Alteration of GABA transporter expression in the rat cerebral cortex following needle puncture and colchicine injection

(cid:14) . In the adult cerebral cortex, GABA transporters GATs are expressed by both neurons and astrocytes. GAT-1 immunoreactivity is found in axon terminals of GABAergic neurons and astrocytes, while GAT-3 immunolabeling occurs only in the latter. The present study was designed to determine whether the expression of GAT-1 and GAT-3 in the adult rat cerebrum changes after needle lesion and colchicine infusion. Following a needle puncture or a saline injection, immunolabeling for GAT-1 and GAT-3 was slightly increased in an area around the needle track. Not only was the neuropil labeling for both GATs increased, but also a few neuronal somata were found to be immunoreactive for GAT-1. Colchicine injections induced a striking increase in immunolabeling for both GATs in the neuropil in an area adjacent to the needle path and surrounding it. A homologous region of the contralateral hemisphere also showed a moderate increase of immunoreactivity in the neuropil for both GATs. Furthermore, this contralateral site showed many neuronal somata immunolabeled for (cid:14) . GAT-1. These changes were mainly detected during the first 5 days following intracortical lesions. These results indicate that 1 the upregulation of GAT-1 and GAT-3 in cortical interneurons and astrocytes is caused by both mechanical and chemical factors associated (cid:14) . with the injections; 2 increased GAT-1 and GAT-3 expression contralateral to the site of colchicine injection is mediated by (cid:14) . transcellular signaling across the corpus callosum; and 3 the lesion-induced GAT expression may play a protective role by helping to balance excitatory and inhibitory neuronal activities. q 1999 Elsevier Science B.V. All rights reserved.


Ž
. GABA transporters GATs are recently identified translemmal proteins in the brain and peripheral tissue w x 4,9,17,32 .They are found in neurons and glial cells in the nervous system, and functionally have been shown to clear GABA from the synaptic cleft and extracellular space w x 23,25,26,51 , though they may also be involved in the secretion of GABA from neurons in a Ca 2q -independent, w x nonvesicular manner in certain circumstances 2,3,31 .Thus, it is suggested that GATs play an important role in determining the magnitude and duration of GABA's neurotransmission as well as other activities.To date, four GATs have been cloned and they are classified as GAT-1, GAT-2, GAT-3 and GAT-4 according to their differential amino acid sequences and pharmacological properties w x 4,9,17,32,35 .For example, GAT-1 displays the pharmacological features typical of a 'neuronal transporter' in that its GABA uptake is strongly inhibited by cis-3-Ž .aminocyclohexane carboxylic acid ACHC but not by b-alanine.On the other hand, GAT-2 and GAT-3 are considered to be 'glial transporters' because GABA uptake via these two GATs is inhibited by b-alanine but not ACHC.GABA uptake by GAT-4 is inhibited by neither w x agent 4,9,25,32 .
The cellular and subcellular localization of GATs in the brain and several sensory organs has been recently studied, and it appears that GAT-1 and GAT-3 are the major GATs w in the nervous system 5,9,11,21-24,36-39,42,43,45, x 46,51,53,54 .In the adult mammalian cerebral cortex, GAT-1 mRNA is expressed in the cell bodies of GABAergic interneurons and astrocytes as determined by in situ w x hybridization 36,42,43 , whereas its protein as shown in immunocytochemical preparations is mainly found in axon w x terminals of these neurons and astrocytic processes 36,45 , though sparse immunoreaction products are detectable on the Golgi complex of some interneurons and in the somata w x of astrocytes at the electron microscopic level 45 .Unlike GAT-1, GAT-3 is only found in glial elements, i.e., the 0006-8993r99r$ -see front matter q 1999 Elsevier Science B.V. All rights reserved.
While the distribution of GATs in the normal brain has been extensively studied, little is known about how the w x expression of GATs is regulated 13,15,20,41 .We have recently shown an increase in immunoreactivity for GAT-1 and GAT-3 in the superior colliculus following deafferentation from a distant lesion of the optic nerve, suggestive of an upregulation of these GATs following terminal w x degeneration 54 .In the present study, we analyzed whether the immunoreactivity for GAT-1 and GAT-3 in the cerebral cortex changes following colchicine infusion, based upon several considerations.First, since colchicine w x is commonly used to block axonal transport 47 , we were interested in determining whether it enhances GAT-1 immunolabeling in the somata of mature cortical interneurons.Second, colchicine is somewhat neurotoxic and has been used to create chemical lesions in certain brain w x regions 19,34 .Thus, we wanted to determine whether colchicine neurotoxicity alters the immunoreactivity of GAT-1 and GAT-3 in cortical neurons and glial cells.Sterile saline was used as the vehicle for colchicine and the first control for this experiment examined the possible effect of this vehicle.In addition, as needle penetration can also cause tissue damage, a second control was used to verify if and how the mechanical lesion associated with the needle puncture contributed to cortical expression of GATs.Unilateral injections were made to the cerebral cortex, using the contralateral hemisphere as an intrinsic control for the injected cortex.A preliminary report of this study w x was presented elsewhere 48 .

