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Alterations of AMPA-selected glutamate subtype immunoreactivity in the dentate gyrus after perforant pathway lesion
Brain Research 768 Ž1997. 354–360
Short communication
Alterations of AMPA-selected glutamate subtype immunoreactivity in the dentate gyrus after perforant pathway lesion Katsuyoshi Mizukami, Amanda Mishizin, Milos D. Ikonomovic, Roxanne Sheffield, David M. Armstrong ) Neuroscience Research Center, MCP l Hahnemann School of Medicine, Allegheny-Campus, 320 East North AÕenue, Pittsburgh, PA 15212, USA Accepted 17 June 1997
Abstract Immunocytochemical techniques were employed to examine the changes in immunolabeling of the a-amino-3-hydroxy-5-methyl-4isoaxolepropionate ŽAMPA. receptor subunits GluR1 and GluR2r3 within the dentate gyrus 1, 3, 7, 14, 30, and 90 days after a unilateral perforant pathway lesion in the rat brain. Completeness of the lesion was confirmed following examination of Nissl-stained tissue sections at all times post-lesion and acetylcholinesterase ŽAChE.-stained sections 14, 30 and 90 days post-lesion, the latter providing evidence of compensatory sprouting of cholinergic fibers in the outer molecular layer of the dentate gyrus. Compared to the non-lesioned hippocampus there was no difference in the staining pattern of AMPA receptor subunits in the dentate gyrus of the deafferented hippocampus 1, 3, 7 and 14 days following lesioning of the perforant pathway. In contrast, 30 and 90 days post-lesion, GluR1 immunolabeling was increased in the outer molecular layer of the dentate gyrus Ži.e., deafferented zone. ipsilateral to lesion. Likewise, GluR2r3 immunolabeling was increased within the same region although the intensity of the response was less than that which was observed for GluR1. These data suggest that the loss of the perforant pathway fibers results in a compensatory increase in GluR1 and to a lesser extent GluR2r3 immunolabeling of the outer molecular layer at 30 and 90 days post-lesion and further suggest that AMPA receptor subunits play a role in perforant pathway signal transduction. q 1997 Elsevier Science B.V. Keywords: AMPA; Receptor; Perforant pathway lesion; Immunocytochemistry; Up-regulation; Hippocampus
Alzheimer’s disease ŽAD. brains are characterized by the presence of senile plaques and neurofibrillary tangles as well as a loss of neurons and synapses. Despite significant pathology, recent research has demonstrated that AD brains are capable of considerable plasticity even during the terminal stages of the disease. For example, the sprouting of AChE-containing fibers into the outer molecular layer of the dentate gyrus has been recognized for many years and is believed to occur in response to disease-related cellular damage of hippocampal-projection neurons in the entorhinal cortex w6,8x. Our previous study of AD brains demonstrated a disease-related increase in immunolabeling of GluR1 and GluR2r3 in regions of the dentate gyrus corresponding to the termination sites of the perforant pathway w2,9x. We hypothesize that this increase in immunolabeling is compensatory in nature and occurs in response to the loss of glutamatergic input via the perforant pathway. In the present study, we examined this
)
Corresponding author. Fax: q1 Ž412. 359-4364.
