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Depletion of connexin43-immunoreactivity in astrocytes after kainic acid-induced lesions in rat brain
Neuroscience Letters, 130 (1991) 120-124 (~; 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100493G
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Depletion of connexin43-immunoreactivity in astrocytes after kainic acid-induced lesions in rat brain J.I. Vukelic 1, T. Y a m a m o t o 1, E.L. Hertzberg 2 and J.I. Nagy 1 IDepartment of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg,Man. (Canada) and ZDepartment of Neuroscience and Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461 (U.S.A.) (Received 14 February 1991; Revised version received 16 May 1991; Accepted 20 May 1991)
Key words.: Astrocyte; Gap junction; Potassium spatial buffering; Connexin43; Immunohistochemistry; Neuronal-glial interaction Recent studies have established that the gap junction protein connexin43 is a major structural component of gap junctions between astrocytes in rat brain. Here, we investigated by immunohistochemical methods the effect of kainic acid-induced neuronal degeneration on connexin43 expression by astrocytes. Stereotaxic injections of kainic acid into the thalamus were found to cause a near total depletion of connexin43-immunoreactivity at the lesion site. Areas depleted of connexin43 corresponded to those exhibiting substantial neuronal loss and intense gliosis. These results implicate a neuronal contribution to the regulation of connexin43 expression by astrocytes and, hence, to local control of the potassium spatial buffering capacity afforded by astrocyte gap junctions.
Astrocytes in the central nervous system (CNS) are extensively coupled by intercellular communication channels formed by gap junctions [1, 12, 13]. By allowing the passage of small molecules and ions throughout the astrocytic syncytial network created by these channels, gap junctions between astrocytes are thought to coordinate the metabolic activities of these cells. Among the better established functions of astrocytes, and one in which their attendant gap junctions play a major role, is potassium spatial buffering wherein ion accumulated by these cells from the extracellular space is redistributed within the continuity of junctionally coupled cytoplasmic compartments of neighbouring astrocytes [20, 23, 24]. That gap junctions mediate such intercellular traffic has been well established [1, 10, 19]. Recent studies have identified a number of gap junction proteins, often termed connexins, which have significant regions of homology and similar overall structural features. The most notable regions of divergence exist in cytoplasmically disposed domains, presumably the site of action of cell-specific modulatory activities [2, 7-9, 16, 17]. Immunohistochemical analysis of the distribution of these connexins in the rat CNS [5, 14, 15, 21, 25-27] have demonstrated that astrocytes throughout the brain express connexin43. However, our LM and EM analyses have revealed a striking regional heterogeneity in connexin43Correspondence: J.I. Nagy, Department of Physiology, Faculty of Medicine, University of Manitoba, 770 Bannatyne Avenue, Winnipeg, Man. Canada R3E 0W3.
immunoreactivity as well as a restricted intracellular localization of this protein. These observations suggested that connexin43 production by astrocytes may be locally regulated. Inasmuch as local demands for potassium spatial buffering and, hence, for astrocytic gap junctions is likely determined by the extracellular ionic milieu generated by active neurones, we hypothesized that this regulation may have a neuronal component. To test this possibility, we investigated the effect of kainic acid-induced neuronal destruction on connexin43 expression by astrocytes. Five male Spragu~Dawley rats weighing 275-300 g were deeply anaesthetized, mounted in a stereotaxic apparatus and injected unilaterally with 5 nmol of kainic acid dissolved in 0.5 /11 of 0.9% saline. The injections were aimed at the thalamus and were given over a period of 10 rain after which the syringe needle (30 gauge) was left in place for an additional 10 min. The coordinates used were AP, +6.7: ML, + 1.7; and DV, +4.3 according to the atlas of Paxinos and Watson [18]. Two animals were similarly injected with saline vehicle. The thalamus was chosen for injection in order to allow assessment of the effect of kainic acid on various thalamic nuclei which contain differential densities of connexin43-immunoreactivity. After a postoperative survival time of 7 days, the animals were deeply anaesthetized with chloral hydrate and perfused transcardially with fixative and postfixed as previously decribed [27]. The brains were then cryoprotected in 50 mM sodium phosphate buffer, pH 7.4, containing 25% sucrose and 10% glycerol. Sections
