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On the cellular localization and distribution of carbonic anhydrase II immunoreactivity in the rat brain
BRAIN RESEARCH ELSEVIER
Brain Research 676 (1995) 10-24
Research report
On the cellular localization and distribution of carbonic anhydrase II immunoreactivity in the rat brain L.F. Agnati
a
B. Tinner
b
W.A. Staines
d
K. V~i~in~inen c, K. Fuxe b, *
a Department of Human Physiology, University ofModena, Modena, Italy b Department ofNeuroscience, Karolinska Institute, 171 77 Stockholm, Sweden c Department of Anatomy, University of Oulu, Oulu, Finland d Department of Anatomy and Neurobiology, University of Ottawa, Ottawa, Canada Accepted 6 December 1994
Abstract
Evidence is provided that carbonic anhydrase-II is localized in the central nervous system to wide spread systems of oligodendrocytes and restricted astroglia populations, involving both fiber bundles and neuropil. It is suggested that CO 2 formed in activated axons may, via carbonic anhydrase-II, give rise to protons controlling the excitability of surrounding neuropil. Thus, CO2 may represent an important, highly diffusible, signal in brain, involved in the tonic control of neuronal activity.
Keywords: Oligodendroglia; Astroglia; CO2; Brain; Carbonic anhydrase II
1. Introduction
Carbonic anhydrase (CA; EC 4.2.1.1; carbonate dehydratase) is a monomeric zinc metalloenzyme that catalazyses the reversible hydratation of carbon dioxide [28]. Seven distinct CA isozymes (CA-I, CA-VII) have been cloned and characterized on the basis of their susceptibility to inhibitors, tissue distribution and subcellular localization [23,27,29]. To date CA-II, CA-Ill and CA-IV have been reported to be present in the central nervous system (CNS). CA-II is the principal form and is present mainly in oligodendrocytes and, to some extent, also in astrocytes as found in the mouse brain [5,13]. There is also evidence for the presence of CA-II in dorsal root ganglia neurons [6,7] and, perhaps, also in neurons of the CNS [20]. Low levels of the CA-Ill transcript have been described in mouse brain [31] and more recently CA-IV has been demonstrated in capillary endothelial cells by means of immunohistochemistry and immunoelectron
* Corresponding author. Fax: (46) (8) 337 941. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993(95)00026-7
microscopy carried out on sections from rat and mouse brain [14]. The nucleotide sequence of a cDNA encoding rat brain CA-II and its amino acid sequence has also been reported [25]. This enzyme plays an important role in several organs taking part in different physiological processes, such as ion transport, acid base regulation, transport of carbon dioxide from cells, and secretion of cerebrospinal fluid. However, the functional implications of its widespread distribution in the CNS is still largely unknown. By means of a well characterized polyclonal rabbit antirat CA-II antiserum [26,30], it is now possible to study the localization and distribution of CA-II immunoreactivity (IR) in the rat brain and herewith clarify its cellular localization in the rat brain. Using as a marker for oligodendrocytes a monoclonal antibody against myelin-associated glycoprotein M A G 513 [22], we demonstrate its localization within the oligodendrocyte population, while with the G F A P antibody [3] it was also found within discrete astroglial cell populations. In a separate experiment, endothelin-1 (ET-1) was injected into the rat neostriatum to induce ischemic lesions [10,12] and CA-II was used to follow the oligodendrocyte response within the ischemic focus.
L.F. Agnati et al. / Brain Research 676 (1995) 10-24
Finally, on the basis of the present findings and of data of other groups [8,9,17,19] a possible new functional role for this enzyme in the brain function is suggested.
2. Materials and methods
Twenty 2-month-old and 10 17-day postnatal male rats were perfused at room temperature with 0.01 M PBS (phosphate buffer saline) followed by an ice-cold fixative solution (200 ml) containing 4% paraformal-
11
dehyde and 0.2% picric acid in 0.1 M phosphate buffer pH 6.9. After 90 min of postfixation the brains were transferred to a 10% sucrose Solution in 0.1M phosphate buffer containing 0.01% sodium azide. Coronal 10-/~m sections were cut on a cryostat and incubated with the CA-II antibody at a dilution of 1:800 overnight at 4°C. After rinses in 0.01 mM phosphate buffer saline the sections were incubated with donkey antirabbit FITC linked whole antibody (Amersham) for 40 min at 37° C. The secondary antibody was used at a concentration of 1:20 in 0.01 M phosphate-buffered saline containing 0.3% Triton. After washing the sec-
Fig. 1. Fluorescence photomicrographs of a coronal section through the parietofrontal cortex in a 17-day-old male rat, using a two-colour immunofluorescence procedure. The arrows point to coexistence of carbonic acid anhydrase (CA-II) IR (FITC fluorescence) and myelin-associated glycoprotein (MAG) IR (Texas red fluorescence) in oligodendrocyte cell bodies.
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L.F. Agnati et al. / Brain Research 676 (1995) 10-24
tions were mounted in 90% glycerine in PBS containing 0.1% phenylendiamine to reduce fading. A double indirect immunofluorescence protocol was used to evaluate the coexistence of CA-II and an oligodendrocyte marker, the MAG 513 [1,22]. Sections were incubated with a mixture of rabbit anti CA-II (1:800) and mouse anti MAG (1:500) or mouse anti glial fibrillary acidic protein (GFAP; Boehringer-Mannheim; 1:25) overnight at 4°C. After washing, a mixture of donkey anti-rabbit FITC (Amersham, 1:20) and biotinylated sheep anti-mouse IgG (Amersham, 1:50) was applied and incubated for approximately 40 min at 37° C. The sections were once again washed and incubated in streptavidin-Texas red (Amersham) diluted 1:100 for 40 min at 37° C. After washing the sections were coverslipped with glycerol containing 0.1% phenylendiamine to reduce fading. In a separate experiment endothelin-1 (ET-1) (1 ~g/0.5/zl) was injected into the rat neostriatum of the adult rat using stereotaxic procedures as previously described to induce an ischemic lesion within the injected area [10]. The lesions were analyzed for CA-II IR 7 days following the ET-1 induced lesion.
