Increased interaction of dopamine-immunoreactive varicosities with GABA neurons of rat medial...

SYNAPSE 231237-245 (1996)

Increased Interaction of Dopamine- Immunoreactive Varicosities With GABA Neurons of Rat Medial Prefkontal Cortex Occurs During the Postweanling Period

FRANCINE M. BENES, STEPHEN L. VINCENT, RAYMOND MOLLOY, AND YUSUF KHAN Department of Psychiatry (I?M.B., S.L. V) and Program in Neuroscience (I?M.B.), Harvard Medical School,

Boston, Massachusetts 02115, and Mailman Research Center, Laboratory for Structural Neuroscience, McLean Hospital, Belmont, Massachusetts 021 78 (I?M.B., S.L.V, R.M., YK.)

KEY WORDS Dopamine, GABA, Medial prefrontal cortex, Development, Nonpyra- midal cells, Pyramidal cells

ABSTRACT A double immunofluorescence technique has been used to assess postnatal maturational changes in the extent to which dopamine-immunoreactive (DA-IR) varicose fibers form contacts with y-aminobutyric acid (GABAI-immunoreactive (IR) neuronal cell bodies in rat medial prefrontal cortex (mPFCx). Two separate measures of interaction, the percentage of GABA-IR cell bodies having DA-IR varicosities in apposition and the number of such profiles in contact with any given GABA-IR cell, were assessed. Between birth and adulthood, there was a progressive linear increase (r = 0.75, P 5 0.0005) in the percentage of GABA-IR cell bodies having at least one DA-IR varicosity in apposition. While the number of varicosities in contact with any given GABA cell body showed very little change during the preweanling period, later during the postweanling interval, this parameter increased in a curvilinear fashion toward adult levels (r = 0.81, P 5 0.0005). Taking together these latter two measures, an index of interaction was found to be 1.8 times higher when adult animals were compared to postweanling rats, and 2.5 times higher when compared to preweanling rats. Overall, these results are consistent with the view that there are late postnatal changes in the extent to which midbrain DA afferents interact specifically with GABAergic interneurons in rat mPFCx. 0 1996 Wiley-Liss, Inc.

INTRODUCTION It has long been suspected that the development of

corticolimbic regions of human brain may continue well beyond birth (Benes, 1989; Benes et al., 1994; Flechsig, 1920; Yakovlev and Lecours, 1967). Recently, this idea has received increased attention with the growing real- ization that normal maturational changes probably play an important role in the appearance of various neuropsychiatric diseases at specific stages of postnatal life (Benes, 1993; Weinberger, 1987). Consistent with this concept, many studies in rodent brain have demonstrated that there are significant postnatal changes in several key neurotransmitter systems (for comprehen- sive reviews, see Johnston, 1988; Parnavelas et al., 1988). For examle, the y-amino-butyric acid (GABA) system shows increased levels of glutamate decarboxyl- ase activity (GAD) during the first 2-3 postnatal weeks (Coyle and Enna, 1976), and these changes are paralleled by a marked increment in the number of GABA 0 1996 WILEY-LISS. INC.

terminals that form axosomatic contacts with pyramidal cell bodies (Vincent and Benes, 1995). Dopaminergic projections to rat medial prefrontal cortex (mPFCx) have also been found to increase progressively beyond the weanling stage through to the adult (Kalsbeek et al., 1988; Verney et al., 1982). Since recent studies have demonstrated that appositions exist between dopamine-immunoreactive (DA-IR) varicosities and GABA- immunoreactive (GABA-IR) cell bodies (Berger and Verney, 1984) and are quite common (Benes et al., 19931, it is relevant to consider whether a progressive increase in the interaction of these two neural systems may be occurring postnatally (Benes, 1995). Such maturational changes could be relevant to our understanding of

Received and accepted July 26, 1995. Address reprint requests to Dr. Francine M. Benes, Laboratory for Structural

Neuroscience, McLean Hospital, 115 Mill Street, Belmont, MA 02178.

