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Taurine, GABA and GFAP immunoreactivity in the developing and adult rat optic nerve
Brain Research, 596 (1992) 124-132
124
© 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
BRES 18260
Taurine, GABA and GFAP immunoreactivity in the developing and adult rat optic nerve Norma Lake Departments of Physiology and Ophthalmology, McGill Unicersity, Montreal, Que. (Canada) (Accepted 16 June 1992)
Key words: Taurine; y-Aminobutyric acid; Glial fibrillary acidic protein; Rat; Optic nerve; Immunocytochemistry
The Iocalizations of taurine, y-aminobutyric acid (GABA) and glial fibrillary acidic protein (GFAP) within the developing rat optic nerve were determined using immunocytochemical techniques on tissues from animals ranging in age from embryonic day 20 to postnatal 28 days. Mature nerves from 3-4-month-old adults were also examined. At the younger ages, taurine immunoreactivity was intense and localized specifically to the optic nerve axons, but by postnatal day 15 and thereafter its predominant localization was in macroglia. Some of these glia were astrocytes as indicated by the specific marker, GFAP. GABA immunoreactivity was present at the same time as taurine but was found only in macroglia. In mature nerves the patterns of taurine, GABA and GFAP distribution (within glia) were highly similar.
INTRODUCTION The non-protein amino acids, taurine and GABA are widely distributed in mature central nervous tissue and their potent inhibitory actions on neurons are well known 14. Their role during development is less well understood although it is becoming clear that a number of 'neurotransmitter' substances regulate neuronal development and plasticity for example 26, and both taurine and GABA are present in releasable pools in growth cones of early postnatal brain 42. There have been a number of descriptions of developmental abnormalities in the CNS (retina, visual cortex and cerebellum) of taurine-deficient mammals where the deficits involve failure of neuronal migration and differentiation, and/or abnormal glial-neuronal interactions (reviewed in ref. 41). The taurine-deficient cellular element(s) have not been identified in most of these models. In the retina development of the usual complement of photoreceptor cell types and normal outer plexiform syaaptic connections is disrupted by neonatal lesioning of horizontal cells 27 which contain both G A B A 27 and taurin¢ 4. Our studies of developing rabbit and rat retina have
identified prominent taurine immunoreactivity (taurine-IR) in the nerve fibre layer (axons of ganglion cells) in the perinatal period 4'!7. The present report describes the immunocytochemical localizations of taurine and GABA in the developing rat optic nerve, and their correlations with the astrocyte-specific marker, GFAP ~. A preliminary report has appeared 2~. In adult human za and rat z2 optic nerves we have previously reported localization of taurine-lR within glial cells, and here describe a similar locale for GABA-IR. In the optic nerve of mature rats taurine deficiency is associated with significant shrinkage and losses of optic nerve axons and decreases in myelin thickness t6,ta,tg. MATERIALS AND METHODS Pregnant Sprague-Dawley rats were obtained from Charles River (Quebec) and were maintained under dim cyclic lighting (12/12) and fed standard rat chow (Purina). Gestation is about 22 days in the rat. The optic nerves of rat pups were examined on the following days: embryonic (E) 20, day of birth (designated postnatal day 0), postnatal days 7, 15, 21, 28 and 40. A minimum of 3 pups per time point and 1 nerve per animal was used. Optic nerves from 3-4-month-old rats were also processed. For the embryonic tissues the mothers were anaesthesized with sodium pentobarbital (60 mg/kg i.p.) and the pups removed by cesarian section. Two methods of fixation were employed in anaesthesized pups (50 mg sodium pentobarbital/kg
Correspondence: N. Lake, McGiH University, Depar|ment of Physiology, 3655 Drummond Street, Montreal, Que., Canada H3G 1Y6. Fax: (1)
(514) 398-7452.
125 i.p.): (a) intracardiac perfusion preceded by warm heparinized saline to wash out blood, followed by nerve dissection, or (b) the skull was rapidly opened, the forebrain retracted and fixative applied directly to the nerves from behind the eye to the chiasm, with repeated application during the dissection. The piece of dissected nerve (one nerve per animal) was then placed in the fixative (3.5% glutaraldehyde in 0.1 M sodium cacodylate and 0.06 M magnesium chloride) i
I E20
for 1 hour prior to further processing and embedding in Epon, as described previously 2°.
lmmunocytochemistry Conjugates of taurine or GABA with bovine serum albumin (BSA, Fraction V, Sigma Chemical) were prepared and used as
PN7
100 I
!
Fig. 1. Histological sections of the optic nerve at various stages of development. These transverse 1.0/~m sections were stained with Toluidine blue. At E 20 and PN 7 bundles of primarily unmyelinated axons within the nerve are partially separated by the darkc~ staining gliai cells and their processes. Several blood vessels (v) are seen in the interior and at the periphery which is wrapped by several layers of leptomeningeal membranes. The PN 15 section is at the same magnification as E 20 and PN 7. The sections of PN 21 and adult nerves are at higher power (same scale for both). By PN 21 myelinated fibres are seen and there is a profusion of lighter staining glial cell processes. In the a¢,ult many fibres have increased in diameter to a size readily resolved at this magnification. At least two types of gila are distinguishable by the sta~,~.ingdensity of their nuclei: light, probable astrocytes (a) and dark, probable oligodendrocytes (o).