Animals and surgery
Experiments were performed on male and female adult Ž .albino rats Sprague-Dawley weighing 250-350 g.The Ž animals were anesthetized with sodium pentobarbital 50 .mgrkg, i.p. and placed on a stereotaxic frame.A hole Ž .
Ž .was drilled on the left side of the skull at 4.5 mm lateral and 4.5-5.5 mm posterior to the bregma.A microliter Ž .syringe a705, Hamilton, NV that was empty, or loaded Ž with 7 ml sterile saline, or with 7 mg colchicine Sigma, .St. Louis diluted in 7 ml saline, was positioned above the predrilled hole and then advanced into the cerebrum 4.0 w x mm 40 .For colchicine injections, the solution was infused at a constant rate for 3 min and the needle remained in position for another 5 min before it was withdrawn.The skin incision was sutured and cleaned.After recovery, the operated animals were returned to the vivarium, and were Ž .Ž .Ž .allowed to survive for 1 n s 3 , 2 n s 3 , 5 n s 3 , 10 Ž .Ž .n s 2 , or 15 n s 2 days before perfusion.

Tissue preparation
Operated animals were deeply re-anesthetized at the above-indicated time points with sodium pentobarbital and perfused transcardially with 0.02 M phosphate buffered Ž .saline PBS, pH 7.4 followed by 4% paraformaldehyde Ž .and 0.2% glutaraldehyde in phosphate buffer pH 7.4 .The brains were then removed and postfixed in the perfusion solution for 4-8 h at 48C, and transferred to cold PBS for several hours to a few days.Brain blocks were prepared to contain the posterior cerebrum and diencephalon.For orientation purposes, a portion of the ventrolateral cortex contralateral to the lesion side was removed.The brain blocks were then sectioned coronally at 40 mm with a vibratome, and the sections that included the injection site were collected in tissue-culture wells in cold PBS.They were further processed for immunocytochemistry and Nissl staining with cresyl violet.

Immunocytochemistry
Free-floating sections were stained for GAT-1 and GAT-3, respectively, using the standard avidin-biotin Ž .complex ABC method.At first, endogenous peroxidase activity was bleached with a 20-min rinse in 0.05% H O 2 2 in PBS.Non-specific background staining was lowered Ž .with a 2-h incubation in 5% normal horse serum NHS at room temperature.The sections were further incubated in a PBS solution containing 5% NHS, 0.3% Triton X-100 and Ž .rabbit anti-GAT-1 or GAT-3 1:5000 at 48C for 24 h and with agitation.After several rinses with PBS, the tissue was reacted in 1% anti-mouserrabbit IgG with 5% NHS for 1 h at room temperature, followed by 1% ABC solution for another hour.The bound antibodies were visualized with 0.05% DAB and 0.005% H O .Three 10-min rinses 2 2 with PBS were used between all incubations.The primary antibodies were kindly provided by Dr. w x N.C.Brecha 36,37 and their specificity was verified in previous studies.Other immunoreactive reagents were purchased from Vector Laboratories.In the present experiments, processing the sections with the primary antibodies omitted, or replaced by normal horse serum, yielded no specific immunolabeling.