0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 7 9 7 - X
hypothesis by employing immunocytochemical techniques and examining within the dentate gyrus the alterations in the staining pattern of the AMPA receptor subunits GluR1; GluR2r3 following unilateral lesions of the perforant pathway. These latter subunits, in contrast to GluR4, are abundantly expressed in rat hippocampus w10x, thus are the focus of the current investigation. Male Sprague–Dawley rats weighing approximately 250 g were anesthetized with 360 mgrkg of chloral hydrate and placed in a stereotaxic apparatus. The skull was exposed and a hole measuring approximately 4.0 mm = 4.0 mm was drilled at a position corresponding to 1.0–5.0 mm right of the midline and 0.0–4.0 mm anterior to lambda. The dura was removed and the underlying cortex and corpus collosum were suctioned using a fine tip blunt needle attached to a vacuum pump. Under visual guidance, the presubiculum and angular bundle, including the fibers of the perforant pathway, were then suctioned. Subsequently, the cavity was packed with Gelfoam ŽUpjohn., the scalp closed with Autoclips ŽRoboz., and the rats returned to their home cages. At 1, 3, 7, 14, 30, and 90 days
K. Mizukami et al.r Brain Research 768 (1997) 354–360
post-lesion Žeach n s 6., animals were killed humanely under deep anesthesia by transcardial perfusion with 4% paraformaldehyde in 100 mM phosphate buffer. Following 24 h post-fixation the brains were placed into 30% sucrose for 3 days. Brains were cut to a thickness of 40 m m in a horizontal plain and stored in cryoprotectant solution until processed for immunocytochemistry as described w1,2,9x. Throughout the hippocampus, at least four near-adjacent sections were immunolabeled using polyclonal antibodies against GluR1 or GluR2r3 receptor subunits ŽChemicon. w17x. Both primary antibodies were diluted 1 : 4000 in Tris-saline containing 1% goat serum and 0.25% Triton X-100. The final dilution of the antibody was determined following experiments in which a number of dilutions were tested Ži.e., dilution curve.. It was the goal of these latter experiments to determine a dilution which allowed for adequate delineation of hippocampal subregions but was not so robust to obscure our ability to detect subtle differences in staining intensity. Importantly, sections representative of each of the six survival times were processed together in order to control for any variability in the immunocytochemical procedure. As a control for nonspecific staining, sections were incubated with initial incubation media minus the primary antibody and otherwise processed as described. For each brain at least four sections were stained for Nissl substance or processed for AChE histochemistry, in order to verify the extent of the lesion. Controls consisted of the contralateral Ži.e., non-lesioned hippocampus. as well as sham-operated animals Ž n s 1rtime point.. The latter were characterized by lesions of the overlying cortex but without involvement of the angular bundle. For each sham-operated brain, at least four sections were immunolabeled using GluR1 and GluR2r3 antibodies. In controls, intense GluR1 and GluR2r3 immunolabeling was observed throughout the neuropil of the molecular layer of the dentate gyrus. Notably, this layer was among the most intensely labeled brain region within these horizontal sections. In controls, the staining intensity within the inner and outer portions of the molecular layer was comparable and thus it was impossible to distinguish one portion from another. In lesioned brains, the completeness of our lesions was assessed following examination of Nissl-stained tissue sections 1, 3, 7, 14, 30 and 90 days post-lesion, and by the presence of intense AChE fibers within the outer molecular layer of the dentate gyrus 14, 30, 90 days post-lesion ŽFig. 1c,d.. Only those rats with complete lesions of the perforant pathway were included in our analysis. Examination of rats 1, 3, 7 and 14 days post-lesion revealed a staining pattern in the lesioned dentate gyrus which was no different from that observed in the contralateral Ži.e., non-lesioned. dentate gyrus and in the dentate gyrus of sham-operated rats. In contrast, at 30 days postlesion, we observed in the outer portion of the molecular layer Ži.e., the projection zone of the perforant pathway. an
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increase in the intensity of GluR1 immunolabeling relative to the inner portion. The increase in immunolabeling within a restricted region of the molecular layer resulted in our ability to readily differentiate between the inner and outer portions of the dentate gyrus. GluR2r3 immunolabeling was also more intense within the outer molecular layer compared to the inner portion although the extent to which these two regions differed was more modest than that observed for GluR1. Of importance, the staining intensity within the inner portion of the molecular layer appeared no different than that seen in inner and outer portions of the molecular layer of contralateral dentate gyrus and from both dentate gyri of the sham-operated rats. Ninety days post-lesion, we observed a similar if not more robust pattern of immunolabeling as was seen at 30 days ŽFig. 2c,d, Fig. 3c,d.. Like the 30 days post-lesion rats the increase in GluR1 immunolabeling was more pronounced than for GluR2r3 ŽFig. 2c,d.. No changes in the pattern or intensity of immunolabeling were observed within any other brain region at any of the times points examined. Collectively, these data support a lesion-induced increase in GluR1 and GluR2r3 immunolabeling within the outer portion of the molecular layer 30 and 90 days following lesioning of the perforant pathway. In interpreting these data it is important to recognize that perforant pathway lesion does result in some shrinkage of the molecular layer of the dentate gyrus. This shrinkage could account for increases in immunolabeling considering that the reduction in tissue volume likely results