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Long term changes in brain cholinergic markers and nerve growth factor levels after partial immunolesion
Brain Research 801 Ž1998. 190–197
Research report
Long term changes in brain cholinergic markers and nerve growth factor levels after partial immunolesion Zezong Gu, Juan Yu 1, J. Regino Perez-Polo
)
Department of Human Biological Chemistry and Genetics, The UniÕersity of Texas Medical Branch at GalÕeston, 301 UniÕersity BlÕd., GalÕeston, TX 77555-0652, USA Accepted 26 May 1998
Abstract There are deficits in cholinergic basal forebrain neurons ŽCBFNs. in the aged brain and patients suffering Alzheimer’s disease associated with a partial loss of the CBFNs. To mimic this partial loss and assess its long term effects on residual cholinergic activity and resultant target-derived nerve growth factor ŽNGF. levels, we produced a partial immunolesion to CBFNs with 192 IgG-saporin, an immunotoxin selectively taken up by p75 NTR-bearing neurons. We measured two cholinergic markers, choline acetyltransferase ŽChAT. and acetylcholinesterase ŽAChE. activity, and NGF protein levels at 10 days, 1, 6 and 12 months postlesion. There were no significant changes in the cholinergic markers and the NGF protein levels in the sham-treated animal controls during the one year experiment. Ten days after 192 IgG-saporin treatment, ChAT activity decreased to 35–50% of controls in the olfactory bulb, hippocampus, and cortex. There was a minor but significant recovery of ChAT activity one year after the immunolesion in the hippocampus. Changes in AChE activity mirrored the ChAT changes but were less robust. There were transient increases in NGF protein levels in the hippocampus and cortex that returned to basal levels at 6 months and 12 months postlesion, respectively. In summary, partial immunolesions resulted in partial region-specific and time-dependent recoveries of cholinergic activity in the target areas of the basal forebrain after a partial elimination of CBFNs and a return to basal levels of NGF protein consistent with the hypothesis that the remaining CBFNs compensated for losses of ChAT and NGF due to changes in cholinergic innervation of basal forebrain target areas. q 1998 Elsevier Science B.V. All rights reserved. Keywords: 192 IgG-saporin; Acetylcholinesterase; Choline acetyltransferase; Cholinergic basal forebrain neuron; Immunolesion; Nerve growth factor; Regeneration
1. Introduction The cholinergic basal forebrain neurons ŽCBFNs. are located in the medial septum, diagonal band of Broca and nucleus basalis of Meynert and are involved in functions related to cognition and memory w4,21,35,49x. The nerve growth factor ŽNGF. is mostly synthesized in hippocam-
Abbreviations: Acetylcholinesterase, AChE; Brain-derived neurotrophic factor, BDNF; Choline acetyltransferase, ChAT; Cholinergic basal forebrain neuron, CBFN; Nerve growth factor, NGF; Phosphate buffered saline, PBS ) Corresponding author. Fax: q 1-409-772-8028; E-mail: [email protected] 1 Current address: St. Barnabas Medical Center, Livingston, NJ, USA. 0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 5 7 9 - 4
pus, neocortex and olfactory bulb, which are the target regions of CBFNs, and is internalized at the projection terminals after binding to NGF receptors, to be then retrogradely transported to the cell bodies of CBFNs w2,24x. NGF provides critical trophic support to CBFNs Žfor reviews, see Refs. w20,25,27,33,44,51x. that express high levels of the NGF receptors, TrkA and the low affinity neurotrophin ŽNT. receptor p75 NTR w14,15,22,23,42x. NGF has been shown to stimulate the synthesis of acetylcholine ŽACh. synthesizing enzyme, choline acetyltransferase ŽChAT. activity w16,19x. Cholinergic deficits in the aged and Alzheimer’s disease ŽAD. patients have been well documented. ChAT activity is consistently decreased by 50–95% in the cortex and hippocampus of AD patients w6,7,34,56x. There are also significant decreases in high-affinity choline uptake
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ŽHACU., in presynaptic muscarinic receptor binding, and in ACh and acetylcholinesterase ŽAChE. levels in the affected cortex and hippocampus w28,31,37,41x. The decrease of these cholinergic markers is associated with a marked loss of CBFNs w50x, and is closely correlated with the extent of AD neuropathology and the severity of cognitive impairment w6,30,34x. Attempts to study the role of neurotrophic factors on cholinergic function, such as learning and memory, have been based on the use of a wide variety of lesion models. Lesions have been produced either by physical means Žradio frequency lesion, electrolytic lesion, and fimbria– fornix transection. or by the injection of relatively specific neurotoxic substances. Four different injection strategies have been used: Ž1. injections of excitatory amino acids ŽEAAs. into basal forebrain ŽBF. w8,9x, Ž2. injections of aziridinium ion of ethyl choline mustard ŽAF64A. into the BF or CBFN target areas w11,17x, Ž3. intracerebral or systemic injections of murine monoclonal antibodies against AChE w3,36x. Although all these paradigms showed a uniform transient increase in NGF synthesis, it is not clear if these resulted from lesion-induced effects on CBFNs or non-cholinergic signaling pathways. Ž4. An alternative approach is to use the cytotoxin saporin coupled to the monoclonal antibody against low affinity neurotrophin receptor p75 NTR , 192 IgG w52,54,55x. Behavioral, biochemical, and morphological studies show that there are selective and robust effects of the 192 IgG-saporin on CBFNs in a stable and dose-dependent manner. Thus, 192 IgG-saporin produces significant long-lasting increases in NGF protein in the hippocampus, cortex and olfactory bulb and large decreases of ChAT activity w5,26,38,39,43,45– 47,54,55x. The injection of 192 IgG-saporin into the medial septum produces dose-related deficits in a variable-delay radial arm maze task. Injections of 192 IgG-saporin decrease the number of correct choices and increase the number of errors in the delayed non-match to sample task. These behavioral deficits are associated with dose-dependent decreases in pre-synaptic high affinity choline uptake in the hippocampus, cingulate and entorhinal cortex, the terminal fields of the medial septum w46x. Thus, the injection of 192 IgG-saporin into the medial septum produces cholinergic hypofunction and dose-dependent working memory deficits. Our previous work w40,53x showed that after a partial immunolesion to the rat CBFNs, transplants of mouse 3T3 NG Fq fibroblasts engineered to vigorously secrete NGF can up-regulate several components of CBFN function. Also, we have shown that the immunolesion-induced increase in NGF protein levels is absent in aged rats when compared to their young counterparts. Here, we applied the partial immunolesion paradigm by injecting the immunotoxin, 192 IgG-saporin, into the cerebral ventricles in order to assess the effects of a partial loss of CBFNs on residual cholinergic activity and target-derived nerve growth factor ŽNGF. levels over time.