121 were cut on a sliding microtome at a thickness of 20 p m and washed overnight in 0.1 M sodium phosphate buffer at p H 7.4 (PB) containing 0.9% saline and 0.3% Triton X-100 (PBST). The sections were incubated for 72 h with a rabbit anti-connexin43 [26, 27] antibody diluted 1:2000 in PBST containing 1% bovine serum albumin (PBSTBSA), washed in PBST for 1 h, incubated for 2 h at r o o m temperature with anti-rabbit I g G (Sternberger-Meyer) diluted 1:20 in PBST-BSA, washed for 1 h in PBST, incubated for 2 at r o o m temperature with rabbit-PAP (Sternberger-Meyer) diluted 1:100 with PBST-BSA and then washed for 20 min in PBST and for 40 min in 50 m M Tris-HCl buffer, p H 7.4. The sections were then incubated in Tris-HC1 buffer containing 0.02% 3,3'-diaminobenzidine and 0.005% hydrogen peroxide, mounted onto slides from 30% gelatin/alcohol, dehydrated and
coverslipped with Lipshaw medium. Alternate sections were stained for Nissl substance with thionin. The antibody used to immunolabel connexin43 was generated against a synthetic peptide corresponding to amino acids 346-363 of connexin43 [2]. The specificity of this antibody has been previously demonstrated by Western blot and absorption control procedures [26, 27]. As described in detail elsewhere [27], connexin43immunoreactivity (connexin43-IR) is heterogeneously distributed in rat brain and often, individual brain regions are clearly demarcated by differential densities of immunostaining. In sections through the thalamus of kainic acid-injected brains, connexin43-IR on the control side had an appearance similar to that seen in normal unoperated and in vehicle-injected control animals. With respect to areas of interest here, staining was most
L
¢ Fig. 1. Photomicrographs of a section at a midthalamic level of rat brain showing the effect of a unilateral intrathalamic injection of kainic acid on connexin43-IR. A: low magnification photomontage showing the left control and the right injected side. Connexin43-IR is depleted in the area of the lesion (arrow) which encompassesportions of the ventromedial (VM), ventrolateral (VL) and ventral posteromedial and posterolateral (VP) thalamic nuclei. B,C: higher magnifications of an area within the VL on the control (B) and lesioned (C) side. A, × 20; B,C, x 170.
122 intense in the reticular thalamic nucleus (RTN) and moderate in the ventrolateral (VL), ventromedial (VM) and ventral posterolateral and posteromedial (VP) thalamic nuclei (Fig. IA). Connexin43-IR consisted of punctate immunolabelling (Fig. 1B) which has a characteristic flocculent appearance at low magnification. On the kainic acid-injected side, connexin43-IR was depleted in the area of the lesion encompassing portions of VL, VM and VP (Fig. 1A, C) and staining density was barely above that which we have previously obtained with preimmune serum [27]. In addition, there was some reduction in immunostaining in the ventral half of the R T N . For a comparison of areas depleted of connexin43 with those displaying neuron loss, a Nissl-stained section adjacent to the one in Fig. 1 is shown in Fig. 2. The lesion site is characterized by infiltration with numerous,
densely packed reactive glial cells outlined by an area of intense thionin staining and occupies portions of VL, VM and VP (Fig. 2A). Neurones were totally absent within this area of massive gliosis (Fig. 2B,C) and reduced in numbers within a 100-200 /tm perimeter extending beyond it. Some neurone loss was also evident in the ventral portion of the R T N . F r o m the shape of the intensely Nissl-stained area and from visual inspection of corresponding blood vessels around the lesion site in Figs. 1A and 2A, the connexin43-depleted region is seen to correspond closely to the area of total or substantial neurone loss. The present results show that 7 days after an intracerebral injection of kainic acid there is a dramatic reduction of connexin43-IR at the injection site. That this reduction represents a loss of connexin43 in astrocytes can be
Fig. 2. Photomicrographs of a Nissl-stained section adjacent to the one shown in Fig. 1. The fields shown here in A, B and C correspond exactly to those in A, B and C of Fig. 1, respectively.The kainic acid injection site within the thalamus (arrow in A) is outlined by a densely stained area of gliosis. Comparisons of neuronal and glial densities in the control (B) and lesioned (C) side demonstrate a near total loss of neurons and an increased number of glial cells in the latter. A, x 20; B,C, x 170.
