• Abstract
  

• Introduction
  

• Materials and Methods
  

• Results
  

• Discussion
  

• Acknowledgments
  

• References
  

ABSTRACT

Recent evidence has suggested that GABAergic neurons in rat cortex may receive a convergence of monoaminergic inputs (Gellman and Aghajanian, 1993). To extend an earlier study in which dopamine-immunoreactive (IR) fibers were noted to form frequent, nonrandom appositions with GABA-IR cell bodies (Benes et al., 1993), an immunofluorescent technique has been developed for the colocalization of glutamate decarboxylase (GAD), tyrosine hydroxylase (TH) and serotonin (5HT) in single sections of rat medial prefrontal cortex (mPFCx). The primary antisera consisted of a polyclonal raised in rabbit against GAD67, a monoclonal raised in mouse against TH, and a polyclonal raised in goat against 5HT. Initially, the frequency with which 5HT- and TH-IR varicosities independently form contacts with pyramidal vs nonpyramidal cell bodies was assessed in sections processed with a single immunoperoxidase technique and counterstained with cresyl violet. Overall, many pyramidal and nonpyramidal somata were found to be in apposition with both 5HT- and TH-IR varicosities in layers II and layer VI of mPFCx. The "observed" numbers of 5HT- and TH-IR varicosities in contact with cell bodies or in neuropil were compared to the "predicted" numbers that were computed from the areal percentage of these respective compartments. Using a Poisson analysis based on the Chi-square distribution, it was found that there were many more 5HT- and TH-IR varicosities in apposition with cresyl violet- stained cell bodies in layer II ( = 93.7, p = 0.0001 and = 37.2, p = 0.0001, respectively) and VI ( = 19.5, p = 0.0001 and = 18.7, p = 0.0001, respectively) than random interactions would predict. The probability that both 5HT- and TH-IR varicosities would be simultaneously in contact with any given cresyl violet-stained cell body was calculated as the product of the individual "predicted" values. This so-called convergence probability was found to be surprisingly high for both pyramidal and nonpyramidal cell types in layer II (23% and 36%, respectively) and layer VI (25% and 26%, respectively).

For the triple immunofluorescence (IF) colocalization studies, the secondary antibodies employed were raised in donkey (against rabbit, mouse, and goat) and were conjugated to FITC (GAD67), TRITC (TH) and AMCA (5HT), respectively. A digital confocal imaging system was used to visualize serial Z-axis images of the three fluorescent emissions and, following the application of a deconvolution subroutine, corresponding planes of the respective images were placed in co-registration. As predicted from the convergence probabilities computed from the single immunoperoxidase studies, TH- and 5HT-IF varicosities were commonly found in apposition with the same GAD67-IF cell body. In addition, pyramidal neuron cell bodies were often visualized as a result of a "tri-vergence" of TH-, 5HT- and GAD-IF varicosities/bouton that formed a network of apparent appositions around the periphery of the ghost cell. These latter profiles appeared to show "presynaptic" appositions of TH- and 5HT-IF varicosities with GAD-IF bouton. Overall, these findings are consistent with the idea that catecholaminergic and serotonergic fibers converge on a significant proportion of GABAergic cell bodies, and some of these latter fibers may even engage in presynaptic interactions with GABAergic inputs to pyramidal cell somata. © 1996 Neuroscience-Net.