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2. Materials and methods 2.1. Animals and surgical procedures Thirty-three 3-month-old male Sprague–Dawley rats weighing 250–300 gm ŽHarlan Sprague–Dawley, Indianapolis, IN. were used in this study. Procedures, dosages, location of injections, as well as time intervals required for the full development of the immunolesions were based on published work w39,40,53–55x or determined in a preliminary experiment using the same 192 IgG-saporin Ž192 IgG-SAP. lot used in these experiments ŽChemicon, Temecula, CA. where efficacy of immunolesion was monitored using AChE histochemistry. Animals were anaesthetized with ketamine Ž60 mgrkg i.m.. and Nembutal Ž40 mgrkg i.p.., and placed in a Kopf stereotaxic apparatus. The skull was exposed and two small burr holes were made using a dental drill. A Hamilton syringe with a 26-gauge needle was used for injection. A stock solution of 192 IgG-SAP was stored in frozen aliquots until just before use. Aliquots were then thawed and diluted to 0.65 mgrml in sterile phosphate buffered saline ŽPBS.. Rats were stereotaxically injected with 6 ml of either 192 IgG-saporin or vehicle, 10 mM sterile PBS at a rate of 1 mlrmin to the intracerebral ventricles Ži.c.v.. using the following coordinates: AP, y0.8; ML, "1.2; DV, y3.4, mm respectively w32x. 1.30 mg of 192 IgG-saporin was injected bilaterally to produce a CBFN partial immunolesion ŽPIL. and 4.5 mg of same immunotoxin delivered unilaterally was used to produce a total immunolesion ŽTIL. to CBFNs. After each injection the needle was left in place for 5 min to allow diffusion. The needle was then slowly retracted, the wound was bone-waxed and sutured, and the animal allowed to recover on a heating pad. No special post-operative care was required after 192 IgGsaporin lesions. All animals were sacrificed by decapitation under deep halothane anaesthesia and the brains were rapidly removed onto an ice-cold plate. Brain regions including the basal forebrain area, olfactory bulbs, bilateral hippocampus, and bilateral parieto-occipital cortex were dissected on ice. The tissues were immediately frozen on dry ice and stored at y808C until processing. In order to study the long-term effects of the CBFNs, four groups of animals were sacrificed at 10 days, 1, 6 and 12 Ž N s 4. months after PIL, one group of TIL animals Ž N s 4. at 1 month, and one group of control animals at 3 months and another at 15 months as controls for the one year duration of some experiments. Five animals were in each group unless otherwise indicated. 2.2. NGF immunoassay Tissue samples were prepared in three volumes of cold extraction buffer containing 0.4 M NaCl, 0.05% Žvolrvol. Tween 20, 0.5% Žwtrvol. bovine serum albumin ŽBSA.,
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0.1 mM phenylmethylsulfonyl fluoride Žfrom 1 M dimethylsulphofide stock solution., 0.1 mM benzethionium chloride, and 20 KIrml of aprotinin, pH 7.4. The homogenates were centrifuged at 14,000 g for 20 min, and the supernatants further diluted 1:1 with tissue buffer containing 10 mM CaCl 2 and 0.1% Triton X-100, pH 7.0 prior to NGF quantification. The procedure involves the following major steps: Ž1. coat 96 well EIA plates ŽNunc. with the monoclonal anti-NGF solution ŽBoehringer-Mannheim, 0.25 mgrml. 2 h at 378C, Ž2. block with 0.5% BSA 1r2 h at 378C, Ž3. incubate overnight at 48C with the samples to be tested or with standard solutions Žpurified mouse NGF diluted in 1:1 extraction and tissue buffer., Ž4. incubate with the anti-NGF solution ŽBoehringer-Mannheim, 4 mgrml. 4 h at 378C, Ž5. add substrate solution Ž40 mg of chlorophenolred b-galactopyranoside in 20 ml substrate buffer containing 100 mM HEPES, 150 mM NaCl, 2 mM MgCl 2 , 1% BSA, pH 7.0. and react for 1 1r2 h at 378C, Ž6. measure against substrate solution at 570 nm using an ELISA-Reader. Between each major step, the plate was washed three times with cold wash buffer containing 50 mM Tris–HCl, 200 mM NaCl, 10 mM CaCl 2 , 0.1% Triton X-100, pH 7.0.
density was read at 405 nm. The reaction mixture comprised the following: 0.20 mM DTNB ŽEllman’s reagent, Sigma, D-8130., 0.56 mM acetylthiocholine iodide and 39 mM phosphate buffer ŽpH 7.2.. To exclude the co-reaction of pseudocholinesterase the reaction mixture was added 10y5 M iso-OMPA. 2.5. Statistical analysis All the data are presented as the mean " S.E.M. The statistical significance of ChAT and AChE activities, and NGF levels among experimental groups was calculated by One-way ANOVA with post-hoc Fisher’s LSD analysis. The criteria for significance were set as P - 0.05.
3. Results Since a one year time period was included in the time course measurements, we determined whether ChAT activity or NGF protein levels were different for 3-month-old sham control groups as compared to 15-month-old sham
2.3. ChAT actiÕity A 4 ml aliquot of each homogenate prepared for NGF immunoassays was used in AChE assay Žsee the following. and ChAT assay according to the radiometric method of Fonnum Ž1975. w12x. Homogenates were further diluted as 1:30 ratio with homogenization buffer. Triplicate samples of 15 ml from diluted homogenates were used in the assay. The reaction mixture comprised the following: 600 mM NaCl, 0.2 mM eserine sulfate, 40 mM disodium EDTA, 100 mM phosphate buffer ŽpH 7.4., 18 mM choline chloride, 0.39 mM acetyl-CoA and 80 mM w14 Cxacetyl-CoA Ž0.3 mCirmmol.. Triplicate samples of each homogenate were incubated with 15 ml of reaction mixture at 378C for 40 min to allow for the synthesis of w14 Cxacetylcholine ŽACh.. Six samples of water, lacking additional radio labeled acetyl-CoA, were also assayed to serve as sample blanks. To terminate the reaction, 2.1 ml of 10 mM cold phosphate buffer ŽpH 7.4. and 0.5 ml of 0.5% cold sodium tetraphenylboron in acetonitrile were added, and the samples were vortex-mixed to extract the w14 CxACh-tetraphenylboron. The w14 Cx measurements were made with a scintillation counter ŽBeckman LS 7500 Liquid Scintillation System.. 2.4. AChE actiÕity The enzymatic activity was determined, as previously described w29x, by the method of Ellman et al. Ž1961. w10x with some modifications w1x. In brief, triplicate samples of 20 ml from 1:30 diluted homogenates were incubated with 250 ml of reaction mixture at 378C for 30 min and optical
Fig. 1. ChAT activity and NGF protein levels in different brain areas of sham-treated rats at two ages Ž3 vs. 15 months. one month after PBSvehicle injection into the intracerebral ventricles Ži.c.v... Values are means"S.E.M., N s 5, One-way ANOVA with post-hoc Fisher’s LSD analysis.