Injection site and oÕerÕiew of immunolabeling
Pilot experiments were performed to determine the appropriate volume of vehicle and the amount of colchicine to be used.Briefly, 5-10 ml of vehicle were found to be enough to carry the needed amount of colchicine.The response of the cortex that was injected with this amount of saline was almost identical to that in the cortex punctured with an empty needle, which in both cases appeared as a regional and slight increase in immunostaining for GAT-1 and GAT-3.Injections of 3-7 mg colchicine induced a much greater increase in immunoreactivity in a larger area than that caused by the needle puncture alone or with saline injections.In all of these three conditions, cell loss was only observed within the needle track.Higher doses of colchicine often resulted in structural distortion and cell loss in a larger area within and outside the needle track.As a result, 7 ml vehicle and 7 mg colchicine were chosen to be used for most animals in our experiments.
The pattern of immunoreactivity in the cerebral cortex Ž was basically similar for the three short time points 1, 2 .and 5 days postoperation examined in the present study.We will first describe the pattern of immunolabeling observed in the rats with 2-day survival time.Observations from the rats survived for 10 and 15 days will be described at the end of this section.

ImmunoreactiÕity in the unaffected cortex: normal distribution pattern
The normal distribution pattern of immunoreactivity for GAT-1 and GAT-3 in the adult rat cerebral cortex including the hippocampal formation has been described in detail by ourselves and other investigators in previous studies w x 36,37,42,43,45,53 .In the present study, cortical regions with a normal pattern of immunolabeling were observed in most cortical areas from rats receiving a puncture lesion or saline injection.In both cases, the unaffected regions included the entire contralateral hemisphere and most parts Ž .
Ž .Fig. 3. Low magnification light microphotographs showing an altered pattern of immunoreactivity for GAT-1 A and GAT-3 B in both hemispheres following an unilateral intracortical colchicine injection, 2 days postoperation.The needle track in the ipsilateral hemisphere is indicated with arrows.The cortical regions with changes in immunoreactivity are bordered by arrowheads.Note the remarkable upregulation of GAT-1 and GAT-3 within and outside the needle track in the ipsilateral side, and a noticeable increase in a contralateral neocortical region that is symmetric to the affected area on the injected side.Scale bar s 1 mm. of the ipsilateral cortex except for a zone surrounding the needle path.In the rats injected with colchicine, the unaffected cortical regions were smaller than those in other animals, and were medial and ventral to the affected areas Ž .see below in both the ipsilateral and contralateral hemispheres.For GAT-1, a normal pattern of immunostaining is shown in Fig. 1A-C, which was obtained from the contralateral cortex of a rat with a saline injection at this same level.In the neocortex, GAT-1 immunoreactivity was present in the neuropil of all six layers, with little immunola-beling in the white matter.Unlabeled cell bodies were outlined by immunolabeled puncta in layers II-VI, and Ž .were most distinct in the supragranular layers Fig. 1B .Axonal plexuses surrounding the somata and initial segments, respectively, of neocortical pyramidal neurons were

ImmunoreactiÕity in the ipsilateral cortex of control rats
Both the needle puncture and saline-injected control rats showed similar changes in immunoreactivity for GAT-1 and GAT-3 in the injected hemisphere.A cortical area ranging from 100 mm to 500 mm in diameter surrounding the needle track showed a slight increase in immunostaining for both GATs.The degree of the upregulation Ž .was generally greater for GAT-1 Fig. 1D-F

Ž
. Fig. 2B .Both GAT-1 and GAT-3 showed increased immunostaining of the neuropil.However, for GAT-1 but not GAT-3, a few immunoreactive cell bodies were detected within the affected region of the neocortex and Ž .hippocampus Fig. 1D,E, Fig. 2B .In the neocortex, GAT-1 immunoreactive neuronal somata were found in all layers Ž .except for layer I and the white matter Fig. 1D .They Ž .were round, small to medium size 10-15 mm , and lacked Ž .identifiable dendritic or axonal processes Fig. 1E .In the hippocampus, GAT-1 immunoreactive cell bodies were often observed in stratum pyramidale of CA1, and some of Ž .them had immunolabeled dendrites Fig. 1F .