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INTRODUCTION

Various studies have emphasized the important role that cortical GABAergic neurons probably play in the regulation of pyramidal cell activity (Houser et al., 1984; Krnjevic, 1987). Recently, it has become apparent that these inhibitory interneurons may themselves be modulated by dopaminergic, serotonergic, and noradrenergic inputs, and some GABA neurons may even receive a convergence of all three of these monoaminergic afferent fiber systems (Gellman and Aghajanian, 1993). To date, immunocytochemical studies conducted both at the light (Benes et al., 1993) and electron (Verney et al., 1991) microscopic levels have confirmed that dopamine-immunoreactive (IR) varicosities form appositions with GABA-IR cell bodies. These latter contacts appear to be quite common and cannot be explained simply by random interactions (Benes et al., 1993). Moreover, the finding of both D₁ and D₂ dopamine receptor mRNA (Huntley et al., 1992) and binding activity (Vincent et al., 1993; Vincent et al., 1995) on nonpyramidal cells in rat mPFCx provides support for the idea that these appositions may be functional in nature. Similarly, the fact that both pharmacologic (Sheldon and Aghajanian, 1991) and microscopic (Morilak et al., 1993) studies have linked 5HT₂ receptor binding activity to inhibitory interneurons in pyriform cortex (Morilak et al., 1993; Sheldon and Aghajanian, 1991) suggests that serotonergic inputs may also exert physiological action on GABA cells. In rat mPFCx, DA fibers are most densely distributed in layers V and VI (Lindvall and Bjorklund, 1984; Emson and Koob, 1978), while 5HT fibers are abundant both in superficial and deep laminae of this region (Lidov et al., 1980; Reader, 1981). Based on these laminar distributions, a convergence of these latter two monoaminergic systems onto individual GABAergic cells could potentially be a common occurrence in the deeper layers of mPFCx. Unlike the interaction of dopamine afferents with cortical GABA neurons (Verney et al., 1991; Benes et al., 1993), there has not as yet been anatomic confirmation that 5HT afferents form appositions with GABAergic cell bodies in rat mPFCx. Accordingly, it is not as yet known whether a convergence of both DA and 5HT varicose fibers onto a single GABAergic interneuron can be demonstrated in this region using microscopic approaches. To investigate these questions, a technique has been developed to simultaneously visualize tyrosine hydroxylase- (TH-), 5HT- and GAD-immunofluorescence (IF) in rat mPFCx. Together with single immunoperoxidase localizations, this colocalization approach has been used to explore whether 5HT and TH afferents form nonrandom, convergent appositions with GABAergic neurons in rodent cortex.

 

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MATERIALS AND METHODS

Animal Perfusion Protocol
Adult male CD rats (Charles River Breeding Laboratories, Wilmington, MA) were anesthetized with sodium pentobarbital (65 mg/kg body weight), and 0.5 ml heparin (conc. 143USP units/10 ml) was injected into the left ventricle. 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 animals. For immunoperoxidase preparations, the rats were perfused intracardially with 100 ml 0.1 M phosphate buffer, pH 7.4, containing 0.9% NaCl (PBS) for 5 minutes, followed by 100 ml of 4% paraformaldehyde in PBS at pH 7.4 for 5 minutes, and 600 ml of 4% paraformaldehyde in 0.1 M borate buffer at pH 11.0 for 25 minutes. For immunofluorescence microscopy, the rats were perfused intracardially with 100 ml of PBS for 5 minutes, followed by 100 ml of 4% paraformaldehyde in PBS at pH 6.5 for 5 minutes, and 600 ml of 4% paraformaldehyde in 0.1 M borate buffer at pH 11.0 for 25 minutes. For both single and triple immunolocalizations, the perfused brains were immediately removed and postfixed in 4% paraformaldehyde in borate buffer, pH 11.0 for 90 minutes at room temperature. Serial sections (50 µm thick) were cut on a vibratome and immersed in ice-cold PBS.

Immunocytochemical Procedures
Immunoperoxidase Labeling: The following steps were carried out at room temperature on a motor driven rocking platform. Free floating sections were washed 3 X 10 minutes in PBS and were then incubated in 0.03% H₂O₂ in PBS for 30 minutes, washed 3 X 5 minutes in PBS containing an additional 1% normal serum (5HT: donkey; TH: horse) and 0.25% Triton-X-100 (PBS-TX) and then incubated for 90 minutes in PBS-TX containing 10% normal serum. The sections were then stored overnight at 4° C in PBS-TX containing either anti-TH raised in mouse (Chemicon MAB-318) at a titration of 1/1000 or anti-5HT raised in rabbit (INCSTAR Stock No. 20080) at a titration of 1/8000. On day 2, the free-floating tissue sections were washed 3 X 5 minutes in PBS-TX plus 1% normal serum, and then incubated for 90 minutes in PBS-TX plus 1% normal serum containing the appropriate secondary antibodies (Jackson ImmunoResearch Laboratories, Inc) that included: biotinylated donkey-anti-rabbit (5HT) or biotinylated horse-anti-mouse (TH), at a titer of 1/200. The tissue sections were washed 3 X 5 minutes in PBS-TX plus 1% normal serum, followed by incubation in avidin-biotin complex (Vector Labs) in PBS-TX plus 1% normal serum for 90 minutes. Following 3 X 5 minutes in 0.1 M PBS, the sections incubated in 0.025% DAB with 0.005% H₂O₂ solution for 10 minutes and were mounted on acid-cleaned gelatin-coated slides. Mounted tissue sections were osmicated for 5 minutes in 0.01% OsO₄, counterstained with 0.1% cresyl violet, dehydrated through a graded series of ethanol and xylene, and coverslipped using Cytoseal mounting medium.