238 F.M. BENES ET AL.

schizophrenia because this disorder has a characteristic onset during late adolescence (Kraepelin, 1919) and is now believed to involve defects of both dopaminergic (Carlsson, 1978a,b; Meltzer and Stahl, 1976; Seeman, 1981) and GABAergic (Benes et al., 1991, 1992; Reyn- olds et al., 1990; Roberts, 1972; Simpson et al., 1989) neurotransmission in corticolimbic brain regions. AC- cordingly, late postnatal changes in the interaction of these latter two systems could potentially play an important role, not only in the acquisition of normal cognitive functions that depend upon midbrain dopaminergic modulation (Brozoski et al., 1979), but also in the unique onset of schizophrenia during late adolescence (Benes, 1995).

To explore this question further, a double-immunofluorescence technique has been employed to colocalize both DA and GABA in rat mPFCx at different postnatal stages. Using a previously described technique (Benes et al., 1993), a semiquantitative analysis has been used to assess the extent to which DA-IR varicosities form appositions with GABA-IR cell bodies.

MATERIALS AND METHODS Animals

Timed-pregnant CD rats were purchased from Charles River Breeding Laboratories (Wilmington, MA). Following parturition, the litter size was adjusted to 10 pups using a combination of males and females, and each litter was housed in a separate cage with their respective mother. After postnatal day 24 (P24), the pups were weaned from their mothers and then housed in groups (4 pups/cage). All animals were maintained on a 12-hour light-dark cycle and were fed ad libitum with standard food pellets and water.

Tissue preparation Hypothermia anesthesia was used for P5 pups by

immersing them in a bed of crushed ice for approximately 3 min or until they no longer exhibited an aver- sive response to pressure on their distal hind limb. This procedure was performed in accordance with a protocol approved by the McLean Hospital Institutional Animal Care and Use Committee and was carried out by a trained person who continuously monitored the status of the pups. Between P6 and P18, rats were anesthe- tized with sodium pentobarbital at a dose of 25 mgkg body weight, and between P19 and adulthood, with a dose of 50 mgkg body weight. The animals were per- fused intracardially with a series of three solutions using a variable-speed peristaltic pump. First, a solution containing 1% Na-metabisulfite in 0.05 M sodium cacodylate-HC1, pH 6.2, was run through the perfusion cath- eter, followed by 5% glutaraldehyde and 1% Na-metabisulfite in 0.1 M cacodylate buffer, pH 7.4. Finally, a solution of 0.9% NaCl and 1% Na-metabisulfite in 0.05 M Tris-HC1 buffer, pH 7.6, was delivered through the system. All perfusates were maintained at room tem-

perature. The brains were immediately removed and briefly stored in the final buffer solution.

Immunocytochemical procedures

Immunoperoxidase localization of dopamine Following perfusion furation, the brains were cut in

the coronal plane at a level immediately rostral to the optic chiasm, and the rostral pole containing mPFCx was sectioned on a vibratome at a thickness of 40 pm. Sections were immunostained using in a polyclonal antibody raised in rabbit against dopamine (INCSTAR, Stillwater, MN) at a titer of 11500. Initially, the sections were washed 3 X 10 min in 0.05 M Tris-HC1 buffer at pH 7.6, containing 0.9% NaCl and 1% Na-metabisulfite, followed by preincubation in 10% normal donkey serum (NDS; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and 1% Na-metabisulfite in 0.05 M Tris at pH 7.4, for 30 min. The sections were then incubated overnight at room temperature in 11500 anti-dopamine in 0.05 M Tris-0.9% NaC1, and 0.3% Triton X-100 (TNT), also containing 1% Na-metabisulfite and 1% NDS. The sections were then washed 3 x 10 min in TNT and incubated for 2 h in 11100 biotinylated donkey anti-rabbit (Jackson ImmunoResearch Labs) in TNT. The sections were washed 3 X 10 min in 0.05 M Tris-0.9% NaCl and immersed in avidin-biotin complex (Vector Labora- tories, Inc., Burlingame, CA) in TNT for 2 h at room temperature. The sections were then washed 3 x 10 min in 0.05 M Tris-0.9% NaC1, followed by incubation in 0.05% diaminobenzidine and 0.01% HzOz in 0.05 M Tris at pH 7.4, for 5 min. The sections were then washed in 0.05 M Tris at pH 7.4, mounted on gelatin-coated glass slides, dehydrated in a graded series of ethanol and xylene and coverslipped with Permount. Some sections were counterstained with 0.1% cresyl violet prior to dehydration and coverslipping.

