GABA transporter-1 (GAT-1) immunoreactivity in the cat periaqueductal gray matter

Neuroscience Letters 250 (1998) 123–126

GABA transporter-1 (GAT-1) immunoreactivity in the cat periaqueductal gray matter Paolo Barbaresi a ,*, Giancarlo Gazzanelli b, Manuela Malatesta b a

Institute of Human Physiology, University of Ancona, Ancona, Italy b Institute of Histology, University of Urbino, Urbino, Italy

Received 17 April 1998; received in revised form 29 May 1998; accepted 29 May 1998

Abstract Using light- and electron-microscopic immunocytochemistry, we investigated the regional distribution and the ultrastructural localization of GAT-1, a prominent GABA transporter, in the cat PAG. Light microscopic observations indicate that GAT-1immunoreactive elements are particularly dense in PAG-DL and form a pair of longitudinal columns extending in the intermediate region of this structure. At electron-microscopic level, GAT-1 immunoreactivity was present in axon terminals forming symmetric synapses and in the distal processes of astroglial cells. These data further confirm the existence of longitudinal columns within PAG. They also indicate that GAT-1 could influence the action of GABA on its receptors, probably regulating the magnitude and duration of GABA’s synaptic action on PAG neurons, and suggest that astrocytes may play an important role in this process.  1998 Elsevier Science Ireland Ltd. All rights reserved

Keywords: GABA transporters; PAG columns; Immunocytochemistry; Electron microscopy; Axon terminals; Astrocytic processes

Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter of the vertebrate central nervous system (CNS), plays a prominent role in regulating various types of function in the periaqueductal gray matter (PAG) [16]. GABAergic neurons and terminals have been well characterized in PAG using immunocytochemistry to visualize GABA [16] and its synthetic enzyme, glutamic acid decarboxylase (GAD) [3]. GABA, released from presynaptic vesicles by exocytosis, crosses the synaptic cleft, binds to receptors [7] located postsynaptically and is then removed from the cleft. The removal of the transmitter can be achieved either by enzymatic degradation or by reuptake into presynaptic neurons. The GABA uptake system is made up of a class of membrane proteins that translocate GABA in a Na + and Cl − dependent manner from the synaptic cleft into axon terminals and surrounding glial cells [6]. * Corresponding author. Istituto di Fisiologia Umana, Universita` di Ancona, Via Tronto, 10/A, Torrette di Ancona-I-60020 Ancona, Italy. Tel.: +39 71 2206054; fax: +39 71 2206052; e-mail: [email protected]

Cloning studies have identified four GABA transporters, termed GAT-1, GAT-2, GAT-3 and betain glycine transporter (BGT-1), with different pharmacological properties and tissue distribution indicating differences in their functional properties [6,8]. Previous studies with specific polyclonal antibodies to GABA transporters have shown that GAT-1 is present predominantly in neurons [11,14,17], GAT-2 in the ependymal cells [11,17], and GAT-3 in astrocytes [11,15,17]. The present study of the GABA uptake system in PAG investigates the regional distribution and the cellular localization of GAT-1 in three adult cats using a specific affinity-purified antibody. Animals were anesthetized with Nembutal (33 mg/kg, i.p.) and perfused transcardially with saline followed by a mixture containing 4% paraformaldehyde in 0.1 phosphate buffer (PB; pH 7.4). The brainstems were removed and postfixed for 8 h in the same fixative. Blocks containing the PAG were cut in coronal sections (30 mm thick) on a Vibratome and collected in serial groups of two. The first series was transferred to phosphate-buffered saline (PBS)

0304-3940/98/$19.00  1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00449- 2

