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Entorhinal lesions result in shrinkage of the outer molecular layer of rat dentate gyrus leading subsequently to an apparent increase of glutamate decarboxylase and cytochrome oxidase activities
Neuroscience Letters, 39 (1983) 255-260
255
Elsevier Scientific Publishers Ireland Ltd.
ENTORHINAL LESIONS RESULT IN SHRINKAGE OF THE OUTER MOLECULAR LAYER OF RAT DENTATE GYRUS LEADING SUBSEQUENTLY TO AN APPARENT INCREASE OF G L U T A M A T E DECARBOXYLASE A N D CYTOCHROME OXIDASE ACTIVITIES
G.P. WAGNER I'*, W.H. OERTEL z and J.R. WOLFF I"**
~Department of Anatomy, University of G6ttingen, Kreutzbergring 36, D-3400 G6ttingen and 2Neurological Clinic, University of Munich, D-8000 Miinchen (F.R.G.) (Received October 21st, 1982; Revised version received April 30th, 1983; Accepted June 10th, 1983)
Key words: neuroplasticity - dentate gyrus - entorhinal deaffe~'entation .- glutamate decarboxylase cytochrome oxidase - rat
In intact dentate gyrus, glutamatq decarboxylase immunoreactivity (GAD) and cytochrome oxida,.,e activity (CyO) showed different distribution patterns. Entorhinal lesions caused increases of GAD and CyO in the outer molecular layer (OML) of the ipsilateral side. Submicro~opical localization of these enzymes did not change, except for CyO labeling more astrocytic mitochondtia. The increase in numerical density of GAD puncta correlated quantitatively with shrinkage of OML, whereas in the whole molecular layer the number of GAD puncta remained unchanged. Hence, the localized increase of enzyme activities and lysosomes is apparently related to shrinkage of OML, but does not indicate plasticity of GABAergic neurons.
Unilateral destruction of rat entorhinal cortex causes degeneration of about 86070 of all synapses in the outer and middle zone of the molecular layer in the ipsilateral dentate gyrus [2]. The middle zone is rapidly reinnervated by commissural and associational fibers from the hippocampus proper, whereas the outer zone shows a complex and long-lasting reaction [9, l l, 19, 23]. Glutamate decarboxylase (GAD) is a specific marker for GABAergic interneurons in the hippocampus [22]. At 1-4 weeks after deafferentation of the dentate gyrus the enzyme activity was shown to have been increased in the molecular layer. This increase was interpreted as a transneuronal neuroplastic response of local GABAergic neurons [6, 13]. Since in striate cortex the distribution of GAD immunoreactivity covaries with that of cytochrome oxidase (CyO) activity (monkey [7], rat [24]), in the present study the long-term effects of removing entorhinai afferents on .-.he histochemical *Present address: Max-Planck-lnstitut fiir Virusforschung, Molekularbiologische aLbteilung, Spemannstr. 35, D-7400 Tiibingen, F.R.G. **Author for correspondence. 0304-3940/83/$ 03.00 Q 1983 Elsevier Scientific Publishers Ireland Ltd.
256
architecture of the dentate gyrus were reinvestigated. In 8 adult Sprague-Dawley rats the entorhinal cortex was unilaterally removed by aspiration under combined anesthesia with Ketanest and Rompun. After 120-150 days survival the animals v~-ere sacrificed by cardiac perfusion with 4% formaldehyde in phosphate buffer (0.1 M, pH 7.4). Serial 40 tLm sections through the septal region of the hippocampus ,,',ere prepared either with a Vibratome or a freezing microtome. Four unoperated animals and the contralateral hemispheres served as controls. The location of GADlike immunoreactivity [181 (from hereon termed GAD-immunoreactivity) was visualized by the unlabeled antibody enzyme method [20] using the GAD-antiserum 53 [151. Cv() activity was demonstrated by the diaminobenzidine method [20, 251. 1he activity of acetyicholinesterase (ACHE), a marker for septal afferents [8], was demonstrated by Koclle's acetylthiocholine method, modified according to Naik [4]. The distribution of lysosomes and terminal degeneration was visualized by Gallvas' impregnation method {51. G kl)-immunoreactive puncta were counted in rows of squares (400 ~,me, magnification 900 × ) which were oriented radially in the dorsal blade of the dentate gyru,,. Additionally. the thickness of the tnolecular layer and outer lnolecular laver were measured. ('ontralatcral to the entort'tinal lesion the binding pattern of G A D antibodies in the dentate gyrus (Fig. la) and the thickness of the molecular layer were identical to that in :.nimals without lesion [!, 171. This pattern also resembles the distribution