ImmunoreactiÕity in the ipsilateral cortex of colchicine-injected rats
The neocortical and hippocampal regions injected with colchicine displayed a striking increase in the immuno-Ž .reactivity for GAT-1 and GAT-3 Fig. 3 .The affected area was much larger and the amount of expression was much greater than that in the region around the injected cortex of the control rats.Thus, the affected region in colchicine-injected rats was as much as 1r4 to 1r3 of the Ž cortical area of the entire ipsilateral hemisphere Fig.
. 3A,B .The strongest immunolabeling was present at the needle track and its vicinity, and the intensity of immunoreactivity reduced gradually from this central zone toward the periphery.
At higher magnification, the cellular distribution of this intense immunolabeling was confined to the neuropil for Ž .Ž .both GAT-1 Fig. 4A-C and GAT-3 Fig. 5A-C .Thus, no neuronal somata were observed in the injection site and adjacent areas.The immunonegative cell bodies of pyramidal cells were distinctly outlined by strong neuropil immunolabeling for GAT-1 and less so for GAT-3.However, the size and laminar distribution pattern of these somal profiles were basically the same as those in the unaffected cortex, though the increase of neuropil immunoreactivity Ž resulted in an elevated contrast in the affected region Fig.

ImmunoreactiÕity in the contralateral cortex of colchicine-injected rats
In contrast to both the needle puncture and saline-injected controls, rats with colchicine injections had a homologous region of the contralateral hemisphere that showed an increased expression of GAT-1 and GAT-3.This contralateral region was spatially symmetric to the region with intense GAT expression in the ipsilateral Ž .cortex Fig. 3 .The general staining intensity in this homologous zone was higher than that in the other portions of this hemisphere, but was much lighter than the regions around the injection site.
At higher magnification, it was found that the GAT-1 immunoreactive changes in the homologous area of the contralateral side involved both neuronal somata and neu-Ž .ropil Fig. 4D-F .Thus, GAT-1 immunolabeled cell bodies were distributed across the cortex, but peaked in layers Ž .III and IV parietal cortex at the injection level .The immunoreaction products were concentrated in the cytoplasm and the nucleus of the cell body.The processes of these cells were mostly unlabeled.If present, they were very short and probably the proximal dendrites.In contrast to GAT-1, the increased immunolabeling for GAT-3 in the homologous zone of the contralateral hemisphere was Ž .solely associated with neuropil Fig. 5D-F .
The change in immunostaining in the hippocampal formation of the contralateral hemisphere was less than that of the neocortex.For GAT-1, a slight increase of neuropil immunostaining was detectable in the CA1 region deep to the cortical area that showed alterations in GAT expression Ž .Fig. 4E , but no immunoreactive cell bodies were observed.For GAT-3, no immunoreactive changes were de-Ž .tectable over the hippocampal formation Fig. 3B .

ImmunoreactiÕity at later surÕiÕal time points
At 10 days, the increased immunoreactivity in the needle puncture or saline-injected controls became very localized to the injection site at the white matter in the ipsilat-Ž .eral hemisphere Fig. 6A .For the colchicine-injected rats, the immunostaining in the neuropil of the contralateral hemisphere became equivalent to that found in the unaffected regions, and the GAT-1 immunoreactive cell bodies Ž .became undetectable Fig. 6C .However, in the injected side a small cortical area close to the needle track still Ž .expressed increased immunostaining for GAT-1 Fig. 6B,C Ž .and GAT-3 not shown in the neuropil as compared to other regions at this stage.By 15 days, it returned to Ž .baseline level not shown .

Discussion
The present study revealed that the immunoreactivity for two GABA transporters, GAT-1 and GAT-3, is upregulated in the adult rat cerebral cortex following colchicine infusion.The increased expression appears not only in the injected cortical region, but also in its homologous area in the contralateral hemisphere.Needle punctures and saline infusions also cause a certain amount of upregulation for both GATs but it is limited to the area surrounding the injection site, suggesting that the mechanical lesion associated with needle penetration is causing a local alteration of GAT expression.For GAT-1, the elevated expression is found to be associated with both glia and neurons, and is most prominent for colchicine-injected rats.The possible mechanisms and biological significance for the postlesion upregulation of glial and neuronal GATs observed in the present study in the adult cerebral cortex are discussed below.