in a concomitant increase in packing density of neuronal elements including AMPA-labeled fibers. However, a number of factors argue against this latter possibility. First, previous studies demonstrate that the majority of the shrinkage of the dentate gyrus occurs within the first 2 weeks post-lesion w3,4x. During the first 2 weeks post-lesion we observed no changes in pattern or intensity of immunolabeling for GluR1 or GluR2r3. Rather the most pronounced effects were observed weeks, if not months after the majority of the shrinkage has occurred. Notably, the increase in immunolabeling occurs in the outer portion of the molecular layer and corresponds to the principal site of deafferentation of glutamatergic perforant pathway fibers. An additional factor contributing to changes in the staining intensity is the lesion-induced proliferation of fibrous astrocytes within the deafferented zone w5x. These data, together with the observation that AMPA receptor subunits are present on astrocytes w11x, suggest that the lesioned-induced proliferation of AMPA-bearing astrocytes might be contributing to some of the observed increase in GluR1 and GluR2r3 labeling in the outer molecular layer. However, arguing against this latter notion is the facts that astrocytic proliferation is observed at times much earlier than those of peak AMPA receptor immunolabeling Ži.e., by 4 days post-lesion., and that an increase in GFAP immunoreactivity within the deafferented zone disappears by 30 days post-lesion w5x. Moreover, work in our labora-
356 K. Mizukami et al.r Brain Research 768 (1997) 354–360 Fig. 1. Photomicrographs showing near adjacent hippocampal sections of rat following 90 day survival of perforant pathway lesion. a, b illustrate the intact Ži.e., contralateral. hippocampus while c, d illustrate the lesioned Ži.e., ipsilateral. hippocampus. Nissl-stained tissue sections Ža, c. reveal the cytoarchitecture of the hippocampal formation and entorhinal cortex and the location and extent of the lesion. Perforant pathway ablation was further confirmed following AChE histochemistry and the demonstration of intense AChE fibers within the outer molecular layer of the dentate gyrus Žd..
K. Mizukami et al.r Brain Research 768 (1997) 354–360 Fig. 2. Photomicrographs showing GluR1 immunolabeling in the hippocampus contralateral Ža, b. and ipsilateral Žc, d. to the lesion 90 days post-lesion. Within the outer molecular layer, GluR1 immunolabeling is more robust than in the inner portion of the molecular layer Žc, d.. 357
358 K. Mizukami et al.r Brain Research 768 (1997) 354–360 Fig. 3. Photomicrographs showing GluR2r3 immunolabeling in the hippocampus contralateral Ža, b. and ipsilateral Žc, d. to the lesion 90 days post-lesion. Within the molecular layer GluR2r3 immunolabeling is more robust in the outer portion compared to the inner portion.
K. Mizukami et al.r Brain Research 768 (1997) 354–360
tory Žunpublished observation. suggests that GluR4, rather than GluR1 and GluR2r3, is largely associated with reactive astrocytes. Although the precise mechanism underlying the changes in the intensity of GluR1 and GluR2r3 within the outer portion of the molecular layer is not entirely clear, the fact that the most pronounced changes in the intensity of immunolabeling occur within a region of the dentate gyrus corresponding to the zone of principle deafferentation suggests that this response is occurring as a result of the loss of glutamatergic input following perforant pathway lesions. In the present study, the most robust changes in immunolabeling occurred after relatively long survival times Ži.e., 30 and 90 days.. In interpreting these data it is important to bear in mind that the majority of the AMPA receptor subunits in the molecular layer are present on the dendrites of granule cells w14,15x and that the outer portions of these dendrites are post-synaptic to glutamatergic perforant pathway fibers. Following removal of perforant pathway fibers the granule cells undergo morphologic changes characterized by reductions in the total length of their dendritic trees, decreases in dendritic diameter, and declines of total spine density in the denervated zone. These alterations are, however, largely transient and by 30 days post-lesion the dendrites resume much of their pre-lesion appearance w3x. Notably, despite the marked plasticity of granule cell dendrites, including their return to normal pre-lesion morphology, the termination zone of the perforant pathway remains deficient in glutamate for as long as 8 months post-lesion w13x. These data suggest that even following extensive reinnervation of the termination zone of the perforant pathway the replacement boutons are deplete in their use of glutamate as their neurotransmitter. Thus, in light of the persistent loss of glutamatergic input, it is only reasonable that the denervated dendrites attempt to compensate for the loss of glutamate by up-regulating post-synaptic glutamate receptors. These results are consistent with those of Ulas et al. w16x who, using autoradiographic techniques, showed that following perforant pathway lesions the density of quisqualate receptors in the ipsilateral molecular layer was increased 30 and 60 days post-lesion. Collectively, these data suggest that the increase in receptor level is likely compensatory in nature and is occurring in an effort to maintain an appropriate level of excitatory input onto dentate gyrus granule cells and in so doing, contributes to the functional maintenance of these cells w2,9x. In addition to their localization on dendrites and soma of granule cells, it is known that AMPA receptors are present on the cell bodies and processes of GABAergic interneurons within the dentate gyrus w14x. Since GABAergic fibers also have been documented to sprout following perforant pathway lesion w7,12x, it too is reasonable to consider that these fibers are a source of innervation of the molecular layer and also may be contributing to the increase in GluR immunolabeling.