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237
of GAD enzyme activity as determined by biochemical methods [13, 21 ] and of binding sites for GABA [21. On the other hand, ipsilateral to entorhinal lesions, the distribution of GAD immunoreactivity was changed, even after 4 - 5 months. Instead of a shallow gradient of staining intensity a clearly bilaminar pattern was seen within the molecular layer (Fig. la, b). Whereas staining intensity in the 'inner molecular layer' (IML) remained unchanged, it had strongly increased in the 'outer molecular layer' (OML). Electron microscopically GAD immunoreactivity was almost exclusively located in axon terminals (Fig. 2a, b) and only occasionally found in axons, dendrites and neuronal somata. Quantitative evaluation of the numerical density of GAD-positive points in the dorsal blade of the deafferented dentate gyrus confirmed the difference between OML and IML. In the IML the numerical density was not significantly different between deafferented and control dentate gyrus, while in the ipsilateral OML the density was 54.1070 (S.D. + 1.7) higher than in the contralateral OML. Since the ipsilateral OML was, however, 53.2°70 (S.D. + 0.96) thinner than the contralateral OML, there was no significant increase of the total number of GAD-positive points in the whole molecular layer (P <0.005 according to sign test, n = 8). The AChE activity (not shown) matched the changed distribution pattern of GAD immunoreactivity also at much longer survival times than shown by Lynch et al. [10]. Similarly, the present study revealed that high concentrations of lysosomes persist in OML even 5 months after deafferentation (not shown; see also ref. 1 1). The distribution of CyO activity in the dentate gyrus contralateral to the lesion (Fig. lc) resembled that of CyO in intact animals (see also ref. 16), but not that of GAD (Fig. la). Hence, in the intact archicortex of rat, the distribution patterns of CyO and GAD do not seem to correlate (Fig. la, c) as in the intact neocortex [7, 24]. However, after entorhinal lesions the distribution of CyO activity resembled strikingly the bilaminar pattern of GAD (Fig. lb, d). Electron microscopically CyO activity in the ipsilateral OML was mainly found in mitochondria of dendrites, while it was low in mitochondria of presynaptic elements (arrows in Fig. 2c), just as in intact animals [16]. In addition, some CyO activity was seen in mitochondria of certain astrocytes (Fig. 2d) which, according to structural criteria (plump processes with numerous ribosomes, filaments and glycogen granules), often resembled reactive astrocytes. About 1 week after antorhinal lesions GAD immunoreactivity begins to increase in the OML of dentate gyrus [6]. According to biochemical data GAD activity increases up to 33% after about 3 months [19], while the present immunohistochemical data show an increase of 54% by 5 months. This suggests that the GAD increase is based on a long-lasting process which might even be progressive. An increase of GABAergic innervation, generally assumed to be inhibitory in nature [1], cannot be explained as a compensation for a loss of excitatory input. Our data are also not compatible with the idea that local GABAergic neurons respond
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to the destruction of the perforant pathway with sprouting or redistribution between IML and OML of their axon tern'finals [6, 13]. Rather, it became evident that the overall number of GABAergic terminals remains constant in the molecular layer, i.e. their density is not regulated down to control values during thc shrinkage of OML. This in contrary t o the tendency of excitatory synapses which re-establish normal numerical densities in most cases of neuroplastic responses studied (for rcvicv, see ref. 3). The coindicence between augmented CvO activity and GAI) immu~oreacti,,itv following entorhinal lesions is not easily reconciled with the discrepancies of distribution in the intact dentate gyrus. Electron microscopy demonstrated that Cy(~ is not a marker for GAD-positive profilcs. Hcncc, a direct causal relationship does not seem to exist. Correlated changes in the distribution of s~tcll diverse enzymes as ACHE, CyO, G A D and of lysosomcs may, ho~evcr, be duc to change