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controls experiencing intracerebral ventricular Ži.c.v.. delivery of vehicle ŽPBS.. Fig. 1 shows that there were no significant differences in ChAT activity or NGF protein content in the different brain areas 1 month after PBS injections into 3- and 15-month-old rat ventricles. These control values were therefore considered indistinguishable. When rats were exposed to partial immunolesions, there were 50–65% decreases in ChAT activity in the hippocampus, cortex and olfactory bulb 10 days after the 192
Fig. 3. Time course of changes in AChE activity in the target areas of the cholinergic basal forebrain neurons ŽCBFNs. after partial immunolesion. The AChE activity was expressed as the optical density of the colorimetric reading at 405 nm Ž N s 5 for each time point except N s 4 for 1 year postlesion.. Values are means"S.E.M., ) Ž P - 0.05. different time points after the lesion vs. sham controls and a Ž P - 0.05. vs. 10 days postlesion respectively, One-way ANOVA with post-hoc Fisher’s LSD analysis.
IgG-saporin treatment ŽFig. 2.. There was a minor but significant recovery of ChAT activity one year after the immunolesion in the hippocampus. In the olfactory bulb, ChAT activity remained at 56% of sham levels for up to one year postlesion. Measurement of NGF protein levels in these same animals showed transient increases in NGF levels in the hippocampus and cortex with returns to basal levels at 6 and 12 months postlesion, respectively, and significant long-term increases in NGF protein levels in the olfactory bulb with the highest levels reached 6 months after lesion. Thus, there was a prolonged partial recovery of cholinergic activity and a return of NGF protein levels
Fig. 2. Time course of changes in ChAT activity and NGF protein levels in the target areas of the cholinergic basal forebrain neurons ŽCBFNs. after partial immunolesion. ChAT activity and NGF protein levels are expressed as percentage of the values for vehicle-delivered shams Ž N s 5 for each time point except N s 4 for 1 year postlesion.. Values are means"S.E.M., ) Ž P - 0.05. different time points after the lesion vs. sham controls and a Ž P - 0.05. vs. 10 days postlesion respectively, One-way ANOVA with post-hoc Fisher’s LSD analysis.
Fig. 4. Time course of changes in ChAT activity and NGF protein levels in the basal forebrain after partial immunolesion. ChAT activity and NGF protein levels were expressed as percentage of the corresponding vehicle-delivered sham values Ž N s 5 for each time points except N s 4 for 1 year postlesion.. Values are means"S.E.M., ) P - 0.05 vs. sham controls, One-way ANOVA with post-hoc Fisher’s LSD analysis.
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to basal levels in the CBFN target areas after the partial elimination of CBFNs in a time-dependent and regionspecific fashion. The activity of AChE, the rate-limiting enzyme in acetylcholine ŽACh. hydrolysis, dramatically decreased after the partial elimination of CBFNs, consistent with the ongoing cholinergic degeneration in the basal forebrain ŽFig. 3.. There was a significant recovery of AChE activity in the olfactory bulb, but not in the hippocampus and cortex regions.
The levels of ChAT activity in the basal forebrain decreased Žabout 20%. 30 days postlesion before returning to sham control values. The magnitude of the observed changes was less than that in the target areas of CBFNs. NGF protein levels in the immunolesioned basal forebrain increased transiently by 10 days postlesion and had returned to normal levels by 30 days ŽFig. 4.. As expected total immunolesions resulted in more robust decreases in ChAT and AChE, and increases in NGF in the different brain regions ŽFig. 5. when compared to their partially immunolesioned counterparts reflecting a dose dependent effect on all tissues affected by immunolesion.
4. Discussion
Fig. 5. ChAT and AChE activities and NGF protein levels in different brain areas one month after partial Ž1.3 mg of 192 IgG-saporin. or total Ž4.5 mg of 192 IgG-saporin. immunolesion. Values are means"S.E.M., ) Ž P - 0.05. vs. sham controls and a Ž P - 0.05. vs. partial lesioned groups respectively, One-way ANOVA with post-hoc Fisher’s LSD analysis, N s 5 for sham control and partial lesioned groups and N s 4 for total lesioned group.
There were no significant changes in the expression of the two cholinergic markers used here in the sham control animals during a one year period. These results would support the hypothesis that physiological cholinergic function of the basal forebrain does not gradually decline in adulthood but rather that age-associated decreases are expressed late in life. This is not surprising insofar as there are no reports of dramatic differences in cognitive behavior between 3- and 15-month-old rats. In one study w48x carried out during acquisition, the T maze alternation task was shown to be sensitive to the effects of aging, i.e., the choice accuracy of 24-month-old rats was significantly less worse than either 3- or 9-month-old rats Ž P - 0.05., whereas the choice accuracy of 3- and 9-month-old rats did not differ significantly from each other. Expression of the two cholinergic markers used here was drastically reduced in those brain regions normally innervated by CBFNs as early as 10 days after the 192 IgG-saporin-mediated partial lesions. The decreased expression of ChAT and AChE activity in the target areas of the CBFNs is consistent with the loss of CBFN nerve terminals in the hippocampus, cortex, and olfactory bulb. Yu et al. w55x demonstrated that ChAT activity is progressively decreased in the septum, hippocampus, cortex and olfactory bulb during the first 11 days after immunotoxin injection and remained at those level for 5 months. There were no significant changes in ChAT activity in the cerebellum and a small decrease at 30 days in the striatum which are no or less p75 NTR positive cholinergic neurons. Other studies w18,39x have shown that several groups of non-cholinergic neurons in the basal forebrain ŽGABAergic septal neurons, Calbindin-D28K and NADPHd positive neurons. and one group of p75 NTR negative cholinergic neurons are not affected by the immunotoxin. The concomitant increase in NGF protein levels in these brain areas is evidence that the specific deafferentation of the CBFNs, in the absence of other direct interruptions of non-cholinergic neurotransmitter functions or direct lesions to any neurons in the hippocampus Žhippocampal neurons
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do not express p75 NTR ., triggers accumulation of NGF protein in those brain regions synthesizing NGF. Such an accumulation of NGF has been reported for total immunolesions as constant over time up to 5 months postlesion w55x. Given that cholinergic deafferentation alone has no effect on NGF mRNA levels in the hippocampus, cortex, and olfactory bulb and that the observed increases in NGF levels for a partial immunolesion are transient ŽFig. 2., it is likely that the return of NGF protein levels in target areas between 6 and 12 months postlesion reflect the restoration of retrograde transport of NGF by the residual CBFNs and therefore suggest that synaptic reorganization is carried out successfully after the deafferenting consequences of a partial immunolesion. Such an interpretation is consistent with the hypothesis that the lesion-induced increases in NGF suggest a role for NGF in postlesion remodeling. It is interesting that proximal sites witnessed a return to basal values earlier than sites more distant from the CBFNs. Reports of deafferentation associated with trauma, trauma alone, or deafferenting lesions that include a traumatic component, direct involvement of other neurotransmitter systems, or inflammatory responses have been shown to stimulate both NGF and BDNF mRNA and protein levels in contrast to the more restricted effects of cholinergic immunolesions on NGF accumulation but not synthesis, consistent with the hypothesis that the retrograde transport of NGF is largely responsible for cholinergic regulatory events and changes in basal levels of NGF synthesis may more directly reflect regulation of stress response gene products directly affecting neuronal commitment to apoptosis and inflammatory responses. It has been proposed that reinnervation by deafferented neurons after partial cholinergic lesions is due to lesion-induced increases in the release of NGF w13,40x. There was a transient and relatively smaller response in cholinergic marker activities in the basal forebrain after the partial immunolesions perhaps reflecting compensatory changes by the 50% residual CBFNs unaffected by the immunolesion ŽFig. 4.. There was a pronounced and very transient 150% increase in NGF protein levels that did not persist beyond 10 days postlesion ŽFig. 4. and could explain the delay in the decreases in ChAT activity in the CBFNs as compared to the more sudden decreases in cholinergic markers in the target areas for the CBFNs ŽFig. 2..