GAT upregulation in astrocytes as a response to neural injury
The first important finding of the present study is a localized alteration of GAT-1 and GAT-3 immunoreactivity in neocortical and hippocampal regions associated with the mechanical lesion made by needle penetration.A careful histological examination showed that neuronal damage was limited to the needle track, because the cortical thickness as well as the size, morphology and distribution pattern of the neurons in the surrounding cortex remained Ž virtually normal in immunocytochemical and Nissl not .shown preparations.However, the cortical area that displayed an increase of GAT immunolabeling extended beyond the needle track and was not limited to only the site with cell loss caused by the injection needle.Furthermore, this change in immunostaining was reversible with time.Thus, the lesion-induced upregulation of GAT-1 and GAT-3 immunoreactivity observed in the present study may be triggered by other factors than neuronal death.For example, the mechanical lesion caused by needle penetration w x may damage neuronal processes 6,12,18,30,33 , resulting in degenerating axons and activated astrocytes in the area displaying increased GAT expression.In fact, early studies Ž had shown that following a needle 2.4 mm external .diameter lesion in the brain, the astrocytes within a zone up to 1200 mm from the wound edge were activated and w x underwent proliferation 6 .The mitotic activity of the focal astrocytes was increased between 1-6 days after the w x needle lesion 6 .These activated astrocytes became phagocytes that could remove cell debris, including degenw x erating axon terminals 1 .It is possible that these activated astrocytes are involved in the upregulation of GAT-1 and GAT-3 in the neuropil of the cortex around the needle track.
Pertinent to this hypothesis are data from our recent study that showed increased immunolabeling for GAT-1 and GAT-3 in the rat superior colliculus after optic nerve w x transection 54 .This change in GAT expression was limited to the retinal-recipient layers of the superior colliculus and was temporally associated with axon terminal degeneration following retinal ganglion cell axotomy.Electron microscopic data indicated that the increased immunolabeling was found in hypertrophied astrocytes that were frequently associated with degenerating ganglion w x cell axons 54 .
Why do colchicine injections induce a much greater upregulation of GAT-1 and GAT-3 expression in the neuropil than needle punctures or saline injections?First, it may be that colchicine is stressful to cortical neurons, and thus induces a more severe neuronal damage and a stronger glial response than needle puncture does.In fact, cell death occurs in certain vulnerable neuronal populations, such as dentate granule cells, following a similar dose of colchicine w x injection into the cortex 19,34 .Another possibility is that colchicine can promote cell division, especially of astro-w x cytes 6,10 .For example, an intracerebral colchicine infusion increases astrocytic proliferation by 2-fold more than w x that caused by needle lesion alone 6 .Thus, the upregulation of GAT expression in the neuropil of the colchicineinjected cortex is probably enhanced due to astrocytic proliferation.In fact, immature astrocytes express concentrated GAT-1 and GAT-3 in the developing rat cerebral w x w x cortex 53 and cerebellar cortex own unpublished data .