126 immunogens in rabbits 2°. Serum was harvested, purified and checked by enzyme linked immunosorbent assay (ELISA) for specificity and titre. Any crossreactivity to BSA, or other amino acid-BSA conjugates was removed by adsorption, until the ELISA was satisfactory. In addition to anti-taurine and anti-GABA sera, we used a commercial antibody to GFAP, (Dakopatts) which has been shown to have no anti-vime~tin reactivity 43. lmmunoreactivity in 1 t~m sections was visualized using a peroxidase anti-peroxidase (PAP) method or FITC-immunofluorescence observed with a brightfield or epifluorescent Nikon system and photographed using Kodak Pan X ASA 32 or Kodak Tmax A~'A 400 films. The techniques we used are described in more detail in previous reports 2°'23. Control sections were processed in parallel using antisera pre-adsorbed with the appropriate taurine- or GABA-BSA conjugate, or by omission of the primary antibody. RESULTS
Fig. 1 shows transverse sections of the optic nerve about 1-2 mm from its exit from the eye. These examples span the ages that were examined. At E 20 nerve diameter is about 200 /zm, and this first decreases and then increases during the initial 3 weeks after birth, to attain the adult diameter of about 1 mm. At E 20 the retinal ganglion cell axons are unmyelinated and separated into bundles by thin processes of glial cells whose perikarya are prominent. At PN 7 the total nerve diameter is somewhat smaller due to the normally occurring axonal death and loss. By PN 15 although the number of nerve fibres has decreased by two thirds from E 20 7, the nerve cross-sectional area has increased about 4 fold due to glial cell proliferation, increases in axon diameter, and the onset of myelination. About 10% of the axons are myelinated ~3 but this is not resolved at this magnification. Between PN 15 and PN 21 glial cell processes become more highly developed and about half of the nerve fibres are myelinated. In the adult most of the axons are myeli. hated and both dark and lightly-staining glial somata are observed.
lmmunocytochemical observations Fig. 2 shows transverse sections of the optic nerve from a late stage embryo (E 20) processed for taurine-,
Fig.2. Taurine-IR, GFAP-IR and GABA-IR in embryonic day 20. it. this and all the succeeding figures (except for Fig. 5 top panel) photomicrographs have been made of sections processed for specific immunofluorescence (white). These are three adjacent sections (scale bar is the same for all). While marked taurine-lR fills all the axons it is absent from glial cells, blood vessels and the leptomeningeal membranes. GFAP-IR, an astrocytic marker is relatively sparse but is found particularly in the glia limitans, a structure at the periphery of the nerve (white arrow) and in serpentine processes which extend towards the nerve interior and encircle the blood vessels (v). GABAIR fills the gliai cells (and precursors) more diffusely and extensively than GFAP-IR, but appears to overlap the GFAP distribution in its staining of the gila limitans, perivascular structures, and fine serpentine processes.
GABA- and GFAP-immunofluorescence. There are striking differences in the distributions: taurine-IR is localized solely to the bundles of axons, whereas the astrocytic marker, GFAP-IR, is confined to the astroglial processes forming the glia limitans, a barrier structure at the pe~'iphery of the nerve, and some fine
127 serpentine processes extending from there towards the interior of the nerve and encircling the blood vessels. GABA-IR is absent from axons, but fills the cytoplasm of glial cells throughout the nerve including their nuclear regions and thin serpentine processes which expand to surround the blood vessels, and to form the gila limitans. At this and other developmental stages omission of the primary antibody, or pre-adsorption of anti-taurine or anti-GABA serum with the appropriate taurine or GABA conjugates resulted in no background reactivity: the absence of sufficient immunofluorescence to activate the automatic exposure system even with ASA 400 and exposure times up to 10 minutes. In contrast the immunofluorescent pictures reproduced here required exposures of 2 to 20 seconds under the same conditions. At PN 7 taurine-IR remains confined to ganglion cell axons, but compared to E 20 the axon bundles appear to be more striated by glial processes unreacrive for taurine-IR (see Fig. 3). This glial developmental change is confirmed by the distribution of GFAP-IR which shows up a much denser array of astrocytic processes in the interior of the nerve as well as at the periphery, surrounding the axon bundles and encircling the numerous blood vessels. GABA-IR (not shown) is found within the somata and processes of glial cells throughout the nerve as before. By the end of the second PN week there has been a dramatic shift in the distribution of taurine-IR. As seen in Fig. 4 of PN 15 nerve, taurine immunofluorescence is now most prominent in glial perikarya, rather than the axon bundles. The patterns of GABA-IR and taurine-IR are highly similar at this stage and there is a range of immunofluorescent labelling within the clusters of glial cells. Some glial nuclei are highly labelled in a diffuse manner while others show only bright nuclear outlines, and others are unreactive. The GFAP-IR is more filamentous in appearance than the diffuse IR of the amino acids, and GFAP-IR is excluded from the astrocyte nuclei. Rat pups mature very rapidly in the initial 3 postnatal weeks and are weaned at about PN 21 or 22. Fig. 5 shows taurine-IR in the optic nerve at this time, visualized using the PAP method and th~~,FITC method. The pattern of taurine-IR in either of these sections has many common features with that for GFAP-iR in the bottom panel: dense IR in the gila limitans and in multibranched radial glial processes. However, while taurine-IR is located within the glial nuclear region, these nuclei are totally unreactive for GFAP-IR and appear as dark 'ghost' profiles. GABA-IR (not shown) is found solely within gila, and resembles taurine-IR distribution.
Fig. 3. Taurine-IR and GFAP-IR in adjacent sections in the 7-day-old rat optic nerve. Scale bar is the same for both panels. Taurine-lR is intense and restricted to axons as seen in E 20 nerve. GFAP-IR processes have become more profuse and form a distinct gila limitans at the nerve periphery and encircle the interior blood vessels, though not those lying external to the axons (e.g. white V, bottom right).