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inferred from our previous demonstration that this protein is localized within astrocyte processes in many areas of the brain including the thalamic structures which were chosen here for kainic acid injections [26, 27]. In view of the widespread occurrence of connexin43 in normal astrocytes, the near total depletion of connexin43-IR by kainic acid is even more striking given the massive gliosis that is well known to accompany kainic acid-induced neuronal degeneration [4]. Our results are consistent with the notion, derived from our observations of connexin43 in normal material and from considerations of its involvement in the support of neurons vis-a-vis potassium spatial buffering, that neurones may somehow exert regulatory control over the expression of this protein in astrocytes. They are also consistent with the growing recognition of a multiplicity of neuronal-glial interactions [6, 23]. There are, of course, many other possible explanations for the disappearance of connexin43-IR at a kainic acid lesion site. Some of these are as follows. (1) The protein may still be present, but in a form no longer recognized by the antibody. This possibility remains to be tested by biochemical analysis of connexin43 in lesioned tissue. (2) Reactive astrocytes may not have the capacity to produce connexin43. The apparent failure of reactive astrocytes to manufacture connexin43, be this an intrinsic property or a further manifestation of a neuronal dependence, implies an absence of gap junctional coupling between these cells and their exclusion from the normal astrocytic syncytial network. This feature of reactive astrocytes may have far reaching functional implications with respect to the ability of these cells to maintain local metabolic environments. It may also need to be considered in the context of results derived from studies of astrocytes in kainic acid-lesioned tissues [11]. On the other hand, the possibility that reactive astrocytes are in fact gap junctionally coupled via a gap junction protein other than connexin43 cannot be excluded. (3) The loss of connexin43 in astrocytes may be due to a direct action of kainic acid on these cells inasmuch as they have been shown to be responsive to excitatory amino acids and probably contain glutamate receptors including that of the kainate type [3, 22]. However, we have found (unpublished observations) similar depletions of connexin43-immunoreactivity by other experimental methods that cause neuronal degeneration. The authors wish to thank M. Sawchuk for excellent technical assistance. This work was supported by grants from the Canadian Medical Research Council (MRC) and the University of Manitoba Faculty of Medicine Fund to J.I.N. and by a grant (GM 30667) from the National Institutes of Health and an Irma T. Hirschl Career
Scientist Award to E.L.H.J.I.N. is a recipient o f a MRC Scientist Award. 1 Bennett, M.V.L. and Goodenough, D.A., Gap junctions, electrotonic coupling, and intercellular communication, Neurosci. Res. Prog. Bull., 16 (1978) 373-485. 2 Beyer, E.C., Paul, D.L. and Goodenough, D.A., Connexin43: a protein from rat heart homologous to a gap junction protein from liver, J. Cell Biol., 105 (1987) 2621-2629. 3 Cornell-Bell, A.H., Finkbeiner, S.M., Cooper, M.S. and Smith, S.J., Glutamate induces calcium waves in cultured astrocytes: longrange glial signaling, Science, 247 (1990) 470-473. 4 Coyle, J.I. and Schwartz, R., The use of excitatory amino acids as selective neurotoxins. In A. Bj6rklund and T. H6kfelt (Eds.), Handbook of Chemical Neuroanatomy, Vol. 1, Methods in Chemical Neuroanatomy, Elsevier, Amsterdam, 1983, pp. 508-527. 