Immunofluorescence Processing: Free floating sections were washed 4 X 10 minutes in PBS, followed by 4 X 5 minutes in PBS containing an additional 1% sodium chloride and 0.5% Triton-X-100 (PBS-TX). The sections were then immersed for 90 minutes in 5% normal donkey serum (NDS) and 3% bovine serum albumin (BSA) in PBS-TX, then incubated overnight at 4° C in PBS-TX containing 5% NDS and 3% BSA and the appropriate primary antisera. The primary antibodies included: anti-GAD₆₇ raised in rabbit (Chemicon AB-108) at a titration of 1/500; anti- TH from mouse hybridoma (Chemicon MAB-318) at a titration of 1/1000; and anti-5HT raised in goat (INCSTAR 20079) at a titration of 1/2000. In some experiments anti-GAD₆₅ from mouse hybridoma (Boehringer Mannheim, Stock No. 1522 825) at a titration of 1/8000 was employed to visualize GAD containing terminals in conjunction with anti-TH raised in rabbit (Chemicon AB-151) at a titration of 1/4000 and anti-5HT from goat (INCSTAR 20079) at a titration of 1/1000. On day 2, the free floating tissue sections were washed 4 X 10 minutes in PBS-TX, then incubated for 90 minutes in 1% NDS in PBS-TX containing the appropriate secondary antibodies (Jackson ImmunoResearch Laboratories, Inc) at a titration of 1/100. For triple localization studies, FITC- conjugated donkey anti-rabbit (GAD₆₇), TRITC-conjugated donkey anti-mouse (TH), and AMCA-conjugated donkey anti-goat (5HT) were employed as secondary antibodies. For the localization of GAD₆₅ containing terminals, TRITC-conjugated donkey anti-mouse was used in combination with FITC-conjugated donkey anti-rabbit (TH-containing fibers) and AMCA- conjugated donkey anti-goat (5HT-containing fibers). The tissue sections were washed 4 X 10 minutes in PBS-TX containing 1% NDS, followed by an additional wash in PBS for 5 minutes. The tissue sections were then mounted on acid-cleaned gelatin-coated slides, air dried and stored overnight in a light-tight drawer and coverslipped with freshly made mounting medium containing 10% polyvinyl alcohol, 10% glycerol, and 0.1% p-phenylenediamine in 0.05 M Tris-HCl, at pH 8.1.

Microscopic Analyses
Bright Field Microscopy: Using brightfield optics on a Leitz Diaplan light microscope, images were obtained using a 40X oil immersion objective to visualize either 5HT- or TH-IR varicosities that were in close apposition with cresyl violet stained neuronal cell bodies. A Leitz Laborlux 12 microscope was interfaced with a BIOQUANT OS/2 Image Analysis System via a Dage MTI CCD 72 video camera and a DTK 486 PC computer. Adult rat specimens were prepared on postnatal days 62 and 63 and used for the quantitative analysis of 5HT- and TH-IR varicosities in rat mPFCx. For each of six fields in layers II and VI of the CG1 subregion of rat mPFCx (Neafsey, 1993), a cursor was used to trace around the perimeter of every "in-focus" cell body to determine its areal size (µm²). Touch counting was used to determine the number of IR varicosities that were either in contact with the "in-focus" cell bodies or present in the surrounding neuropil at the same level of focus. A varicosity was considered to be in contact with a cresyl violet counterstained cell body if 1) the varicosity and cell body were both crisply visualized in the same plane of focus; 2) there appeared to be direct apposition between the varicosity and the cell body; and 3) there was no apparent space lying between the varicosity and the cell body. For each microscopic field, the percent total number of either TH- or 5HT-IR varicosities in contact with neuronal cell bodies versus the percent total varicosities in neuropil was determined. In addition, the total areal percent for the neuronal cell bodies and the surrounding neuropil were obtained for each field. A "predicted" number of varicosities based on the areal percent for cell body and neuropil compartments were as determined. A Poisson analysis based on the Chi-square distribution (Benes et al., 1993) was used to assess whether there was a significant difference between the "observed" and "predicted" numbers of 5HT- or TH-IR varicosities present on neurons or in the neuropil.