5. Conclusion In summary, partial immunolesions result in dose-dependent, region-specific and time-dependent losses in cholinergic activities in the innervation target areas of the CBFNs followed by long term recovery of activity over a 6 month to one year period. There are transient accumulations of NGF in the deafferented target regions consistent
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with impairment of retrograde transport of NGF to the basal forebrain and eventual recovery due to reinnervation of the target areas. The observed time course of changes in both the measured cholinergic markers and the levels of NGF protein are quite different for the CBFNs directly impacted by the 192 IgG-saporin injections and their projection areas in the hippocampus, cortex, and olfactory bulb. The differential time courses would suggest that different recuperative and regenerating processes are at work in the different regions. Furthermore, the responses to partial immunolesions would suggest that both ChAT and NGF levels respond to the state of cholinergic innervation of basal forebrain target areas and that there are recuperative processes in adult rats that might prove useful for the determination of the extent of plasticity of cholinergic function.
Acknowledgements This study was supported in part by NINDS Grant NS 33288 to JR P-P.
References w1x J. Andra, ¨ Z. Lojda, A histochemical method for the demonstration of acetylcholinesterase activity using semipermeable membranes, Histochemistry 84 Ž1986. 575–579. w2x C.E. Bandtlow, R. Heumann, M.E. Schwab, H. Thoenen, Cellular localization of nerve growth factor synthesis by in situ hybridization, EMBO J. 6 Ž1987. 891–899. w3x A.J. Bean, Z. Xu, S.Y. Chai, S. Brimijoin, T. Hokfelt, Effect of intracerebral injection of monoclonal acetylcholinesterase antibodies on cholinergic nerve terminals in the rat central nervous system, Neurosci. Lett. 133 Ž1991. 145–149. w4x V. Bigl, N.J. Woolf, L.L. Butcher, Cholinergic projections from the basal forebrain to frontal, parietal, temporal, occipital, and cingulate cortices: a combined fluorescent tracer and acetylcholinesterase analysis, Brain Res. Bull. 8 Ž1982. 727–749. w5x A.A. Book, R.G. Wiley, J.B. Schweitzei, Specificity of 192 IgGsaporin for NGF receptor-positive cholinergic basal forebrain neurons in the rat, Brain Res. 590 Ž1992. 350–355. w6x D.M. Bowen, C.B. Smith, P. White, A.N. Davison, Neurotransmitter-related enzymes and indices of hypoxia in senile dementia and other abiotrophies, Brain 99 Ž1976. 459–496. w7x P. Davies, Neurotransmitter-related enzymes in senile dementia of the Alzheimer’s type, Brain Res. 171 Ž1979. 319–327. w8x A.J. Dekker, D.J. Conner, L.J. Thal, The role of cholinergic projections from the nucleus basalis in memory, Neurosci. Biobehav. Rev. 15 Ž1991. 299–317. w9x S.B. Dunnett, B.J. Everitt, T.W. Robbins, The basal forebrain-cortical cholinergic systems: interpreting the functional consequences of excitotoxic lesions, Trend. Neurosci. 14 Ž1991. 494–501. w10x G.L. Ellman, K.D. Courtney, V. Andres Jr., R.M. Featherstone, A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol. 7 Ž1961. 88–95. w11x A. Fisher, I. Hanin, Potential animal models for senile dementia of Alzheimer’s type, with emphasis on AF64A induced cholinotoxicity, Ann. Rev. Pharmacol. Toxicol. 26 Ž1986. 161–181.