3. Results
3.1. On the cellular localization of CA-H IR in the developing rat brain As shown in Figs. 1, 2 and 3 on postnatal day 17 CA-II IR was localized to cells demonstrating antiMAG like IR. This finding definitively identifies CA-II as localized to oligodendrocytes at this stage of development. 3.2. On the cellular localization of CA-H IR in the adult brain As seen in Figs. 4, 5 and 6, CA-II IR is again colocated within cell bodies which are immunoreactive for MAG. In this more mature state (2 month of age), the level of expression of MAG within the oligodendrocyte cell soma is diminished, while CA-II remains a robust marker of this cell type. A fine plexus of CA-II immunoreactive processes is present within the gray matter, and densely packed, parallel CA-II immunoreactive processes are found within the myelinated
Fig. 2. Fluorescence photomicrographs of a coronal section through caudate putamen in a 17-day-old male rat, using a two-colour immunofluorescence procedure. The carbonic acid anhydrase (CA-II) IR is visualized by FITC fluorescence and the myelin-associated glycoprotein (MAG) immunoreactivity by Texas Red fluorescence. Arrows indicate oligodendrocyte cell bodies containing both markers.
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L.F. Agnati et al. / Brain Research 676 (1995) 10-24
bundles, marking the myelinating processes of the oligodendrocytes. Figs. 7, 8 and 10, demonstrate the only regions where a colocalization of CA-II-like I R with G F A P I R could be unequivocally demonstrated. Thus, this enzyme is not a general constituent of the astroglial population. The pattern of distribution of C A - I I I R in cell bodies and processes is very similar in most of the areas analyzed (cerebral cortex, hippocampal formation, hypothalamus, midbrain, pons and medulla oblongata and the cerebellar cortex).
3.3. C A - H IR in neostriatum following an ET-1 induced lesion 7 days earlier
Cresyl violet staining showed the disappearance of nerve cells within the lesioned area of the neostriatum [10]. The core of the lesion was characterized by a disappearance of CA-II immunoreactive cell bodies and processes. This was not apparent in the outer zone of the lesion, close to the unlesioned part of the neostria-
CAti
day 17
/
'
J
Fig. 3. Fluorescence photomicrographs of a coronal section through the nucleus accumbens in a 17-day-old male rat using a two-colour immunofluorescence procedure. The carbonic acid anhydrase (CA-II) IR is visualized by FITC fluorescence and the myelin-associated glycoprotein (MAG) immunoreactivityby Texas Red fluorescence. Arrows indicate oligodendrocytes containing both markers. Many of the MAG immunoreactive processes do not show CA-II IR.
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L.F. Agnati et al. // Brain Research 676 (1995) 10-24
L.F. Agnati et al. / Brain Research 676 (1995) 10-24
15
Fig. 4. Fluorescence photomicrographs of coronal sections through the dorsal dentate gyrus in an adult rat brain after incubation with a rabbit polyelonal carbonic acid anhydrase (CA-II) (A,B) antiserum and control serum (C). Scattered CA-II immunoreactive cell bodies are seen, especially in the hilus (H). After incubation with control serum none of the fluorescent structures are seen. CA-II antiserum preabsorbed with carbonic acid anhydrase II (500 u.g/ml antiserum at a concentration of 1:10), was used as a control. Bregma level: -5.2mm.
tum (Figs. 10-12). In this Itatter region, CA-II immunoreactive processes were still in evidence, although severely reduced in numbe,r and in intensity. The CA-II immunoreactive cell bodies, however, often showed increased immunoreactivity, but did not colocalize with GFAP IR (Fig. 12).
4. Discussion In accord with previous findings [13,15,16,21] on the localization of CA-II based on histochemistry, the present immunocytochemical results provide evidence for a very dominant localization of CA-II IR within oligo-
CA II
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MAG
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Fig. 5. Double immunolabelling of a coronal section of the dorsal hippocampus, CA1 area, in adult rat brain using a rabbit polyclonal antiserum against carbonic acid anhydrase II (CA-II) and a mouse monoclonal myelin-associated glycoprotein (MAG) antibody. Arrow indicate an oligddendrocvte cell body contaiaing both markers. Bregma level: -4.5mm.
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L.F. Agnati et al. / Brain Research 676 (1995) 10-24
dendrocytes and their processes throughout the brain of the rat. It was found that CA-II-like IR was colocalized with a marker for oligodendrocytes, M A G [1] and as such the presence of CA-II IR appears to be a general property of oligodendrocytes within the developing and the adult rat brain. It should be noted that the CA-II immunoreactive processes in the oligodendrocytes were not only found in association with myelinated boundles, but also within the neuropil indicating a relationship with prefasciculated myelinating processes, unmyelinated fibres or oligodendroglial processes unrelated to axonal processes. A restricted num-
ber of cells in the outer layer of the cerebral cortex including the hilus of the dentate gyrus and in the substantia nigra showed both CA-II and G F A P IR, indicating the existence of CA-II IR also in restricted populations of astrocytes. The findings in the cortical gray matter are in agreement with previous findings showing localization of CA-II IR to astroglial cells probably of the protoplasmatic type [4,5]. Of particular note was the demonstration of GFAP-positive/CA-IIpositive processes surrounding the Purkinje cell bodies. Thus, it seems possible that the Bergrnann glia, which surround the Purkinje cells, may play a special role in
Fig. 6. Double immunolabelling of a coronal section of the caudate putamen in the adult rat brain using a rabbit polyclonal antiserum against
carbonic acid anhydrase II (CA-II) and a mouse monoclonalmyelin-associatedglycoprotein(MAG) antibody. Arrows indicate oligodendrocytes containing both markers. Brcgma level: 1.00 ram.