Fig. 6 shows the pattern of taurine-IR, GFAP-IR and GABA-IR in fully mature optic nerve. Taurine-IR and GABA-IR distributions are highly similar, labelling the gila limitans, and, throughout the nerve, radiating glial processes and glial cell nuclei, often with outline staining. The GFAP-IR pattern is dense and similar to that for the amino acids, except that the glial nuclei fail to label. DISCUSSION
Determinants of optic nerve diameter During the developmental stages that we have made our observations there are a number of processes that contribute to the overall size and composition of the optic nerve: axonal death, glial proliferation and differentiation, axonal growth, and myelinafian. At late embryonic stages the optic nerve contains about 325,000
128 unmyelinated retinal ganglion cell axons, which is about three times as many fibres as the adult nerve. Twothirds of these fibres degenerate between E 20 and PN 10 7 which tends to reduce nerve size (for example compare E 20, PN 7 of Fig. 1). However, most of the remaining fibres increase in diameter, in association initially with glial ensheathment beginning at PN 6, and then with compact myelin formation, beginning at PN 813. About 90% of the axons become myelinated by maturing oligodendroglia during the period from PN 8 to PN 40, and 97% of adult fibres are myelinated. The size distribution of unmyelinated fibres changes little during development. In addition to axons, the macroglia, of which there are several subtypes, are the other major component of the optic nerve, while microglia are few in number. Electron microscopic and [3H]thymidine autoradiographic studies of the rat optic n e r v e 35'36 indicate that some astroglia can be detected by embryonic day 15 or 16, but the majority are generated in the first postnatal week and differentiate gradually over the first 2 to 3 weeks into protoplasmic astrocytes or the more numerous, but later appearing, fibrous astrocyte subtype. There are about twice as many oligodendrocytes as astrocytes in the mature nerve 44. These begin their final division just before the onset of myelination (PN 6 or 7) but the majority are generated during the second postnatal week, after the crest of astrocyte divisionas'a6. Myelinogenesis peaks around PN 18 and is substantially reduced by PN 30 s. These glial subtypes are distinguished by morphological, physiological and antigenic phenotypes which change and evolve through various stages of development and differentiation. Clear descriptions of subtypes have come from studies using cell cultures derived from developing optic nerve, for example 3°'31'3s. However there are problems and controversies about confirming and extending the findings and schema from in vitro culture systems that may express potentialities which are difficult to demonstrate as actualities in vivo or in situ. This has been reviewed in a recent symposium on glial-neuronal interactions t.
Immunocytochemical findings: GFAP and GABA At E 20 the astrocytic marker GFAP is seen in glial
Fig. 4. Taurine-lR, GFAP-IR and GABA-IR in the 15-day-old rat optic nerve. Scale bar is the same for all. Taurine-IR is no longer axonal but instead closely resembles GABA-IR: both are found at different intensities within clusters of glial cells, sometimes filling the somata, sometimes just outlining them, sometimes absent. Taurine-IR and GABA-IR glial processes are seldom prominent at this stage, although the GFAP-IR stains many fine astroglial processes surrounding vessels and forming the gila limitans.
processes (not somata) and the distribution is somewhat more prominent at the nerve periphery. In contrast, GABA-IR is more extensive and appears to fill all the cellular structures within the nerve that are not taurine-IR axon bundles (see Fig. 2), with the exception of the blood vessel lumens. While some of the GABA-IR glial cells at the periphery may be astrocytes as deduced by the overlap with GFAP-IR, it is more
129 difficult to speculate on the identity of the GABA-IR cells of the nerve interior if one ventures past the designation of glioblast. At later times (PN 7, 15 and beyond) oligodendrocytes and fibrous (type-2) astrocytes are candidates for some of the GABA-IR glial cells that we observe. In rat CNS cultures these cell types have been shown to avidly take up labelled GABA, while protoplasmic (type-l) astrocytes were less active 1°'11'33. Recently brief reports by Sakatani and colleagues 12'34 have described in postnatal rats modulation of the conduction properties of optic nerve axons via GABA g and GABA B receptors. It is likely that the GABA-IR glial cells that we see are a source of this non-synaptic modulation. It is usually assumed that CNS GABA production occurs only in a select population of neurons, since that is where the synthetic enzyme, glutamic acid decarboxylase (GAD), has been located. Glial cells are presumed to acquire their GABA through removal processes (inactivation of neuronally released GABA) and to metabolize it to non-neuroactive glutamine (reviewed in ref. 40). The source of the glial GABA-IR that we observe from E 20 to maturity is not clear, since we do not see GABA-IR in optic axons. In 1 or 3 week cultures derived from PN 0 or 7 rat optic nerves Barres et al. 3 have found GABA-IR in oligodendrocytes, type-2 (fibrous) astrocytes and O-2A progenitor cells, whereas type-1 (protoplasmic) astrocytes were usually negative. It is interesting that they described this GABA at. dependent on glial synthesis from putrescine in the medium via a synthetic pathway alternative to one using GAD.
Taurine immunoreactivity
Fig. 5. Taurine-IR and (3FAP-IR in 21-day-old rat optic nerve. Scale bar is the same for all panels. The top panel shows taurine-IR visualized with the PAP technique (dark immunoproduct) whereas the middle panel shows the results of visualization using the immunofluorescence method (white immunoproduct). Both panels show marked taurine-lR within glial cells of the interior nerve and at the periphery (gila limitans). Somata and nuclear IR is prominent. The GFAP-IR in astrocytes (bottom panel) shows a labelling distribution similar to the taurine panels except that astroglial nuclei are not stained.