196
Z. Gu et al.r Brain Research 801 (1998) 190–197
w12x F. Fonnum, A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem. 24 Ž1975. 407–409. w13x F.H. Gage, A. Bjorklund, U. Stenevi, Reinnervation of the partially deafferented hippocampus by compensatory collateral sprouting from spared cholinergic and noradrenergic afferents, Brain Res. 268 Ž1983. 39–47. w14x R.B. Gibbs, J.T. McCabe, C.R. Buck, M.V. Chao, D.M. Pfaff, Expression of NGF receptor in the rat forebrain detected with in situ hybridization and immunohistochemistry, Mol. Brain Res. 6 Ž1989. 275–287. w15x R.B. Gibbs, D.W. Pfaff, In situ hybridization detection of trkA mRNA in brain; distribution, colocalization with p75 NG FR and up regulation by nerve growth factor, J. Comp. Neurol. 341 Ž1994. 324–339. w16x H. Gnahn, F. Hefti, R. Heumann, M.E. Schwab, H. Thoenen, NGF-mediated increase of choline acetyltransferase ŽChAT. in the neonatal rat forebrain: evidence for a physiological role of NGF in the brain, Dev. Brain Res. 9 Ž1983. 45–52. w17x I. Hanin, The AF64A model of cholinergic hypofunction: an update, Life Sci. 58 Ž1996. 1955–1964. w18x S. Heckers, T. Ohtake, R.G. Wiley, D.A. Lappi, C. Geula, M.-M. Mesulam, Complete and selective cholinergic denervation of rat neocortex and hippocampus but not amygdala by an immunotoxin against the p75 NGF receptor, J Neurosci. 14 Ž1994. 1271–1289. w19x F. Hefti, A. Dravid, J. Hartikka, Chronic intraventricular injections of nerve growth factor elevate hippocampal choline acetyltransferase activity in adult rats with partial septo-hippocampal lesions, Brain Res. 293 Ž1984. 305–311. w20x F. Hefti, B. Knusel, P.A. Lapchak, Protective effects of nerve growth factor and brain-derived neurotrophic factor on basal forebrain cholinergic neurons in adult rats with partial fimbrial transections, Prog. Brain Res. 98 Ž1993. 257–263. w21x Z. Henderson, Responses of basal forebrain cholinergic neurons to damage in the adult brain, Prog. Neurobiol. 48 Ž1996. 219–254. w22x D.M. Holtzman, Y. Li, L.F. Parada, S. Kinsma, C.-K. Chen, J.S. Valleta, J. Zhou, J.B. Long, W.C. Mobley, p140 trk mRNA marks NGF-responsive forebrain neurons: evidence that trk gene expression in induced by NGF, Neuron 9 Ž1992. 465–478. w23x E.M. Johnson, M. Taniuchi, Nerve growth factor ŽNGF. receptors in the central nervous system, Biochem. Pharmacol. 36 Ž1987. 4189– 4195. w24x E.M. Johnson, M. Taniuchi, H.B. Calrk, J.E. Springer, S. Koh, M. Tayrien, R. Loy, Demonstration of the retrograde transport of nerve growth factor in the peripheral and central nervous system, J. Neurosci. 7 Ž1987. 923–928. w25x S. Korsching, The role of nerve growth factor in the CNS, Trends Neurosci. 11 Ž12. Ž1986. 570–573. w26x G. Leanza, O.G. Nilsson, R.G. Wiley, A. Bjorklund, Selective lesioning of the basal forebrain cholinergic system by intraventricular 192 IgG-saporin: behavioral, biochemical and stereological studies in the rat, Eur. J. Neurosci. 7 Ž1995. 329–343. w27x C.A. Lucidi-Phillipi, F.H. Gage, The neurotrophin hypothesis and the cholinergic basal forebrain projection, Prog. Brain Res. 98 Ž1993. 241–256. w28x D.C. Mash, D.D. Flynn, L.T. Potter, Loss of M2 muscarine receptors in the cerebral cortex in Alzheimer’s disease and experimental cholinergic denervation, Science 228 Ž1985. 1115–1117. w29x H. Michalek, S. Fortuna, A. Pintor, Age-related differences in brain cholinesterase and muscarinic receptor sites in two strains of rats, Neurobiol. Aging 10 Ž1989. 143–148. w30x D. Neary, J.S. Snowden, D.M. Mann, D.M. Bowen, N.R. Sims, B. Northen, P.O. Yates, A.N. Davison, Alzheimer’s disease: a correlative study, J. Neurol. Neurosurg. Psychiatr. 49 Ž1986. 229–237. w31x J. Pascual, A. Fontan, J.J. Zarranz, J. Berciano, J. Florez, A. Pazos, High-affinity choline uptake carrier in Alzheimer’s disease: implications for the cholinergic hypothesis of dementia, Brain Res. 552 Ž1991. 170–174.
w32x G. Paxinos, C. Watson, The rat brainatlas in stereotaxic coordinates, 2nd edn., Academic Press, Sydney, 1986. w33x J.R. Perez-Polo, Roles of trophic factors in neuronal aging, Adv. Exp. Med. Biol. 265 Ž1990. 101–116. w34x E.K. Perry, B.E. Tomlinson, G. Blessed, K. Bergmann, P.H. Gilbson, R.H. Perry, Correlation of cholinergic abnormalities with senile plaques and mental test scores in senile dementia, Br. Med. J. 2 Ž1978. 1457–1459. w35x D.L. Price, V.E. Koliatsos, R.C. Clatterbuck, Cholinergic systems: human diseases, animal models, and prospects for therapy, Prog. Brain Res. 98 Ž1993. 51–60. w36x Z. Rakonczay, P. Hammond, S. Brimijoin, Lesion of central cholinergic system by systemically administered acetylcholinesterase antibodies in newborn rats, Neuroscience 54 Ž1993. 225–238. w37x J.A. Richter, E.K. Perry, B.E. Tomlinson, Acetylcholine and choline levels in post mortem human brain tissue: preliminary observations in Alzheimer’s disease, Life Sci. 26 Ž1980. 1683–1689. w38x S. Roßner, J.R. Perez-Polo, R.G. Wiley, V. Bigl, Differential expression of immediate early genes in distinct layers of rat cerebral cortex after selective immunolesion of the forebrain cholinergic system, J. Neurosci. Res. 38 Ž1994. 282–293. w39x S. Roßner, R. Schliebs, J.R. Perez-Polo, R.G. Wiley, V. Bigl, Differential changes of cholinergic markers from selected brain regions after specific immunolesion of the rat cholinergic basal forebrain system, J. Neurosci. Res. 40 Ž1995. 31–43. w40x S. Roßner, J. Yu, D. Pizzo, K. Werrbach-Perez, R. Schliebs, V. Bigl, J.R. Perez-Polo, Effects of intraventricular transplantation of NGFsecreting cells on cholinergic basal forebrain neurons after partial immunolesion, J. Neurosci. Res. 45 Ž1996. 40–56. w41x R.J. Rylett, M.J. Ball, E.H. Colhoun, Evidence for high affinity choline transport in synaptosomes prepared from hippocampus and neocortex of patients with Alzheimer’s disease, Brain Res. 289 Ž1983. 169–175. w42x T.L. Steininger, B.H. Wainer, R. Klein, M. Barbacid, H.C. Palfrey, High-affinity nerve growth factor receptor ŽTrk. immunoreactivity is localized in cholinergic neurons of the basal forebrain and striatum in the adult rat brain, Brain Res. 62 Ž1993. 330–335. w43x E.M. Torres, T.A. Perry, A. Blokland, L.S. Wilkinson, R.G. Wiley, D.A. Lappi, S.B. Dunnett, Behavioral, histochemical and biochemical consequences of selective immunolesions in discrete regions of the basal forebrain cholinergic system, Neuroscience 63 Ž1994. 95–122. w44x H. Thoenen, C. Bandtlow, R. Heumann, The physiological function of nerve growth factor in the central nervous system: comparison with the peripheral, Rev. Physiol. Biochem. Pharmacol. 109 Ž1987. 145–178. w45x J.J. Waite, M.L. Wardlow, A.C. Chen, D.A. Lappi, R.G. Wiley, Time course of cholinergic and monoaminergic changes in rat brain after immunolesioning with 192 IgG-saporin, Neurosci. Lett. 169 Ž1994. 154–158. w46x T.J. Walsh, C.D. Herzog, C. Gandhi, R.W. Stachman, R.G. Wiley, Injection of IgG-saporin into the medial septum produces cholinergic hypofunction and dose-dependent working memory deficits, Brain Res. 726 Ž1996. 69–79. w47x G.L. Wenk, J.D. Stoehr, G. Quintana, S. Mobley, R.G. Wiley, Behavioral, biochemical, histological, and electrophysiological effects of 192 IgG-saporin injections in the basal forebrain of rats, J. Neurosci. 14 Ž1994. 5986–5995. w48x G.L. Wenk, J.D. Stoehe, S.L. Mobley, J. Gurney, R.J. Morris, Age-related decrease in vulnerability to excitatory amino acids in the Nucleus Basalis, Neurobiol. Aging 17 Ž1996. 1–7. w49x G.L. Wenk, The nucleus basalis magnocellularis cholinergic system: one hundred years of progress, Neurobiol. Learn. Mem. 67 Ž1997. 85–95. w50x P.J. Whitehouse, D.L. Price, R.G. Struble, A.W. Clark, J.T. Coyle, M.R. DeLong, Alzheimer’s disease and senile dementia—loss of neurons in the basal forebrain, Science 215 Ž1982. 1237–1239.