L.F. Agnati et aL / Brain Research 676 (1995) 10-24
the control of the microenvironment of the Purkinje cell bodies. However, in view of the findings of Ridderstr~k and Hanson [20] it may also be possible that the negative findings of CA-II IR within neurons is due to the low levels of this enr.cme within the neuronal cells. Based on the present distribution map of CA-II IR within the oligodendrocytes and their processes and to distinct astroglia populations of the brain it seems likely that CO 2 released by active axons will rapidly reach CA-II within the processes surrounding the axons. Thus, the morphological analysis would support the view that the CO 2 :formed in the active bundles could rapidly be converted into protons and HCO 3. The consequent pH shifts are capable of modulating ion channels and enzyme activity [17,18,24]. Thus, excitability changes will take place in the network surrounding the bundles according to their firing rate and this control is made very effective by the presence of a CA-II IR within the oligodendrocytes associated to these bundles•
"~,
GFAP
This mechanism could be of particular importance for the neurons of the reticular formation, leading to a tonic activity within not only the pathways involved with sleep/wakefulness control but also within those, e.g., involved with autonomic control and neuroendocrine functions. In conclusion, CO 2 formed either by neuronal metabolism or, via glial CA-II catalytic action, from bicarbonate may represent a highly diffusible signal [2,11] to modulate neuronal networks surrounding fibre bundles and, in some instances, also far located neurons. The findings obtained in the ET-1 lesioned rats demonstrated a much smaller region of focal pathology in which the local ischemia produces a degeneration of the oligodendrocytes and presumably the axons they provide metabolic support to than of the neuronal population within the region of the ischemic anoxic lesion. This suggests either greater metabolic sensitivity of the neurons or else the involvement of neuron specific processes (e.g. glutamate excitotoxicity) in the
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Fig. 7. Double immunolabelling of a coronal section through the outer layers of the parietofrontal cortex in the adult rat brain using a rabbit polyclonal antiserum against carbonic acid anhydrase CA-II and a mouse monoclonal glial fibrillary acidic protein (GFAP) antibody. The CA-II IR (D) is visualized by FITC fluorescence and the GFAP IR by Texas red fluorescence (A,C). Arrows point to cells containing both markers. Panel B shows GFAP IR and CA-II IR in the same microphotograph, using a dual filter system (Zciss) visualizing both FITC fluorescence and Texas Red fluorescencc simultaneously. Bregma level: 1.20 mm.
18
L.F. Agnati et al. / Brain Research 676 (1995) 10-24
CAlm
A i I,
Fig. 8. Fluorescence photomicrograph of coronal sections through hilus areae dentatae in adult rat brain using a two colour immunofluorescence procedure. The glial flbrillary acid protein (GFAP) IR is visualized by Texas Red fluorescence and the carbonic acid anhydrase (CA-II) IR by FITC fluorescence. Arrows indicate cell bodies containing both markers. Bregma level: 1.20 mm.
E T - l - i n d u c e d toxic m i c r o e n v i r o n m e n t , i.e. that this toxic m i c r o e n v i r o n m e n t p r o d u c e d by the ET-1 m o d e l has d e m o n s t r a b l y different spheres of i n f l u e n c e for n e u r o n s a n d the oligodendrocytes s u p p o r t i n g the axons within the same ischemic region. T h e increased C A - I I
I R in oligodendroglial cells located in the o u t e r part of the lesion could arise from a n u p r e g u l a t i o n of this e n z y m e in r e s p o n s e to the anoxic acidosis, a n experim e n t a l d e m o n s t r a t i o n of the ability of o l i g o d e n d r o cytes to sense a n d a d a p t to acid challenge.
Fig. 9. Fluorescence photomicrographs of coronal sections through the substantia nigra in adult rat brain using a two colour immunofluorescence procedure. The glial fibrillary acidic protein (GFAP) IR (A,B) is visualized by Texas red fluorescence and the carbonic acid anhydrase (CA-II) IR by FITC fluorescence (C). Panel A shows a lower magnification for orientation and arrows indicate ceils in the lateral part of the zona compacta containing both markers. SNR, zona retieulata of the substantia nigra. Bregma level: -5.8ram.
L.F. Agnati et al. ~Brain Research 676 (1995) 10-24
19
20
L.F. Agnati et aL / Brain Research 676 (1995) 10-24
Fig. 10. Immunofluorescence photomicrographs of a coronal section through the caudate putamen in adult rat brain 7 days after an ET-l-induced striatal lesion using a rabbit polyclonal antiserum against carbonic acid anhydrase (CA-II) visualized by FITC fluorescence. The rat was intrastriatally injected with ET-1 (1/.rg/0.5/~1). The lesion is located to the left of the border line, which was based on counterstaining with Cresyl violet. Panel A shows the disappearance of myelinated fibre bundles, whereas the CA-II immunoreactive cell bodies but not their processes remain. Panels B and C show the lesioned and unlesioned side in higher magnification. Arrowhead (B) shows degenerating myelinated fibre bundles and arrow (B) indicates the presence of CA-II immunoreactive cell bodies with increased CA-II IR on the lesioned side compared with unlesioned side (C). No CA-II immunoreactive processes are seen in the neuropil in the area of the ET-l-induced lesion. Bregma level: 1.0 mm.