Digital Confocal Microscopy: Tissue sections that were fluorescently labeled for GAD, 5HT and TH were viewed with a 63X oil immersion lens (NA 1.30) coupled to a 1.6X teleconverter on a Leitz Diaplan light microscope equipped with epifluorescence optics and dichroic filters appropriate for fluorescence emissions spectra of fluorescein isothiocynate (FITC), tetramethyl rhodamine isothiocyanate (TRITC) and aminomethylcoumarin acetate (AMCA). To amplify the intensity of the fluorescent images, a Dage MTI SIT-68 high sensitivity video camera was interfaced with a Dage DSP-100 signal processor (DAGE-MTI Inc., Michigan City, IN) and was operated in the automatic mode for black level, kV and gain settings to maintain consistency in light level throughout the acquisition of Z-axis stacks. For the three fluorochrome labeled probes, digital images were created by interfacing this system with an Apple Macintosh Quadra-950 computer with BDS-IMAGE processing software (ONCOR Imaging Systems, Inc, Baltimore, MD). Three Z-series stacks representing the respective fluorescent images for FITC, TRITC and AMCA were acquired using 5-µm-step intervals. Three consecutive focal planes were obtained above, in and below the plane of focus for a given GAD-IF cell body. Each of the captured images was stored as a 512 X 512 8-bit pixel image file, and then processed with a "deconvolve" subroutine to mathematically correct image distortion effects produced by fluorescent background flare. The three deconvolved "in-focus" images were then co-registered using pseudo-color processing to distinguish the different fluorescent/emissions.

 

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RESULTS

Immunoperoxidase Staining of 5HT- and TH-IR fibers
Visual inspection of sections processed with single immunoperoxidase staining indicated that there were abundant numbers of both 5HT- (Fig. 1-left) and TH-IR (Fig. 1-right) fibers throughout the expanse of rat mPFCx. The 5HT-IR fibers tended to be most dense in layers I-III and least dense in upper portions of layer V. TH-IR fibers, on the other hand, were most dense in layers V and VI and showed a gradual decline in density toward layer II. Some TH-IR fibers followed a long straight vertical course toward layer I where they then traveled horizontally. These latter fibers seemed to have fewer varicose regions, than those observed in layers V and VI. When sections counterstained with cresyl violet were viewed at a higher magnification, both 5HT- and TH-IR varicosities were found to be in apposition with cresyl violet stained cell bodies (Fig. 2A and B, respectively).

 

Figure 1. Low-power, dark field photomicrographs of 5HT (left) and TH (right) immunoperoxidase-stained fibers in rat mPFCx. Cortical laminae are denoted by the Roman numerals on the left. Bar = 100 µm.

 

Figure 2. High-power, bright field photomicrographs of 5HT (A) and TH (B) immunoperoxidase-stained fibers in apposition with cresyl violet stained cell bodies in layer II of rat mPFCx. Arrows indicate varicosities in close apposition to cell bodies. Bar = 10 µm.

As shown in Table I, the proportion of 5HT-IR varicosities that were in apposition with neuronal cell bodies in layers II and VI was 35% and 18%, respectively, while the majority (65% and 82%, respectively) were in the neuropil of these two laminae. For TH-IR varicosities, a similar pattern was observed, although the percentages associated with cell bodies (23% and 13%, respectively) was somewhat lower than those observed for 5HT-IR varicosities (Table I). As shown in Table II, the total areal percent for neuronal cell bodies represented in the sample fields used in this analysis was much lower (approximately 8-10%) than for neuropil (approximately 90-92%). Using these latter percentages and a random distribution model (Table III), the "predicted" frequencies for finding 5HT-IR varicosities on cell bodies in layers II and VI was determined to be 11% and 8%, respectively, while the "observed" frequencies (35% and 18%, respectively) were two to three times higher than predicted ( = 93.7, p = 0.0001 and = 19.5, p = 0.0001, respectively). Similarly, the "predicted" frequencies for finding TH-IR varicosities in apposition with cell bodies in layers II and VI (10% and 8%, respectively) was significantly lower than the respective "observed" values ( = 37.2, p = 0.0001 and ( = 18.7, p = 0.0001, respectively). Thus, the frequency with which 5HT- and TH-IR varicosities were in contact with cell bodies in both layers II and VI is much greater than would be expected on the basis of a random effects model.