5 Dermietzel, R., Traub, O., Hwang, T.K., Beyer, E., Bennett, M.V.L., Spray, D.C. and Willecke, K., Differential expression of three gap junction proteins in developing and mature brain tissues, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 10148-10152. 6 Hansson, E., Astroglia from defined brain regions as studied with primary cultures, Prog. Neurobiol., 30 (1988) 369-397. 7 Hertzberg, E.L., A detergent-independent procedure for the isolation of gap junctions from rat liver, J. Biol. Chem., 259 (1984) 9936-9943. 8 Heynkes, R., Kozjek, G., Traub, O. and Willecke, K., Identification of a rat liver eDNA and mRNA coding for the 28 kDa gap junction protein, FEBS Lett., 205 (1986) 56~0. 9 Kumar, N.M. and Gilula, N.B., Cloning and characterization of human and rat liver cDNAs coding for a gap junction protein, J. Cell Biol., 103 (1986) 767-776. I0 Loewenstein, W.R., Junctional intercellular communication: the cell-to-cell membrane channel, Physiol. Rev., 61 ( 1981) 829-913. 11 MacVicar, B.A., Crichton, S.A., Burnard, D.M. and Tse, F.W.Y., Membrane conductance oscillations in astroeytes induced by phorbol ester, Nature, 329 (1987) 242-243. 12 Massa, P.T. and Mugnaini, E., Cell junctions and intramembrane particles of astrocytes and oligodendrocytes: a freeze-fracture study, Neuroscienee, 7 (1982) 523-538. 13 Mugnaini, E., Cell junctions of astroeytes, ependyma, and related cells in the mammalian central nervous system, with emphasis on the hypothesis of a generalized functional syncytium of supporting cells. In S. Fedoroff and A. Vernadakis (Eds.), Astrocytes, Vol. 1, Academic Press, New York, 1986, pp. 329-371. 14 Nagy, J.I., Yamamoto, T., Shiosaka, S., Dewar, K.M., Whittaker, M.E. and Hertzberg, E.L., Immunohistochemical localization of gap junction protein in rat CNS: a preliminary account. In E.L. Hertzberg and R.G. Johnson (Eds.), Modern Cell Biology, Vol. 7, Alan R. Liss, New York, 1988, pp. 357-389. 15 Nagy, J.I., Yamamoto, T., Sawchuk, M.A., Nanee, D.M. and Hertzberg, E.L., Quantitative immunohistochemical and biochemical correlates of connexin43 localization in rat brain, Glia, in press. 16 Nicholson, B.R., Dermietzel, R., Teplow, D., Traub, O., Willecke, K. and Revel, J.-P., Two homologous protein components of hepatic gap junctions, Nature, 329 (1987) 732-734. 17 Paul, D.L., Molecular cloning of eDNA for rat liver gap junction protein, J. Cell Biol., 103 (1986) 123-134. 18 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1986. 19 Peracchia, C., Structural correlates of gap junction permeation, Int. Rev. Cytol., 66 (1980) 81-146. 20 Ransom, B.R. and Carlini, W.G., Eleetrophysiological properties of astrocytes. In S. Fedoroff and A. Vernadakis (Eds.), Astrocytes, Vol. 2, Academic Press, New York, 1986, pp. 1-49.
124 21 Shiosaka, S., Yamamoto, T., Hertzberg, E.L. and Nagy, J.I., Gap junction protein in rat hippocampus: correlative light and electron microscope immunohistochemical localization, J. Comp. Neurol., 281 (1989) 282 297. 22 Usowicz, M.M., Gallo, V. and Cull-Candy, S.G., Multiple conductance channels in type-2 cerebellar astrocytes activated by excitatory amino acids, Nature, 339 (1989) 381%383. 23 Walz, W., Role of glial cells in the regulation of the brain ion microenvironment, Prog. Neurobiol., 33 (1989) 309-333. 24 Walz, W. and Hertz, L., Functional interactions between neurons and astrocytes. II. Potassium homeostasis at the cellular level, Prog. Neurobiol., 20 (1983) 133-183.