Double-immunofluorescence technique As previously described (Benes et al., 19931, the sec-

tions were processed in a medium containing polyclonal antibody raised in rabbit against DA (INCSTAR) at a titler of 1/100 and a monoclonal antibody raised in mouse against GABA (Biohit Systems, Inc., San Diego, C A Clone #5A9) at a titer of 11500. The secondary antibodies employed for the colocalization of DA and GABA included fluorescein isothiocyanate (F1TC)-conjugated donkey anti-rabbit and tetramethyl-rhodamine isothiocyanate (TR1TC)-conjugated donkey anti-mouse (Jack- son ImmunoResearch Labs), respectively. The sections were then incubated for approximately 16 h a t room temperature in a medium containing 11100 anti-dopamine and 11500 anti-GABA in TNT, 1% Na-metabisulfite and 1% NDS. The sections were then washed 3 x 10 min in TNT and incubated for 2 h, in 1/100 FITC-conjugated donkey anti-rabbit and 1/100 TRITC-conjugated donkey anti-mouse secondary antisera in TNT con-

DEVELOPMENT OF DA GABA INTERACTIONS 239

taining 1% NDS. This was followed by 3 x 10 min washes in TNT and a brief wash in doubled distilled water. The sections were then mounted on gelatin- coated glass slides and coverslipped with a solution containing 10% polyvinyl alcohol, 10% glycerol and 0.1% p-phenylenediamine in 0.05 M Tris-HC1 at pH 8.1, to stabilize the fluorescent emission against quenching.

Microscopic analyses of double-immunolabeled sections

Semiquantitative estimates for the frequency of interaction between DA-immunofluorescent (-IF) fibers and GABA-IF cell bodies were performed using a method previously described (Benes et al., 1993). Briefly, the sections were visualized with a 40x objec- tive lens and a 1.25X teleconverter on a Leitz Diaplan microscope equipped with epifluorescence optics, a Dage MTI SIT 68 high sensitivity video camera, a DSP- 100 image processing unit and a Sony still video re- corder. Using a rhodamine filter, the full dorsal to ventral extent of each layer in ra t mPFCx was examined, and each GABA-IF cell body was localized and its image recorded. Without changing the level of focus or the stage position, the FITC-specific filter was then employed to visualize and record the image of DA-IF fibers present in the same field. These corresponding pairs of microscopic images were rated as to whether each GABA-containing cell body had one or more DA-IR varicosities in direct apposition with the cell body within the same plane of focus. An apposition was defined as a point of contact where there was no apparent space or gap between the labeled cell and fiber elements. The data were expressed as two separate parameters: (a) the percent total GABA cell bodies in contact with DA- IF fibers, and (b) as the number of DA-IF varicosities in apposition with any given GABA-IF cell body. The data were collected separately for animals at postnatal time points ranging from P5 through the first week of adult life (P60-P67) and expressed as bivariate scatterplots with age as the independent variable. Re- gression analyses using either first- or second-order polynomial equations were used to test whether the relationship between the two different measures of interaction and age were significant. In addition, a contingency table analysis (3 x 5) was used to compare the percentage of GABAcells with 0,1,2,3,4, or 5+ varicosities in contact during the preweanling, postweanling and adult period. The number of DA-IR varicosities per GABA cell at these same stages (with two degrees of freedom) was analyzed using a Kruskal-Wallis test.