L.F. Agnati et al. ~Brain Research 676 (1995) 10-24
21
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Fig. 11. Immunofluorescence photomicrograph of coronal sections through caudate putamen in adult rat brain 7 days after ET-1 (1 / z g / 0 . 5 / t l ) induced striatal lesions using a rabbit polyclonal antiserum against carbonic acid anhydrase (CA-II), visualized by FITC fluorescence. Different magnifications of the outer part of the lesion (to the right of the border line) show the swelling and increased IR of the CA-II immunoreactive cell bodies. The arrow points to the CA-II immunoreactive processes extending from the CA-II immunoreactive cell bodies towards the degenerating myelinated fber bundles. Bregma level: 1.0 ram.
L.F. Agnati et al. / Brain Research 676 (1995) 10-24
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L.F. Agnati et al. /Brain Research 676 (1995) 10-24
23
Fig. 12. Fluorescence photomicrographs of coronal sections through caudate putamen in adult rat brain 7 days after an ET-1 (1 ~g/0.5/zl) induced striatal lesion using a two-colour immunofluorescence procedure. The glial fibrillary acid protein (GFAP) IR is visualized by Texas red fluorescence and the carbonic acid anhydrase II (CA-II) IR by FITC fluorescence. Arrows in panel A indicate the presence of CA-II immunoreactive cell bodies with increased CA-II IR within the lesion and at the border of the lesion. The same arrows in panel B show that the CA-II immunoreactive cell bodies do not contain or colocalize GFAP IR. Panel C shows GFAP IR and CA-II IR in the same microphotograph using a dual filter system (Zeiss) visualizing both FITC and Texas Red fluorescence simultaneously. Bregma level: 1.0 mm.
Acknowledgements This work has been supported by a grant from the Swedish Medical Research Council and Petrus and Augusta Hedlunds Stiftelse. L.F.A. is the recipient of a Nobel Fellowship. We are grateful to Mrs. Anne Edgren for excellent secretarial assistance.
References [1] Afar, D., Salzer, J., Roder, J., Braun, P. and Bell, J., Differential phosphorylation of myelin-associated glycoprotein isoforms in cell culture, J. Neurochem., 55 (1990) 1418-1426. [2] Agnati, L., Fuxe, K., Zoli, M., Zini, I., Toffano, G. and Ferraguti, F., A correlation analysis of the regional distribution of central enkephalin and /3-eadorphin immunoreactive terminals and of opiate receptor in adult and old male rats. Evidence of the existence of two mairt types of communications in the central nervous system: the volume transmission and the wiring transmission, Acta Physiol. Scand., 128 (1986)201-207. [3] Bignami, A. and Dahl, D., Differentiation of astrocytes in the cerebellar cortex and the pyramidal tracts of the newborn rat. An immunofluorescence study with antibodies to a protein specific to astrocytes, Brain Res., (1973) 393-402. [4] Cammer, W., Sacchi, R. and Sapirstein, V., Immunocytochemical localization of carbonic anhydrase in the spinal cords of normal and mutant (shh,erer) adult mice with comparisons among fixation methods, J. Histochem. Cytochem., 33 (1985) 45-54. [5] Cammer, W. and Tansey, F., The astrocytes as a locos of carbonic anhydrase in the brains of normal and dysmyelinating mutant mice, J. Comp. Neurol., 275 (1988) 65-75. [6] Carr, P., Yamamoto, T., Karmy, G., Baimbridge, K. and Nagy, J., Analysis of parvalbumin and calbindin D28k-immunoreactive neurons in dorsal root ganglia of rat in relation to their cytochrome oxidase and carbonic anhydrase content, Neuroscience, 33 (1989) 363-371. [7] Carr, P., Yamamoto, T., Staines, W., Whittaker, M. and Nagy, J., Quantitative histochemical analysis of cytochrome oxidase in rat dorsal root ganglia and its co-localization with carbonic anhydrase, Neuroscience, 33 (1989) 351-362. [8] Chesler, M., The regulation and modulation of pH in the nervous system, Prog. Neurobiol., 34 (1990) 401-427. [9] Chesler, M. and Kalia, K., Modulation of pH by neuronal activity, Trends Neurosci., 15 (1992) 396-402. [10] Fuxe, K., Kurosawa, M., Ciatra, A., Goiny, M., Hallstr6m, A., Ros6n, L., Ungerstedt, U. and Agnati, L., Endothelin-l-induced lesions in the brain as a new model of focal ischemia. In P. Conn (Eds.), Methods in Neurosciences, Vol. 7: Transplantations, Lesions, and Surgical Techniques, Academic Press, Orlando, 1991, pp. 256-282. [11] Fuxe, K. and Agnati, L., Two principal modes of electrochemio