25 Yamamoto, T., Shiosaka, S., Whittaker, M.E., Hertzberg, E.L. and Nagy, J.I., Gap junction protein in rat hippocampus: light microscope immunohistochemical localization, J. Comp. Neurol., 281 (1989) 269-281. 26 ¥amamoto, T., Ochalski, A., Hertzberg, E.L. and Nagy, J.l., LM and EM immunolocalization of the gap junctional protein connexin43 in rat brain, Brain Res., 508 (1990) 313-319. 27 Yamamoto, T., Ochalski, A., Hertzberg, E.L. and Nagy, J.l., On the organization of astrocytic gap junctions in rat brain as suggested by LM and EM immunohistochemistry of connexin43 expression, J. Comp. Neurol., 302 (1990) 853 883.
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Depletion of connexin43-immunoreactivity in astrocytes after kainic acid-induced lesions in rat brain J.I. Vukelic 1, T. Y a m a m o t o 1, E.L. Hertzberg 2 and J.I. Nagy 1 IDepartment of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg,Man. (Canada) and ZDepartment of Neuroscience and Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461 (U.S.A.) (Received 14 February 1991; Revised version received 16 May 1991; Accepted 20 May 1991)
Key words.: Astrocyte; Gap junction; Potassium spatial buffering; Connexin43; Immunohistochemistry; Neuronal-glial interaction Recent studies have established that the gap junction protein connexin43 is a major structural component of gap junctions between astrocytes in rat brain. Here, we investigated by immunohistochemical methods the effect of kainic acid-induced neuronal degeneration on connexin43 expression by astrocytes. Stereotaxic injections of kainic acid into the thalamus were found to cause a near total depletion of connexin43-immunoreactivity at the lesion site. Areas depleted of connexin43 corresponded to those exhibiting substantial neuronal loss and intense gliosis. These results implicate a neuronal contribution to the regulation of connexin43 expression by astrocytes and, hence, to local control of the potassium spatial buffering capacity afforded by astrocyte gap junctions.
Astrocytes in the central nervous system (CNS) are extensively coupled by intercellular communication channels formed by gap junctions [1, 12, 13]. By allowing the passage of small molecules and ions throughout the astrocytic syncytial network created by these channels, gap junctions between astrocytes are thought to coordinate the metabolic activities of these cells. Among the better established functions of astrocytes, and one in which their attendant gap junctions play a major role, is potassium spatial buffering wherein ion accumulated by these cells from the extracellular space is redistributed within the continuity of junctionally coupled cytoplasmic compartments of neighbouring astrocytes [20, 23, 24]. That gap junctions mediate such intercellular traffic has been well established [1, 10, 19]. Recent studies have identified a number of gap junction proteins, often termed connexins, which have significant regions of homology and similar overall structural features. The most notable regions of divergence exist in cytoplasmically disposed domains, presumably the site of action of cell-specific modulatory activities [2, 7-9, 16, 17]. Immunohistochemical analysis of the distribution of these connexins in the rat CNS [5, 14, 15, 21, 25-27] have demonstrated that astrocytes throughout the brain express connexin43. However, our LM and EM analyses have revealed a striking regional heterogeneity in connexin43Correspondence: J.I. Nagy, Department of Physiology, Faculty of Medicine, University of Manitoba, 770 Bannatyne Avenue, Winnipeg, Man. Canada R3E 0W3.