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In the present study, we observed a more pronounced increase in GluR1 immunolabeling than GluR2r3 following perforant pathway lesion. These observations are consistent with our previous studies of human brains in which we observed a greater intensity of GluR1 immunolabeling in the molecular layer compared to GluR2r3 w2,9x. Since excitation of the distal apical dendrites of the principal cells by entorhinal input may be accompanied by calcium influx into the dendrites w10x, it is noteworthy that those subunits which contribute to the passage of Ca2q through the ion channel Ži.e., GluR1. display a more robust response than those subunits which impede the flow of calcium Ži.e., GluR2r3.. Thus, the up-regulated GluR1 to a greater extent than GluR2r3 compensates effectively for deficit of glutamatergic input following perforant lesion. In summary, our results suggest that the loss of perforant pathway fibers results in the compensatory up-regulation of postsynaptic glutamate receptor subunits GluR1 and GluR2r3 30 and 90 days post-lesion. Furthermore, our findings support the notion that in AD the increase in immunolabeling within the termination zone of the perforant pathway may too be in response to the loss of glutamatergic perforant pathway fibers.
Acknowledgements This work was supported by NIH grant AG 08206 and a grant provided by the Allegheny Singer Research Institute, Pittsburgh, PA.
References w1x D.M. Armstrong, M.D. Ikonomovic, R. Sheffield, R.J. Wenthold, AMPA-selective glutamate receptor subtype immunoreactivity in the entorhinal cortex of non-demented elderly and patients with Alzheimer’s disease, Brain Res. 639 Ž1994. 207–216. w2x D.M. Armstrong, M.D. Ikonomovic, AMPA-selective glutamate receptor subtype immunoreactivity in the hippocampal dentate gyrus of patients with Alzheimer disease, Mol. Chem. Neuropathol. 28 Ž1996. 59–64. w3x A. Caceres, O. Steward, Dendritic reorganization in the denervated dentate gyrus of the rat following entorhinal cortical lesions: a Golgi and electron microscopic analysis, J. Comp. Neurol. 214 Ž1983. 387–403. w4x J.M. Conner, B. Fass-Holmes, S. Varon, Changes in nerve growth factor immunoreactivity following entorhinal cortex lesions: possible molecular mechanism regulating cholinergic sprouting, J. Comp. Neurol. 345 Ž1994. 409–418. w5x F.H. Gage, P. Olejniczak, D.M. Armstrong, Astrocytes are important for sprouting in the septohippocampal circuit, Exp. Neurol. 102 Ž1988. 2–13. w6x J.W. Geddes, D.T. Monaghan, C.W. Cotman, I.T. Lott, R.C. Kim, H.G. Chui, Plasticity of hippocampal circuitry in Alzheimer’s disease, Science 230 Ž1985. 1179–1181. w7x D. Goldowitz, S.R. Vincent, J.Y. Wu, T. Hokfelt, Immunohisto¨ chemical demonstration of plasticity in GABA neurons of the adult rat dentate gyrus, Brain Res. 238 Ž1982. 413–420. w8x B.T. Hyman, L.J. Kromer, G.W. Van Hoesen, Reinnervation of the
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K. Mizukami et al.r Brain Research 768 (1997) 354–360 hippocampal perforant pathway zone in Alzheimer’s disease, Ann. Neurol. 21 Ž1987. 259–267. M.D. Ikonomovic, R. Sheffield, D.M. Armstrong, AMPA-selective glutamate receptor subtype immunoreactivity in the hippocampal formation of patients with Alzheimer’s disease, Hippocampus 5 Ž1995. 469–486. C. Leranth, Z. Szeidemann, M. Hsu, G. Buzsaki, ´ AMPA receptors in the rat and primate hippocampus: a possible absence of GluR2r3 subunits in most interneurons, Neuroscience 70 Ž1996. 631–652. C. Matute, K. Gutierrez-Igarza, C. Rıo, ´ ´ R. Miledi, Glutamate receptors in astrocytic end-feet, NeuroReport 5 Ž1994. 1205–1208. J.V. Nadler, C.W. Cotman, G.S. Lynch, Biochemical plasticity of short-axon interneuron: increased glutamate decarboxylase activity in the denervated area of rat dentate gyrus following entorhinal lesion, Exp. Neurol. 45 Ž1974. 403–413. J.V. Nadler, E.M. Smith, Perforant path lesion depletes glutamate