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containing 10% normal goat serum (NGS) and 0.3% Triton X-100 and then incubated overnight in primary antibody directed against the predicted C terminus [10] of rat GAT1 (GAT-1588–599; Chemicon International, 1:700 to 1:1000) in PBS containing 10% NGS. Sections were then rinsed in PBS and transferred in biotinylated goat-anti-rabbit IGg (1:100; 1 h; Vector Labs.), washed again in PBS and reacted with Elite ABC Kit (1:100; Vector, Burlingame, CA). Sections were further rinsed in PBS (15 min) and placed in 0.05 M Tris buffer containing 0.1% of diaminobenzidine tetrahydrochloride (DAB) and 0.01% H2O2. After 10 min of incubation and a final rinse in Tris buffer, slices were mounted on subbed slides, air-dried, dehydrated in ethanol, immersed in xylene and coverslipped. The specificity of GAT-1 antiserum was tested in two adjacent sections in every 20. One section was absorbed as previously described [14] and the other was reacted by omitting the primary antiserum. No immunoreaction was observed in either. The second series of sections, used for electron microscopy, underwent a mild ethanol pretreatment before the immunocytochemical session. The primary antibody was diluted 1:700 and Triton X-100 was not added. Subsequently, sections were postfixed for 1 h in cold (4°C) 1% OsO4, dehydrated in a graded series of ethanol, infiltrated in Epon and flat-embedded between two sigmacote-coated strips of aclar. Plastic-embedded sections were examined with a stereomicroscope. Small PAG regions were trimmed out and mounted on preformed resin blocks. Six tissue blocks for each animal were taken from different anteroposterior levels and from different PAG subdivisions and sectioned on an ultramicrotome. Each series comprised four to six serial ultrathin sections separated by 1–1.5 mm. Ultrathin sections were mounted in a single-coated copper grid (about 17 grids for each block) and stained with uranyl acetate and lead citrate. One or two sections every grid were examined with a Philips CM-10 electron microscope (about 300 ultrathin sections observed). Specific GAT-1 immunoreactivity (GAT-1IR) was particularly dense on the lateral edge of the dorsolateral PAG column (PAG-DL; Fig. 1), along its intermediate third.

Fig. 1. Low-power photomicrographs showing the distribution of GAT-1IR at two different rostrocaudal levels of intermediate PAG (A,B, rostral and caudal, respectively). PAG-DM, dorsomedial PAG; PAG-DL, dorsolateral PAG; PAG-L, lateral PAG; PAG-VL, ventrolateral PAG; AQ, aqueduct; Su3C, supraoculomotor cap; 3, oculomotor nucleus. Scale bar, 1 mm.

Fig. 2. High-power photomicrographs from 2-mm-thick sections from the dorsal (A) and the dorsolateral PAG (B) showing GAT-1-immunopositive puncta (arrowheads) close to somata and proximal dendrites of PAG neurons of varying morphology. Scale bar, 10 mm.

Labeling gradually diminished at the caudal- and rostralmost midbrain levels. Outside the dorsolateral column, GAT-1IR was uniformly distributed throughout the other PAG subdivisions (Fig. 1) at all rostrocaudal levels. Dense labeling was found also in the supraoculomotor cap, interstitial nucleus of Cajal, nucleus of Darkschewitsch and the Edinger–Westphal nucleus. GAT-1IR was in punctate structures surrounding the somata and dendrites of PAG neurons (Fig. 2). In each PAG subdivision, electron-microscopic analysis revealed that GAT-1IR was mostly associated with axonal and glial profiles, whereas neuronal perikarya and dendrites were completely free of labeling (Fig. 3). In axon terminals, the reaction product was heterogeneously distributed with dense accumulations associated with the plasma membrane. The overwhelming majority of labeled axon terminals formed symmetric synapses with unlabeled cell bodies (Fig. 3B) and dendrites (Fig. 3D–F). In rare cases, asymmetric synapses were observed between immunoreactive boutons and unlabeled dendrites (Fig. 3C). The immunolabeling filled the entire profile of distal astrocytic processes (Fig. 3A,C–F), identified by their irregular contours, which tended to wrap surrounding neuronal elements. Astrocytic cell bodies, oligodendrocytes and microglial cells were unlabeled. PAG is not a homogeneous structure and recent behavioral, functional and anatomical studies have suggested that it is organized into four longitudinal neuronal columns (dorsolateral, dorsomedial, lateral and ventrolateral PAG) lying for varying distances along its rostrocaudal extension [2,4]. This pattern of GAT-1 distribution further supports previous findings of the existence of longitudinal columns within PAG [2,4]: GAT-1IR was present in punctate structures which were particularly dense on the lateral edge of the dorsolateral column, extending for several millimeters in intermediate PAG. This distribution appears consistent with that reported in the rat by Yasumi and co-workers [21] who, using in-situ hybridization histochemistry for GAT-1 mRNA, found a strong hybridization signal over dorsolateral PAG. The distribution pattern of GAT-1IR is also in

