences were detected only for layer V with the cocaineexposed animals having a reduced level of grains/cell area (Table 2). ACC layer V is larger than the other ACC layers and is sparsely packed with cells. 3.4. Effects of prenatal cocaine exposure on rabbits aged P60

Fig. 2. Representative sense/antisense control g2 hybridization experiment. The top panel (A) and bottom panel (B) were generated using equivalent amount of 35S-labeled sense or antisense probe, respectively, corresponding to the rabbit g2 GABAA receptor subunit. The P20 coronal sections were hybridized and washed in parallel, then exposed to film in the same cassette.

in ACC layer were significant for all three GABAA receptor subunit mRNAs: a1 [F (4,16) /90.4, P B/ 0.001], b2 [F (4,24) /151.5, P B/0.001], and g2 [F (4,24) /48.4, P B/0.001]. Post hoc analysis using unpaired t-tests revealed significant differences between prenatal treatments and GABAA receptor subunit mRNA levels for a1 and b2, but not for g2, in specific laminae of ACC (Fig. 5, panels A, C, and E). Thus, although mRNA levels for each subunit were highest in lamina II for both saline- and cocaine-exposed animals, the difference between lamina II and lamina III is diminished in cocaine-exposed animals. This apparent

The distribution of mRNAs for the a1, b2, and g2 subunits in ACC of rabbits aged P60 is different from that at P20. The difference between lamina II and lamina III is no longer as distinct as it was in the P20 material in either saline- or cocaine-exposed animals, and the levels of all three subunit mRNAs now appear to be highest in ACC layers II, III, and V. ANOVA confirmed that differences in ACC layer were significant for all three GABAA receptor subunit mRNAs: a1 [F (4,24) /47.5, P B/0.001], b2 [F (4,24) /61.1, P B/ 0.001], and g2 [F (4,24) /27.1, P B/0.001]. However, prenatal cocaine exposure no longer had any effect on the distribution of the three GABAA receptor subunit mRNAs across the ACC layers (see Fig. 5, panels B, D, and F). Thus, no significant differences were found between cocaine- and saline-exposed animals in a1, b2, or g2 GABAA receptor subunit mRNA levels in any of the laminae of ACC nor for ACC layer II/III OD ratios (Fig. 4B).

4. Discussion Our results indicate that following prenatal cocaine exposure, the expression of selected GABAA receptor subunit mRNAs is differentially regulated and that these effects are lamina specific. These differences are observed at P20, but not at P60. Thus, they appear to be transient effects which are prominent at P20, the time of maximal plasticity in the formation, refinement, and stabilization of synaptic connections (Murphy, 1984). While additional data with these and additional subunit probes collected at different ages are needed to fully interpret these observations, the data are consistent with our previous reports (Wang et al., 1995a,b, 1996; Levitt et al., 1997; Murphy et al., 1997b) that prenatal cocaine exposure has significant effects on the development and function of the inhibitory GABAergic circuitry in the

J.S. Shumsky et al. / Journal of Chemical Neuroanatomy 24 (2002) 243 /255

249

Fig. 3. Differential GABAA subunit expression in P20 rabbits prenatally exposed to saline or cocaine. Shown are pseudocolored autoradiograms of the ACC layers for the a1 (A and B) and b2 (C and D) subunit mRNAs. The ACC is located below the laminae marking label. mRNA expression levels have a clearer laminar specificity in the saline exposed animals (A and C) then in the cocaine exposed offspring (B and D). Color scale is shown in the inset in panel A. Hotter colors reflect greater expression level. Note the color assignment is linear relative to OD calibration. Scale bar/500 mm.

ACC of rabbits. However, this data is distinct in that it describes a transient effect in GABAergic subunit mRNA levels, in contrast to long-term increases in GABA and parvalbumin immunoreactivity that have been detected across similar postnatal ages (Wang et al., 1995a,b, 1996; Levitt et al., 1997; Murphy et al., 1997b).