Digital microscopy A digital processing sytem was used to generate im-

ages in which both DA-IF fibers and GABA-IF cell bodies were simultaneously localized. The sections were processed microscopically in a manner similar to that described above, but with the addition of a Z-axis stage

controller inferfaced with an Apple Macintosh@ Quadra- 950 computer, equipped with digital image processing software (ONCOR, Inc., Gaithersburg, MD). Serial images of through-focus planes for both GABA-IF cell bodies and their associated DA-IF fibers were collected and corrected for background flare and image blur. A coregistration subroutine was used to superimpose corresponding pairs of images representing DA-IF fibers and GABA-IF cell bodies.

RESULTS During the first two weeks of postnatal life (Fig. 1,

P l l ) , there were very few DA-IR varicose fibers that could be detected in rat mPFCx. During the third postnatal week, the density of such fibers showed discern- ible increases (Fig. 1, PZO), and this pattern seemed to continue until adulthood (Fig. 1, P45, P52 and adult). As described by others (Lindvall and Bjorklund, 1978), the density of DA-containing fibers in rat mPFCx was greatest in layer VI and showed a progressive decrease toward layer I where very few fibers were observed. Noradrenergic fibers, on the other hand, show a rather different laminar distribution that is characterized by the presence of long, vertical fibers travelling both verti- cally toward layer I and horizontally within this lamina of rat mPFCx (Lindvall and Bjorklund, 1984). Thus, it seems unlikely that appreciable numbers of noradrenergic axons were included in the analyses described below, particularly since the antibody preparation employed in this study is 50 times less selective for norepi- nephrine conjugates (Geffard et al., 1984).

The pattern of staining described above for DA-IR fibers could be distinguished at all postnatal stages examined, and the increase of fiber density that occurred postnatally did not progress in a distinct “inside- out” manner. The size of the DA-IR varicosities did increase from approximately 1.2 pm at P20 to approximately 2.4 pm by P60; however, this change in size did not account for the increase in the density of fibers because the dimensions of the varicosities were quite small relative to the thickness of the sections (40 pm). Using a correction for particle size in which the numerical density, N,, is divided by section thickness, t, plus the average diameter of the particles, D, (Abercrombie, 1946; Weibel, 1979), the density of fibers in postweanling mPFCx was found to be approximately 3.5-fold higher than in that of preweanling rats. For sections in which single immunoperoxidase processing was com- bined with cresylviolet staining (Fig. 2), DA-containing varicose fibers were observed throughout the neuropil, but very commonly, such fibers seemed to course toward neuronal cell bodies. Many cell bodies had one or more varicosities in close apposition, and such contacts were observed in both superficial (Fig. 2A, C and E) and deep (Fig. 2B, D and F) laminae at all stages of postnatal life examined, although the density of varicosities in layers I1 and I11 was characteristically quite low.


I

I I

V

VI

WM P I 1 P20 P45 Adult

Fig. I. Low power, darkfield photomicrograph montages of dopamine-immunoreactive (DA-IR) varicose fibers in rat mPFCx a t five different postnatal ages (P11, P20, P45, P52, and adult). At P11, there is a paucity of DA-containing fibers in the cortical mantle, but by P20, such fibers become considerably more common. At days P45 and P52, DA-IR varicose fibers show a typical laminar distribution, with

the highest density occurring in deeper layers V and VI and a progressive decrease in density occurring in a gradient fashion toward layer I. In adult rats, this unique laminar distribution is still present in spite of a marked increase in the density of fibers in each of the layers. Roman numerals indicate cortical layers. WM, subcortical white mat- ter. Bar = 100 pm.