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Fig. 3. Electron micrographs demonstrating the subcellular localization of GAT-1IR in the cat PAG. (A) Labeled astrocytic processes (arrowheads) surrounding unlabeled neurons and dendrites. (B) Labeled axon terminal (arrows) synapsing on unlabeled cell body. (C) Two axon terminals, one of which shows GAT-1IR (asterisk), making asymmetric synapses on an unlabeled dendrite. Arrowheads indicate a labeled thin astrocytic process surrounding both an unlabeled dendrite and a labeled axon terminal. (D–F) Labeled axon terminals synapsing on unlabeled dendrites. Axt, axon terminal; Den, dendrite; Nuc, nucleus; ncl, nucleolus. Scale bar (A,B), 5 mm; (C–F) 0.5 mm.

agreement with that of GABAergic neurons, particularly numerous in dorsolateral PAG [16]. This electron microscopic study shows that GAT-1IR is expressed in both axon terminals and distal astroglial processes. Labeled axon terminals contained oval, flattened synaptic vesicles and formed symmetric synapses. Consistently with previous studies [14,17] terminals with these features are usually associated with the presence of GABA, suggesting that GABA transporters play an important role in terminating GABA’s synaptic action, reaccumulating released transmitter into presynaptic GABAergic axon terminals [6] (or glial elements; see below). This action is particularly intense in dorsolateral PAG, were we

found the highest density of GAT-1-positive elements. In addition, this region contains a high concentration of GABAA receptors [7]. Overall, these data suggest to us that the GABA uptake system could play an important role in controlling the duration of GABA’s action on those PAG neurons that have GABAA receptors [16]. Although previous papers indicate that the presynaptic inhibition associated with GABAB receptors is extremely rare in PAG [16], we cannot rule out the possibility that the GABA uptake systems regulate the paracrine action of GABA on GABAB receptors [12] located on excitatory axon terminals [9,12,20]. The presence in dorsolateral PAG of a high number of GABAB receptors [7] and of a dense excitatory input

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from several areas of the CNS [5] supports this hypothesis. In rare cases, GAT-1IR was observed in the axon terminals forming asymmetric synapses. This finding, though in contrast with the function of GABA transporters, is in agreement with a previous study [14]. In the cerebral cortex, GAT-1IR was observed in a small number of pyramidal neurons [14] which make only asymmetric synapses. Moreover, a previous report described the presence of glutamate transporters in non-glutamatergic neurons, including GABAergic cerebellar Purkinje cells [19]. These data suggest that in the CNS there are neurons expressing transporters for neurotransmitters different from those released by their own axon terminals. Although GAT-1 rapidly clears GABA from the extracellular space, the localization of GAT-1IR is consistent with the possibility of the release of GABA from axon terminals into the extracellular space according to a Ca2 + -independent, non-vesicular mechanism [1]. Moreover, GAT-1IR was expressed by distal processes of glial cells that were often adjacent to GAT-1-immunoreactive axon terminals. This arrangement suggests that the GABA released from inhibitory terminals could be simultaneously taken up by both its own terminals and surrounding glial processes. GAT-1IR was also observed in glial processes placed distant from axon terminals, thus suggesting that glial uptake could have an important role of limiting the diffusion of GABA into adjacent synapses present on GABA responsive neurons [8]. GAT-1 expression in distal astrocytic processes has been reported in many other regions of the CNS. For instance, immunoreactivity is expressed in glial cells of the cerebral cortex [14], hippocampus [17] and cerebellum [18]. All these findings are in line with previous autoradiographic and biochemical studies showing that the GABA uptake system is expressed by glial cells too [13]. This work was supported by funds from MURST (40% and 60%). We thank Dr. S. Cinti (Institute of Human Anatomy, University of Ancona, Italy) for use of the electron microscope; Dr. S. Modena for reviewing the English, Mr. M. Gradara for excellent photographic assistance, and Dr. F. Conti for the supply of peptide for immunoabsorption. [1] Attwell, D., Barbour, B. and Szatkowski, M., Non vesicular release of neurotransmitter, Neuron, 11 (1993) 401–407. [2] Bandler, R., Carrive, P. and Depaulis, A., Emerging principles of organization of the midbrain periaqueductal gray matter. In A. Depaulis and R. Bandler (Eds.), The Midbrain Periaqueductal Gray Matter, Plenum Press, New York, 1991, pp. 1–8. [3] Barbaresi, P. and Manfrini, E., Glutamate decarboxylase-immunoreactive neurons and terminals in the periaqueductal gray of the rat, Neuroscience, 27 (1988) 183–191. [4] Beitz, A.J., Periaqueductal gray. In G. Paxinos (Ed.), The Rat Nervous System, second edn., Academic Press, New York, 1995, pp. 173–182.

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