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Cholinergic neurons in the rat hippocampus do not compensate for the loss of septohippocampal cholinergic fibers
Neuroscienee Letters, 87 (1988) 18 22 Elsevier Scientific Publishers Ireland Ltd.
18
NSL 05234
Cholinergic neurons in the rat hippocampus do not compensate for the loss of septohippocampal cholinergic fibers M. Frotscher Institute o]'Anatom.v, Johann WolJgang Goethe University, Frank[urt am Mare ( F.R.G. (Received 1 December 1987; Accepted 23 December 1987)
Keywords."
Cholinergic neuron; Medial septum; Hippocampus; Neuronal degeneration: Sprouting; lmmunocytochemistry
In recent studies a small number of choline acetyltransferase (ChAT)-immunoreactive, supposedly cholinergic, neurons intrinsic to the rat hippocampus have been described. Here we report that thesc neurons arc not capable of sprouting in response to removal of the cholinergic input to the hippocampus from the medial septum/diagonal band complex. One m o n t h after unilateral transection of the fimbria-fornix an almost complete lack of cholinergic fibers persists in all layers of the dorsal hippocampus and t'~scia dentata ipsilatera] to the lesion when compared to the contralateral hippocampus or to unlesioned control rats. These results indicate that the well-known phenomenon of collateral sprouting in response to partial deafferentation is a specific process that spares a distinct group of chofinergic cells in the rat hippocampus.
The finding that cholinergic neurons in the basal forebrain degenerate in Alzheimer's disease (e.g. ref. 5) has intensified research on the degeneration and regeneration of these cells. Thus, it was observed that transection of the fimbria-fornix, which contains the septohippocampal cholinergic fibers, results in retrograde degeneration of the parent cell bodies in the medial septum/diagonal band complex [10]. This cell loss could be substantially attenuated by infusion of nerve growth factor (NGF) into the lateral ventricle [11, 13, 16]. In the target area of the septohippocampal pathway, in the various layers of the hippocampus proper and fascia dentata, there is a dramatic loss of cholinergic fibers and terminals following fimbria-fornix transections or septal lesions as revealed by acetylcholinesterase (ACHE) histochemistry and immunostaining for choline acetyitransferase (CHAT) [14]. A large number of studies have demonstrated that such a partial deafferentation of the hippocampus induces collateral sprouting of other afferent fibers to the hippocampal neurons (e.g. ref. 4). It seems logical to assume that a transection of the choliCorrespondence: M. Frotscher, Institute of Anatomy, Johann Wolfgang Goethe University, TheodorStern-Kai 7, D-6000 Frankfurt am Main, F.R.G. 0304-3940/88/$ 03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd.
19 nergic fibers from the medial septum would induce the cholinergic neurons in the hippocampus [9, 12, 14] to sprout because these neurons which express the same type of transmitter would best be suited to replace the lost septal afferents. The present study was carried out to test this hypothesis. In 9 male adult SpragueDawley rats the fimbria-fornix was transected unilaterally by a knife cut while the animals were deeply anesthesized with Nembutal (30 mg/kg). Following a survival time of 32 days - a time span which is generally sufficient to allow for collateral sprouting [4] - these animals as well as 4 unlesioned control rats were anesthesized and fixed by transcardial perfusion with a fixative containing 4% paraformaldehyde, 0.08% glutaraldehyde and 15% saturated picric acid in 0.1 M phosphate buffer (pH 7.3) [15]. After postfixation in glutaraldehyde-free fixative for 3 h, sections of the septal region and of the dorsal portions of the right and left hippocampus were cut on a Vibratome (Shandon). Following rinsing in phosphate buffer, the sections were freeze-thaw treated, again washed in phosphate buffer, and immunostained for C h A T as described in more detail elsewhere [7]. The following antibodies and dilutions were used: monoclonal antibody against C h A T from rat-mouse hybridoma (type I, Boehringer Mannheim G m b H , F.R.G. [6]) 1:9 for 48 h at 4°C, anti rat IgG (Miles, Naperville, IL) 1:40, 1.5 h, 20°C; rat peroxidase-antiperoxidase (PAP) complex (Sternberger-Meyer Immunocytochemicals, Jarrettsville, MD) 1:40, 2h, 20°C. All dilutions were made in 0.1 M phosphate buffer containing 1% normal rabbit se-
Fig. 1. Vibratome section of the right and left medial septal region immunostained for CHAT. Normal distribution of cholinergic neurons on the right side. Massive loss of immunoreactiveneurons on the contralateral side due to retrograde neuronal degeneration following fimbria-fornixtransection 1 month before sacrifice. Bar= 100/lm.