Specimens of rat mPFCx processed with the double- immunostaining technique showed DA-IF varicosities both in neuropil and in apposition with GABA-IF cell bodies. Overall, the number of DA-IF varicosities seemed to increase between the preweanling period (Fig. 3, P20) and the early stages of the postweanling period (Fig. 3, P25). There appeared to be increasing numbers of such varicosities forming contacts with GAJ3A-IF neurons as the postweanling period pro- gressed, and this was most apparent a t the beginning of the adult period (Fig. 3, P60). When double-IF prepa- rations were subjected to a blind, semiquantitative analysis, a progressive linear increase in the per- centange of GABA-IF cell bodies (Fig. 4, upper panel) with apposed DA-IR varicosities occurred between PO

and P60, and best fit a first order polynomial equation (r = 0.75, P = 0.0005). During the preweanling period, any given GABA-IR cell body, on average, showed approximately one such varicosity in apposition. During the postweanling period, however, the number of DA- IR varicosities (Fig. 4, lower panel) in contact with GABA-IR cell bodies showed a curvilinear rise through P60 (r = 0.81, P = 0.0005) when a second-order polynomial equation was employed. An index of interaction was computed by mutliplying the percentage of GABA cells having apposed dopamine varicosities by the number of such varicosities in contact with any given GABA- containing neuron. For postweanling rats, this index was 1.5 times higher than that for preweanling animals (Table I). By adulthood, this index increased 1.8 times


Fig. 2. Nomarksi photomicrographs of dopamine-immunoreactive (DA-IR) varicose fibers in specimens of rat mPFCx at three different postnatal ages (P20, P45, P60). The upper tier is from layer I1 (A, C and E), while the lower tier is from layer VI (B, D and F) for P20 (A, B), P45 (C, D) and P60 (E, F), respectively. Cell bodies of neurons

(asterisks) and glial (GI have been visualized with cresyl violet coun- terstaining. At all developmental time points shown, DA-IR fibers course through the neuropil and oRen several varicosities are in apposition with a single neuronal cell body. Bar = 20 pm.

with respect to postweanling rats and 2.5 times when compared to that for the preweanling period (Table I).

vides support for the idea that there are significant postnatal increases in the dopaminergic projections to rat mPFCx. This apparent ingrowth of ventral tegmen- tal afferents (VTA) is associated with an increased interaction of DA varicosities with intrinsic GABAergic neurons, particularly during the postweanling period when there are increases not only in the percentage of GABA-

DISCUSSION As previously described by other laboratories (Kals-

beek et al., 1988; Verney et al., 1982), this study pro-


Fig. 3. A digital confocal photomicrograph of a double-immunofluorescence localization of DA fibers (yellow) and GABA cell bodies (green) in layer VI of rat mPFCx at three different postnatal ages (P20, P25 and P66). By P20, DA-IR varicosities are already forming contacts with GABAergic cell bodies (arrows). Immediately after weaning (P25),

IR cell bodies having DA varicosities in apposition, but also the number of DA varicosities engaging in contact with any given GABA cell body.

At the ultrastructural level, DA-containing varicosities have been shown to form synapses with shafts and spines of distal dendritic branches, although some also form contacts with neuronal somata having a morphological appearance similar to that of inhibitory basket cells (Goldman-Rakic et al., 1989). Although the contacts that occur on neuronal cell bodies involve varicose portions of the fibers (Benes et al., 1993) where the mechanisms for the synthesis, storage, release and re- uptake of DA are probably concentrated (Beaudet and Descarries, 19781, the appositions formed with GABA cell bodies are typically nonsynaptic in nature and do not show glial processes interposed between them (Verney et al., 1990). Consistent with this, apreliminary study in our lab, conducted at the ultrastructural level, has revealed that approximately half (51.5%) of all axo- nal profiles apposed to neuronal cell bodies in layer VI of rat mPFCx: (a) are separated from the perikaryal plasma membrane by a distance of 20 nm, (b) show no interposition of glial processes, (c) demonstrate no evidence of membrane specializations, and (d) contain abundant numbers of spherical vesicles (unpublished data). Many of these profiles also contain dense-core vesicles which are characteristically seen in monoami- nergic terminations (Beaudet and Descarries, 1984). Taken together, these findings suggest that monoami- nergic fibers may commonly engage in nonsynaptic junctional contacts with neuronal cell bodies. Although most dopamine-containing varicosities do appear to make at least one synaptic contact with postsynaptic

however, there is not as yet an obvious change in the extent of interaction, although some varicosities may he forming contacts with a dendriticshaft (arrowhead). ByP66, however, there are several varicosities in apposition with the GAJ3A-containing cell body and the interaction seems to have markedly increased. Magnification = X 1,650.

dendritic profiles (Seguela et al., 1988), it is not known if this occurs on cell bodies as well.