Since taurine-IR is found in macroglia in optic nerves from adult rats 22, it would not be surprising to find it expressed in gila of the developing nerve. In this study one of the most striking findings is the selective localization of taurine-IR in axons at E 20 and PN 7, with a shift to macroglia at subsequent developmental ages. [Since these are not quantitative techniques, one cannot exclude the presence of taurine in axons at later times, or in glia at earlier times, but at any one time the taurine content difference between the neuronal/ glial compartments is apparently relatively large.] In contrast, during this same period GABA-IR was detected only in glial cells. The biological significance of the taurine within the axons of the optic nerve is not yet clear. The time period (E 20 to PN 7) corresponds roughly to the critical period of establishment and refinement of retino-geniculate connections. Studies of the axonal
130 transport of exogenous labelled taurine injected into the eye have indicated that taurine is transported along the optic nerve and is found in the lateral geniculate nucleus (LGN) in a non-protein fraction 29, i.e. unlike other amino acids it is not first incorporated into newly synthesized protein by ganglion cells, and then transported. The magnitude of this taurine transport appears to be much higher in neonatal animals than at later developmental stages or in adults29; however the data were not adjusted for the loss of 60% of axons which occurs postnatally 7. We presume taurine is also present in the growth cones and axonal arbors of retinal efferents in the LGN and other central terminal fields, since it is present in amino acid assays of neonatal LGN s and neonatal rat brain growth cones 42. It appears to be releasable by depolarizing stimuli from these growth cones 42 and may, among other actions, stimulate the expression of transmitter receptor molecules 2. Additionally there is evidence which suggests that taurine may play a role in the stabilization and/or proliferation of cytoskeletal elements both in the nerve and its terminal arbors. Stimulatory effects of exogenous taurine on the number and elongation of neurites and growth cones have been reported in goldfish ganglion cells regenerating following optic nerve crush 24'2s and in cultures of mouse neuroblastoma cells 39. Ir~ the latter studies taurine effects included proliferation of cytoskeletal elements noted at the ultrastructural level. It is conceivable that early in development endogenous taurine could be released along the axonal trunks
and influence neighbouring axons and/or glial cells. After the second postnatal week taurine-IR shifted to and may be released from glia. While taurine re!ease from cultured astrocytes has been described (see ref. 23), to my knowledge release of taurine from axonal trunks has not been demonstrated. However, depolarization of optic nerve axons by taurine, that is partially sensitive to strychnine and bicuculline has been reported 37.
Developmental changes in taurine immunoreactivi~
Fig. 6. Taurine-IR, GFAP-IR and GABA-IR in the adult rat optic nerve. Scale bar is the same for all panels. Taurine and GABA immunofluorescence patterns appear very similar: in glial cells and their radiating processes throughout the nerve, notably the gila limitans, and in outline staining of glial nuclei. Some of these glial cells may be astrocytes since the marker GFAP is distributed in a similar manner, with the exception that glial nuclei are unreactive.
We have previously noted in the retina, where differentiation of various cell types and regions occurs sequentially, that transient or permanent expression of taurine-IR tracks differentiation ~7(and in preparation). In the optic nerve the distribution of taurine-IR switches from a predominantly neuronal (axonal) locale to a predominantly glial locale between PN 7 and PN 15. This is a time period when gliai cells are showing maturational changes, with the initial appearances of fibrous astrocytes and a wave of oligodendrocyte proliferation and myelinogenesis. At this time changes in
131
axolemmal specializations and axonal-glial relationships are thought to underlie changes in conduction velocity and properties in the postnatal developing optic nerve 9a3. In addition, potassium ion handling within the nerve shows marked changes during this time 32 due in part perhaps to maturation of astroglial ion pumping properties which respond to and remove extracellular potassium to capillaries ~s'2s. It has yet to be determined if the expression of taurine-IR and any of these activities are causally or only casually related. Ackno~.~edgements. This work was supported by a grant from the Medical Research Council of Canada and funds from the Department of Ophthalmology, McGill University. I am ~rateful to Carole Verdone-Smith for excellent technical assistance.
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Value and Mechanisms of Action, Plenum Press, New York, 1992, pp. 303-307. 17 Lake, N., De Marte, L. and Verdone-Smith, C., Immunocytochemical localization of taurine in the developing rat retina, Invest. Ophthalmol. Iris. Sci., 30 (1989)ARVO Suppl. 122. 18 Lake, N., Malik, N. and Battista, G., Taurine deficiency leads to loss of optic nerve fibres in tile rat, Soc. Neurosci. Abstr., 14 (1988) 358. 19 Lake, N., Malik, N. and De Marte, L., Taurine depletion leads to loss of optic nerve axons, Vis. Res., 28 (1988) 1071-1076. 20 Lake, N. and Verdone-Smith, C., Immunocytochemical loeahz~,. tion of taurine in the mammalian retina, Curr. Eye Res., 8 (!989) 163-173. 21 Lake-Castillo, N. and Verdone-Smith, C., Immunocytochemical localization of taurine in the optic nerve of the developing rat, Invest. Ophthal. Vis. Sci., 31 (1990) ARVO Suppl. 159. 22 Lake, N. and Verdone-Smith, C., Immunocytochemical localization of taurine within glial cells in the optic nerve of adult albino rats, Curr. Eye Res., 9 (1991) 1115-1120. 23 Lake, N., Verdone-Smith, C. and Brownstein, S., Immunocytochemical localization of taurine and gliai fibriilary acidic protein in human optic nerve, Visual Neurosci., 8 (1992) 25i-255. 24 Lima, L., Drujan, B. and Matus, P., Taurine and neuritic growth from goldfish retinal explants. In H. Pasantes-Morales and D. Redburn (Eds.), Extraceilular and lntracet!'~!ar Messengers in the Vertebrate Retina, Aian R. Liss, New York, 1989, pp. 105-116. 25 Lima, L., Matus, P. and Drujan, B., The interaction of substrate and taurine modulates the outgrowth from regenerating goldfish retinal explants, Int. J. Dev. Neurosci., 7 (1989) 375-382. 26 Lipton, S.A. and Kater, S.B., Neurotransmitter regulation of neuronal outgrowth, plasticity and survival, Trends NeuroscL, 12 (1989) 265-270. 