4.1. Functional implications of differential regulation of specific GABAA receptor subunits A major goal in studies that examine the distribution of GABAA receptor subunits in the CNS, is to determine the functional significance of changes in

250

J.S. Shumsky et al. / Journal of Chemical Neuroanatomy 24 (2002) 243 /255

tions (Mhatre and Ticku, 1994; Devaud et al., 1995; Land et al., 1995; Micheva and Beaulieu, 1997). We initially selected the a1, b2 and g2 subunits for study as these are the most abundantly expressed (Benke et al., 1991) and they form the basis for most of the functional GABAA receptors in the adult mammalian cerebral cortex (Huntsman et al., 1994; Wisden et al., 1988). In addition, the genes corresponding to these three subunits are organized within the same gene cluster in both mice and humans (Russek, 1999). In our study of P20 rabbits, the most striking effect of prenatal cocaine was the 20% decrease in levels of the b2 subunit mRNA in lamina II of the ACC. In recombinantly expressed receptors, b subunits influence both current amplitudes and barbiturate binding (Bureau and Olsen, 1990; Sigel et al., 1990). In P20 animals, a smaller (13%) but significant increase in mRNA levels of the a1 subunit was found in lamina III. a subunits are known to modify binding affinities for various types of BZDs and, in combination with b and g units, to affect the sensitivity of the receptor to GABA (Pritchett et al., 1989a,b; Pritchett and Seeburg, 1990; Puia et al., 1991). No significant changes were seen in mRNA levels for the g2 subunit. The absence of similar differences in the grain counts suggests that the observed variation is not restricted to subunit mRNA confined to the soma. Our present study does not address whether prenatal cocaine exposure may have disrupted transcriptional or translational levels of subunit regulation or to what extent compensatory changes may have affected potentially altered receptors or their function. Additional physiological measurements will need to be performed prior to assessing the extent to which these changes also result in changes at the level of assembled receptors and the extent of their functional significance.

4.2. Laminar specificity of the effects of in utero cocaine exposure on GABAA receptor subunits Fig. 4. Effects of prenatal cocaine exposure on mean OD ratio of ACC laminae II/III9/S.E.M. for a1, b2, and g2 GABAA receptor subunit mRNAs in rabbits aged P20 (A) and P60 (B). Significant reductions in OD ratio for cocaine-exposed animals were identified using unpaired t tests (*, P B/0.05; $, P B/0.10).

subunit levels and in receptor composition, and to determine the extent to which such changes may underlie critical developmental events or pathological condi-

The altered expression of a1 and b2 subunits, summarized above, were restricted to laminae II and III of ACC in rabbits exposed prenatally to cocaine. These laminae play vital roles in intracortical processing (for reviews, see Benes, 1993; Vogt et al., 1993), integrating inputs from other ipsilateral cortical areas, including the DA-rich lateral prefrontal cortex, and callosal inputs from the contralateral cortex. The columnar organization of the cortex also provides for

Fig. 5. Effects of prenatal cocaine exposure on a1, b2, and g2 GABAA receptor subunit mRNAs in rabbits aged P20 (A, C, E) and P60 (B, D, F). Equivalent ROIs for the five laminae of ACC were delineated on the histologic image and their corresponding optical densities were measured on the aligned autoradiographic image. Data were collected from four sequential sections from four pairs of saline or cocaine-exposed animals, with each pair matched for cortical level and processed together. Two-way ANOVA (prenatal treatment/ACC layer) was performed with ACC layer taken as a repeated measure. Values indicate mean OD9/S.E.M. Post hoc analysis was performed using unpaired t -tests (**, P B/0.01; *, P B/0.05).