DA varicosities engaging in nonsynaptic interactions with GABAergic somata could be capable of exerting a significant modulatory influence on the activity of these cells. Electrophysiological studies suggest that dopamine may indeed function as a neuromodulator of other classical transmitters in the prefrontal cortex (Bunney and Chiodo, 1984). For example, dopamine markedly reduces the excitatory effect of acetylcholine on cortical neurons for up to 5 min following cessation of a 20-30 sec application (Reader et al., 1979). Similarly, destruc- tion of the dopamine projections to frontal cortex results in a decrease of not only the inhibitory effect of enkephalin, but also the antagonism of this effect by antipsychotic drugs (Palmer and Hoffer, 1980). Based on these electrophysiological studies, it seems plausible that a neuromodulatory effect of dopamine could have a significant impact on the interaction of mPFCx with other regions with which it is connected. In this regard, it is noteworthy that most cortical cells showing a suppres- sion of firing in response to DA are also excited by inputs from the medial dorsal nucleus of the thalamus, a response that is in turn suppressed by simultaneous stimulation ofVTA neurons (Theirry et al., 1988). Thus, a modulatory action of dopamine on target cells in mPFCx of adult rat may play an important role in the integration of this region with other key areas, such as the thalamus. During postnatal development, it is likely that this modulatory role may become progressively more prominent as dopamine fibers gradually increase their penetration of mPFCx.

Postnatal increases of dopaminergic projections to rat


2.5 - 2-25 . - -

a! .- $2 2-

58 1.5.

~a 1.75 .

Interaction of Dopamine Fibers With GABA Neurons In Rat mPFC During Postnatal Development

Layer V I VI r = 0.81 0

loo 1 Layer V I VI r = 0.753

L 8 sp 1.

5 8 .75 - z '= p .5-

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Fig. 4. Bivariate plots of the interaction of dopamine-immunoreactive (DA-IR) varicosities with GABA-IR cell bodies a t different postnatal ages. Upper panel: The percentage of GABA-IR cell bodies having a DA-IR varicosity in apposition. Lower panel: The number of DA- IR varicosities per GABA-IR cell. Between P5 and P60, there is a

mPFCx (Kalsbeek et al., 1988; Verney et al., 1982) are paralleled by an increase of D2 receptor binding activity, that begins prenatally (Bruinink et al., 1983) and con- tinues through the fourth postnatal week (Deshn et al., 1981). Interestingly, administration of 6-OH-dopamine prevents this latter increase of D, receptor binding (Deskin et al., 1981), and this effect is associated with dystrophic changes in the basal dendrites of pyramidal neurons (Kalsbeek et al., 1988). Similar lesions in primate prefrontal cortex result in an impaired perfor- mance of the spatial delayed alternation task (Brozoski et al., 1979). Since GAEiAergic interneurons are essen- tial to discriminative functions, such as the orientation selectivity of neurons in visual cortex (Sillito, 19841, it is tempting to speculate that the increased interaction of dopaminergic afferents with GABAergic neurons in rat mPFCx may contribute to the acquisition of adult patterns of information processing. Interestingly, GABAergic neurons in cortex also show significant maturational changes during the postnatal period. For example, GAI3A-accumulating cells of occipital cortex show a progressive increase in numerical density until P11 (Chronwwall and Wolff, 1980). In contrast, the con- centration of GABA and the specific activity of GAD (Coyle and Enna, 1976), as well as GABA receptor binding activity (Coyle and Enna, 1976; Palacios et al., 1979) and its messenger RNA (Gambarana et al., 19901, all increase until the third postnatal week. In rat mPFCx, between P5 and P15, there is a progressive decline in the density of GABA-containing cell bodies as the surrounding neuropil expands (Vincent et al., 1995). The expansion of neuropil in rat mPFCx probably involves, in part, an increase in the number of GABA terminals surrounding pyramidal neurons, a process that contin- ues in the superficial layers until P25 (Vincent et al., 1995) when the efficacy of GABAergic synaptic trans- mission becomes optimal (Luhmann and Prince, 1991).