cal communicaton in the brain: volume versus wiring transmission. In K. Fuxe and L. Agnati (Eds.), Advances in Neuroscience, Vol. 1. Volume Transmission in the Brain, Novel Mechanisms for Neural Transmission, Raven Press, New York, 1991, pp. 1-9. [12] Fuxe, K., Kurosawa, N., Cintra, A., HaUstr6m, A., Goiny, M., Ros6n, L., Agnati, L.F. and Ungerstedt, U., Involvement of local ischemia in endothelin-1 induced lesions of the neostriatum of the anaesthetized rat, Exp. Brain Res., 88 (1992) 131-139. [13] Ghandour, M., Langley, O., Vincendon, G. and Gombos, G., Immunochemical and immunohistochemical study of carbonic anhydrase II in adult rat cerebellum: a marker for oligodendrocytes, Neuroscience, 5 (1980) 559-571. [14] Ghandour, M., Langley, O., Zhu, X., Waheed, A. and Sly, W., Carbonic anhydrase IV on brain capillary endothelial cells: a marker associated with blood-brain barrier, Proc. Natl. Acad. Sci. USA, 89 (1992) 6823-6827. [15] Kumpulainen, T. and Korhonen, L., Immunohistochemical localization of carbonic anhydrase isoenzyme C in the central and peripheral nervous system of the mouse, J. Histochem. Cytochem., 30 (1982) 283-292. [16] Langley, O., Ghandour, M., Vincendon, G. and Gombos, G., Carbonic anhydrase: an ultrastructural study in rat cerebellum, Histochem. J., 12 (1980) 473-483. [17] Moody, W., Effects of intracellular H + on the electrical properties of excitable cells, Annu. Rev. Neurosci., 7 (1984) 257-278. [18] Morad, M., Ditcher, M. and Tang, C.-M., The NMDA activated current in hippocampal neurons is highly senstive to [H + ], Soc. Neurosci. Abstr., 14 (1988) 791. [19] Olney, J., Zorumski, C., Price, M. and Labruyere, J., L-Cysteine, a bicarbonate-sensitive endogenous excitotoxin, Science, 248 (1990) 596-599. [20] Ridderstr~le, Y. and Hanson, M., Histochemical study of the distribution of carbonic anhydrase in the rat brain, Acta Physiol. Scand., 124 (1985) 557-564. [21] Roussel, G., Delaunoy, J.-P., Nussbaum, J. and Mandel, P., Demonstration of a specific localization of carbonic anhydrase C in the glial cells of rat CNS by an immunohistochemical method, Brain Res., 160 (1979) 47-55. [22] Salzer J.L, Roder, J., Braun, P.E. and Bell, J.S., Differential phosphorylation of myelin associated glycoprotein isoforms in cell cultures, J. Neurochem., 55 (1990) 1418-1426. [23] Spicer, S., Ge, Z., Tashian, R.E., Hazen-Marten, D.J. and Schuite, B.A., Comparative distribution of carbonic anhydrase isozymes III and II in rodent tissues, Am. J. Anat., 187 (1990) 55-64. [24] Spray, D., Harris, A. and Bennet, L., Gap junctional conductance is a simple and sensitive function of intracellular pH, Science, 211 (1981) 712-715. [25] Stolle, C., McGowan, M., Heim, R., Varia, M. and Neubauer, J., Nucleotide sequence of a cDNA encoding rat brain carbonic anhydrase II and its deduced amino acid sequence, Gene, 109 (1991) 265-267. [26] Sundquist, K., Leppilampi, M., J~irvelin, K., Kumpulainen, T. and V~iiin~inen, H., Carbonic anhydrase isoenzymes in peripheral monocytes, marcophages and osteoclasts, Bone, 8 (1987) 33-39.
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[27] Tashian, R. and Hewett-Emmett, D., Organization of the mouse and human carbonic anhydrase II genes, Ann. N Y Acad. Sci., 429 (1984) 309-323. [28] Tashian, R., The carbonic anhydrase: widening perspectives on their evolution, expression and function, BioEssays, 10 (1989) 186-192. [29] Tashian, R., Genetics of the mammalian carbonic anhydrases, Adv. Genet., 30 (1992) 321-356. [30] V~i~in~inen, H., Valve, E., M~ikel~i, S., Nevalainen, M. and
H~irk6nen, P., Hormone regulation of carbonic anhydrase II expression. In F. Botre, G. Gros and B. Storey (Eds.), Carbonic Anhydrase, VCH, Weinheim, 1991, pp. 345-351. [31] Wade, R., Gunning, P., Eddy, R., Shows, T. and Kedes, L., Nucleotide sequence, tissue specific expression, and chromosome location of human carbonic anhydrase III: the human CA III gene is located on the same chromosome as the closely linked CAI and CAII genes, Proc. Natl. Acad. Sci. USA, 83 (1986) 9571-9575.
Brain Research 676 (1995) 10-24
Research report
On the cellular localization and distribution of carbonic anhydrase II immunoreactivity in the rat brain L.F. Agnati
a
B. Tinner
b
W.A. Staines
d
K. V~i~in~inen c, K. Fuxe b, *
a Department of Human Physiology, University ofModena, Modena, Italy b Department ofNeuroscience, Karolinska Institute, 171 77 Stockholm, Sweden c Department of Anatomy, University of Oulu, Oulu, Finland d Department of Anatomy and Neurobiology, University of Ottawa, Ottawa, Canada Accepted 6 December 1994
Abstract
Evidence is provided that carbonic anhydrase-II is localized in the central nervous system to wide spread systems of oligodendrocytes and restricted astroglia populations, involving both fiber bundles and neuropil. It is suggested that CO 2 formed in activated axons may, via carbonic anhydrase-II, give rise to protons controlling the excitability of surrounding neuropil. Thus, CO2 may represent an important, highly diffusible, signal in brain, involved in the tonic control of neuronal activity.