in the regulation of tissut" volume, i:actors in,~oixcd in this regulation are largely unknoven. Shrinkage and degeneration could explain both (!)correlated increases of several c~./,,'me activities ;tnd (2) a disproportional increase of ('v() activity depending t'~ the ( ' r e positive mitochondria of reactive astroc.v'~e,;, involved in the removal of degeneration products [41. it is obvious thai more inforn~ation in required about the long-lasting dcvcncration in O M l , before ,,vc can achicxc a better tmdcrstanding of the t~curoplastic rcspotlsc in fascia tlcntata that follov~"; cntorhit~al lesions. Supported by l)euts,.:hc l:c~rschting,,gcmcinschafl
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260 8 Lewis, P.R. and Shute, C.C.D., The cholinergic limbic system: Projections to the hippocampal formation, medial cortex, nuclei of the ascending cholinergic reticular system, and the subfornical organ and supraoptic crest, Brain, 90 (1967) 521-540. 9 Lynch, G., Rose, G., Gall, C. and Cotman, C.W., The response of the dentate gyrus to partial deafferentation, in M. Santini (lEd.), Golgi Centennial Symposium, Raven Press, New York, 1975, pp. 305-318. 10 Lynch, G., Matthews, D., Mosko, S., Park, T. and Cotman, C.W., Induced acetylcholinesterase-rich layer in the dentate gyrus following entorhinal lesions, Brain Res., 42 (1977) 311-318. I ! Matthews, D.A., Cotman, C.W. and Lynch, G., An electron microscopic study of lesion-induced synaptogenesis in the dentate gyrus of the adult rat. !. Magnitude and time course of degeneration, Brain Res., 115 (1976) 1-21. 12 Matthews, D.A., Cotman, C.W. and Lynch, G., An electron microscopic study of lesion-induced synaptogenesis in the dentate gyrus of the adult rat. II. Re-appearance of morphologically normal synaptic contacts, Brain Res., 115 (1976) 23-41. 13 Nadler, J.V., Cotman, C.W. and Lynch, G.S., Biochemical plasticity of short-axon interneurons: Increased glutamate decarboxylase activity in the denervated area of the rat dentate gyrus following entorhinal lesion, Exp. Neurol., 45 (1974) 403-413. 14 Naik, N.T., Technical variations in Koelle's histochemical method for demonstrating cholinesterase activity, Quart. J. micr. Sci., 104 (1963) 89-100. 15 Oertel, W.H., Schmechel, D.E., Mugnaini, E., Tappaz, M.L. and Kopin, i.J., Immunocytochemical localization of glutamate decarboxylase in the rat cerebellum with a new antiserum, Neuroscience, 6 (1982) 2715-2735. 16 Ribak, C.E., "[he histochemical localization of cytochrome oxidase in the dentate gyrus of the rat hippocampus, Brain Res., 212 (1981) 169--174. 17 Ribak. C.I:.., Vaughn, J.E. and Saito, K., Immunocytochcmical localization of glutamic acid decarboxylase in neuronal somata follov.ing colchicine inhibition of axonal transport. B~'ain Res., 140 (1978) 315 --332. i 18 Ros~ier..I., Serum monospecifity" a prerequisite for reliable immunohistochemi,:al localization of neuronal markers including choline acetyltransferase, Neuroscience, 6 (1981)989--991. 19 Seligman, A.M., Karnovsky, M.J., Wasserkrug, H.L. and Hanker, J.S., Nondroplet ultrastructural 3emon~tration of cytochrome oxidase activity with a pol.vmerizing osiophilic reagent, diaminobenAdine (DAB), .I. Cell Biol., 38 (1968) 1-14. 2i) Sternberger, L.A., Immunochemistry, 2nd edn., John Wiley and Sons, Inc., New York, 1979, 354 pp. 21 Stornt-Mathisen, .I. and l'onnum, F., Quantitative histochenfistry of glutamate decarboxylase in the rat hippocampal region, J. Neurochem., 18 (1971) 1105 i!!1. 22 Storm-Mathi~en, J., Glutamate decarboxylase in the rat hippocampal region after lesions of the afferent fiber systems. Evidence that the enzyme is located in intrinsic neurons, Brain Res., 40 (1972) 215 -235. 23 Storm-Mathisen, J., Distribution of the components of the GABA system in neuronal tissue: cerebellum and hippocampus- effects of a.xotomy. In E. Roberts, T.N. Chase and D.B. Tower (Eds.), GABA in Nervous System Function, Raven Press, New York, 1976, pp. 149-168. 24 Wolff, J.R., Oertel, W. and Wolff, A., Covariance of inhibitory GABA-ergic and excitatory synapses in the visual cortex of rats, 77. Vers. Anat. Ges., Hannover, 1982, p. 29 (;~bstract). 25 Wong-Rilcy, M.T.T., Changes in the visual system of monocularly sutured or emtcleated cats demonstrable with c.vtochrome oxidase histochemistry, Brain Res., 171 (1979) 11-28.
255
Elsevier Scientific Publishers Ireland Ltd.
