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.
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).
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.
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.
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.
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.
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.
REFERENCES
Beaudet, A., and Descarries, L. (1978) The monoamine innervation of rat cerebral cortex:
Synaptic and nonsynaptic axon terminals. Neurosci. 3:851-860.
Benes, F.M., McSparren, J., Bird, E.D. (1991) Vincent SL, SanGiovanni JP. Deficits in small
interneurons in prefrontal and anterior cingulate cortex of schizophrenic and schizoaffective
patients. Arch. Gen. Psychiat. 48:996-1001.
Benes, F.M., Vincent, S.L., Alsterberg, G., Bird, E.D., SanGiovanni, J.P. (1992) Increased
GABA-A receptor binding in superficial layers of cingulate cortex in schizophrenics. J. Neurosci.
12:924-929.
Benes, F.M. (1995) Is there a neuroanatomic basis for schizophrenia? The Neuroscientist. 1:104-
115.
Benes, F.M., Vincent, S.L., Molloy, R. (1993) Dopamine-immunoreactive axon varicosities form
nonrandom contacts with GABA-immunoreactive neurons in rat medial prefrontal cortex. Synapse.
15:285-295.
Bergson, C., Ladislav, M., 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.
Emson, P.C., Koob, G.F. (1978) The origin and distribution of dopamine-containing afferents to rat
frontal cortex. Brain Res. 142:249-267.
Gellman, R.L., Aghajanian, G.K. (1993) Pyramidal cells in piriform cortex receive a convergence of
inputs from monoamine activated GABAergic interneurons. Brain Res. 600:63-73.
Houser, C.R., Vaughn, J.E., Hendry, S.H.C., Jones, E.G., Peters, A. (1984) GABA neurons in
the cerebral cortex. In: Jones, E.G., Peters, A., Eds. Cerebral Cortex. New York: Plenum Press.
2:63-89.
Huntley, G.K., Morrison, J.H., Prikhozhan, A., & Sealfon, S.C. (1992) Localization of multiple
dopamine receptor subtype mRNAs in human and monkey motor cortex and striatum. Molecular
Brain Research. 15:181-188.
Krnjevic, K. (1987) GABAergic inhibition in the neocortex. Journal of Mind and Behavior. 8(4):
537-548.
Lidov, H.G.W., Grzanna, R., Molliver, M.E. (1980) The serotonin innervation of the cerebral
cortex in the rat - an immunocytochemical analysis. Neurosci. 5:207-227.
Lindvall, O., Bjorklund, A. (1984) General organization of cortical monoamine systems. In:
Descarries L.; Reader, T. R.; Jasper, H. H., Eds. Monoamine Innervation of Cerebral Cortex.
New York: Alan R. Liss. 9-40.
Meltzer, H.Y., Matsubara, S., Lee, J.C. (1989) Classification of typical and atypical antipsychotic
drugs on the basis of dopamine D-1, D-2 and serotonin-2 pKi values. J. Pharmacol. Exp. Revs.
43:587-604.
Meltzer, H.Y., Stahl, S.M. (1976) The dopamine hypothesis of schizophrenia: A review. Schiz.
Bull. 2:9-76.
Morilak, D.A., Garlow, S.J., Ciaranello, R.D. (1993) Immunocytochemical localization and
description of neurons expressing 5-HT2 receptors in the rat brain. Neurosci. 54:701-717.
Neafsey, E.J. (1993) Anterior cingulate cortex in rodents: connections, visceral control functions,
and implications for emotion. In: Vogt, B.A., Gabriel, M. Eds. Neurobiology of Cingulate Cortex
and Limbic Thalamus: A Comprehensive Handbook. Boston, Birkhauser, 6:206-222.
Penit-Soria, J., Audinat, E., Crepel, F. (1987) Excitation of rat prefrontal cortical neurons by
dopamine: an in vitro electrophysiological study. Brain Res. 425:363-374.
Reader, T.A. (1981) Distribution of catecholamines and serotonin in the rat cerebral cortex:
Absolute levels and relative proportions. J. Neural Trans. 50:13-27.
Retaux, S., Besson, M.J., Penit-Soria, J. (1991a) Opposing effects of dopamine
D2 receptor
stimulation on the spontaneous and the electrically evoked release of [3H]GABA on rat prefrontal
cortex slices. Neurosci. 42:61-71.
Retaux, S., Besson, M.J., Penit-Soria, J. (1991b) Synergism between
D1 and D2 dopamine
receptors in the inhibition of the evoked release of [3H]GABA in the rat prefrontal cortex. Neurosci.
43:323-329.
Seeman, P. (1981) Brain dopamine receptors. Pharmacol. Rev. 32:229-313.
Seguela, P., Watkins, K.C. Descarries, L. (1988) Ultrastructural features of dopamine axon
terminals in the anteromedial and suprarhinal cortex of rat. J. Comp. Neurol. 289:11-22.
Sheldon, P. W., Aghajanian, G.K. (1991) Excitatory responses to serotonin (5-HT) in neurons of
the rat piriform cortex: evidence for mediation by 5-HT1C receptors in pyramidal
cells and 5-HT2
receptors in interneurons. Synapse. 9:208-218.
Verney, C., Alvarez, C., Gerrard, M., Berger, B. (1991) Ultrastructural double-labeling study of
dopamine terminals and GABA-containing neurons in rat anteromedial cortex. Eur. J. Neurosci.
2:960-972.
Vincent, S.L., Khan, Y., Benes, F.M. (1993) Cellular distribution of dopamine
D1 and D2 receptors
in rat medial prefrontal cortex. J.Neurosci. 13:2551-2564.
Vincent, S.L., Khan, Y., Benes, F.M. (1995) Cellular colocalization of dopamine
D1 and D2
receptors in rat medial prefrontal cortex. Synapse. 19:112-120.