J.S. Shumsky et al. / Journal of Chemical Neuroanatomy 24 (2002) 243 /255

251

J.S. Shumsky et al. / Journal of Chemical Neuroanatomy 24 (2002) 243 /255

252

Table 2 Grain density per cell area was counted for each layer of ACC on matched slides for a1 and b2 mRNAs from P20 animals prenatally exposed to cocaine (n  4) or saline (n 4) Prenatal treatment

Subunit mRNA

Layer I

Layer II

Layer III

Layer V

Layer VI

Cocaine Saline

a1 a1

0.12890.008 0.14090.030

0.16690.007 0.17790.009

0.14990.014 0.21690.028

0.16690.008 0.21290.027

0.14290.005 0.17290.017

Cocaine Saline

b2 b2

0.07890.016 0.12190.033

0.12090.017 0.20990.054

0.13990.047 0.16890.021

0.13190.023** 0.27490.024

0.14990.032 0.22090.047

Values indicate mean grains per cell area9S.E.M. In contrast to OD results, unpaired t -tests revealed no significant differences in ACC layers between prenatal treatment groups, except for a significant reduction in layer V of P20 offspring prenatally exposed to cocaine (**, P B 0.01).

integration, in laminae II and III, of inputs from lamina V and lamina VI pyramidal neurons. We observed a reduction in b2 mRNA levels in ACC laminae V in P20 animals prenatally exposed to cocaine. This reduction was confined to the somatic compartment, and was not significant when dendritic and axonal compartments were included through the OD measure. We observed no other effects of prenatal cocaine exposure on mRNA levels of a1, b2 or g2 subunits in laminae V and VI. The infragranular layers provide the major output from ACC, and changes here could have major functional consequences. Thus, while it is possible that the majority of the effects of prenatal cocaine exposure on a1, b2 and g2 subunits are restricted to the supragranular layers, interpretation of the changes in laminae V must await further studies. One possibility is that the effects of prenatal cocaine exposure on GABAA receptor subunit expression might target other subunits in the infragranular layers. Likely candidates are the a4 and a5 subunits. In visual cortex (VC) of primates (Huntsman et al., 1994), the a4 and a5 subunits are expressed at relatively higher levels in laminae V and VI. Furthermore, these subunits are developmentally regulated and their mRNAs are more prominent in the immature cortex (Laurie et al., 1992b; Poulter et al., 1992). Another possibility is that the effects of prenatal cocaine exposure on GABAA receptor subunit expression in the infragranular layers may appear earlier than P20-the age we selected for our study. Lamina II is the last cortical layer to mature, and we observed the most significant changes in Lamina II in P20 animals. The ‘inside-out’ sequence of development of cortical laminae (Rakic, 1972, 1977) suggests the possibility that, at earlier ages, changes might be apparent in deeper laminae. Finally, it is important to note that although mRNA expression is normally limited to the soma, the vertical organization of neocortex is such that it is not necessarily a reflection of the highest concentration of the receptors. GABAA receptor immunoreactivity is detectable in both pyramidal and nonpyramidal neurons (Hendry et al., 1990). The pyramidal neurons of laminae V and VI have apical dendrites which can extend through all cortical layers.

4.3. Transient versus long-term effects The effects of prenatal cocaine exposure on a1 and b2 receptor subunit mRNAs were not permanent: they were present at P20, but not at P60. In this regard, our data on GABAA receptor subunit expression differ from our other data on the effects of cocaine on the GABAergic system, where increases in GABA and parvalbumin immunoreactivity specific to ACC have been identified (Wang et al., 1995a,b, 1996; Levitt et al., 1997; Murphy et al., 1997b). The cocaine-associated changes in GABA immunoreactivity were observed in the neonate and maintained into adulthood. Furthermore, the decreased coupling of DA D1-like receptors from their G protein, which we hypothesize is the central element underlying the effects of cocaine on the GABA system, is also maintained into adulthood (Wang et al., 1995a,b; Friedman et al., 1996; Friedman, 1998). Transient abnormalities, especially if they occur during a critical developmental period, can have long-lasting effects on cortical organization (for review see Goodman and Shatz, 1993), but we can only speculate as to how transient alterations in the expression of specific receptor subunit mRNA might play a role in the longterm synaptic organization of the GABAergic system. 4.4. Developmental interactions of DA and GABA receptor systems The alterations in mRNA levels for the a1 and b2 GABAA receptor subunits associated with prenatal cocaine exposure are region specific. They are observed in the DA-rich ACC, but not in the DA-poor VC (Shumsky et al., 1998). This regional specificity is consistent with our hypothesis that the effects are mediated by cocaine’s effects on the DA system (Levitt et al., 1997). The evidence that the DA D1 receptor system is dysfunctional in the cocaine-exposed offspring adulthood (Wang et al., 1995a,b; Friedman et al., 1996; Friedman, 1998), that D1 receptors promote GABA release (Beauregard and Ferron, 1991; Aceves et al., 1992; Campbell et al., 1993; Cameron and Williams, 1994), and that GABA levels may regulate the expres-