20
rum and 0.1% NAN3. Between each incubation step the sections were thoroughly rinsed in several changes of phosphate buffer. The immunostaining for ChAT was visualized by incubating the sections in diaminobenzidine (DAB) and H202 (0.07% DAB and 0.002% H202 in 0.1 M Tris buffer, pH 7.6) for 5-10 min. Following osmication and dehydration the sections were embedded fiat in Araldite [7]. Vibratome sections of the septal region of controls revealed numerous cholinergic cells in the medial septal nucleus and in the nuclei of the diagonal band as known from previous studies (e.g. refs. 1, 2). This characteristic distribution was similarly observed in the experimental animals on the side contralateral to the fimbria-fornix transection (Fig. 1). In contrast, there was a dramatic reduction in the number of
Fig. 2. Vibratome sections of dorsal hippocampus immunostained for CHAT. a: fascia dentata on nonlesioned side. Normal distribution of fine varicose cholinergic fibers in the vicinity of the granule cell layer (g). h, hilus; m, molecular layer, b: same area as in (a) on the contralateral (lesioned) side. An almost complete lack of cholinergic fibers persists 1 month after fimbria-fornix transection whereas a CHATimmunoreactive neuron just above the granule cell layer is stained, c and d: ChAT-immunoreactive neurons in characteristic location in stratum lacunosum-moleculare of CA 1. c: non-lesioned side. d: ipsilateral to fimbria-fornix transection. Bar = 50 ~tm.
21 immunoreactive cells on the lesioned side (Fig. 1) most likely due to retrograde degeneration of the cholinergic septohippocampal neurons [10]. Pronounced differences between the lesioned and non-lesioned sides were also noted in the hippocampal sections of the experimental animals (Fig. 2a, b). Comparable to the unoperated control rats numerous ChAT-immunoreactive fibers and terminals were observed on the non-lesioned side, mainly concentrated close to the cell layers of pyramidal neurons and granule cells (Fig. 2a). Here the fine, sometimes varicose, fibers formed a dense network around cell bodies and proximal dendrites [3, 7, 8]. On the lesioned side this characteristic fiber staining was almost completely absent (Fig. 2b). Only rarely single immunoreactive fibers were observed. Hippocampal cholinergic neurons, on the other hand, which most often occurred in stratum lacunosum-moleculare o f CA1 [9], were seen in hippocampal sections from either side (Fig. 2b-d). I was unable to detect any differences between ChAT-immunoreactive neurons on the lesioned and intact side. As described in a previous study [9], CHATimmunoreactive neurons in the hippocampus were smaller and less intensely immunostained when compared with cholinergic neurons in the basal forebrain and neostriatum. No immunostaining of fibers and cell bodies was seen in control sections from operated and non-operated animals which were reacted with all but the primary anti-ChAT antibody. The present results have confirmed previous studies on anterograde and retrograde degeneration of septohippocampal cholinergic neurons [I0, 14]. I regard it as a main result of the present study that the removal of the septohippocampal input does not lead to any reactive changes detectable by ChAT immunostaining in the intrinsic hippocampal cholinergic neurons. The almost complete absence of ChAT-immunoreactive fibers 1 month after fimbria-fornix transection strongly suggests that these cells are not capable of substituting for the lost cholinergic input from the medial septum. A large number of studies on collateral sprouting in the hippocampus in response to partial deafferentation have shown that reactive synaptogenesis takes place as early as in the first postoperative week (e.g. ref. 4). It is thus hard to imagine that these cells would only start to sprout after a postoperative survival time longer than 1 month. It is in line with the low density of fiber staining that there is no detectable increase in immunoreactivity of cell bodies and dendrites of the hippocampal cholinergic neurons in the deafferented hippocampus which, if present, would have indicated a compensatory increase in the activity of the transmitter-synthesizing enzyme ChAT in these cells. It appears that the capacity of central neurons to sprout in response to partial deafferentation varies and may also depend on the nature of the afferents removed. Work is in progress to study other fiber systems for their capacity to sprout after removal o f the septohippocampal input. The author wishes to thank E. Thielen and E. Schreiber for technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 45). I Amaral, D.G. and Kurz, J., An analysisof the originsof the cholinergicand noncholinergicprojections to the hippocampalformation of the rat, J. Comp. Neurol., 240 (1985) 37-59.