Differential regulation of GAT-1 expression in neuronal somata
A novel finding in the present study is that colchicine infusions caused an increased expression of GAT-1 and GAT-3 in a homologous region of the contralateral hemisphere.It is important to note that this region showed light microscopically detectable levels of GAT-1 immunoreactivity within the somata of many cortical neurons.Our results indicated that the laminar pattern, size and somal shape of these somata resembled the features of neocortiw x cal GABAergic interneurons 44,55 .This finding is consistent with the fact that GAT-1 mRNA occurs in w x GABAergic neurons in the mature cerebrum 36,42,43 .Neuropil immunolabeling is also enhanced in this selective contralateral cortical area.As GAT-3 is only contained in w x astrocytes 37,45 , it is obvious that glial cells in this region also contribute to the increased neuropil immunostaining for GATs, including GAT-1.
The upregulation of GAT-1 and GAT-3 in interneurons and glial cells in the contralateral hemisphere cannot be explained by a direct effect of colchicine applied to the other hemisphere because of the regional selectivity of the immunoreactive change.Colchicine's effect on blocking w x axonal transport 10,47 cannot explain a glial upregulation of GATs and an occurrence of GAT-1 only in the cell bodies of interneurons, because only a few GABAergic neurons in the adult rat have callosal projections and they w x are mainly localized to the supragranular layers 16,27 .In the present study, GAT-1 immunoreactive somata are numerous and distributed throughout the cortical depth.
The increased neuropil immunolabeling in the contralateral side could be a response of the astrocytes in the vicinity of the callosally projecting axons undergoing retrograde degeneration caused by the injections.On the other hand, the 'reactive' upregulation of GAT-1 in the neocortical interneurons contralateral to colchicine injections is likely related to principal neurons in the ipsilateral hemisphere or those in the contralateral side that project across the corpus callosum.These neocortical pyramidal cells are probably activated by these injections, and then send a signal to GABAergic interneurons, perhaps astrow x cytes as well 13 , to induce the alteration of GAT expression in these latter cells, which is mediated by activation of glutamate receptors andror the cAMP signal pathway w x 13,15,20,41 .The reason why there is no upregulation of GATs in GABAergic somata of the contralateral hip-( ) X.-X.Yan, C.E. Ribak r Brain Research 816 1999 317-328 327 pocampal formation could be because hippocampal pyramidal cells have a limited point-to-point commissural prow x jection 8,29 .
A similar explanation can be drawn for the expression of GAT-1 in the neuronal somata of the cerebral cortex with a needle puncture or saline injection because these treatments enhance neuronal activity around the needle path.This notion is supported by the finding of Fos expression in local neuronal and non-neuronal cells followw x ing needle puncture 12 .One result of the present study that puzzles us the most involves the lack of GAT-1 immunolabeling in interneurons in the colchicine-injected hemisphere.It is unlikely that GABAergic interneurons in this region are dying after colchicine infusion because previous studies used colchicine to label these neurons for w x GAD 47 .A possible explanation for the absence of GAT-1 upregulation in GABAergic neurons around the colchicine-injected cortex may be, again, because this chemical is somewhat toxic to local neurons.As a result, the GAT-1 regulatoryrsynthetic system in these interneurons may be somewhat distorted by this neurotoxicity.

