?-aminobutyric acid (GABA) is the most extensively studied neurotransmitter of all amino acid transmitters in the central nervous system (CNS). It is probably the principal inhibitory neurotransmitter in the CNS and has a widespread distribution in the adult brain. Moreover, GABA is also present in peripheral tissues where it may act as a transmitter. In the past few years, a number of studies have focused on the localization of GABA receptors in different parts of the CNS and peripheral nervous system (PNS), including sensory ganglia. GABA has been shown to be involved in the generation of dorsal root reflexes via GABA(A) receptors. The Cl^?- and Ca²⁺-dependent negative cross-talk between cationic P2X and anionic GABA(A) receptors of dorsal root ganglia (DRG) neurons inhibits afferent excitation to the spinal cord as GABA and ATP are coreleased within the dorsal horn have demonstrated the presence of GABA(B) receptor subunit GABA(B1) and GABA(B2) mRNA and their corresponding subunit proteins in DRG neurons. Moreover, the findings of McCarson and Enna (1999) indicate activity-dependent differential regulation of GABA(B1) and (B2) receptor gene expression in spinal sensory systems in response to chemogenic nociceptive activation.

Whereas the role of GABA receptors in sensory transmission is relatively well-understood, there is not as much data on the presence of GABA-containing primary afferents. GABA-immunostaining has been demonstrated in neurons in the mesencephalic trigeminal nucleus in rat and cat. The mesencephalic trigeminal nucleus is considered to be a morphological equivalent of primary sensory neurons in cranio-spinal ganglia. Studies on the presence of GABA in sensory ganglia in other species are restricted to snail, chicken, and rat. Some of these studies are quantitative, focusing on the number and size of GABAergic neurons in the investigated structures. Only scattered data are available concerning the role of GABA in the PNS.

The present study reports on the presence of GABA in feline trigeminal ganglia (TrG), nodose ganglia (NG), and DRG, and the possible involvement of GABA in sensory transmission is discussed. For that reason, quantitative immunohistochemistry was applied. Part of this data were published in abstract form.

Five adult cats of both sexes (body weight 2.5–3.5 kg) were deeply anesthetized with Ketanest (50 mg/kg i.p.; Pfizer, New York, NY, USA) and perfused through the ascending aorta with 100 ml 1% heparin in cold 0.9% NaCl, followed by 2 l fixative containing 4% paraformaldehyde and 0.3% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, at room temperature for 20 min. After perfusion, TrG, NG and DRG L_1–2 were removed, postfixed in 4% paraformaldehyde at 4°C for 5–6 h, and then immersed overnight at 4°C in 0.01 M Tris-buffer, pH 7.4, containing 20% sucrose for cryoprotection. The ganglia were embedded in TissueTek OCT compound (Miles, Elkhart, NI, USA), frozen and 20 ?m-thick sections were cut in a cryostat at ?20°C. The sections were separated into five series. After rinsing in 0.1 M phosphate buffer saline (PBS), pH 7.4, each complete serie of one-in-five longitudinal sections over the entire thickness of the ganglion were investigated. Immunohistochemical staining was performed using the avidin–biotin method on free-floating sections. The primary antibody, rabbit anti-GABA (Sigma, St. Louis, MO, USA), diluted 1:1000 in a solution that blocks nonspecific antibody binding, and promotes penetration of the antibody into the tissue, containing 1 ml 0.05% of the preservative thimerosal (Fluka, Buchs, Switzerland), 1 ml 0.1% bovine serum albumin, 1 ml 10% normal goat serum, 1 ml 0.01% sodium azide and 6 ml 0.1 M PBS, pH 7.4, for 24 h at room temperature. After rinsing in PBS, sections were incubated with the secondary antibody, biotinylated goat anti-rabbit IgG (Vector, Burlingame, CA, USA) diluted 1:160 in PBS containing 1% normal goat serum and 0.1% Triton X-100, for 6 h at room temperature. After washing the sections, the ABC complex (6.25 ?l/ml of each compound in PBS; Vector) was applied. Following rinsing, peroxidase activity was visualized using 0.05% diaminobenzidine (Sigma) and 0.003% H₂O₂ in 0.05 Tris-HCl buffer, pH 7.4 for 20 min at room temperature. Finally, sections were dehydrated in a graded series of alcohol and xylene, and embedded in Entellan (Merck, Darmstadt, Germany). Neurons were considered to be GABA positive when staining was clearly stronger than background staining. Negative controls included sections that were incubating in the absence of the primary antibody or in the presence of nonimmune normal serum in the same dilution as the primary antibodies, as well as antigen–antibody preabsorption experiments with the native antigen (10 ?g/ml) at 4°C for 48 h. In all cases, negative controls resulted in complete absence of staining.

Quantitative assessment of GABA expression was performed by analysis of all parts of sensory ganglia according to Coggeshall and Lekan (1996). Three–five sections from each series were selected, and images were generated using a X16 objective. Quantitative analysis of immunostaining was performed with a VIDAS 25 image analyser (Kontron, Eching, Germany). Numbers obtained in all series of sections of each animal were averaged, and the percentage of GABA-positive neurons was calculated. The Mann–Whitney U test and the Student’s t-test were applied for comparison of mean values. Differences between mean values were considered to be statistically significant when p<0.05.

Immunostaining of GABA was observed in all ganglia examined in this study. Significant numbers of neurons exhibited GABA immunostaining with varying intensity. The labeled neurons were scattered throughout the ganglia and arranged in a sporadic fashion. GABA-positive neurons were large primary sensory neurons (>50 ?m in diameter) as well as medium (30–50 ?m) to small ones (<30 ?m). All neurons were round to oval in appearance. The intensity of immunostaining varied from weak to strong in the different neuronal subpopulations. The most intensely-stained cells were small-to-medium sized whereas large ganglion neurons were not markedly stained. Immunostained varicose and nonvaricose neuronal fibres were found surrounding both unstained and immunostained ganglion cells of all sizes in a basket-like manner. In addition, occasionally-stained fibres were scattered between columns of ganglion cells. In the neuropil, GABA-containing varicose neuronal fibres, some branching, were detected. GABA-positive structures were not observed in control sections.

Large populations of different types of neurons are present in the CNS that use GABA as neurotransmitter. In the spinal cord of rat and cat, it appears that GABA mediates pre- and postsynaptic inhibition. In monkeys, GABA was found to inhibit both the pre- and postsynaptic conductance of nociceptive signals from primary afferent fibres to dorsal horn sensory cells, including spinothalamic tract neurons. Data obtained in cat as presented here extend previous observations implicating that GABA-mediated inhibitory processes are involved in the control of nociceptive modulatory circuitries in the spinal cord. In addition, recent studies have suggested a role for GABA transporters in nociception and demonstrated a selective analgesic activity of some GABA transport inhibitors.

Althoug GABA is the principal inhibitory neurotransmitter in the brain and spinal cord, its presence and function in primary sensory neurons are still obscure. As is generally accepted, the major amino acid transmitter of the pseudo-unipolar sensory ganglion neurons is glutamate, which is an excitatory neuromediator. The question arises then whether primary sensory neurons can utilize the inhibitory amino acid GABA. However, so far GABAergic primary sensory neurons have only been reported in mollusks, and rodents. There are no data in the literature on GABA immunostaining in mammals other than rodents.