immunoreactivity as well as a restricted intracellular localization of this protein. These observations suggested that connexin43 production by astrocytes may be locally regulated. Inasmuch as local demands for potassium spatial buffering and, hence, for astrocytic gap junctions is likely determined by the extracellular ionic milieu generated by active neurones, we hypothesized that this regulation may have a neuronal component. To test this possibility, we investigated the effect of kainic acid-induced neuronal destruction on connexin43 expression by astrocytes. Five male Spragu~Dawley rats weighing 275-300 g were deeply anaesthetized, mounted in a stereotaxic apparatus and injected unilaterally with 5 nmol of kainic acid dissolved in 0.5 /11 of 0.9% saline. The injections were aimed at the thalamus and were given over a period of 10 rain after which the syringe needle (30 gauge) was left in place for an additional 10 min. The coordinates used were AP, +6.7: ML, + 1.7; and DV, +4.3 according to the atlas of Paxinos and Watson [18]. Two animals were similarly injected with saline vehicle. The thalamus was chosen for injection in order to allow assessment of the effect of kainic acid on various thalamic nuclei which contain differential densities of connexin43-immunoreactivity. After a postoperative survival time of 7 days, the animals were deeply anaesthetized with chloral hydrate and perfused transcardially with fixative and postfixed as previously decribed [27]. The brains were then cryoprotected in 50 mM sodium phosphate buffer, pH 7.4, containing 25% sucrose and 10% glycerol. Sections
121 were cut on a sliding microtome at a thickness of 20 p m and washed overnight in 0.1 M sodium phosphate buffer at p H 7.4 (PB) containing 0.9% saline and 0.3% Triton X-100 (PBST). The sections were incubated for 72 h with a rabbit anti-connexin43 [26, 27] antibody diluted 1:2000 in PBST containing 1% bovine serum albumin (PBSTBSA), washed in PBST for 1 h, incubated for 2 h at r o o m temperature with anti-rabbit I g G (Sternberger-Meyer) diluted 1:20 in PBST-BSA, washed for 1 h in PBST, incubated for 2 at r o o m temperature with rabbit-PAP (Sternberger-Meyer) diluted 1:100 with PBST-BSA and then washed for 20 min in PBST and for 40 min in 50 m M Tris-HCl buffer, p H 7.4. The sections were then incubated in Tris-HC1 buffer containing 0.02% 3,3'-diaminobenzidine and 0.005% hydrogen peroxide, mounted onto slides from 30% gelatin/alcohol, dehydrated and
coverslipped with Lipshaw medium. Alternate sections were stained for Nissl substance with thionin. The antibody used to immunolabel connexin43 was generated against a synthetic peptide corresponding to amino acids 346-363 of connexin43 [2]. The specificity of this antibody has been previously demonstrated by Western blot and absorption control procedures [26, 27]. As described in detail elsewhere [27], connexin43immunoreactivity (connexin43-IR) is heterogeneously distributed in rat brain and often, individual brain regions are clearly demarcated by differential densities of immunostaining. In sections through the thalamus of kainic acid-injected brains, connexin43-IR on the control side had an appearance similar to that seen in normal unoperated and in vehicle-injected control animals. With respect to areas of interest here, staining was most
L
¢ Fig. 1. Photomicrographs of a section at a midthalamic level of rat brain showing the effect of a unilateral intrathalamic injection of kainic acid on connexin43-IR. A: low magnification photomontage showing the left control and the right injected side. Connexin43-IR is depleted in the area of the lesion (arrow) which encompassesportions of the ventromedial (VM), ventrolateral (VL) and ventral posteromedial and posterolateral (VP) thalamic nuclei. B,C: higher magnifications of an area within the VL on the control (B) and lesioned (C) side. A, × 20; B,C, x 170.