22 2 Bialowas, J. and Frotscher, M., Choline acetyltransferase-immunoreactive neurons and terminals in the rat septal complex: A combined light and electron microscopic study, J. Comp. Neurol., 259 (1987) 298 307. 3 Clarke, D.J., Cholinergic innervation of the rat dentate gyrus: A light and electron microscopical study using a monoclonal antibody to choline acetyltransferase, Brain Res., 360 (1985) 349 354. 4 Cotman, C.W. and Nadler, J.V., Reactive synaptogenesis in the hippocampus. In C.W. Cotman (Ed.), Neuronal Plasticity, Raven, New York, 1978, pp. 227 271. 5 Coyle, J.T., Price, D.L. and DeLong, M.R., Alzheimer's disease: a disease of cortical cholinergic innervation, Science, 219 (1983) 1184 1189. 6 Eckenstein, F. and Thoenen, H., Production of specific antisera and monoclonal antibodies to choline acetyltransferase. Characterization and use for identification of cholinergic neurons, EMBO J., 1 (1982) 363-368, 7 Frotscher, M. and Leranth, C., Cholinergic innervation of the rat hippocampus as revealed by choline acetyltransferase immunocytochemistry: A combined light and electron microscopic study, J. Comp. Neurol., 239 (1985) 237 246. 8 Frotscher, M. and Leranth, C., The cholinergic innervation of the rat fascia dentata: identification of target structures on granule cells by combining choline acetyltransferase immunocytochemistry and Golgi impregnation, J. Comp. Neurol., 243 (1986) 58-70. 9 Frotscher, M., Schlander, M. and Leranth, C., Cholinergic neurons in the hippocampus: a combined light and electron microscopic immunocytochemical study in the rat, Cell Tissue Res., 246 (1986) 293 301. 10 Gage, F.H., Wictorin, K., Fischer, W., Williams, L.R., Varon, S. and Bj6rklund, A., Retrograde cell changes in medial septum and diagonal band following fimbria-fornix transection: quantitative temporal analysis, Neuroscience, 19 (1986) 241 255. 11 Hefti, F., Nerve growth factor promotes survival of septal cholinergic neurons after limbrial transections, J. Neurosci., 6 (1986) 2155-2162. 12 Houser, C.R., Crawford, G.D., Barber, R.P., Salvaterra, P.M. and Vaughn, J.E., Organization and morphological characteristics of cholinergic neurons: an immunocytochemical study with a monoclonal antibody to choline acetyltransferase, Brain Res., 266 (1983) 97 119. 13 Kromer, L.F., Nerve growth factor treatment after brain injury prevents neuronal death, Science, 235 (1987) 214-216. 14 Matthews, D.A., Salvaterra, P.M., Crawford, G.D., Houser, C.R. and Vaughn, J.E., An immunocytochemical study of choline acetyltransferase-containingneurons and axon terminals in normal and partially deafferented hippocampal formation, Brain Res., 402 (1987) 3(~43. 15 Somogyi, P. and Takagi, H., A note on the use of picric acid-paraformaldehyde-glutaraldehydcfixative for correlated light and electron microscopic immunocytochemistry, Neuroscience, 7 (1982) 1779 1784. 16 Williams, L.R., Varon, S., Peterson, G.M., Wictorin, K., Fischer, W., Bj6rklund, A. and Gage, F.H., Continuous infusion of nerve growth factor prevents basal forebrain neuronal death after fimbria fornix transection, Proc. Natl. Acad. Sci. USA, 83 (1986)9231 9235.