27 Messersmith, E.K. and Redburn, D.A., Kainic acid lesioning alters outer plexiform development in neonatal rabbit retina, Int. J. Dev. Neurosci., 8 (1990)447-461. 28 Newman, E.A., Frambach, D.A. and Odette, L.L., Control of extracellular potassium levels by retinal glial cell K + siphoning, Science, 225 (1984) 1174-1175. 29 Politis, M.J. and Ingoglia, N.A., Axonai transport of taurine along neonatal and young adult rat optic axons, Brain Re]., 166 (1979) 221-231. 30 Raft, M.C., Glial cell diversification in the rat optic nerve, Science, 243 (1989) 1450-1455. 31 Raft, M.C., Abney, E.A., Cohen, J., Lindsay, R. and Noble, M., Two types of astrocytes in cultures of developing rat white matter: differences in morphology, surface gangliosides and growth characteristics, J. NeuroscL, 3 (1983) 1289-1300. 32 Ransom, B.R., Connors, B.W. and Kunis, D., Activity dependent K + accumulation in rat optic nerve: a developmental study, Soc. Neurosci. Abstr., 7 (1981) 901. 33 Reynolds, R. and Herschowitz, N., Selective uptake of neuroactire amino acids by both oligodendrocytes and astrocytes in primary dissociated cultures: a possible role for oligodendrocytes in neurotransmitter metabolism, Brain Res., 371 (1986) 253-266. 34 Sakatani, K., Black, J.A. and Kocsis, J.D., Activation of GABAA receptors by endogenous GABA release in developing~rat optic nerve, Soc. Neurosci. Abstr., 17 (1991) 526. j 35 Skoff, R., Price, D. and Stocks, A., Electron microsco~c autoradiographic studies of gliogenesis in the rat optic nrf, . 1. Cell proliferation, J. Comp. Neurol., 169 (197.6) 291-312.,/ 36 Skoff, R., Price, D. and Stocks, A., Electron micr,~,=opic autoradiographic studies of gliogenesis in the rat optic n ~ e . 2. Time of origin, J. Comp. Neurol., 169 (1976) 313-333. 37 Simmonds, M.A., Neuronal responses to taurine are distinct from those to ),-aminobutyric acid and glycine in rat cuneate nucleus and optic nerve, Br. J. Pharmacol., 78 (1983) 19P. 38 Sonthein~er, H., Minturn, .I.E., Black, J.A., Ransom, B.R. and Waxman, S.G., Two types of Na.currents in cultured rat optic nerve astrocytes: chang.~ ~ with time in culture and with age of culture derivation, J. Ne,~rosci. Res., 30 (1991) 275-287. 39 Spoerri, P.E., Caple, C.G. and Roisen, F.J., Taurine-induced
132 neuronal differentietion: The influence of calcium and the ganglioside GMI. b,t. J. Dev. Neurosci., 8 (1990) 491-503. 40 Storm-Mathisen, J., Ottersen, O.P., Fu-Long, T., Gundersen, V., Laake, J.H. and Nordbo, G., Metabolism and transport of amino acids studied by immunocytochemistry, Med. Biol., 64 (1986) 127-132. 4! Sturman, J.A., Taurine in development, J. Nutr., 118 (1988) 1169-1176. 42 Taylor, J., Docherty, M. and Gordon-Weeks, P.R., GABAergic
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© 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
BRES 18260
Taurine, GABA and GFAP immunoreactivity in the developing and adult rat optic nerve Norma Lake Departments of Physiology and Ophthalmology, McGill Unicersity, Montreal, Que. (Canada) (Accepted 16 June 1992)
Key words: Taurine; y-Aminobutyric acid; Glial fibrillary acidic protein; Rat; Optic nerve; Immunocytochemistry
The Iocalizations of taurine, y-aminobutyric acid (GABA) and glial fibrillary acidic protein (GFAP) within the developing rat optic nerve were determined using immunocytochemical techniques on tissues from animals ranging in age from embryonic day 20 to postnatal 28 days. Mature nerves from 3-4-month-old adults were also examined. At the younger ages, taurine immunoreactivity was intense and localized specifically to the optic nerve axons, but by postnatal day 15 and thereafter its predominant localization was in macroglia. Some of these glia were astrocytes as indicated by the specific marker, GFAP. GABA immunoreactivity was present at the same time as taurine but was found only in macroglia. In mature nerves the patterns of taurine, GABA and GFAP distribution (within glia) were highly similar.
INTRODUCTION The non-protein amino acids, taurine and GABA are widely distributed in mature central nervous tissue and their potent inhibitory actions on neurons are well known 14. Their role during development is less well understood although it is becoming clear that a number of 'neurotransmitter' substances regulate neuronal development and plasticity for example 26, and both taurine and GABA are present in releasable pools in growth cones of early postnatal brain 42. There have been a number of descriptions of developmental abnormalities in the CNS (retina, visual cortex and cerebellum) of taurine-deficient mammals where the deficits involve failure of neuronal migration and differentiation, and/or abnormal glial-neuronal interactions (reviewed in ref. 41). The taurine-deficient cellular element(s) have not been identified in most of these models. In the retina development of the usual complement of photoreceptor cell types and normal outer plexiform syaaptic connections is disrupted by neonatal lesioning of horizontal cells 27 which contain both G A B A 27 and taurin¢ 4. Our studies of developing rabbit and rat retina have
identified prominent taurine immunoreactivity (taurine-IR) in the nerve fibre layer (axons of ganglion cells) in the perinatal period 4'!7. The present report describes the immunocytochemical localizations of taurine and GABA in the developing rat optic nerve, and their correlations with the astrocyte-specific marker, GFAP ~. A preliminary report has appeared 2~. In adult human za and rat z2 optic nerves we have previously reported localization of taurine-lR within glial cells, and here describe a similar locale for GABA-IR. In the optic nerve of mature rats taurine deficiency is associated with significant shrinkage and losses of optic nerve axons and decreases in myelin thickness t6,ta,tg. MATERIALS AND METHODS Pregnant Sprague-Dawley rats were obtained from Charles River (Quebec) and were maintained under dim cyclic lighting (12/12) and fed standard rat chow (Purina). Gestation is about 22 days in the rat. The optic nerves of rat pups were examined on the following days: embryonic (E) 20, day of birth (designated postnatal day 0), postnatal days 7, 15, 21, 28 and 40. A minimum of 3 pups per time point and 1 nerve per animal was used. Optic nerves from 3-4-month-old rats were also processed. For the embryonic tissues the mothers were anaesthesized with sodium pentobarbital (60 mg/kg i.p.) and the pups removed by cesarian section. Two methods of fixation were employed in anaesthesized pups (50 mg sodium pentobarbital/kg
Correspondence: N. Lake, McGiH University, Depar|ment of Physiology, 3655 Drummond Street, Montreal, Que., Canada H3G 1Y6. Fax: (1)
(514) 398-7452.