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content of fascia dentata synaptosomes, Neurosci. Lett. 25 Ž1981. 275–280. R.S. Petralia, R.J. Wenthold, Light and electron immunocytochemical localization of AMPA-selective glutamate receptors in the rat brain, J. Comp. Neurol. 318 Ž1992. 329–354. S.J. Siegel, W.G. Jansen, J.W. Tullai, S.W. Rogers, T. Moran, S.F. Heinemann, J.H. Morrison, Distribution of the excitatory amino acid receptor subunits GluR2Ž4. in monkey hippocampus and colocalization with subunits GluR5-7 and NMDAR1, J. Neurosci. 15 Ž1995. 2707–2719. J. Ulas, D.T. Monaghan, C.W. Cotman, Plastic response of hippocampal excitatory amino acid receptors to deafferentation and reinnervation, Neuroscience 34 Ž1990. 9–17. R.J. Wenthold, N. Yokotani, K. Doi, K. Wada, Immunochemical characterization of the non-NMDA glutamate receptor using subunit-specific antibodies, J. Biol. Chem. 267 Ž1992. 501–507.
Short communication
Alterations of AMPA-selected glutamate subtype immunoreactivity in the dentate gyrus after perforant pathway lesion Katsuyoshi Mizukami, Amanda Mishizin, Milos D. Ikonomovic, Roxanne Sheffield, David M. Armstrong ) Neuroscience Research Center, MCP l Hahnemann School of Medicine, Allegheny-Campus, 320 East North AÕenue, Pittsburgh, PA 15212, USA Accepted 17 June 1997
Abstract Immunocytochemical techniques were employed to examine the changes in immunolabeling of the a-amino-3-hydroxy-5-methyl-4isoaxolepropionate ŽAMPA. receptor subunits GluR1 and GluR2r3 within the dentate gyrus 1, 3, 7, 14, 30, and 90 days after a unilateral perforant pathway lesion in the rat brain. Completeness of the lesion was confirmed following examination of Nissl-stained tissue sections at all times post-lesion and acetylcholinesterase ŽAChE.-stained sections 14, 30 and 90 days post-lesion, the latter providing evidence of compensatory sprouting of cholinergic fibers in the outer molecular layer of the dentate gyrus. Compared to the non-lesioned hippocampus there was no difference in the staining pattern of AMPA receptor subunits in the dentate gyrus of the deafferented hippocampus 1, 3, 7 and 14 days following lesioning of the perforant pathway. In contrast, 30 and 90 days post-lesion, GluR1 immunolabeling was increased in the outer molecular layer of the dentate gyrus Ži.e., deafferented zone. ipsilateral to lesion. Likewise, GluR2r3 immunolabeling was increased within the same region although the intensity of the response was less than that which was observed for GluR1. These data suggest that the loss of the perforant pathway fibers results in a compensatory increase in GluR1 and to a lesser extent GluR2r3 immunolabeling of the outer molecular layer at 30 and 90 days post-lesion and further suggest that AMPA receptor subunits play a role in perforant pathway signal transduction. q 1997 Elsevier Science B.V. Keywords: AMPA; Receptor; Perforant pathway lesion; Immunocytochemistry; Up-regulation; Hippocampus
Alzheimer’s disease ŽAD. brains are characterized by the presence of senile plaques and neurofibrillary tangles as well as a loss of neurons and synapses. Despite significant pathology, recent research has demonstrated that AD brains are capable of considerable plasticity even during the terminal stages of the disease. For example, the sprouting of AChE-containing fibers into the outer molecular layer of the dentate gyrus has been recognized for many years and is believed to occur in response to disease-related cellular damage of hippocampal-projection neurons in the entorhinal cortex w6,8x. Our previous study of AD brains demonstrated a disease-related increase in immunolabeling of GluR1 and GluR2r3 in regions of the dentate gyrus corresponding to the termination sites of the perforant pathway w2,9x. We hypothesize that this increase in immunolabeling is compensatory in nature and occurs in response to the loss of glutamatergic input via the perforant pathway. In the present study, we examined this
)
Corresponding author. Fax: q1 Ž412. 359-4364.