18
NSL 05234
Cholinergic neurons in the rat hippocampus do not compensate for the loss of septohippocampal cholinergic fibers M. Frotscher Institute o]'Anatom.v, Johann WolJgang Goethe University, Frank[urt am Mare ( F.R.G. (Received 1 December 1987; Accepted 23 December 1987)
Keywords."
Cholinergic neuron; Medial septum; Hippocampus; Neuronal degeneration: Sprouting; lmmunocytochemistry
In recent studies a small number of choline acetyltransferase (ChAT)-immunoreactive, supposedly cholinergic, neurons intrinsic to the rat hippocampus have been described. Here we report that thesc neurons arc not capable of sprouting in response to removal of the cholinergic input to the hippocampus from the medial septum/diagonal band complex. One m o n t h after unilateral transection of the fimbria-fornix an almost complete lack of cholinergic fibers persists in all layers of the dorsal hippocampus and t'~scia dentata ipsilatera] to the lesion when compared to the contralateral hippocampus or to unlesioned control rats. These results indicate that the well-known phenomenon of collateral sprouting in response to partial deafferentation is a specific process that spares a distinct group of chofinergic cells in the rat hippocampus.
The finding that cholinergic neurons in the basal forebrain degenerate in Alzheimer's disease (e.g. ref. 5) has intensified research on the degeneration and regeneration of these cells. Thus, it was observed that transection of the fimbria-fornix, which contains the septohippocampal cholinergic fibers, results in retrograde degeneration of the parent cell bodies in the medial septum/diagonal band complex [10]. This cell loss could be substantially attenuated by infusion of nerve growth factor (NGF) into the lateral ventricle [11, 13, 16]. In the target area of the septohippocampal pathway, in the various layers of the hippocampus proper and fascia dentata, there is a dramatic loss of cholinergic fibers and terminals following fimbria-fornix transections or septal lesions as revealed by acetylcholinesterase (ACHE) histochemistry and immunostaining for choline acetyitransferase (CHAT) [14]. A large number of studies have demonstrated that such a partial deafferentation of the hippocampus induces collateral sprouting of other afferent fibers to the hippocampal neurons (e.g. ref. 4). It seems logical to assume that a transection of the choliCorrespondence: M. Frotscher, Institute of Anatomy, Johann Wolfgang Goethe University, TheodorStern-Kai 7, D-6000 Frankfurt am Main, F.R.G. 0304-3940/88/$ 03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd.
19 nergic fibers from the medial septum would induce the cholinergic neurons in the hippocampus [9, 12, 14] to sprout because these neurons which express the same type of transmitter would best be suited to replace the lost septal afferents. The present study was carried out to test this hypothesis. In 9 male adult SpragueDawley rats the fimbria-fornix was transected unilaterally by a knife cut while the animals were deeply anesthesized with Nembutal (30 mg/kg). Following a survival time of 32 days - a time span which is generally sufficient to allow for collateral sprouting [4] - these animals as well as 4 unlesioned control rats were anesthesized and fixed by transcardial perfusion with a fixative containing 4% paraformaldehyde, 0.08% glutaraldehyde and 15% saturated picric acid in 0.1 M phosphate buffer (pH 7.3) [15]. After postfixation in glutaraldehyde-free fixative for 3 h, sections of the septal region and of the dorsal portions of the right and left hippocampus were cut on a Vibratome (Shandon). Following rinsing in phosphate buffer, the sections were freeze-thaw treated, again washed in phosphate buffer, and immunostained for C h A T as described in more detail elsewhere [7]. The following antibodies and dilutions were used: monoclonal antibody against C h A T from rat-mouse hybridoma (type I, Boehringer Mannheim G m b H , F.R.G. [6]) 1:9 for 48 h at 4°C, anti rat IgG (Miles, Naperville, IL) 1:40, 1.5 h, 20°C; rat peroxidase-antiperoxidase (PAP) complex (Sternberger-Meyer Immunocytochemicals, Jarrettsville, MD) 1:40, 2h, 20°C. All dilutions were made in 0.1 M phosphate buffer containing 1% normal rabbit se-
Fig. 1. Vibratome section of the right and left medial septal region immunostained for CHAT. Normal distribution of cholinergic neurons on the right side. Massive loss of immunoreactiveneurons on the contralateral side due to retrograde neuronal degeneration following fimbria-fornixtransection 1 month before sacrifice. Bar= 100/lm.