Functional considerations for altered GAT expression
GABA is the major inhibitory neurotransmitter in the w x normal adult brain 28 .However, a body of evidence indicates that it exerts an excitatory effect during developw x ment and following neuronal trauma 7,50,52 .GABA's action can be regulated by its high affinity transporters, which take up GABA from the synaptic cleft and extracellular spaces into presynaptic axon terminals and astrocytes, so that inhibitory synaptic transmission is terminated in a timely manner and GABA can be recycled into presynaptic w x terminals 23,26 .In the present study, the upregulation of GAT-1 and GAT-3 is present mainly in the neuropil in the neocortical and hippocampal areas around the needle track.This appearance suggests an enhanced GABA uptake into astrocytes in the cortical regions with a direct lesion.If GABA acts as an excitatory substance in the lesioned cortex, an increased GABA uptake into glial cells would provide a significant neuroprotective effect for the cortical neurons.
On the other hand, the upregulation of GAT-1 in the Ž .somata of cortical interneurons in the ipsilateral puncture Ž .and contralateral colchicine infusion cortex may be to strengthen the GABA uptake ability for GABAergic neurons.The biological significance of this mechanism is perhaps to enhance synaptic and non-synaptic GABA rew x lease 2,3,31,41,49,56 .As a result, cortical inhibitory activity is elevated to antagonize neuronal overexcitation caused either by the stimulation from the needle puncture, or by transneuronal signaling from the transcallosally projecting pyramidal cells that are activated after colchicine injections.Furthermore, since excitation of cortical pyramidal neurons appears to be an inductive factor for GAT-1 w x expression 20,41 , it is possible that a persistent over-activation or excitation of these cerebral principal neurons may somehow deplete GAT-1 or interfere with GAT-1 production in the interneurons.This suggestion may explain why there is a reduction of GABA transporters in w x human temporal lobe epilepsy 14 .
Light micrographs showing the immunoreactivity for GAT-1 in homologous areas of the contralateral A-C and ipsilateral D-F neocortex and hippocampal formation from a rat that survived 2 days after an intracerebral saline injection.The immunolabeling in the contralateral side shows the Ž .normal distribution pattern of this GABA transporter in the adult rat cerebral cortex, with the immunoreactivity exclusively localized to neuropil.A the Ž .laminar pattern in the neocortex.B is an enlargement of a cortical area in layer III to show that immunonegative pyramidal cell bodies and their axon Ž .Ž .initial segments are contacted by basket and chandelier cell terminals arrows .C shows the normal pattern of GAT-1 immunoreactivity in the neuropil of Ž .the hippocampal formation.Following the injection, the cortical areas near the needle track indicated with stars in D and white asterisks in F show a Ž .Ž. Ž. slight increase of immunoreactivity over the neuropil, and a few lightly stained cell bodies arrowheads in both the neocortex E and hippocampus F .Ž .Ž .Ž .E is an enlargement of the boxed region in D to show that the labeled somata arrowheads are small, round, and have features of cortical interneurons.Ž .CA1 and CA3: CA regions of the hippocampus; s.p.: stratum pyramidale; DG: dentate gyrus; GL: granule cell layer.Scale bar s 200 mm for A, C, D , Ž .Ž .100 mm for E, F and 75 mm for B .( ) X.-X.Yan, C.E. Ribak r Brain Research 816 1999 317-328 w Fig. 2. GAT-3 immunoreactivity in homologous areas of the contralateral A and ipsilateral B neocortex and hippocampus from a rat that survived for 2 Ž .Ž .days after an intracerebral saline injection stars .A represents a normal distribution pattern of this GAT in the adult rat cerebral cortex with Ž .immunolabeling occurring exclusively in the neuropil.B shows a slight increase of immunoreactivity in the neuropil of the neocortical and hippocampal Ž .area surrounding the needle track white arrows .IV: layer IV; WM: white matter.Other abbreviations see Fig. 1.Scale bar s 200 mm.( ) X.-X.Yan, C.E. Ribak r Brain Research 816 1999 317-328 X. Yan, C.E. Ribak r Brain Research 816 1999 317-328  321 X. Yan, C.E. Ribak r Brain Research 816 1999 317-328  322 Fig. 4. Higher power views of GAT-1 immunoreactivity in the ipsilateral A-C and contralateral D-F hemispheres 2 days postinjection of colchicine.Ž .Ž .Ž .A, B, D, E illustrate the transitional areas of increased GAT-1 expression from the affected areas to the medial A, D and ventrolateral B, E unaffected Ž regions.The general staining intensity is much greater in the affected area of the ipsilateral side than the contralateral side indicated by small arrows in D, .Ž .E .The upregulated immunostaining is confined to the neuropil in the affected neocortical and hippocampal areas in the ipsilateral side C , whereas that in Ž .Ž .the contralateral side involves both neuropil and cell bodies in the neocortex F .The CA1 region small white arrows deep to the affected neocortex Ž .shows a slight increase of neuropil immunolabeling as compared to other hippocampal regions, but no cell bodies are found E .IV: layer IV.Scale Ž .Ž .bar s 500 mm for A, B, D, E and 100 mm for C, F .
X. Yan, C.E. Ribak r Brain Research 816 1999 317-328 323 Ž found to be GAT-1 immunoreactive in layers IIrIII Fig. .1B .In the hippocampal formation, the cell bodies of pyramidal and granule cells were unlabeled, whereas axon terminals on them and their axon initial segments were Ž .immunoreactive for GAT-1 Fig. 1C .GAT-3 immunoreactivity in the unaffected cortex was diffusely distributed in the neuropil in both neocortical and hippocampal areas Ž .Fig. 2A .Ž .Ž .Fig. 5. Higher magnification light microphotographs showing GAT-3 immunoreactivity in the affected cortex of ipsilateral A-C and contralateral D-F hemispheres 2 days after colchicine injection.Note that the increase of immunolabeling is much greater in the ipsilateral side, and that the elevated staining is exclusively localized to the neuropil on both sides.Deep to the affected neocortex on the ipsilateral side, some small and flat cell bodies with long, thin Ž .processes arrows in C are recognizable in the white matter.They represent immunoreactive astrocytes.VIa and VIb: sublamina of layer VI.Scale Ž .Ž .bar s 200 mm for A, B and 75 mm for B, D .