Z. Gu et al.r Brain Research 801 (1998) 190–197
˚ Seiger, The expression, localization and funcw51x S.R. Whittemore, A. tional significance of b-nerve growth factor in the central nervous system, Brain Res. Rev. 12 Ž1987. 439–464. w52x R.G. Wiley, Neural lesioning with ribosome-inactivating proteins: suicide transport and immunolesioning, Trends Neurosci. 15 Ž1992. 285–290. w53x G. Wortwein, J. Yu, T. Toliver-Kinsky, J.R. Perez-Polo, Responses ¨ of young and aged rat CNS to partial cholinergic immunolesions and NGF treatment, J. Neurosci. Res. 52 Ž1998. 322–333. w54x J. Yu, D.P. Pizzo, L.A. Hutton, J.R. Perez-Polo, Role of the
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cholinergic system in the regulation of neurotrophin synthesis, Brain Res. 705 Ž1995. 247–254. w55x J. Yu, R.G. Wiley, J.R. Perez-Polo, Altered NGF protein levels in different brain areas after immunolesion, J. Neurosci. Res. 43 Ž1996. 213–223. w56x G.S. Zubenko, J. Moossy, A.J. Martinez, G.R. Rao, U. Kopp, I. Hanin, A brain regional analysis of morphologic and cholinergic abnormalities in Alzheimer’s disease, Arch. Neurol. 46 Ž1989. 634– 638.
Research report
Long term changes in brain cholinergic markers and nerve growth factor levels after partial immunolesion Zezong Gu, Juan Yu 1, J. Regino Perez-Polo
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Department of Human Biological Chemistry and Genetics, The UniÕersity of Texas Medical Branch at GalÕeston, 301 UniÕersity BlÕd., GalÕeston, TX 77555-0652, USA Accepted 26 May 1998
Abstract There are deficits in cholinergic basal forebrain neurons ŽCBFNs. in the aged brain and patients suffering Alzheimer’s disease associated with a partial loss of the CBFNs. To mimic this partial loss and assess its long term effects on residual cholinergic activity and resultant target-derived nerve growth factor ŽNGF. levels, we produced a partial immunolesion to CBFNs with 192 IgG-saporin, an immunotoxin selectively taken up by p75 NTR-bearing neurons. We measured two cholinergic markers, choline acetyltransferase ŽChAT. and acetylcholinesterase ŽAChE. activity, and NGF protein levels at 10 days, 1, 6 and 12 months postlesion. There were no significant changes in the cholinergic markers and the NGF protein levels in the sham-treated animal controls during the one year experiment. Ten days after 192 IgG-saporin treatment, ChAT activity decreased to 35–50% of controls in the olfactory bulb, hippocampus, and cortex. There was a minor but significant recovery of ChAT activity one year after the immunolesion in the hippocampus. Changes in AChE activity mirrored the ChAT changes but were less robust. There were transient increases in NGF protein levels in the hippocampus and cortex that returned to basal levels at 6 months and 12 months postlesion, respectively. In summary, partial immunolesions resulted in partial region-specific and time-dependent recoveries of cholinergic activity in the target areas of the basal forebrain after a partial elimination of CBFNs and a return to basal levels of NGF protein consistent with the hypothesis that the remaining CBFNs compensated for losses of ChAT and NGF due to changes in cholinergic innervation of basal forebrain target areas. q 1998 Elsevier Science B.V. All rights reserved. Keywords: 192 IgG-saporin; Acetylcholinesterase; Choline acetyltransferase; Cholinergic basal forebrain neuron; Immunolesion; Nerve growth factor; Regeneration
1. Introduction The cholinergic basal forebrain neurons ŽCBFNs. are located in the medial septum, diagonal band of Broca and nucleus basalis of Meynert and are involved in functions related to cognition and memory w4,21,35,49x. The nerve growth factor ŽNGF. is mostly synthesized in hippocam-
Abbreviations: Acetylcholinesterase, AChE; Brain-derived neurotrophic factor, BDNF; Choline acetyltransferase, ChAT; Cholinergic basal forebrain neuron, CBFN; Nerve growth factor, NGF; Phosphate buffered saline, PBS ) Corresponding author. Fax: q 1-409-772-8028; E-mail: [email protected] 1 Current address: St. Barnabas Medical Center, Livingston, NJ, USA. 0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 5 7 9 - 4
pus, neocortex and olfactory bulb, which are the target regions of CBFNs, and is internalized at the projection terminals after binding to NGF receptors, to be then retrogradely transported to the cell bodies of CBFNs w2,24x. NGF provides critical trophic support to CBFNs Žfor reviews, see Refs. w20,25,27,33,44,51x. that express high levels of the NGF receptors, TrkA and the low affinity neurotrophin ŽNT. receptor p75 NTR w14,15,22,23,42x. NGF has been shown to stimulate the synthesis of acetylcholine ŽACh. synthesizing enzyme, choline acetyltransferase ŽChAT. activity w16,19x. Cholinergic deficits in the aged and Alzheimer’s disease ŽAD. patients have been well documented. ChAT activity is consistently decreased by 50–95% in the cortex and hippocampus of AD patients w6,7,34,56x. There are also significant decreases in high-affinity choline uptake
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ŽHACU., in presynaptic muscarinic receptor binding, and in ACh and acetylcholinesterase ŽAChE. levels in the affected cortex and hippocampus w28,31,37,41x. The decrease of these cholinergic markers is associated with a marked loss of CBFNs w50x, and is closely correlated with the extent of AD neuropathology and the severity of cognitive impairment w6,30,34x. Attempts to study the role of neurotrophic factors on cholinergic function, such as learning and memory, have been based on the use of a wide variety of lesion models. Lesions have been produced either by physical means Žradio frequency lesion, electrolytic lesion, and fimbria– fornix transection. or by the injection of relatively specific neurotoxic substances. Four different injection strategies have been used: Ž1. injections of excitatory amino acids ŽEAAs. into basal forebrain ŽBF. w8,9x, Ž2. injections of aziridinium ion of ethyl choline mustard ŽAF64A. into the BF or CBFN target areas w11,17x, Ž3. intracerebral or systemic injections of murine monoclonal antibodies against AChE w3,36x. Although all these paradigms showed a uniform transient increase in NGF synthesis, it is not clear if these resulted from lesion-induced effects on CBFNs or non-cholinergic signaling pathways. Ž4. An alternative approach is to use the cytotoxin