122 intense in the reticular thalamic nucleus (RTN) and moderate in the ventrolateral (VL), ventromedial (VM) and ventral posterolateral and posteromedial (VP) thalamic nuclei (Fig. IA). Connexin43-IR consisted of punctate immunolabelling (Fig. 1B) which has a characteristic flocculent appearance at low magnification. On the kainic acid-injected side, connexin43-IR was depleted in the area of the lesion encompassing portions of VL, VM and VP (Fig. 1A, C) and staining density was barely above that which we have previously obtained with preimmune serum [27]. In addition, there was some reduction in immunostaining in the ventral half of the R T N . For a comparison of areas depleted of connexin43 with those displaying neuron loss, a Nissl-stained section adjacent to the one in Fig. 1 is shown in Fig. 2. The lesion site is characterized by infiltration with numerous,
densely packed reactive glial cells outlined by an area of intense thionin staining and occupies portions of VL, VM and VP (Fig. 2A). Neurones were totally absent within this area of massive gliosis (Fig. 2B,C) and reduced in numbers within a 100-200 /tm perimeter extending beyond it. Some neurone loss was also evident in the ventral portion of the R T N . F r o m the shape of the intensely Nissl-stained area and from visual inspection of corresponding blood vessels around the lesion site in Figs. 1A and 2A, the connexin43-depleted region is seen to correspond closely to the area of total or substantial neurone loss. The present results show that 7 days after an intracerebral injection of kainic acid there is a dramatic reduction of connexin43-IR at the injection site. That this reduction represents a loss of connexin43 in astrocytes can be
Fig. 2. Photomicrographs of a Nissl-stained section adjacent to the one shown in Fig. 1. The fields shown here in A, B and C correspond exactly to those in A, B and C of Fig. 1, respectively.The kainic acid injection site within the thalamus (arrow in A) is outlined by a densely stained area of gliosis. Comparisons of neuronal and glial densities in the control (B) and lesioned (C) side demonstrate a near total loss of neurons and an increased number of glial cells in the latter. A, x 20; B,C, x 170.
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inferred from our previous demonstration that this protein is localized within astrocyte processes in many areas of the brain including the thalamic structures which were chosen here for kainic acid injections [26, 27]. In view of the widespread occurrence of connexin43 in normal astrocytes, the near total depletion of connexin43-IR by kainic acid is even more striking given the massive gliosis that is well known to accompany kainic acid-induced neuronal degeneration [4]. Our results are consistent with the notion, derived from our observations of connexin43 in normal material and from considerations of its involvement in the support of neurons vis-a-vis potassium spatial buffering, that neurones may somehow exert regulatory control over the expression of this protein in astrocytes. They are also consistent with the growing recognition of a multiplicity of neuronal-glial interactions [6, 23]. There are, of course, many other possible explanations for the disappearance of connexin43-IR at a kainic acid lesion site. Some of these are as follows. (1) The protein may still be present, but in a form no longer recognized by the antibody. This possibility remains to be tested by biochemical analysis of connexin43 in lesioned tissue. (2) Reactive astrocytes may not have the capacity to produce connexin43. The apparent failure of reactive astrocytes to manufacture connexin43, be this an intrinsic property or a further manifestation of a neuronal dependence, implies an absence of gap junctional coupling between these cells and their exclusion from the normal astrocytic syncytial network. This feature of reactive astrocytes may have far reaching functional implications with respect to the ability of these cells to maintain local metabolic environments. It may also need to be considered in the context of results derived from studies of astrocytes in kainic acid-lesioned tissues [11]. On the other hand, the possibility that reactive astrocytes are in fact gap junctionally coupled via a gap junction protein other than connexin43 cannot be excluded. (3) The loss of connexin43 