125 i.p.): (a) intracardiac perfusion preceded by warm heparinized saline to wash out blood, followed by nerve dissection, or (b) the skull was rapidly opened, the forebrain retracted and fixative applied directly to the nerves from behind the eye to the chiasm, with repeated application during the dissection. The piece of dissected nerve (one nerve per animal) was then placed in the fixative (3.5% glutaraldehyde in 0.1 M sodium cacodylate and 0.06 M magnesium chloride) i
I E20
for 1 hour prior to further processing and embedding in Epon, as described previously 2°.
lmmunocytochemistry Conjugates of taurine or GABA with bovine serum albumin (BSA, Fraction V, Sigma Chemical) were prepared and used as
PN7
100 I
!
Fig. 1. Histological sections of the optic nerve at various stages of development. These transverse 1.0/~m sections were stained with Toluidine blue. At E 20 and PN 7 bundles of primarily unmyelinated axons within the nerve are partially separated by the darkc~ staining gliai cells and their processes. Several blood vessels (v) are seen in the interior and at the periphery which is wrapped by several layers of leptomeningeal membranes. The PN 15 section is at the same magnification as E 20 and PN 7. The sections of PN 21 and adult nerves are at higher power (same scale for both). By PN 21 myelinated fibres are seen and there is a profusion of lighter staining glial cell processes. In the a¢,ult many fibres have increased in diameter to a size readily resolved at this magnification. At least two types of gila are distinguishable by the sta~,~.ingdensity of their nuclei: light, probable astrocytes (a) and dark, probable oligodendrocytes (o).
126 immunogens in rabbits 2°. Serum was harvested, purified and checked by enzyme linked immunosorbent assay (ELISA) for specificity and titre. Any crossreactivity to BSA, or other amino acid-BSA conjugates was removed by adsorption, until the ELISA was satisfactory. In addition to anti-taurine and anti-GABA sera, we used a commercial antibody to GFAP, (Dakopatts) which has been shown to have no anti-vime~tin reactivity 43. lmmunoreactivity in 1 t~m sections was visualized using a peroxidase anti-peroxidase (PAP) method or FITC-immunofluorescence observed with a brightfield or epifluorescent Nikon system and photographed using Kodak Pan X ASA 32 or Kodak Tmax A~'A 400 films. The techniques we used are described in more detail in previous reports 2°'23. Control sections were processed in parallel using antisera pre-adsorbed with the appropriate taurine- or GABA-BSA conjugate, or by omission of the primary antibody. RESULTS
Fig. 1 shows transverse sections of the optic nerve about 1-2 mm from its exit from the eye. These examples span the ages that were examined. At E 20 nerve diameter is about 200 /zm, and this first decreases and then increases during the initial 3 weeks after birth, to attain the adult diameter of about 1 mm. At E 20 the retinal ganglion cell axons are unmyelinated and separated into bundles by thin processes of glial cells whose perikarya are prominent. At PN 7 the total nerve diameter is somewhat smaller due to the normally occurring axonal death and loss. By PN 15 although the number of nerve fibres has decreased by two thirds from E 20 7, the nerve cross-sectional area has increased about 4 fold due to glial cell proliferation, increases in axon diameter, and the onset of myelination. About 10% of the axons are myelinated ~3 but this is not resolved at this magnification. Between PN 15 and PN 21 glial cell processes become more highly developed and about half of the nerve fibres are myelinated. In the adult most of the axons are myeli. hated and both dark and lightly-staining glial somata are observed.
lmmunocytochemical observations Fig. 2 shows transverse sections of the optic nerve from a late stage embryo (E 20) processed for taurine-,
Fig.2. Taurine-IR, GFAP-IR and GABA-IR in embryonic day 20. it. this and all the succeeding figures (except for Fig. 5 top panel) photomicrographs have been made of sections processed for specific immunofluorescence (white). These are three adjacent sections (scale bar is the same for all). While marked taurine-lR fills all the axons it is absent from glial cells, blood vessels and the leptomeningeal membranes. GFAP-IR, an astrocytic marker is relatively sparse but is found particularly in the glia limitans, a structure at the periphery of the nerve (white arrow) and in serpentine processes which extend towards the nerve interior and encircle the blood vessels (v). GABAIR fills the gliai cells (and precursors) more diffusely and extensively than GFAP-IR, but appears to overlap the GFAP distribution in its staining of the gila limitans, perivascular structures, and fine serpentine processes.