20
rum and 0.1% NAN3. Between each incubation step the sections were thoroughly rinsed in several changes of phosphate buffer. The immunostaining for ChAT was visualized by incubating the sections in diaminobenzidine (DAB) and H202 (0.07% DAB and 0.002% H202 in 0.1 M Tris buffer, pH 7.6) for 5-10 min. Following osmication and dehydration the sections were embedded fiat in Araldite [7]. Vibratome sections of the septal region of controls revealed numerous cholinergic cells in the medial septal nucleus and in the nuclei of the diagonal band as known from previous studies (e.g. refs. 1, 2). This characteristic distribution was similarly observed in the experimental animals on the side contralateral to the fimbria-fornix transection (Fig. 1). In contrast, there was a dramatic reduction in the number of
Fig. 2. Vibratome sections of dorsal hippocampus immunostained for CHAT. a: fascia dentata on nonlesioned side. Normal distribution of fine varicose cholinergic fibers in the vicinity of the granule cell layer (g). h, hilus; m, molecular layer, b: same area as in (a) on the contralateral (lesioned) side. An almost complete lack of cholinergic fibers persists 1 month after fimbria-fornix transection whereas a CHATimmunoreactive neuron just above the granule cell layer is stained, c and d: ChAT-immunoreactive neurons in characteristic location in stratum lacunosum-moleculare of CA 1. c: non-lesioned side. d: ipsilateral to fimbria-fornix transection. Bar = 50 ~tm.
21 immunoreactive cells on the lesioned side (Fig. 1) most likely due to retrograde degeneration of the cholinergic septohippocampal neurons [10]. Pronounced differences between the lesioned and non-lesioned sides were also noted in the hippocampal sections of the experimental animals (Fig. 2a, b). Comparable to the unoperated control rats numerous ChAT-immunoreactive fibers and terminals were observed on the non-lesioned side, mainly concentrated close to the cell layers of pyramidal neurons and granule cells (Fig. 2a). Here the fine, sometimes varicose, fibers formed a dense network around cell bodies and proximal dendrites [3, 7, 8]. On the lesioned side this characteristic fiber staining was almost completely absent (Fig. 2b). Only rarely single immunoreactive fibers were observed. Hippocampal cholinergic neurons, on the other hand, which most often occurred in stratum lacunosum-moleculare o f CA1 [9], were seen in hippocampal sections from either side (Fig. 2b-d). I was unable to detect any differences between ChAT-immunoreactive neurons on the lesioned and intact side. As described in a previous study [9], CHATimmunoreactive neurons in the hippocampus were smaller and less intensely immunostained when compared with cholinergic neurons in the basal forebrain and neostriatum. No immunostaining of fibers and cell bodies was seen in control sections from operated and non-operated animals which were reacted with all but the primary anti-ChAT antibody. The present results have confirmed previous studies on anterograde and retrograde degeneration of septohippocampal cholinergic neurons [I0, 14]. I regard it as a main result of the present study that the removal of the septohippocampal input does not lead to any reactive changes detectable by ChAT immunostaining in the intrinsic hippocampal cholinergic neurons. The almost complete absence of ChAT-immunoreactive fibers 1 month after fimbria-fornix transection strongly suggests that these cells are not capable of substituting for the lost cholinergic input from the medial septum. A large number of studies on collateral sprouting in the hippocampus in response to partial deafferentation have shown that reactive synaptogenesis takes place as early as in the first postoperative week (e.g. ref. 4). It is thus hard to imagine that these cells would only start to sprout after a postoperative survival time longer than 1 month. It is in line with the low density of fiber staining that there is no detectable increase in immunoreactivity of cell bodies and dendrites of the hippocampal cholinergic neurons in the deafferented hippocampus which, if present, would have indicated a compensatory increase in the activity of the transmitter-synthesizing enzyme ChAT in these cells. It appears that the capacity of central neurons to sprout in response to partial deafferentation varies and may also depend on the nature of the afferents removed. Work is in progress to study other fiber systems for their capacity to sprout after removal o f the septohippocampal input. The author wishes to thank E. Thielen and E. Schreiber for technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 45). I Amaral, D.G. and Kurz, J., An analysisof the originsof the cholinergicand noncholinergicprojections to the hippocampalformation of the rat, J. Comp. Neurol., 240 (1985) 37-59.