saporin coupled to the monoclonal antibody against low affinity neurotrophin receptor p75 NTR , 192 IgG w52,54,55x. Behavioral, biochemical, and morphological studies show that there are selective and robust effects of the 192 IgG-saporin on CBFNs in a stable and dose-dependent manner. Thus, 192 IgG-saporin produces significant long-lasting increases in NGF protein in the hippocampus, cortex and olfactory bulb and large decreases of ChAT activity w5,26,38,39,43,45– 47,54,55x. The injection of 192 IgG-saporin into the medial septum produces dose-related deficits in a variable-delay radial arm maze task. Injections of 192 IgG-saporin decrease the number of correct choices and increase the number of errors in the delayed non-match to sample task. These behavioral deficits are associated with dose-dependent decreases in pre-synaptic high affinity choline uptake in the hippocampus, cingulate and entorhinal cortex, the terminal fields of the medial septum w46x. Thus, the injection of 192 IgG-saporin into the medial septum produces cholinergic hypofunction and dose-dependent working memory deficits. Our previous work w40,53x showed that after a partial immunolesion to the rat CBFNs, transplants of mouse 3T3 NG Fq fibroblasts engineered to vigorously secrete NGF can up-regulate several components of CBFN function. Also, we have shown that the immunolesion-induced increase in NGF protein levels is absent in aged rats when compared to their young counterparts. Here, we applied the partial immunolesion paradigm by injecting the immunotoxin, 192 IgG-saporin, into the cerebral ventricles in order to assess the effects of a partial loss of CBFNs on residual cholinergic activity and target-derived nerve growth factor ŽNGF. levels over time.
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2. Materials and methods 2.1. Animals and surgical procedures Thirty-three 3-month-old male Sprague–Dawley rats weighing 250–300 gm ŽHarlan Sprague–Dawley, Indianapolis, IN. were used in this study. Procedures, dosages, location of injections, as well as time intervals required for the full development of the immunolesions were based on published work w39,40,53–55x or determined in a preliminary experiment using the same 192 IgG-saporin Ž192 IgG-SAP. lot used in these experiments ŽChemicon, Temecula, CA. where efficacy of immunolesion was monitored using AChE histochemistry. Animals were anaesthetized with ketamine Ž60 mgrkg i.m.. and Nembutal Ž40 mgrkg i.p.., and placed in a Kopf stereotaxic apparatus. The skull was exposed and two small burr holes were made using a dental drill. A Hamilton syringe with a 26-gauge needle was used for injection. A stock solution of 192 IgG-SAP was stored in frozen aliquots until just before use. Aliquots were then thawed and diluted to 0.65 mgrml in sterile phosphate buffered saline ŽPBS.. Rats were stereotaxically injected with 6 ml of either 192 IgG-saporin or vehicle, 10 mM sterile PBS at a rate of 1 mlrmin to the intracerebral ventricles Ži.c.v.. using the following coordinates: AP, y0.8; ML, "1.2; DV, y3.4, mm respectively w32x. 1.30 mg of 192 IgG-saporin was injected bilaterally to produce a CBFN partial immunolesion ŽPIL. and 4.5 mg of same immunotoxin delivered unilaterally was used to produce a total immunolesion ŽTIL. to CBFNs. After each injection the needle was left in place for 5 min to allow diffusion. The needle was then slowly retracted, the wound was bone-waxed and sutured, and the animal allowed to recover on a heating pad. No special post-operative care was required after 192 IgGsaporin lesions. All animals were sacrificed by decapitation under deep halothane anaesthesia and the brains were rapidly removed onto an ice-cold plate. Brain regions including the basal forebrain area, olfactory bulbs, bilateral hippocampus, and bilateral parieto-occipital cortex were dissected on ice. The tissues were immediately frozen on dry ice and stored at y808C until processing. In order to study the long-term effects of the CBFNs, four groups of animals were sacrificed at 10 days, 1, 6 and 12 Ž N s 4. months after PIL, one group of TIL animals Ž N s 4. at 1 month, and one group of control animals at 3 months and another at 15 months as controls for the one year duration of some experiments. Five animals were in each group unless otherwise indicated. 2.2. NGF immunoassay Tissue samples were prepared in three volumes of cold extraction buffer containing 0.4 M NaCl, 0.05% Žvolrvol. Tween 20, 0.5% Žwtrvol. bovine serum albumin ŽBSA.,
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0.1 mM phenylmethylsulfonyl fluoride Žfrom 1 M dimethylsulphofide stock solution., 0.1 mM benzethionium chloride, and 20 KIrml of aprotinin, pH 7.4. The homogenates were centrifuged at 14,000 g for 20 min, and the supernatants further diluted 1:1 with tissue buffer containing 10 mM CaCl 2 and 0.1% Triton X-100, pH 7.0 prior to NGF quantification. The procedure involves the following major steps: Ž1. coat 96 well EIA plates ŽNunc. with the monoclonal anti-NGF solution ŽBoehringer-Mannheim, 0.25 mgrml. 2 h at 378C, Ž2. block with 0.5% BSA 1r2 h at 378C, Ž3. incubate overnight at 48C with the samples to be tested or with standard solutions Žpurified mouse NGF diluted in 1:1 extraction and tissue buffer., Ž4. incubate with the anti-NGF solution ŽBoehringer-Mannheim, 4 mgrml. 4 h at 378C, Ž5. add substrate solution Ž40 mg of chlorophenolred b-galactopyranoside in 20 ml substrate buffer containing 100 mM HEPES, 150 mM NaCl, 2 mM MgCl 2 , 1% BSA, pH 7.0. and react for 1 1r2 h at 378C, Ž6. measure against substrate solution at 570 nm using an ELISA-Reader. Between each major step, the plate was washed three times with cold wash buffer containing 50 mM Tris–HCl, 200 mM NaCl, 10 mM CaCl 2 , 0.1% Triton X-100, pH 7.0.
density was read at 405 nm. The reaction mixture comprised the following: 0.20 mM DTNB ŽEllman’s reagent, Sigma, D-8130., 0.56 mM acetylthiocholine iodide and 39 mM phosphate buffer ŽpH 7.2.. To exclude the co-reaction of pseudocholinesterase the reaction mixture was added 10y5 M iso-OMPA. 2.5. Statistical analysis All the data are presented as the mean " S.E.M. The statistical significance of ChAT and AChE activities, and NGF levels among experimental groups was calculated by One-way ANOVA with post-hoc Fisher’s LSD analysis. The criteria for significance were set as P - 0.05.