J.S. Shumsky et al. / Journal of Chemical Neuroanatomy 24 (2002) 243 /255

sion of specific GABAA receptor subunits (Schousboe and Redburn, 1995) all suggest the role of the DA system in developmental abnormalities of the GABAergic system in this animal model. Furthermore, there is abundant evidence of interactions between the DA and GABA systems (Bernath and Zigmond, 1989; Goeders et al., 1990; Verney et al., 1990; Beauregard and Ferron, 1991; Retaux et al., 1991; Aceves et al., 1992; Koob, 1992; Campbell et al., 1993; Lindefors, 1993; Santiago et al., 1993; Cameron and Williams, 1993, 1994; Bergson et al., 1995). A wealth of new evidence points to the important and changing role that the GABA system plays in cortical development and cortical plasticity (for reviews see Levitt et al., 1997; Micheva and Beaulieu, 1997). As one of the earliest developing neurotransmitter systems, the neurotrophic role of GABA has been well established (Meier et al., 1991). The transient stage when GABA, acting via GABAA receptors, has a depolarizing action is observed throughout the CNS, and the special role of GABA in synapse stabilization during this time has been emphasized, especially with reference to the glutamatergic system and NMDA receptors (Ben-Ari et al., 1997). The potential contributions of the GABAergic system to synaptic plasticity are further emphasized by evidence that the GABA system is not only one of the first to develop but is also characterized by a prolonged period of plasticity during the neonatal period (for review, see Micheva and Beaulieu, 1997) and even in the mature CNS (Huntsman et al., 1994). Our data further document the susceptibility of the GABAergic system to perturbations of normal development and reveal that such perturbations can differentially affect the expression of specific GABAA receptor subunit mRNAs. The localization of these effects in a DA-rich area of the cerebral cortex further emphasize and confirm the importance of interactions between GABA and DA during development.

Acknowledgements Some of the data has previously been presented in abstract form by Grayson et al., 1996. Soc. Neurosci. Abstr. 22:1941, and Wu et al., 1997. Soc. Neurosci. Abstr. 23:103. Methods of animal care and sacrifice were approved by our Institutional Animal Care and Use Committee in accordance with NIH guidelines. The authors would like to thank Louise Bertrand, Jun Xia, and Hong Yan for their excellent technical assistance. Supported by P01DA06871 (E.H.M.), RO1DA11266-01 (E.H.M. and D.R.G), K04NS01647 (D.R.G.), and P41RR01638 (J.N.).