ENTORHINAL LESIONS RESULT IN SHRINKAGE OF THE OUTER MOLECULAR LAYER OF RAT DENTATE GYRUS LEADING SUBSEQUENTLY TO AN APPARENT INCREASE OF G L U T A M A T E DECARBOXYLASE A N D CYTOCHROME OXIDASE ACTIVITIES
G.P. WAGNER I'*, W.H. OERTEL z and J.R. WOLFF I"**
~Department of Anatomy, University of G6ttingen, Kreutzbergring 36, D-3400 G6ttingen and 2Neurological Clinic, University of Munich, D-8000 Miinchen (F.R.G.) (Received October 21st, 1982; Revised version received April 30th, 1983; Accepted June 10th, 1983)
Key words: neuroplasticity - dentate gyrus - entorhinal deaffe~'entation .- glutamate decarboxylase cytochrome oxidase - rat
In intact dentate gyrus, glutamatq decarboxylase immunoreactivity (GAD) and cytochrome oxida,.,e activity (CyO) showed different distribution patterns. Entorhinal lesions caused increases of GAD and CyO in the outer molecular layer (OML) of the ipsilateral side. Submicro~opical localization of these enzymes did not change, except for CyO labeling more astrocytic mitochondtia. The increase in numerical density of GAD puncta correlated quantitatively with shrinkage of OML, whereas in the whole molecular layer the number of GAD puncta remained unchanged. Hence, the localized increase of enzyme activities and lysosomes is apparently related to shrinkage of OML, but does not indicate plasticity of GABAergic neurons.
Unilateral destruction of rat entorhinal cortex causes degeneration of about 86070 of all synapses in the outer and middle zone of the molecular layer in the ipsilateral dentate gyrus [2]. The middle zone is rapidly reinnervated by commissural and associational fibers from the hippocampus proper, whereas the outer zone shows a complex and long-lasting reaction [9, l l, 19, 23]. Glutamate decarboxylase (GAD) is a specific marker for GABAergic interneurons in the hippocampus [22]. At 1-4 weeks after deafferentation of the dentate gyrus the enzyme activity was shown to have been increased in the molecular layer. This increase was interpreted as a transneuronal neuroplastic response of local GABAergic neurons [6, 13]. Since in striate cortex the distribution of GAD immunoreactivity covaries with that of cytochrome oxidase (CyO) activity (monkey [7], rat [24]), in the present study the long-term effects of removing entorhinai afferents on .-.he histochemical *Present address: Max-Planck-lnstitut fiir Virusforschung, Molekularbiologische aLbteilung, Spemannstr. 35, D-7400 Tiibingen, F.R.G. **Author for correspondence. 0304-3940/83/$ 03.00 Q 1983 Elsevier Scientific Publishers Ireland Ltd.
256
architecture of the dentate gyrus were reinvestigated. In 8 adult Sprague-Dawley rats the entorhinal cortex was unilaterally removed by aspiration under combined anesthesia with Ketanest and Rompun. After 120-150 days survival the animals v~-ere sacrificed by cardiac perfusion with 4% formaldehyde in phosphate buffer (0.1 M, pH 7.4). Serial 40 tLm sections through the septal region of the hippocampus ,,',ere prepared either with a Vibratome or a freezing microtome. Four unoperated animals and the contralateral hemispheres served as controls. The location of GADlike immunoreactivity [181 (from hereon termed GAD-immunoreactivity) was visualized by the unlabeled antibody enzyme method [20] using the GAD-antiserum 53 [151. Cv() activity was demonstrated by the diaminobenzidine method [20, 251. 1he activity of acetyicholinesterase (ACHE), a marker for septal afferents [8], was demonstrated by Koclle's acetylthiocholine method, modified according to Naik [4]. The distribution of lysosomes and terminal degeneration was visualized by Gallvas' impregnation method {51. G kl)-immunoreactive puncta were counted in rows of squares (400 ~,me, magnification 900 × ) which were oriented radially in the dorsal blade of the dentate gyru,,. Additionally. the thickness of the tnolecular layer and outer lnolecular laver were measured. ('ontralatcral to the entort'tinal lesion the binding pattern of G A D antibodies in the dentate gyrus (Fig. la) and the thickness of the molecular layer were identical to that in :.nimals without lesion [!, 171. This pattern also resembles the distribution
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Fig. I. l.rontal .,,ections through tile molecular and granular layers of the septal part of the dentate gyrus of the rat. 120-150 days after the etuorhinal lesion. l'he granular cell layer is located near the lower border of the picture,,, a and c ,,how the coutralateral side and b and d the ipsilateral side. a and b: the ,.ti,,ttlbutiotl ot glutamate decarbo\yla,,e immurloreacti',ity (GAD). c and d: tile distribution of cvtochtotne o\idalse (('y(')) activitv. Note the diflereucc in .,,raining intensity near the hippocampal fissure (~ hire at ro,,~ head:) of tile normal dentate g.~ ru.s tat and c). [!ntorhinal lesions induced a laminated pattern of L;AI) and (.'v(} in the molecular l,:~cr {b and d~.