 

TABLE I: Number of 5HT and TH Varicosities on Neuron Cell Bodies and in Neuropil of Rat mPFCx
	Cortical Layer	Number (%) Varicosities Cell Bodies	Number (%) Varicosities Neuropil	Total Number Varicosities
5HT	Layer II Layer VI	189 (35%) 92 (18%)	344 (65%) 429 (82%)	533 521
TH	Layer II Layer VI	131 (23%) 177 (13%)	429 (77%) 1177 (87%)	560 1354

 

TABLE II: Total Areal Percentage of Neurons in Sample Fields of Rat mPFCx
	Cortical Layer	Cell Area in µm2	Field Area in µm2	% Cell Somata	% Neuropil
5HT	Layer II Layer VI	30365.15 24483.78	292350.6 292350.6	10% 8%	90% 92%
TH	Layer II Layer VI	28677.26 23381.47	292350.6 292350.6	10% 8%	90% 92%

 

TABLE III: analysis of the randomness of 5HT and TH varicosity interactions with cell bodies and in neuropil
		5HT		TH
Cortical Layer		Neuropil	Cell Body	Neuropil	Cell Body
Layer II	Observed Predicted	344 (65%) 477 (89%)	189 (35%) 56 (11%)	429 (77%) 505 (90%)	131 (23%) 55 (10%)
Layer VI	Observed Predicted	429 (82%) 477 (92%)	92 (18%) 44 (8%)	1177 (87%) 1245 (92%)	177 (13%) 108 (8%)

5HT

Layer II: ( = 93.7     p = 0.0001)
Layer VI: ( = 19.5     p = 0.0001)

 

TH

Layer II: ( = 37.2     p = 0.0001)
Layer VI: ( = 18.7     p = 0.0001)

 

Based upon previously published data (Vincent et al., 1993), neuronal cell bodies less than 100 µm² in area were identified as being primarily those of nonpyramidal cells and, to a lesser extent, pyramidal neurons, while those greater than or equal to 100 µm² were found to be almost exclusively those of pyramidal neurons (Tables IV and V). When cell bodies were broken down according to these size criteria, the percentage of large neurons ( 100 µm²) versus small neurons (<100 µm²) with 5HT-IR varicosities in apposition with their somata was identical in layer II (54% and 53%, respectively), while in layer VI a slightly higher percentage of small neurons had apposed varicosities (38% and 44%, respectively) (Table IV). For TH-IR fibers (Table V), the percentages were of similar magnitude to those for 5HT-IR fibers, although the latter were slightly lower in layer VI when compared to the former.

 

TABLE IV: Relative size distribution of Nissl-stained neurons with and without 5HT varicosities in layers II and VI of rat mPFCx
	Cortical Layer		Large Neurons (100 µm2)	Small Neurons (<100 µm2)	Total Cells
II	Neurons with Varicosities	116 (54%)	23 (53%)	139 (54%)
	Neurons with no Varicosities	100 (46%)	20 (47%)	120 (46%)
	Total Neurons	216 (83%)	43 (17%)	259
VI	Neurons with Varicosities	65 (38%)	19 (44%)	84 (39%)
Neurons with no Varicosities	108 (62%)	24 (56%)	132 (61%)
Total Neurons	173 (80%)	43 (20%)	216

 

TABLE V: Relative size distribution of Nissl-stained neurons with and without TH varicosities in layers II and VI of rat mPFCx
Cortical Layer		Large Neurons (100 µm2)	Small Neurons (<100 µm2)	Total Cells
II	Neurons with Varicosities	91 (43%)	21 (68%)	112 (46%)
	Neurons with no Varicosities	123 (57%)	10 (32%)	133 (54%)
	Total Neurons	214 (87%)	31 (13%)	245
VI	Neurons with Varicosities	102 (67%)	37 (58%)	139 (64%)
Neurons with no Varicosities	50 (33%)	27 (42%)	77 (36%)
Total Neurons	152 (70%)	64 (30%)	216

 

The probability that any given cell body would have direct contact with both a 5HT- and a TH-IR varicosity was calculated by multiplying the percentage of large or small neurons having 5HT-IR varicosities in apposition by the percent of the same cell type with TH-IR varicosities in contact. As shown in Table VI, this so-called probability of convergence for large neurons in layers II and VI was 23% and 25%, respectively, while that for small neurons was 36% and 26%, respectively.