22 2 Bialowas, J. and Frotscher, M., Choline acetyltransferase-immunoreactive neurons and terminals in the rat septal complex: A combined light and electron microscopic study, J. Comp. Neurol., 259 (1987) 298 307. 3 Clarke, D.J., Cholinergic innervation of the rat dentate gyrus: A light and electron microscopical study using a monoclonal antibody to choline acetyltransferase, Brain Res., 360 (1985) 349 354. 4 Cotman, C.W. and Nadler, J.V., Reactive synaptogenesis in the hippocampus. In C.W. Cotman (Ed.), Neuronal Plasticity, Raven, New York, 1978, pp. 227 271. 5 Coyle, J.T., Price, D.L. and DeLong, M.R., Alzheimer's disease: a disease of cortical cholinergic innervation, Science, 219 (1983) 1184 1189. 6 Eckenstein, F. and Thoenen, H., Production of specific antisera and monoclonal antibodies to choline acetyltransferase. Characterization and use for identification of cholinergic neurons, EMBO J., 1 (1982) 363-368, 7 Frotscher, M. and Leranth, C., Cholinergic innervation of the rat hippocampus as revealed by choline acetyltransferase immunocytochemistry: A combined light and electron microscopic study, J. Comp. Neurol., 239 (1985) 237 246. 8 Frotscher, M. and Leranth, C., The cholinergic innervation of the rat fascia dentata: identification of target structures on granule cells by combining choline acetyltransferase immunocytochemistry and Golgi impregnation, J. Comp. Neurol., 243 (1986) 58-70. 9 Frotscher, M., Schlander, M. and Leranth, C., Cholinergic neurons in the hippocampus: a combined light and electron microscopic immunocytochemical study in the rat, Cell Tissue Res., 246 (1986) 293 301. 10 Gage, F.H., Wictorin, K., Fischer, W., Williams, L.R., Varon, S. and Bj6rklund, A., Retrograde cell changes in medial septum and diagonal band following fimbria-fornix transection: quantitative temporal analysis, Neuroscience, 19 (1986) 241 255. 11 Hefti, F., Nerve growth factor promotes survival of septal cholinergic neurons after limbrial transections, J. Neurosci., 6 (1986) 2155-2162. 12 Houser, C.R., Crawford, G.D., Barber, R.P., Salvaterra, P.M. and Vaughn, J.E., Organization and morphological characteristics of cholinergic neurons: an immunocytochemical study with a monoclonal antibody to choline acetyltransferase, Brain Res., 266 (1983) 97 119. 13 Kromer, L.F., Nerve growth factor treatment after brain injury prevents neuronal death, Science, 235 (1987) 214-216. 14 Matthews, D.A., Salvaterra, P.M., Crawford, G.D., Houser, C.R. and Vaughn, J.E., An immunocytochemical study of choline acetyltransferase-containingneurons and axon terminals in normal and partially deafferented hippocampal formation, Brain Res., 402 (1987) 3(~43. 15 Somogyi, P. and Takagi, H., A note on the use of picric acid-paraformaldehyde-glutaraldehydcfixative for correlated light and electron microscopic immunocytochemistry, Neuroscience, 7 (1982) 1779 1784. 16 Williams, L.R., Varon, S., Peterson, G.M., Wictorin, K., Fischer, W., Bj6rklund, A. and Gage, F.H., Continuous infusion of nerve growth factor prevents basal forebrain neuronal death after fimbria fornix transection, Proc. Natl. Acad. Sci. USA, 83 (1986)9231 9235.