237
of GAD enzyme activity as determined by biochemical methods [13, 21 ] and of binding sites for GABA [21. On the other hand, ipsilateral to entorhinal lesions, the distribution of GAD immunoreactivity was changed, even after 4 - 5 months. Instead of a shallow gradient of staining intensity a clearly bilaminar pattern was seen within the molecular layer (Fig. la, b). Whereas staining intensity in the 'inner molecular layer' (IML) remained unchanged, it had strongly increased in the 'outer molecular layer' (OML). Electron microscopically GAD immunoreactivity was almost exclusively located in axon terminals (Fig. 2a, b) and only occasionally found in axons, dendrites and neuronal somata. Quantitative evaluation of the numerical density of GAD-positive points in the dorsal blade of the deafferented dentate gyrus confirmed the difference between OML and IML. In the IML the numerical density was not significantly different between deafferented and control dentate gyrus, while in the ipsilateral OML the density was 54.1070 (S.D. + 1.7) higher than in the contralateral OML. Since the ipsilateral OML was, however, 53.2°70 (S.D. + 0.96) thinner than the contralateral OML, there was no significant increase of the total number of GAD-positive points in the whole molecular layer (P <0.005 according to sign test, n = 8). The AChE activity (not shown) matched the changed distribution pattern of GAD immunoreactivity also at much longer survival times than shown by Lynch et al. [10]. Similarly, the present study revealed that high concentrations of lysosomes persist in OML even 5 months after deafferentation (not shown; see also ref. 1 1). The distribution of CyO activity in the dentate gyrus contralateral to the lesion (Fig. lc) resembled that of CyO in intact animals (see also ref. 16), but not that of GAD (Fig. la). Hence, in the intact archicortex of rat, the distribution patterns of CyO and GAD do not seem to correlate (Fig. la, c) as in the intact neocortex [7, 24]. However, after entorhinal lesions the distribution of CyO activity resembled strikingly the bilaminar pattern of GAD (Fig. lb, d). Electron microscopically CyO activity in the ipsilateral OML was mainly found in mitochondria of dendrites, while it was low in mitochondria of presynaptic elements (arrows in Fig. 2c), just as in intact animals [16]. In addition, some CyO activity was seen in mitochondria of certain astrocytes (Fig. 2d) which, according to structural criteria (plump processes with numerous ribosomes, filaments and glycogen granules), often resembled reactive astrocytes. About 1 week after antorhinal lesions GAD immunoreactivity begins to increase in the OML of dentate gyrus [6]. According to biochemical data GAD activity increases up to 33% after about 3 months [19], while the present immunohistochemical data show an increase of 54% by 5 months. This suggests that the GAD increase is based on a long-lasting process which might even be progressive. An increase of GABAergic innervation, generally assumed to be inhibitory in nature [1], cannot be explained as a compensation for a loss of excitatory input. Our data are also not compatible with the idea that local GABAergic neurons respond
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to the destruction of the perforant pathway with sprouting or redistribution between IML and OML of their axon tern'finals [6, 13]. Rather, it became evident that the overall number of GABAergic terminals remains constant in the molecular layer, i.e. their density is not regulated down to control values during thc shrinkage of OML. This in contrary t o the tendency of excitatory synapses which re-establish normal numerical densities in most cases of neuroplastic responses studied (for rcvicv, see ref. 3). The coindicence between augmented CvO activity and GAI) immu~oreacti,,itv following entorhinal lesions is not easily reconciled with the discrepancies of distribution in the intact dentate gyrus. Electron microscopy demonstrated that Cy(~ is not a marker for GAD-positive profilcs. Hcncc, a direct causal relationship does not seem to exist. Correlated changes in the distribution of s~tcll diverse enzymes as ACHE, CyO, G A D and of lysosomcs may, ho~evcr, be duc to change in the regulation of tissut" volume, i:actors in,~oixcd in this regulation are largely unknoven. Shrinkage and degeneration could explain both (!)correlated increases of several c~./,,'me activities ;tnd (2) a disproportional increase of ('v() activity depending t'~ the ( ' r e positive mitochondria of reactive astroc.v'~e,;, involved in the removal of degeneration products [41. it is obvious thai more inforn~ation in required about the long-lasting dcvcncration in O M l , before ,,vc can achicxc a better tmdcrstanding of the t~curoplastic rcspotlsc in fascia tlcntata that follov~"; cntorhit~al lesions. Supported by l)euts,.:hc l:c~rschting,,gcmcinschafl
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