 

TABLE VI: Percent of large (100µm2) and small (<100µm2) neurons with varicosities plus probability of convergence
	Cortical Layer	5HT	TH	Probability of Convergence
Large Neurons with varicosities (100 µm2)	Layer II	54%	43%	23%
	Layer VI	38%	67%	25%
Small Neurons with varicosites (<100 µm2)	Layer II	53%	68%	36%
Layer VI	44%	58%	26%

The percentage of 5HT-IR and TH-IR cells in apposition with large and small cell somata in layers II and VI were multiplied by one another to generate a predicted probability of convergence.

CoLocalization of GAD₆₇-, 5HT- and TH-IF Elements
Sections that were simultaneously processed with antibodies against 5HT, GAD₆₇ and TH generally showed a low signal-to-noise ratio for each of the fluorochome-labeled images (Fig. 3, panel set 2). When the respective images were deconvolved with respect to the background fluorescence (panel set 1), there was a marked improvement in the visualization of 5HT-, GAD₆₇- and TH-IF structures (Fig. 3, panel set 4). Following co-registration and pseudocolor processing (Fig. 4 A-D), numerous GAD-IF cell bodies appeared to have both 5HT- and TH-IF fibers coursing toward them and forming appositions with varicose portions of the fiber (Fig. 4 A, B and C). In middle portions of the cortical mantle, where TH-IF varicose fibers were quite abundant, but 5HT-IR fibers were less dense, it was more difficult to find GAD₆₇-IF cell bodies with both fiber types in apposition. In some cases, pyramidal cell "ghosts" could be visualized by virtue of GAD-IF terminal endings forming axosomatic contacts with their cell bodies (Fig. 4 D). For some of these profiles, both 5HT-IF and TH-IF varicosities seemed to be in apposition with GAD-IF bouton (Fig. 4 D); however, direct contacts between TH-IF and 5HT-IF varicosities were not commonly observed.

 

Figure 3. Serial 5 µm Z-axis planes and deconvolution of images taken with the appropriate filters for visualizing 5HT/AMCA, GAD/FITC and TH/TRITC: 1, 2, 3: Above, in and below focus planes respectively. 4. Specific Z-axis plane corrected for the perspective background flares using a deconvolve subroutine. Bar = 10 µm.

 

Figure 4. Co-registered images of 5HT-, GAD- and TH-IR staining: A, B, C: Three deconvolved images showing a convergence of monoaminergic inputs (arrows = 5HT; arrowheads = TH) to GABAergic cell bodies in rat mPFCx. D. Monoaminergic varicosities converge on a pyramidal neuron (P) receiving GAD-IR terminals in rat mPFC. There also appear to be appositions between GAD-IR terminals and 5HT-IR varicosities (*). Similar interactions also seem to occur between GAD-IR terminals and TH-IR varicosities. Bar = 10 µm.

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DISCUSSION

The results of this study have demonstrated that both 5HT- and TH-IR fibers form nonrandom appositions with both pyramidal and nonpyramidal cell bodies in rat mPFCx. Based on the probability of convergence that was computed for these two different fiber types (Table VI), it was not surprising that both 5HT- and TH-IF varicosities were routinely found in apposition with the same GAD-IF cell body using the triple colocalization technique. Thus, the data reported here now provide microscopic evidence in support of the idea that GABAergic interneurons can be simultaneously regulated by more than one monoaminergic system (Gellman and Aghajanian, 1993).