0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 7 9 7 - X
hypothesis by employing immunocytochemical techniques and examining within the dentate gyrus the alterations in the staining pattern of the AMPA receptor subunits GluR1; GluR2r3 following unilateral lesions of the perforant pathway. These latter subunits, in contrast to GluR4, are abundantly expressed in rat hippocampus w10x, thus are the focus of the current investigation. Male Sprague–Dawley rats weighing approximately 250 g were anesthetized with 360 mgrkg of chloral hydrate and placed in a stereotaxic apparatus. The skull was exposed and a hole measuring approximately 4.0 mm = 4.0 mm was drilled at a position corresponding to 1.0–5.0 mm right of the midline and 0.0–4.0 mm anterior to lambda. The dura was removed and the underlying cortex and corpus collosum were suctioned using a fine tip blunt needle attached to a vacuum pump. Under visual guidance, the presubiculum and angular bundle, including the fibers of the perforant pathway, were then suctioned. Subsequently, the cavity was packed with Gelfoam ŽUpjohn., the scalp closed with Autoclips ŽRoboz., and the rats returned to their home cages. At 1, 3, 7, 14, 30, and 90 days
K. Mizukami et al.r Brain Research 768 (1997) 354–360
post-lesion Žeach n s 6., animals were killed humanely under deep anesthesia by transcardial perfusion with 4% paraformaldehyde in 100 mM phosphate buffer. Following 24 h post-fixation the brains were placed into 30% sucrose for 3 days. Brains were cut to a thickness of 40 m m in a horizontal plain and stored in cryoprotectant solution until processed for immunocytochemistry as described w1,2,9x. Throughout the hippocampus, at least four near-adjacent sections were immunolabeled using polyclonal antibodies against GluR1 or GluR2r3 receptor subunits ŽChemicon. w17x. Both primary antibodies were diluted 1 : 4000 in Tris-saline containing 1% goat serum and 0.25% Triton X-100. The final dilution of the antibody was determined following experiments in which a number of dilutions were tested Ži.e., dilution curve.. It was the goal of these latter experiments to determine a dilution which allowed for adequate delineation of hippocampal subregions but was not so robust to obscure our ability to detect subtle differences in staining intensity. Importantly, sections representative of each of the six survival times were processed together in order to control for any variability in the immunocytochemical procedure. As a control for nonspecific staining, sections were incubated with initial incubation media minus the primary antibody and otherwise processed as described. For each brain at least four sections were stained for Nissl substance or processed for AChE histochemistry, in order to verify the extent of the lesion. Controls consisted of the contralateral Ži.e., non-lesioned hippocampus. as well as sham-operated animals Ž n s 1rtime point.. The latter were characterized by lesions of the overlying cortex but without involvement of the angular bundle. For each sham-operated brain, at least four sections were immunolabeled using GluR1 and GluR2r3 antibodies. In controls, intense GluR1 and GluR2r3 immunolabeling was observed throughout the neuropil of the molecular layer of the dentate gyrus. Notably, this layer was among the most intensely labeled brain region within these horizontal sections. In controls, the staining intensity within the inner and outer portions of the molecular layer was comparable and thus it was impossible to distinguish one portion from another. In lesioned brains, the completeness of our lesions was assessed following examination of Nissl-stained tissue sections 1, 3, 7, 14, 30 and 90 days post-lesion, and by the presence of intense AChE fibers within the outer molecular layer of the dentate gyrus 14, 30, 90 days post-lesion ŽFig. 1c,d.. Only those rats with complete lesions of the perforant pathway were included in our analysis. Examination of rats 1, 3, 7 and 14 days post-lesion revealed a staining pattern in the lesioned dentate gyrus which was no different from that observed in the contralateral Ži.e., non-lesioned. dentate gyrus and in the dentate gyrus of sham-operated rats. In contrast, at 30 days postlesion, we observed in the outer portion of the molecular layer Ži.e., the projection zone of the perforant pathway. an