GABA- and GFAP-immunofluorescence. There are striking differences in the distributions: taurine-IR is localized solely to the bundles of axons, whereas the astrocytic marker, GFAP-IR, is confined to the astroglial processes forming the glia limitans, a barrier structure at the pe~'iphery of the nerve, and some fine
127 serpentine processes extending from there towards the interior of the nerve and encircling the blood vessels. GABA-IR is absent from axons, but fills the cytoplasm of glial cells throughout the nerve including their nuclear regions and thin serpentine processes which expand to surround the blood vessels, and to form the gila limitans. At this and other developmental stages omission of the primary antibody, or pre-adsorption of anti-taurine or anti-GABA serum with the appropriate taurine or GABA conjugates resulted in no background reactivity: the absence of sufficient immunofluorescence to activate the automatic exposure system even with ASA 400 and exposure times up to 10 minutes. In contrast the immunofluorescent pictures reproduced here required exposures of 2 to 20 seconds under the same conditions. At PN 7 taurine-IR remains confined to ganglion cell axons, but compared to E 20 the axon bundles appear to be more striated by glial processes unreacrive for taurine-IR (see Fig. 3). This glial developmental change is confirmed by the distribution of GFAP-IR which shows up a much denser array of astrocytic processes in the interior of the nerve as well as at the periphery, surrounding the axon bundles and encircling the numerous blood vessels. GABA-IR (not shown) is found within the somata and processes of glial cells throughout the nerve as before. By the end of the second PN week there has been a dramatic shift in the distribution of taurine-IR. As seen in Fig. 4 of PN 15 nerve, taurine immunofluorescence is now most prominent in glial perikarya, rather than the axon bundles. The patterns of GABA-IR and taurine-IR are highly similar at this stage and there is a range of immunofluorescent labelling within the clusters of glial cells. Some glial nuclei are highly labelled in a diffuse manner while others show only bright nuclear outlines, and others are unreactive. The GFAP-IR is more filamentous in appearance than the diffuse IR of the amino acids, and GFAP-IR is excluded from the astrocyte nuclei. Rat pups mature very rapidly in the initial 3 postnatal weeks and are weaned at about PN 21 or 22. Fig. 5 shows taurine-IR in the optic nerve at this time, visualized using the PAP method and th~~,FITC method. The pattern of taurine-IR in either of these sections has many common features with that for GFAP-iR in the bottom panel: dense IR in the gila limitans and in multibranched radial glial processes. However, while taurine-IR is located within the glial nuclear region, these nuclei are totally unreactive for GFAP-IR and appear as dark 'ghost' profiles. GABA-IR (not shown) is found solely within gila, and resembles taurine-IR distribution.
Fig. 3. Taurine-IR and GFAP-IR in adjacent sections in the 7-day-old rat optic nerve. Scale bar is the same for both panels. Taurine-lR is intense and restricted to axons as seen in E 20 nerve. GFAP-IR processes have become more profuse and form a distinct gila limitans at the nerve periphery and encircle the interior blood vessels, though not those lying external to the axons (e.g. white V, bottom right).
Fig. 6 shows the pattern of taurine-IR, GFAP-IR and GABA-IR in fully mature optic nerve. Taurine-IR and GABA-IR distributions are highly similar, labelling the gila limitans, and, throughout the nerve, radiating glial processes and glial cell nuclei, often with outline staining. The GFAP-IR pattern is dense and similar to that for the amino acids, except that the glial nuclei fail to label. DISCUSSION
Determinants of optic nerve diameter During the developmental stages that we have made our observations there are a number of processes that contribute to the overall size and composition of the optic nerve: axonal death, glial proliferation and differentiation, axonal growth, and myelinafian. At late embryonic stages the optic nerve contains about 325,000
128 unmyelinated retinal ganglion cell axons, which is about three times as many fibres as the adult nerve. Twothirds of these fibres degenerate between E 20 and PN 10 7 which tends to reduce nerve size (for example compare E 20, PN 7 of Fig. 1). However, most of the remaining fibres increase in diameter, in association initially with glial ensheathment beginning at PN 6, and then with compact myelin formation, beginning at PN 813. About 90% of the axons become myelinated by maturing oligodendroglia during the period from PN 8 to PN 40, and 97% of adult fibres are myelinated. The size distribution of unmyelinated fibres changes little during development. In addition to axons, the macroglia, of which there are several subtypes, are the other major component of the optic nerve, while microglia are few in number. Electron microscopic and [3H]thymidine autoradiographic studies of the rat optic n e r v e 35'36 indicate that some astroglia can be detected by embryonic day 15 or 16, but the majority are generated in the first postnatal week and differentiate gradually over the first 2 to 3 weeks into protoplasmic astrocytes or the more numerous, but later appearing, fibrous astrocyte subtype. There are about twice as many oligodendrocytes as astrocytes in the mature nerve 44. These begin their final division just before the onset of myelination (PN 6 or 7) but the majority are generated during the second postnatal week, after the crest of astrocyte divisionas'a6. Myelinogenesis peaks around PN 18 and is substantially reduced by PN 30 s. These glial subtypes are distinguished by morphological, physiological and antigenic phenotypes which change and evolve through various stages of development and differentiation. Clear descriptions of subtypes have come from studies using cell cultures derived from developing optic nerve, for example 3°'31'3s. However there are problems and controversies about confirming and extending the findings and schema from in vitro culture systems that may express potentialities which are difficult to demonstrate as actualities in vivo or in situ. This has been reviewed in a recent symposium on glial-neuronal interactions t.
Immunocytochemical findings: GFAP and GABA At E 20 the astrocytic marker GFAP is seen in glial
Fig. 4. Taurine-lR, GFAP-IR and GABA-IR in the 15-day-old rat optic nerve. Scale bar is the same for all. Taurine-IR is no longer axonal but instead closely resembles GABA-IR: both are found at different intensities within clusters of glial cells, sometimes filling the somata, sometimes just outlining them, sometimes absent. Taurine-IR and GABA-IR glial processes are seldom prominent at this stage, although the GFAP-IR stains many fine astroglial processes surrounding vessels and forming the gila limitans.