These results show good agreement with those reported earlier from a study in which dopamine- and GABA-IF were colocalized in rat mPFCx (Benes et al., 1993). In this previous study, 65% of GABA-containing cell bodies in layer VI were found to be in apposition with dopamine-IR varicosities, while in the current study, 64% of the GABA-containing cell bodies were in apposition with TH-IR varicosities. In layer II, however only 12% of the GABA-IR cell bodies were previously found to be in apposition with the dopamine-IR varicosities (Benes et al., 1993), while in the present report, 46% of the GABA-IR cell bodies showed contacts with TH-IR varicosities (Table V). The more frequent occurrence of contacts in layer II in the present study can probably be explained by the fact that TH is a marker for both dopaminergic and noradrenergic fibers, and while dopamine-IR fibers are quite sparse in superficial layers of rat mPFCx, noradrenergic fibers are relatively abundant, particularly in layers I and II (Lindvall and Bjorklund, 1984). Serotonergic fibers are also densely distributed in superficial laminae (Fig. 1-left) and this is reflected by the fact that a large proportion (54%) of the cell bodies were in apposition with these latter varicosities in layer II (Table IV).

Ultrastructural studies have demonstrated that the varicose swellings of serotonergic and noradrenergic fibers contain an abundant number of synaptic vesicles and are capable of the selective uptake of these transmitters (Beaudet and Descarries, 1978). Although an electron microscopic study in which serial sections were examined had concluded that the majority of varicosities in the neuropil of the anteromedial and suprarhinal cortices of rat brain have at least one subregion making a classic synaptic contact with dendritic structures (Seguela et al., 1988), the junctions of dopaminergic fibers with GABAergic somata have been identified as lacking the membrane specializations typically associated with conventional synapses (Verney et al., 1991). It is noteworthy that D₁ dopamine receptor binding activity in one study was localized to nonpyramidal cells (Vincent et al., 1993), while in another, it was associated with pyramidal neurons (Bergson et al., 1995) of the prefrontal cortex. This apparent discrepancy may be explained by methodologic and specie differences in these two studies, because messenger RNAs for both the D₁ and D₂ subtypes have been localized to both pyramidal and nonpyramidal neurons in prefrontal cortex (Huntley et al., 1992). Consistent with this idea, a large proportion of interneurons in rat mPFCx have been found to express D₁ and D₂ binding activities (Vincent et al., 1995) and studies in which in vitro microdialysis was employed have demonstrated that agonists for both the D₁ and D₂ subtypes are associated with a reduction in the release of ³H-GABA (Penit-Soria et al., 1987; Retaux et al., 1991a; Retaux et al., 1991b). Thus, as suggested by the results reported here, cortical dopamine projections can potentially exert a significant non-synaptic modulatory influence on GABAergic cells in rat mPFCx. With regard to the serotonin system, the 5HT₂ receptor protein has been immunocytochemically localized to presumptive GABAergic interneurons in layer II of the pyriform cortex (Morilak et al, 1993). This latter finding is consistent with the results of an intracellular recording study in which the effect of serotonin on GABA cells was blocked by 5HT₂ antagonists (Sheldon and Aghajanian, 1991).

It is relevant to this discussion that schizophrenia is thought to involve a significant decrease in the number and/or activity of GABAergic interneurons in the anterior cingulate subregion (Benes et al., 1991; Benes et al., 1992). If this hypothesis is correct, an otherwise normal convergence of 5HT and DA fibers on to interneurons could result in a relative increase in the modulatory influence of these two monoaminergic systems on an impaired population of GABAergic cells (Benes, 1995). Typical antipsychotic drugs are believed to reduce the symptoms of psychosis in schizophrenia by selectively blocking D₂ receptors in the mesolimbic and mesostriatal systems (Meltzer and Stahl, 1976; Seeman, 1981). The atypical neuroleptics, on the other hand, show very high affinity for the 5HT₂A receptor, but only moderate affinity for the D₁ and D₂ subtypes (Meltzer et al., 1989). Thus, the greater efficacy of atypical drugs, such as clozapine, in the treatment of schizophrenia could involve the simultaneous blockade of dopaminergic and serotonergic effects on the somata of a compromised pool of GABAergic interneurons.

Taken together, the findings reported here have provided anatomical support for the idea that there is a convergence of catecholaminergic and serotonergic fibers onto GABAergic neurons in the rodent mPFCx. Future studies will be directed toward understanding how a neural circuit of this type may be involved not only in the abnormal corticolimbic processing observed in schizophrenia, but also in the mechanism of action through which atypical antipsychotic drugs exert their unique therapeutic effects.

 

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ACKNOWLEDGMENTS

This work has been supported by NIH grants MH42261, MH00423, MH31154 and an award from the Stanley Foundation.

 

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