355
increase in the intensity of GluR1 immunolabeling relative to the inner portion. The increase in immunolabeling within a restricted region of the molecular layer resulted in our ability to readily differentiate between the inner and outer portions of the dentate gyrus. GluR2r3 immunolabeling was also more intense within the outer molecular layer compared to the inner portion although the extent to which these two regions differed was more modest than that observed for GluR1. Of importance, the staining intensity within the inner portion of the molecular layer appeared no different than that seen in inner and outer portions of the molecular layer of contralateral dentate gyrus and from both dentate gyri of the sham-operated rats. Ninety days post-lesion, we observed a similar if not more robust pattern of immunolabeling as was seen at 30 days ŽFig. 2c,d, Fig. 3c,d.. Like the 30 days post-lesion rats the increase in GluR1 immunolabeling was more pronounced than for GluR2r3 ŽFig. 2c,d.. No changes in the pattern or intensity of immunolabeling were observed within any other brain region at any of the times points examined. Collectively, these data support a lesion-induced increase in GluR1 and GluR2r3 immunolabeling within the outer portion of the molecular layer 30 and 90 days following lesioning of the perforant pathway. In interpreting these data it is important to recognize that perforant pathway lesion does result in some shrinkage of the molecular layer of the dentate gyrus. This shrinkage could account for increases in immunolabeling considering that the reduction in tissue volume likely results in a concomitant increase in packing density of neuronal elements including AMPA-labeled fibers. However, a number of factors argue against this latter possibility. First, previous studies demonstrate that the majority of the shrinkage of the dentate gyrus occurs within the first 2 weeks post-lesion w3,4x. During the first 2 weeks post-lesion we observed no changes in pattern or intensity of immunolabeling for GluR1 or GluR2r3. Rather the most pronounced effects were observed weeks, if not months after the majority of the shrinkage has occurred. Notably, the increase in immunolabeling occurs in the outer portion of the molecular layer and corresponds to the principal site of deafferentation of glutamatergic perforant pathway fibers. An additional factor contributing to changes in the staining intensity is the lesion-induced proliferation of fibrous astrocytes within the deafferented zone w5x. These data, together with the observation that AMPA receptor subunits are present on astrocytes w11x, suggest that the lesioned-induced proliferation of AMPA-bearing astrocytes might be contributing to some of the observed increase in GluR1 and GluR2r3 labeling in the outer molecular layer. However, arguing against this latter notion is the facts that astrocytic proliferation is observed at times much earlier than those of peak AMPA receptor immunolabeling Ži.e., by 4 days post-lesion., and that an increase in GFAP immunoreactivity within the deafferented zone disappears by 30 days post-lesion w5x. Moreover, work in our labora-
356 K. Mizukami et al.r Brain Research 768 (1997) 354–360 Fig. 1. Photomicrographs showing near adjacent hippocampal sections of rat following 90 day survival of perforant pathway lesion. a, b illustrate the intact Ži.e., contralateral. hippocampus while c, d illustrate the lesioned Ži.e., ipsilateral. hippocampus. Nissl-stained tissue sections Ža, c. reveal the cytoarchitecture of the hippocampal formation and entorhinal cortex and the location and extent of the lesion. Perforant pathway ablation was further confirmed following AChE histochemistry and the demonstration of intense AChE fibers within the outer molecular layer of the dentate gyrus Žd..