253

References Aceves, J., Floran, B., Marinez-Fong, D., Benitez, J., Sierra, A., Flores, G., 1992. Activation of D1 receptors stimulates accumulation of g-aminobutyric acid in slices of the pars reticulata of 6hydroxydopamine-lesioned rats. Neurosci. Lett. 145, 40 /42. Beauregard, M., Ferron, A., 1991. Dopamine modulates the inhibition induced by GABA in rat cerebral cortex: an iontophoretic study. Eur. J. Pharmacol. 205, 225 /231. Ben-Ari, Y., Khazipov, R., Leinekugel, X., Caillard, O., Gaiarsa, J.-L., 1997. GABAA, NMDA and AMPA receptors: a developmentally regulated ‘me´nage a´ trois’. Trends Neurosci. 20, 523 /529. Benes, F.M., 1993. Relationship of cingulate cortex to schizophrenia and other psychiatric disorders. In: Vogt, B.A., Gabriel, M. (Eds.), Neurobiology of Cingulate Cortex and Limbic Thalamus. Birkhauser, Boston, pp. 581 /605. Benke, D., Mertens, S., Trzeciak, A., Gillessen, D., Mohler, H., 1991. GABAA receptors display association of g2-subunit with a1- and b2/3 subunits. J. Biol. Chem. 266, 4478 /4483. Bergson, C., Mrzljak, L., Smiley, J.F., Pappy, M., Levenson, R., Goldman-Rakic, P.S., 1995. Regional, cellular and subcellular variations in the distribution of D1 and D5 dopamine receptors in primate brain. J. Neurosci. 15, 7821 /7836. Bernath, S., Zigmond, M.J., 1989. Dopamine may influence GABA release via three separate mechanisms. Brain Res. 476, 373 /376. Bovolin, P., Santi, M.R., Memo, M., Costa, E., Grayson, D.R., 1992. Distinct developmental patterns of expression of the rat a1, a5, g2S and g2L GABAA receptor subunit mRNAs in vivo and in vitro. J. Neurochem. 59, 62 /72. Bureau, M., Olsen, R.W., 1990. Multiple distinct subunits of the gaminobutyric acid-A receptor protein show different ligand-binding affinities. Mol. Pharmacol. 37, 497 /502. Cameron, D.L., Williams, J.T., 1993. Dopamine D1 receptors facilitate transmitter release. Nature 366, 344 /348. Cameron, D.L., Williams, J.T., 1994. Cocaine inhibits GABA release in the VTA through endogenous 5-HT. J. Neurosci. 14, 6763 /6767. Campbell, K., Kalen, P., Wictorin, K., Lundberg, C., Mandel, R.J., Bjorklund, A., 1993. Characterization of GABA release from intrastriatal striatal transplants: dependence on host-derived afferents. Neuroscience 53, 403 /415. Devaud, L.L., Smith, F.D., Grayson, D.R., Morrow, A.L., 1995. Chronic ethanol consumption diferentially alters the expression of gamma-aminobutryic acid A receptor subunit mRNAs in rat cerebral cortex: competitive, quantitative reverse transcriptasepolymerase chain reaction analysis. Mol. Pharmacol. 48, 861 /868. Friedman, E., 1998. D1 dopamine receptor dysfunction in prenatal cocaine exposure. In: Harvey, J.A., Kosofsy, B.E. (Eds.), Cocaine Effects on the Developing Brain, Ann. New York Acad. Sci. 846 238 /246. Friedman, E., Yadin, E., Wang, H.-Y., 1996. Effect of prenatal cocaine on dopamine receptor-G protein coupling in mesocortical regions of the rabbit brain. Neuroscience 70, 739 /747. Fritschy, J.-M., Mohler, H., 1995. GABAA-receptor heterogeneity in the adult rat brain: differential regional and cellular distribution of seven major subunits. J. Comp. Neurol. 359, 154 /194. Fritschy, J.-M., Paysan, J., Enna, A., Mohler, H., 1994. Switch in the expression of rat GABAA-receptor subtypes during postnatal development: an immunohistochemical study. J. Neurosci. 14, 5302 /5324. Goeders, N., Bell, V., Guidroz, A., McNulty, M., 1990. Dopaminergic involvement in the cocaine-induced up-regulation of benzodiazepine receptors in the rat caudate nucleus. Brain Res. 515, 1 /8. Goodman, C.S., Shatz, C.J., 1993. Developmental mechanisms that generate precise patterns of neural connectivity. Cell 72, 77 /98. Grayson, D.R., Bovolin, P., Santi, M.R., 1993. Absolute quantitation of g-aminobutyric acid A receptor subunit messenger RNAs by