Keywords: Oligodendroglia; Astroglia; CO2; Brain; Carbonic anhydrase II
1. Introduction
Carbonic anhydrase (CA; EC 4.2.1.1; carbonate dehydratase) is a monomeric zinc metalloenzyme that catalazyses the reversible hydratation of carbon dioxide [28]. Seven distinct CA isozymes (CA-I, CA-VII) have been cloned and characterized on the basis of their susceptibility to inhibitors, tissue distribution and subcellular localization [23,27,29]. To date CA-II, CA-Ill and CA-IV have been reported to be present in the central nervous system (CNS). CA-II is the principal form and is present mainly in oligodendrocytes and, to some extent, also in astrocytes as found in the mouse brain [5,13]. There is also evidence for the presence of CA-II in dorsal root ganglia neurons [6,7] and, perhaps, also in neurons of the CNS [20]. Low levels of the CA-Ill transcript have been described in mouse brain [31] and more recently CA-IV has been demonstrated in capillary endothelial cells by means of immunohistochemistry and immunoelectron
* Corresponding author. Fax: (46) (8) 337 941. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993(95)00026-7
microscopy carried out on sections from rat and mouse brain [14]. The nucleotide sequence of a cDNA encoding rat brain CA-II and its amino acid sequence has also been reported [25]. This enzyme plays an important role in several organs taking part in different physiological processes, such as ion transport, acid base regulation, transport of carbon dioxide from cells, and secretion of cerebrospinal fluid. However, the functional implications of its widespread distribution in the CNS is still largely unknown. By means of a well characterized polyclonal rabbit antirat CA-II antiserum [26,30], it is now possible to study the localization and distribution of CA-II immunoreactivity (IR) in the rat brain and herewith clarify its cellular localization in the rat brain. Using as a marker for oligodendrocytes a monoclonal antibody against myelin-associated glycoprotein M A G 513 [22], we demonstrate its localization within the oligodendrocyte population, while with the G F A P antibody [3] it was also found within discrete astroglial cell populations. In a separate experiment, endothelin-1 (ET-1) was injected into the rat neostriatum to induce ischemic lesions [10,12] and CA-II was used to follow the oligodendrocyte response within the ischemic focus.
L.F. Agnati et al. / Brain Research 676 (1995) 10-24
Finally, on the basis of the present findings and of data of other groups [8,9,17,19] a possible new functional role for this enzyme in the brain function is suggested.
2. Materials and methods
Twenty 2-month-old and 10 17-day postnatal male rats were perfused at room temperature with 0.01 M PBS (phosphate buffer saline) followed by an ice-cold fixative solution (200 ml) containing 4% paraformal-
11
dehyde and 0.2% picric acid in 0.1 M phosphate buffer pH 6.9. After 90 min of postfixation the brains were transferred to a 10% sucrose Solution in 0.1M phosphate buffer containing 0.01% sodium azide. Coronal 10-/~m sections were cut on a cryostat and incubated with the CA-II antibody at a dilution of 1:800 overnight at 4°C. After rinses in 0.01 mM phosphate buffer saline the sections were incubated with donkey antirabbit FITC linked whole antibody (Amersham) for 40 min at 37° C. The secondary antibody was used at a concentration of 1:20 in 0.01 M phosphate-buffered saline containing 0.3% Triton. After washing the sec-
Fig. 1. Fluorescence photomicrographs of a coronal section through the parietofrontal cortex in a 17-day-old male rat, using a two-colour immunofluorescence procedure. The arrows point to coexistence of carbonic acid anhydrase (CA-II) IR (FITC fluorescence) and myelin-associated glycoprotein (MAG) IR (Texas red fluorescence) in oligodendrocyte cell bodies.
12
L.F. Agnati et al. / Brain Research 676 (1995) 10-24
tions were mounted in 90% glycerine in PBS containing 0.1% phenylendiamine to reduce fading. A double indirect immunofluorescence protocol was used to evaluate the coexistence of CA-II and an oligodendrocyte marker, the MAG 513 [1,22]. Sections were incubated with a mixture of rabbit anti CA-II (1:800) and mouse anti MAG (1:500) or mouse anti glial fibrillary acidic protein (GFAP; Boehringer-Mannheim; 1:25) overnight at 4°C. After washing, a mixture of donkey anti-rabbit FITC (Amersham, 1:20) and biotinylated sheep anti-mouse IgG (Amersham, 1:50) was applied and incubated for approximately 40 min at 37° C. The sections were once again washed and incubated in streptavidin-Texas red (Amersham) diluted 1:100 for 40 min at 37° C. After washing the sections were coverslipped with glycerol containing 0.1% phenylendiamine to reduce fading. In a separate experiment endothelin-1 (ET-1) (1 ~g/0.5/zl) was injected into the rat neostriatum of the adult rat using stereotaxic procedures as previously described to induce an ischemic lesion within the injected area [10]. The lesions were analyzed for CA-II IR 7 days following the ET-1 induced lesion.
3. Results
3.1. On the cellular localization of CA-H IR in the developing rat brain As shown in Figs. 1, 2 and 3 on postnatal day 17 CA-II IR was localized to cells demonstrating antiMAG like IR. This finding definitively identifies CA-II as localized to oligodendrocytes at this stage of development. 3.2. On the cellular localization of CA-H IR in the adult brain As seen in Figs. 4, 5 and 6, CA-II IR is again colocated within cell bodies which are immunoreactive for MAG. In this more mature state (2 month of age), the level of expression of MAG within the oligodendrocyte cell soma is diminished, while CA-II remains a robust marker of this cell type. A fine plexus of CA-II immunoreactive processes is present within the gray matter, and densely packed, parallel CA-II immunoreactive processes are found within the myelinated
Fig. 2. Fluorescence photomicrographs of a coronal section through caudate putamen in a 17-day-old male rat, using a two-colour immunofluorescence procedure. The carbonic acid anhydrase (CA-II) IR is visualized by FITC fluorescence and the myelin-associated glycoprotein (MAG) immunoreactivity by Texas Red fluorescence. Arrows indicate oligodendrocyte cell bodies containing both markers.
13
L.F. Agnati et al. / Brain Research 676 (1995) 10-24
bundles, marking the myelinating processes of the oligodendrocytes. Figs. 7, 8 and 10, demonstrate the only regions where a colocalization of CA-II-like I R with G F A P I R could be unequivocally demonstrated. Thus, this enzyme is not a general constituent of the astroglial population. The pattern of distribution of C A - I I I R in cell bodies and processes is very similar in most of the areas analyzed (cerebral cortex, hippocampal formation, hypothalamus, midbrain, pons and medulla oblongata and the cerebellar cortex).