K. Mizukami et al.r Brain Research 768 (1997) 354–360 Fig. 2. Photomicrographs showing GluR1 immunolabeling in the hippocampus contralateral Ža, b. and ipsilateral Žc, d. to the lesion 90 days post-lesion. Within the outer molecular layer, GluR1 immunolabeling is more robust than in the inner portion of the molecular layer Žc, d.. 357
358 K. Mizukami et al.r Brain Research 768 (1997) 354–360 Fig. 3. Photomicrographs showing GluR2r3 immunolabeling in the hippocampus contralateral Ža, b. and ipsilateral Žc, d. to the lesion 90 days post-lesion. Within the molecular layer GluR2r3 immunolabeling is more robust in the outer portion compared to the inner portion.
K. Mizukami et al.r Brain Research 768 (1997) 354–360
tory Žunpublished observation. suggests that GluR4, rather than GluR1 and GluR2r3, is largely associated with reactive astrocytes. Although the precise mechanism underlying the changes in the intensity of GluR1 and GluR2r3 within the outer portion of the molecular layer is not entirely clear, the fact that the most pronounced changes in the intensity of immunolabeling occur within a region of the dentate gyrus corresponding to the zone of principle deafferentation suggests that this response is occurring as a result of the loss of glutamatergic input following perforant pathway lesions. In the present study, the most robust changes in immunolabeling occurred after relatively long survival times Ži.e., 30 and 90 days.. In interpreting these data it is important to bear in mind that the majority of the AMPA receptor subunits in the molecular layer are present on the dendrites of granule cells w14,15x and that the outer portions of these dendrites are post-synaptic to glutamatergic perforant pathway fibers. Following removal of perforant pathway fibers the granule cells undergo morphologic changes characterized by reductions in the total length of their dendritic trees, decreases in dendritic diameter, and declines of total spine density in the denervated zone. These alterations are, however, largely transient and by 30 days post-lesion the dendrites resume much of their pre-lesion appearance w3x. Notably, despite the marked plasticity of granule cell dendrites, including their return to normal pre-lesion morphology, the termination zone of the perforant pathway remains deficient in glutamate for as long as 8 months post-lesion w13x. These data suggest that even following extensive reinnervation of the termination zone of the perforant pathway the replacement boutons are deplete in their use of glutamate as their neurotransmitter. Thus, in light of the persistent loss of glutamatergic input, it is only reasonable that the denervated dendrites attempt to compensate for the loss of glutamate by up-regulating post-synaptic glutamate receptors. These results are consistent with those of Ulas et al. w16x who, using autoradiographic techniques, showed that following perforant pathway lesions the density of quisqualate receptors in the ipsilateral molecular layer was increased 30 and 60 days post-lesion. Collectively, these data suggest that the increase in receptor level is likely compensatory in nature and is occurring in an effort to maintain an appropriate level of excitatory input onto dentate gyrus granule cells and in so doing, contributes to the functional maintenance of these cells w2,9x. In addition to their localization on dendrites and soma of granule cells, it is known that AMPA receptors are present on the cell bodies and processes of GABAergic interneurons within the dentate gyrus w14x. Since GABAergic fibers also have been documented to sprout following perforant pathway lesion w7,12x, it too is reasonable to consider that these fibers are a source of innervation of the molecular layer and also may be contributing to the increase in GluR immunolabeling.
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In the present study, we observed a more pronounced increase in GluR1 immunolabeling than GluR2r3 following perforant pathway lesion. These observations are consistent with our previous studies of human brains in which we observed a greater intensity of GluR1 immunolabeling in the molecular layer compared to GluR2r3 w2,9x. Since excitation of the distal apical dendrites of the principal cells by entorhinal input may be accompanied by calcium influx into the dendrites w10x, it is noteworthy that those subunits which contribute to the passage of Ca2q through the ion channel Ži.e., GluR1. display a more robust response than those subunits which impede the flow of calcium Ži.e., GluR2r3.. Thus, the up-regulated GluR1 to a greater extent than GluR2r3 compensates effectively for deficit of glutamatergic input following perforant lesion. In summary, our results suggest that the loss of perforant pathway fibers results in the compensatory up-regulation of postsynaptic glutamate receptor subunits GluR1 and GluR2r3 30 and 90 days post-lesion. Furthermore, our findings support the notion that in AD the increase in immunolabeling within the termination zone of the perforant pathway may too be in response to the loss of glutamatergic perforant pathway fibers.
Acknowledgements This work was supported by NIH grant AG 08206 and a grant provided by the Allegheny Singer Research Institute, Pittsburgh, PA.
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