254

J.S. Shumsky et al. / Journal of Chemical Neuroanatomy 24 (2002) 243 /255

competitive polymerase chain reaction. Methods Neurosci. 12, 191 /208. Harris, B.T., Charlton, M.E., Costa, E., Grayson, D.R., 1994. Quantitative changes in a1 and a5 g-aminobutyric acid type A receptor subunit mRNAs and proteins after a single treatment of cerebellar granule neurons with N -methyl-D-aspartate. Mol. Pharmacol. 45, 637 /648. Hendry, S.H.C., Fuchs, J., de Blas, A.L., Jones, E.G., 1990. Distribution and plasticity of immunocytochemically localized GABAA receptors in adult monkey visual cortex. J. Neurosci. 7, 1503 /1519. Howard, C.V., Reed, M.G., 1998. Unbiased Stereology: Three Dimensional Measurement in Microscopy. Springer, New York. Huntsman, M.M., Isackson, P.J., Jones, E.G., 1994. Lamina-specific expression and activity-dependent regulation of seven GABAA receptor subunit mRNAs in monkey visual cortex. J. Neurosci. 14, 2236 /2259. Jones, L., Fischer, I., Levitt, P., 1996. Non-uniform alteration of dendritic development in the cerebral cortex following prenatal cocaine exposure. Cereb. Cortex 43, 439 /453. Khrestchatisky, M., MacLennan, A.J., Chiang, M.-Y., Xu, W., Jackson, M.B., Brecha, N., Sternini, C., Olsen, R.W., Tobin, A.J., 1989. A novel a subunit in rat brain GABAA receptors. Neuron 3, 745 /753. Koob, G.F., 1992. Drugs of abuse: anatomy, pharmacology and function of reward pathways. Trends Pharmacol. Sci. 13, 177 /184. Land, P.W., de Blas, A.L., Reddy, N., 1995. Immunocytochemical localization of GABAA receptors in rat somatosensory cortex and effects of tactile deprivation. Somatosens. Mot. Res. 12, 127 /141. Laurie, D.J., Seeburg, P.H., Wisden, W., 1992a. The distribution of thirteen GABAA receptor subunit mRNAs in the rat brain. III Embryonic and postnatal development. J. Neurosci. 12, 4151 / 4172. Laurie, D.J., Seeburg, P.H., Wisden, W., 1992b. The distribution of thirteen GABAA receptor subunit mRNAs in the rat brain. II. Olfactory bulb and cerebellum. J. Neurosci. 12, 1063 /1076. Levitt, P., Harvey, J.A., Friedman, E., Simansky, K., Murphy, E.H., 1997. Prenatal drug exposure: new evidence for neurotransmitter influences on brain development. Trends Neurosci. 20, 269 /274. Lindefors, N., 1993. Dopaminergic regulation of glutamic acid decarboxylase mRNA expression and GABA release in the striatum: a review. Prog. Neuropsychopharmacol. Biol. Psychiatry 17, 887 /903. MacDonald, R.L., Olsen, R.W., 1994. GABAA receptor channels. Annu. Rev. Neurosci. 17, 569 /602. Marlier, L.N.J.-L., Zheng, T., Tang, J., Grayson, D.R., 1993. Regional distribution in the rat central nervous system of an mRNA encoding a portion of the cardiac sodium/calcium exchanger isolated from cerebellar granule neurons. Mol. Brain Res. 20, 21 /39. Mehta, A.K., Ticku, M.K., 1999. An update on GABAA receptors. Brain Res. Rev. 29, 196 /217. Meier, E., Hertz, L., Schousboe, A., 1991. Neurotransmitters as developmental signals. Neurochem. Int. 19, 1 /15. Memo, M., Bovolin, P., Costa, E., Grayson, D.R., 1991. Regulation of g-aminobutyric acidA receptor subunit expression by activation of N -methyl-D-aspartate-selective glutamate receptors. Mol. Pharmacol. 39, 599 /603. Mhatre, M., Ticku, M.K., 1994. Chronic ethanol treatment upregulates the GABA receptor beta subunit expression. Brain Res. Mol. Brain Res. 23, 246 /252. Micheva, K.D., Beaulieu, C., 1997. Development and plasticity of the inhibitory neocortical circuitry with an emphasis on the rodent barrel field cortex: a review. Can. J. Physiol. Pharmacol. 75, 470 / 478. Murphy, E.H., 1984. Critical periods and the development of the rabbit visual cortex. In: Stone, J., Rappaport, E. (Eds.), Develop-