3.3. C A - H IR in neostriatum following an ET-1 induced lesion 7 days earlier
Cresyl violet staining showed the disappearance of nerve cells within the lesioned area of the neostriatum [10]. The core of the lesion was characterized by a disappearance of CA-II immunoreactive cell bodies and processes. This was not apparent in the outer zone of the lesion, close to the unlesioned part of the neostria-
CAti
day 17
/
'
J
Fig. 3. Fluorescence photomicrographs of a coronal section through the nucleus accumbens in a 17-day-old male rat using a two-colour immunofluorescence procedure. The carbonic acid anhydrase (CA-II) IR is visualized by FITC fluorescence and the myelin-associated glycoprotein (MAG) immunoreactivityby Texas Red fluorescence. Arrows indicate oligodendrocytes containing both markers. Many of the MAG immunoreactive processes do not show CA-II IR.
14
L.F. Agnati et al. // Brain Research 676 (1995) 10-24
L.F. Agnati et al. / Brain Research 676 (1995) 10-24
15
Fig. 4. Fluorescence photomicrographs of coronal sections through the dorsal dentate gyrus in an adult rat brain after incubation with a rabbit polyelonal carbonic acid anhydrase (CA-II) (A,B) antiserum and control serum (C). Scattered CA-II immunoreactive cell bodies are seen, especially in the hilus (H). After incubation with control serum none of the fluorescent structures are seen. CA-II antiserum preabsorbed with carbonic acid anhydrase II (500 u.g/ml antiserum at a concentration of 1:10), was used as a control. Bregma level: -5.2mm.
tum (Figs. 10-12). In this Itatter region, CA-II immunoreactive processes were still in evidence, although severely reduced in numbe,r and in intensity. The CA-II immunoreactive cell bodies, however, often showed increased immunoreactivity, but did not colocalize with GFAP IR (Fig. 12).
4. Discussion In accord with previous findings [13,15,16,21] on the localization of CA-II based on histochemistry, the present immunocytochemical results provide evidence for a very dominant localization of CA-II IR within oligo-
CA II
Adult ?
/
MAG
,
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Fig. 5. Double immunolabelling of a coronal section of the dorsal hippocampus, CA1 area, in adult rat brain using a rabbit polyclonal antiserum against carbonic acid anhydrase II (CA-II) and a mouse monoclonal myelin-associated glycoprotein (MAG) antibody. Arrow indicate an oligddendrocvte cell body contaiaing both markers. Bregma level: -4.5mm.
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L.F. Agnati et al. / Brain Research 676 (1995) 10-24
dendrocytes and their processes throughout the brain of the rat. It was found that CA-II-like IR was colocalized with a marker for oligodendrocytes, M A G [1] and as such the presence of CA-II IR appears to be a general property of oligodendrocytes within the developing and the adult rat brain. It should be noted that the CA-II immunoreactive processes in the oligodendrocytes were not only found in association with myelinated boundles, but also within the neuropil indicating a relationship with prefasciculated myelinating processes, unmyelinated fibres or oligodendroglial processes unrelated to axonal processes. A restricted num-
ber of cells in the outer layer of the cerebral cortex including the hilus of the dentate gyrus and in the substantia nigra showed both CA-II and G F A P IR, indicating the existence of CA-II IR also in restricted populations of astrocytes. The findings in the cortical gray matter are in agreement with previous findings showing localization of CA-II IR to astroglial cells probably of the protoplasmatic type [4,5]. Of particular note was the demonstration of GFAP-positive/CA-IIpositive processes surrounding the Purkinje cell bodies. Thus, it seems possible that the Bergrnann glia, which surround the Purkinje cells, may play a special role in
Fig. 6. Double immunolabelling of a coronal section of the caudate putamen in the adult rat brain using a rabbit polyclonal antiserum against
carbonic acid anhydrase II (CA-II) and a mouse monoclonalmyelin-associatedglycoprotein(MAG) antibody. Arrows indicate oligodendrocytes containing both markers. Brcgma level: 1.00 ram.
L.F. Agnati et aL / Brain Research 676 (1995) 10-24
the control of the microenvironment of the Purkinje cell bodies. However, in view of the findings of Ridderstr~k and Hanson [20] it may also be possible that the negative findings of CA-II IR within neurons is due to the low levels of this enr.cme within the neuronal cells. Based on the present distribution map of CA-II IR within the oligodendrocytes and their processes and to distinct astroglia populations of the brain it seems likely that CO 2 released by active axons will rapidly reach CA-II within the processes surrounding the axons. Thus, the morphological analysis would support the view that the CO 2 :formed in the active bundles could rapidly be converted into protons and HCO 3. The consequent pH shifts are capable of modulating ion channels and enzyme activity [17,18,24]. Thus, excitability changes will take place in the network surrounding the bundles according to their firing rate and this control is made very effective by the presence of a CA-II IR within the oligodendrocytes associated to these bundles•
"~,
GFAP
This mechanism could be of particular importance for the neurons of the reticular formation, leading to a tonic activity within not only the pathways involved with sleep/wakefulness control but also within those, e.g., involved with autonomic control and neuroendocrine functions. In conclusion, CO 2 formed either by neuronal metabolism or, via glial CA-II catalytic action, from bicarbonate may represent a highly diffusible signal [2,11] to modulate neuronal networks surrounding fibre bundles and, in some instances, also far located neurons. The findings obtained in the ET-1 lesioned rats demonstrated a much smaller region of focal pathology in which the local ischemia produces a degeneration of the oligodendrocytes and presumably the axons they provide metabolic support to than of the neuronal population within the region of the ischemic anoxic lesion. This suggests either greater metabolic sensitivity of the neurons or else the involvement of neuron specific processes (e.g. glutamate excitotoxicity) in the
GFAP
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Fig. 7. Double immunolabelling of a coronal section through the outer layers of the parietofrontal cortex in the adult rat brain using a rabbit polyclonal antiserum against carbonic acid anhydrase CA-II and a mouse monoclonal glial fibrillary acidic protein (GFAP) antibody. The CA-II IR (D) is visualized by FITC fluorescence and the GFAP IR by Texas red fluorescence (A,C). Arrows point to cells containing both markers. Panel B shows GFAP IR and CA-II IR in the same microphotograph, using a dual filter system (Zciss) visualizing both FITC fluorescence and Texas Red fluorescencc simultaneously. Bregma level: 1.20 mm.