processes (not somata) and the distribution is somewhat more prominent at the nerve periphery. In contrast, GABA-IR is more extensive and appears to fill all the cellular structures within the nerve that are not taurine-IR axon bundles (see Fig. 2), with the exception of the blood vessel lumens. While some of the GABA-IR glial cells at the periphery may be astrocytes as deduced by the overlap with GFAP-IR, it is more
129 difficult to speculate on the identity of the GABA-IR cells of the nerve interior if one ventures past the designation of glioblast. At later times (PN 7, 15 and beyond) oligodendrocytes and fibrous (type-2) astrocytes are candidates for some of the GABA-IR glial cells that we observe. In rat CNS cultures these cell types have been shown to avidly take up labelled GABA, while protoplasmic (type-l) astrocytes were less active 1°'11'33. Recently brief reports by Sakatani and colleagues 12'34 have described in postnatal rats modulation of the conduction properties of optic nerve axons via GABA g and GABA B receptors. It is likely that the GABA-IR glial cells that we see are a source of this non-synaptic modulation. It is usually assumed that CNS GABA production occurs only in a select population of neurons, since that is where the synthetic enzyme, glutamic acid decarboxylase (GAD), has been located. Glial cells are presumed to acquire their GABA through removal processes (inactivation of neuronally released GABA) and to metabolize it to non-neuroactive glutamine (reviewed in ref. 40). The source of the glial GABA-IR that we observe from E 20 to maturity is not clear, since we do not see GABA-IR in optic axons. In 1 or 3 week cultures derived from PN 0 or 7 rat optic nerves Barres et al. 3 have found GABA-IR in oligodendrocytes, type-2 (fibrous) astrocytes and O-2A progenitor cells, whereas type-1 (protoplasmic) astrocytes were usually negative. It is interesting that they described this GABA at. dependent on glial synthesis from putrescine in the medium via a synthetic pathway alternative to one using GAD.
Taurine immunoreactivity
Fig. 5. Taurine-IR and (3FAP-IR in 21-day-old rat optic nerve. Scale bar is the same for all panels. The top panel shows taurine-IR visualized with the PAP technique (dark immunoproduct) whereas the middle panel shows the results of visualization using the immunofluorescence method (white immunoproduct). Both panels show marked taurine-lR within glial cells of the interior nerve and at the periphery (gila limitans). Somata and nuclear IR is prominent. The GFAP-IR in astrocytes (bottom panel) shows a labelling distribution similar to the taurine panels except that astroglial nuclei are not stained.
Since taurine-IR is found in macroglia in optic nerves from adult rats 22, it would not be surprising to find it expressed in gila of the developing nerve. In this study one of the most striking findings is the selective localization of taurine-IR in axons at E 20 and PN 7, with a shift to macroglia at subsequent developmental ages. [Since these are not quantitative techniques, one cannot exclude the presence of taurine in axons at later times, or in glia at earlier times, but at any one time the taurine content difference between the neuronal/ glial compartments is apparently relatively large.] In contrast, during this same period GABA-IR was detected only in glial cells. The biological significance of the taurine within the axons of the optic nerve is not yet clear. The time period (E 20 to PN 7) corresponds roughly to the critical period of establishment and refinement of retino-geniculate connections. Studies of the axonal
130 transport of exogenous labelled taurine injected into the eye have indicated that taurine is transported along the optic nerve and is found in the lateral geniculate nucleus (LGN) in a non-protein fraction 29, i.e. unlike other amino acids it is not first incorporated into newly synthesized protein by ganglion cells, and then transported. The magnitude of this taurine transport appears to be much higher in neonatal animals than at later developmental stages or in adults29; however the data were not adjusted for the loss of 60% of axons which occurs postnatally 7. We presume taurine is also present in the growth cones and axonal arbors of retinal efferents in the LGN and other central terminal fields, since it is present in amino acid assays of neonatal LGN s and neonatal rat brain growth cones 42. It appears to be releasable by depolarizing stimuli from these growth cones 42 and may, among other actions, stimulate the expression of transmitter receptor molecules 2. Additionally there is evidence which suggests that taurine may play a role in the stabilization and/or proliferation of cytoskeletal elements both in the nerve and its terminal arbors. Stimulatory effects of exogenous taurine on the number and elongation of neurites and growth cones have been reported in goldfish ganglion cells regenerating following optic nerve crush 24'2s and in cultures of mouse neuroblastoma cells 39. Ir~ the latter studies taurine effects included proliferation of cytoskeletal elements noted at the ultrastructural level. It is conceivable that early in development endogenous taurine could be released along the axonal trunks
and influence neighbouring axons and/or glial cells. After the second postnatal week taurine-IR shifted to and may be released from glia. While taurine re!ease from cultured astrocytes has been described (see ref. 23), to my knowledge release of taurine from axonal trunks has not been demonstrated. However, depolarization of optic nerve axons by taurine, that is partially sensitive to strychnine and bicuculline has been reported 37.
Developmental changes in taurine immunoreactivi~
Fig. 6. Taurine-IR, GFAP-IR and GABA-IR in the adult rat optic nerve. Scale bar is the same for all panels. Taurine and GABA immunofluorescence patterns appear very similar: in glial cells and their radiating processes throughout the nerve, notably the gila limitans, and in outline staining of glial nuclei. Some of these glial cells may be astrocytes since the marker GFAP is distributed in a similar manner, with the exception that glial nuclei are unreactive.
We have previously noted in the retina, where differentiation of various cell types and regions occurs sequentially, that transient or permanent expression of taurine-IR tracks differentiation ~7(and in preparation). In the optic nerve the distribution of taurine-IR switches from a predominantly neuronal (axonal) locale to a predominantly glial locale between PN 7 and PN 15. This is a time period when gliai cells are showing maturational changes, with the initial appearances of fibrous astrocytes and a wave of oligodendrocyte proliferation and myelinogenesis. At this time changes in
131
axolemmal specializations and axonal-glial relationships are thought to underlie changes in conduction velocity and properties in the postnatal developing optic nerve 9a3. In addition, potassium ion handling within the nerve shows marked changes during this time 32 due in part perhaps to maturation of astroglial ion pumping properties which respond to and remove extracellular potassium to capillaries ~s'2s. It has yet to be determined if the expression of taurine-IR and any of these activities are causally or only casually related. Ackno~.~edgements. This work was supported by a grant from the Medical Research Council of Canada and funds from the Department of Ophthalmology, McGill University. I am ~rateful to Carole Verdone-Smith for excellent technical assistance.
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