ment of Visual Pathways in Mammals. Alan Liss, New York, pp. 439 /462. Murphy, E.H., Hammer, J.G., Schumann, M.D., Groce, M.Y., Wang, X.-H., Jones, L., Romano, A.G., Harvey, J.A., 1995. The rabbit as a model for studies of in utero cocaine exposure. Lab. Anim. Sci. 45, 163 /168. Murphy, E.H., Chon, J., Darvish-Sefat, F., Diven, K., Schumann, M., Shumsky, J.S., 1997a. Effects of cocaine-induced seizures during pregnancy in the rabbit. Physiol. Behav. 62, 597 /604. Murphy, E.H., Fischer, I., Friedman, E., Grayson, D., Jones, L., Levitt, P., O’Brien-Jenkins, A., Wang, H.-Y., Wang, X.-H., 1997b. Cocaine administration in pregnant rabbits alters cortical structure and function in their progeny in the absence of maternal seizures. Exp. Brain Res. 114, 433 /441. Nissanov, J., McEachron, D.L., 1991. Advances in image processing for autoradiography. J. Chem. Neuroanat. 4, 329 /342. Poulter, M.O., Barker, J.L., O’Carroll, A.-M., Lolait, S.J., Mahan, L.C., 1992. Differential and transient expression of GABAA receptor a subunit mRNAs in the developing rat CNS. J. Neurosci. 12, 2888 /2900. Pritchett, D.B., Seeburg, P.H., 1990. g-aminobutyric acidA receptor a5 subunit creates novel Type II benzodiazepine receptor pharmacology. J. Neurochem. 54, 1802 /1804. Pritchett, D.B., Lu¨ddens, H., Seeburg, P.H., 1989a. Type I and type II GABAA- benzodiazepine receptors produced in transfected cells. Science 245, 1389 /1392. Pritchett, D.B., Sontheimer, H., Shivers, B.D., Ymer, S., Kettenmann, H., Schofield, P.R., Seeburg, P.H., 1989b. Importance of a novel GABAA receptor subunit for benzodiazepine pharmacology. Nature 338, 582 /585. Puia, G., Vicini, S., Seeburg, P.H., Costa, E., 1991. Influence of recombinant gamma-aminobutyric acid-A receptor subunit composition on the action of allosteric modulators of gammaaminobutyric acid-gated Cl currents. Mol. Pharmacol. 39, 691 / 696. Rabow, L.E., Russek, S.J., Farb, D.H., 1995. From ion currents to genomic analysis: recent advances in GABAA receptor research. Synapse 21, 189 /274. Rakic, P., 1972. Mode of cell migration to the superficial layers of fetal monkey neocortex. J. Comp. Neurol. 145, 61 /84. Rakic, P., 1977. Prenatal development of the visual system in the rhesus monkey. Philos. Trans. R. Soc. London (Biol.) 278, 245 / 260. Retaux, S., Besson, M.J., Penit-Soria, J., 1991. Synergism between D1 and D2 dopamine receptors in the inhibition of the evoked release of [3H]GABA in the rat prefrontal cortex. Neuroscience 43, 323 / 329. Rogers, A.W., 1979. Techniques of Autoradiography. Elsevier, New York. Russek, S.J., 1999. Evolution of GABAA receptor diversity in the human genome. Gene 227, 213 /222. Santiago, M., Machado, A., Cano, J., 1993. Regulation of the prefrontal cortical DA release by GABAA and GABAB receptor agonists and antagonists. Brain Res. 630, 28 /31. Schousboe, A., Redburn, D.A., 1995. Modulatory actions of gamma aminobutyric acid (GABA) on GABA Type A receptor subunit expression and function. J. Neurosci. Res. 41, 1 /7. Shivers, B.D., Killisch, I., Sprengel, R., Sontheimer, H., Ko¨hler, M., Schofield, P.R., Seeburg, P.H., 1989. Two novel GABAA receptor subunits exist in distinct neuronal subpopulations. Neuron 3, 327 / 337. Shumsky, J.S., Wu, Y., Murphy, E.H., Nissanov, J., Grayson, D.R., 1998. Prenatal cocaine exposure does not affect selected GABAA receptor subunit mRNA expression in rabbit visual cortex. Ann. New York Acad. Sci. 846, 371 /374.