in astrocytes may be due to a direct action of kainic acid on these cells inasmuch as they have been shown to be responsive to excitatory amino acids and probably contain glutamate receptors including that of the kainate type [3, 22]. However, we have found (unpublished observations) similar depletions of connexin43-immunoreactivity by other experimental methods that cause neuronal degeneration. The authors wish to thank M. Sawchuk for excellent technical assistance. This work was supported by grants from the Canadian Medical Research Council (MRC) and the University of Manitoba Faculty of Medicine Fund to J.I.N. and by a grant (GM 30667) from the National Institutes of Health and an Irma T. Hirschl Career
Scientist Award to E.L.H.J.I.N. is a recipient o f a MRC Scientist Award. 1 Bennett, M.V.L. and Goodenough, D.A., Gap junctions, electrotonic coupling, and intercellular communication, Neurosci. Res. Prog. Bull., 16 (1978) 373-485. 2 Beyer, E.C., Paul, D.L. and Goodenough, D.A., Connexin43: a protein from rat heart homologous to a gap junction protein from liver, J. Cell Biol., 105 (1987) 2621-2629. 3 Cornell-Bell, A.H., Finkbeiner, S.M., Cooper, M.S. and Smith, S.J., Glutamate induces calcium waves in cultured astrocytes: longrange glial signaling, Science, 247 (1990) 470-473. 4 Coyle, J.I. and Schwartz, R., The use of excitatory amino acids as selective neurotoxins. In A. Bj6rklund and T. H6kfelt (Eds.), Handbook of Chemical Neuroanatomy, Vol. 1, Methods in Chemical Neuroanatomy, Elsevier, Amsterdam, 1983, pp. 508-527. 5 Dermietzel, R., Traub, O., Hwang, T.K., Beyer, E., Bennett, M.V.L., Spray, D.C. and Willecke, K., Differential expression of three gap junction proteins in developing and mature brain tissues, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 10148-10152. 6 Hansson, E., Astroglia from defined brain regions as studied with primary cultures, Prog. Neurobiol., 30 (1988) 369-397. 7 Hertzberg, E.L., A detergent-independent procedure for the isolation of gap junctions from rat liver, J. Biol. Chem., 259 (1984) 9936-9943. 8 Heynkes, R., Kozjek, G., Traub, O. and Willecke, K., Identification of a rat liver eDNA and mRNA coding for the 28 kDa gap junction protein, FEBS Lett., 205 (1986) 56~0. 9 Kumar, N.M. and Gilula, N.B., Cloning and characterization of human and rat liver cDNAs coding for a gap junction protein, J. Cell Biol., 103 (1986) 767-776. I0 Loewenstein, W.R., Junctional intercellular communication: the cell-to-cell membrane channel, Physiol. Rev., 61 ( 1981) 829-913. 11 MacVicar, B.A., Crichton, S.A., Burnard, D.M. and Tse, F.W.Y., Membrane conductance oscillations in astroeytes induced by phorbol ester, Nature, 329 (1987) 242-243. 12 Massa, P.T. and Mugnaini, E., Cell junctions and intramembrane particles of astrocytes and oligodendrocytes: a freeze-fracture study, Neuroscienee, 7 (1982) 523-538. 13 Mugnaini, E., Cell junctions of astroeytes, ependyma, and related cells in the mammalian central nervous system, with emphasis on the hypothesis of a generalized functional syncytium of supporting cells. In S. Fedoroff and A. Vernadakis (Eds.), Astrocytes, Vol. 1, Academic Press, New York, 1986, pp. 329-371. 14 Nagy, J.I., Yamamoto, T., Shiosaka, S., Dewar, K.M., Whittaker, M.E. and Hertzberg, E.L., Immunohistochemical localization of gap junction protein in rat CNS: a preliminary account. In E.L. Hertzberg and R.G. Johnson (Eds.), Modern Cell Biology, Vol. 7, Alan R. Liss, New York, 1988, pp. 357-389. 15 Nagy, J.I., Yamamoto, T., Sawchuk, M.A., Nanee, D.M. and Hertzberg, E.L., Quantitative immunohistochemical and biochemical correlates of connexin43 localization in rat brain, Glia, in press. 16 Nicholson, B.R., Dermietzel, R., Teplow, D., Traub, O., Willecke, K. and Revel, J.-P., Two homologous protein components of hepatic gap junctions, Nature, 329 (1987) 732-734. 17 Paul, D.L., Molecular cloning of eDNA for rat liver gap junction protein, J. Cell Biol., 103 (1986) 123-134. 18 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1986. 19 Peracchia, C., Structural correlates of gap junction permeation, Int. Rev. Cytol., 66 (1980) 81-146. 20 Ransom, B.R. and Carlini, W.G., Eleetrophysiological properties of astrocytes. In S. Fedoroff and A. Vernadakis (Eds.), Astrocytes, Vol. 2, Academic Press, New York, 1986, pp. 1-49.
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