© Neuroscience-Net Volume 1, Article #10001

Received April 9, 1996 Accepted for Publication April 17, 1996 Published April 24, 1996

Jill Bolte Taylor ^{1, 3} and Francine M. Benes ^{1, 2, 3}

1. Department of Psychiatry
   

2. Program in Neuroscience, Harvard Medical School, Boston, Massachusetts 02115
   

3. Mailman Research Center, Laboratory for Structural Neuroscience, McLean Hospital, Belmont, Massachusetts 02178

Send correspondence to:

Dr. Francine M. Benes
Laboratory for Structural Neuroscience
Mailman Research Center
McLean Hospital
115 Mill Street
Belmont, MA 02178
(617) 855-2401
FAX: (617) 855-3199
E-mail: benesf@mclean.org

KEY WORDS: TH, 5HT, GAD, cortex

ABSTRACT
(Neuroscience-Net, Volume 1, Article #10001; April 24, 1996)

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.