J.S. Shumsky et al. / Journal of Chemical Neuroanatomy 24 (2002) 243 /255 Sigel, E., Baur, R., Trube, G., Mohler, H., Malherbe, P., 1990. The effect of subunit composition of rat brain GABAA receptors on channel function. Neuron 5, 703 /711. Stanwood, G.D., Washington, R.A., Shumsky, J.S., Levitt, P., 2001. Prenatal cocaine exposure produces consistent developmental alterations in dopamine-rich regions of cerebral cortex. Neuroscience 106, 5 /14. Verney, C., . Alvarez, C., Geffard, M., Berger, B., 1990. Ultrastructural double-labelling study of dopamine terminals and GABAcontaining neurons in rat anteromedial cerebral cortex. Eur. J. Neurosci. 2, 960 /972. Vincent, S.L., Sorenson, I., Benes, F.M., 1991. Localization and high resolution imaging of cortical neurotransmitter compartments using confocal laser scanning microscopy: GABA and glutamate interactions in rat cortex. Biotechniques 11, 628 /633. Vogt, B.A., Sikes, R.W., Vogt, L.J., 1993. Anterior cingulate cortex and the medial pain system. In: Vogt, B.A., Gabriel, M. (Eds.), Neurobiology of Cingulate Cortex and Limbic Thalamus. Birkhauser, Boston, pp. 313 /344. Wang, H.-Y., Runyan, S., Yadin, E., Friedman, E., 1995. Prenatal exposure to cocaine selectively reduces D1 dopamine receptormediated activation of striatal Gs proteins. J. Pharmacol. Exp. Ther. 273, 492 /498.

255

Wang, X.-H., Levitt, P., Grayson, D.R., Murphy, E.H., 1995. Intrauterine cocaine exposure of rabbits: persistent elevation of GABA-immunoreactive neurons in anterior cingulate cortex but not visual cortex. Brain Res. 689, 32 /46. Wang, X.-H., O’Brien-Jenkins, A., Choi, L., Murphy, E.H., 1996. Altered neuronal distribution of parvalbumin in anterior cingulate cortex of rabbits exposed in utero to cocaine. Exp. Brain Res. 112, 359 /371. Wang, X.-H., Zhu, W.J., Corsi, L., Ikonomovic, S., Paljug, W.R., Vicini, S., Grayson, D.R., 1998. Chronic dizocilpine (MK-801) reversibly delays GABAA receptor maturation in cerebellar granule neurons in vitro. J. Neurochem. 71, 693 /704. Wisden, W., Morris, B.J., Darlison, M.G., Hunt, S.P., Barnard, E.A., 1988. Distinct GABAA receptor a subunit mRNAs show differential patterns of expression in bovine brain. Neuron 1, 937 /947. Ymer, S., Schofield, P.R., Draguhn, A., Werner, P., Ko¨hler, M., Seeburg, P.H., 1989. GABAA receptor b subunit heterogeneity: functional expression of cloned cDNAs. EMBO J. 8, 1665 / 1670. Zhu, W.J., Vicini, S., Harris, B.T., Grayson, D.R., 1995. NMDAmediated modulation of GABAA receptor function in cerebellar granule neurons. J. Neurosci. 15, 7692 /7701.