progressive linear increase in the percentage of GABA-IR cell bodies showing appositions with DA-IR varicosities (r = 0.75; P = 0.0005 using a first-order polynomial equation). During this same period, there is, on average, only one DA-IR varicosity found per GABA-IR neuron;

Thus, the maturation of dopaminergic fibers Seems to continue for weeks beyond the stage at which GABA neurons no longer show ontogenetic

however, after weaning (P24), the number ofvaricosities per cell shows a curvilinear rise toward adult levels (r = 0.81, P = 0.0005 using a second order polynomial equation).

changes. This pattern suggests that GABAer& cells could be a passive target for ingrowing dopaminergic fibers, although it is possible that these interneurons

TABLE I. A comparison of dopamine-GABA interactions in layers V and VI of rat mPFCx at various postnatal stages'

% GABA cells in Number of contact with varicosities per Interaction

Postnatal staee n varicosities GABA Cell Index2

Preweanling 6

Adlilt. 4 Postweanling 11

31 2 11 39 2 1 55 ? 14*

1.26 i 0.19 1.38 t 0.21 1.79 2 0.27**

39 54 98

~~~~~~~~

'The preweanling period consisted of P5-PZ4, while the postweanling period consisted of P25-P59. Adult rats included in this study were sacrificed a t P60-P65. The values shown for the % GABA-IR cells and the number of DA-IR varicosities per GABA cell represent the means ? S.E.M. for each stage examined. *For the % GABA cells, a 3 X 5 contingency table analysis generated a xz = 19.9 with P = 0.03. **For the number of DA-IR varicosities per GABA cell, a Kruskal-Wallis test revealed that the H-statistic = 9.1 (df = 2) with P = 0.011. aThe Interaction Index was obtained by multiplying the % GABA cells with DA-IR varicosities by the number of DA-IR varicosities per cell.


might themselves provide a neurotrophic influence on the ingrowth of extrinsic afferent inputs (Spoerri, 1988). In any case, it seems likely that dopaminergic fibers would potentially be capable of exerting an increasing modulatory influence on the activity of inhibitory interneurons, particularly since DA receptors are localized on nonpyramidal cell bodies in rat mPFCx (Vincent et al., 1993,1995). Moreover, both agonists and antago- nists of DA can alter the postsynaptic potentials recorded in GABAergic interneurons in pyriform cortex (Gellman and Aghajanian, 1993), and agonists for the D, receptor have been found to inhibit the evoked release of [3H]GABA (Retaux et al., 1991a,b) and influence the firing of GABAergic neurons (Penit-Soria et al., 1987). It is pertinent to note that a selective increase in the release of DA occurs under stressful conditions (Roth et al., 1988; Thierry et al., 1976) and this response is modulated, at least in part, through GABAergic mechanisms residing within the VTA (Deutch et al., 1989; Deutch and Roth, 1990; Lavielle et al., 1978; Tam and Roth, 1990), although it seems likely that cortical GABA cells may also play a modulatory role (Benes et al., 1993).

Future studies will seek to determine whether sero- tonergic fibers that provide a converging afferent input on GABAergic cells receiving VTA afferents (Gellman and Aghajanian, 1993; Morilak et al., 1993; Sheldon and Aghajanian, 1990; Taylor and Benes, 1994) may also undergo postnatal maturational changes similar to those described here for the cortical DA system.

ACKNOWLEDGMENTS This work has been supported by grants from the

National Institute of Mental Health (MH00423 and MH31154) and the Stanley Foundation. We thank Mary Monismith of Boston Business Graphics, Stoneham, MA, for technical assistance in making the digital image print.

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