18
L.F. Agnati et al. / Brain Research 676 (1995) 10-24
CAlm
A i I,
Fig. 8. Fluorescence photomicrograph of coronal sections through hilus areae dentatae in adult rat brain using a two colour immunofluorescence procedure. The glial flbrillary acid protein (GFAP) IR is visualized by Texas Red fluorescence and the carbonic acid anhydrase (CA-II) IR by FITC fluorescence. Arrows indicate cell bodies containing both markers. Bregma level: 1.20 mm.
E T - l - i n d u c e d toxic m i c r o e n v i r o n m e n t , i.e. that this toxic m i c r o e n v i r o n m e n t p r o d u c e d by the ET-1 m o d e l has d e m o n s t r a b l y different spheres of i n f l u e n c e for n e u r o n s a n d the oligodendrocytes s u p p o r t i n g the axons within the same ischemic region. T h e increased C A - I I
I R in oligodendroglial cells located in the o u t e r part of the lesion could arise from a n u p r e g u l a t i o n of this e n z y m e in r e s p o n s e to the anoxic acidosis, a n experim e n t a l d e m o n s t r a t i o n of the ability of o l i g o d e n d r o cytes to sense a n d a d a p t to acid challenge.
Fig. 9. Fluorescence photomicrographs of coronal sections through the substantia nigra in adult rat brain using a two colour immunofluorescence procedure. The glial fibrillary acidic protein (GFAP) IR (A,B) is visualized by Texas red fluorescence and the carbonic acid anhydrase (CA-II) IR by FITC fluorescence (C). Panel A shows a lower magnification for orientation and arrows indicate ceils in the lateral part of the zona compacta containing both markers. SNR, zona retieulata of the substantia nigra. Bregma level: -5.8ram.
L.F. Agnati et al. ~Brain Research 676 (1995) 10-24
19
20
L.F. Agnati et aL / Brain Research 676 (1995) 10-24
Fig. 10. Immunofluorescence photomicrographs of a coronal section through the caudate putamen in adult rat brain 7 days after an ET-l-induced striatal lesion using a rabbit polyclonal antiserum against carbonic acid anhydrase (CA-II) visualized by FITC fluorescence. The rat was intrastriatally injected with ET-1 (1/.rg/0.5/~1). The lesion is located to the left of the border line, which was based on counterstaining with Cresyl violet. Panel A shows the disappearance of myelinated fibre bundles, whereas the CA-II immunoreactive cell bodies but not their processes remain. Panels B and C show the lesioned and unlesioned side in higher magnification. Arrowhead (B) shows degenerating myelinated fibre bundles and arrow (B) indicates the presence of CA-II immunoreactive cell bodies with increased CA-II IR on the lesioned side compared with unlesioned side (C). No CA-II immunoreactive processes are seen in the neuropil in the area of the ET-l-induced lesion. Bregma level: 1.0 mm.
L.F. Agnati et al. ~Brain Research 676 (1995) 10-24
21
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e
%
Fig. 11. Immunofluorescence photomicrograph of coronal sections through caudate putamen in adult rat brain 7 days after ET-1 (1 / z g / 0 . 5 / t l ) induced striatal lesions using a rabbit polyclonal antiserum against carbonic acid anhydrase (CA-II), visualized by FITC fluorescence. Different magnifications of the outer part of the lesion (to the right of the border line) show the swelling and increased IR of the CA-II immunoreactive cell bodies. The arrow points to the CA-II immunoreactive processes extending from the CA-II immunoreactive cell bodies towards the degenerating myelinated fber bundles. Bregma level: 1.0 ram.
L.F. Agnati et al. / Brain Research 676 (1995) 10-24
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L.F. Agnati et al. /Brain Research 676 (1995) 10-24
23
Fig. 12. Fluorescence photomicrographs of coronal sections through caudate putamen in adult rat brain 7 days after an ET-1 (1 ~g/0.5/zl) induced striatal lesion using a two-colour immunofluorescence procedure. The glial fibrillary acid protein (GFAP) IR is visualized by Texas red fluorescence and the carbonic acid anhydrase II (CA-II) IR by FITC fluorescence. Arrows in panel A indicate the presence of CA-II immunoreactive cell bodies with increased CA-II IR within the lesion and at the border of the lesion. The same arrows in panel B show that the CA-II immunoreactive cell bodies do not contain or colocalize GFAP IR. Panel C shows GFAP IR and CA-II IR in the same microphotograph using a dual filter system (Zeiss) visualizing both FITC and Texas Red fluorescence simultaneously. Bregma level: 1.0 mm.
Acknowledgements This work has been supported by a grant from the Swedish Medical Research Council and Petrus and Augusta Hedlunds Stiftelse. L.F.A. is the recipient of a Nobel Fellowship. We are grateful to Mrs. Anne Edgren for excellent secretarial assistance.
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