3. Results Since a one year time period was included in the time course measurements, we determined whether ChAT activity or NGF protein levels were different for 3-month-old sham control groups as compared to 15-month-old sham
2.3. ChAT actiÕity A 4 ml aliquot of each homogenate prepared for NGF immunoassays was used in AChE assay Žsee the following. and ChAT assay according to the radiometric method of Fonnum Ž1975. w12x. Homogenates were further diluted as 1:30 ratio with homogenization buffer. Triplicate samples of 15 ml from diluted homogenates were used in the assay. The reaction mixture comprised the following: 600 mM NaCl, 0.2 mM eserine sulfate, 40 mM disodium EDTA, 100 mM phosphate buffer ŽpH 7.4., 18 mM choline chloride, 0.39 mM acetyl-CoA and 80 mM w14 Cxacetyl-CoA Ž0.3 mCirmmol.. Triplicate samples of each homogenate were incubated with 15 ml of reaction mixture at 378C for 40 min to allow for the synthesis of w14 Cxacetylcholine ŽACh.. Six samples of water, lacking additional radio labeled acetyl-CoA, were also assayed to serve as sample blanks. To terminate the reaction, 2.1 ml of 10 mM cold phosphate buffer ŽpH 7.4. and 0.5 ml of 0.5% cold sodium tetraphenylboron in acetonitrile were added, and the samples were vortex-mixed to extract the w14 CxACh-tetraphenylboron. The w14 Cx measurements were made with a scintillation counter ŽBeckman LS 7500 Liquid Scintillation System.. 2.4. AChE actiÕity The enzymatic activity was determined, as previously described w29x, by the method of Ellman et al. Ž1961. w10x with some modifications w1x. In brief, triplicate samples of 20 ml from 1:30 diluted homogenates were incubated with 250 ml of reaction mixture at 378C for 30 min and optical
Fig. 1. ChAT activity and NGF protein levels in different brain areas of sham-treated rats at two ages Ž3 vs. 15 months. one month after PBSvehicle injection into the intracerebral ventricles Ži.c.v... Values are means"S.E.M., N s 5, One-way ANOVA with post-hoc Fisher’s LSD analysis.
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controls experiencing intracerebral ventricular Ži.c.v.. delivery of vehicle ŽPBS.. Fig. 1 shows that there were no significant differences in ChAT activity or NGF protein content in the different brain areas 1 month after PBS injections into 3- and 15-month-old rat ventricles. These control values were therefore considered indistinguishable. When rats were exposed to partial immunolesions, there were 50–65% decreases in ChAT activity in the hippocampus, cortex and olfactory bulb 10 days after the 192
Fig. 3. Time course of changes in AChE activity in the target areas of the cholinergic basal forebrain neurons ŽCBFNs. after partial immunolesion. The AChE activity was expressed as the optical density of the colorimetric reading at 405 nm Ž N s 5 for each time point except N s 4 for 1 year postlesion.. Values are means"S.E.M., ) Ž P - 0.05. different time points after the lesion vs. sham controls and a Ž P - 0.05. vs. 10 days postlesion respectively, One-way ANOVA with post-hoc Fisher’s LSD analysis.
IgG-saporin treatment ŽFig. 2.. There was a minor but significant recovery of ChAT activity one year after the immunolesion in the hippocampus. In the olfactory bulb, ChAT activity remained at 56% of sham levels for up to one year postlesion. Measurement of NGF protein levels in these same animals showed transient increases in NGF levels in the hippocampus and cortex with returns to basal levels at 6 and 12 months postlesion, respectively, and significant long-term increases in NGF protein levels in the olfactory bulb with the highest levels reached 6 months after lesion. Thus, there was a prolonged partial recovery of cholinergic activity and a return of NGF protein levels
Fig. 2. Time course of changes in ChAT activity and NGF protein levels in the target areas of the cholinergic basal forebrain neurons ŽCBFNs. after partial immunolesion. ChAT activity and NGF protein levels are expressed as percentage of the values for vehicle-delivered shams Ž N s 5 for each time point except N s 4 for 1 year postlesion.. Values are means"S.E.M., ) Ž P - 0.05. different time points after the lesion vs. sham controls and a Ž P - 0.05. vs. 10 days postlesion respectively, One-way ANOVA with post-hoc Fisher’s LSD analysis.
Fig. 4. Time course of changes in ChAT activity and NGF protein levels in the basal forebrain after partial immunolesion. ChAT activity and NGF protein levels were expressed as percentage of the corresponding vehicle-delivered sham values Ž N s 5 for each time points except N s 4 for 1 year postlesion.. Values are means"S.E.M., ) P - 0.05 vs. sham controls, One-way ANOVA with post-hoc Fisher’s LSD analysis.
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to basal levels in the CBFN target areas after the partial elimination of CBFNs in a time-dependent and regionspecific fashion. The activity of AChE, the rate-limiting enzyme in acetylcholine ŽACh. hydrolysis, dramatically decreased after the partial elimination of CBFNs, consistent with the ongoing cholinergic degeneration in the basal forebrain ŽFig. 3.. There was a significant recovery of AChE activity in the olfactory bulb, but not in the hippocampus and cortex regions.
The levels of ChAT activity in the basal forebrain decreased Žabout 20%. 30 days postlesion before returning to sham control values. The magnitude of the observed changes was less than that in the target areas of CBFNs. NGF protein levels in the immunolesioned basal forebrain increased transiently by 10 days postlesion and had returned to normal levels by 30 days ŽFig. 4.. As expected total immunolesions resulted in more robust decreases in ChAT and AChE, and increases in NGF in the different brain regions ŽFig. 5. when compared to their partially immunolesioned counterparts reflecting a dose dependent effect on all tissues affected by immunolesion.
4. Discussion
Fig. 5. ChAT and AChE activities and NGF protein levels in different brain areas one month after partial Ž1.3 mg of 192 IgG-saporin. or total Ž4.5 mg of 192 IgG-saporin. immunolesion. Values are means"S.E.M., ) Ž P - 0.05. vs. sham controls and a Ž P - 0.05. vs. partial lesioned groups respectively, One-way ANOVA with post-hoc Fisher’s LSD analysis, N s 5 for sham control and partial lesioned groups and N s 4 for total lesioned group.
There were no significant changes in the expression of the two cholinergic markers used here in the sham control animals during a one year period. These results would support the hypothesis that physiological cholinergic function of the basal forebrain does not gradually decline in adulthood but rather that age-associated decreases are expressed late in life. This is not surprising insofar as there are no reports of dramatic differences in cognitive behavior between 3- and 15-month-old rats. In one study w48x carried out during acquisition, the T maze alternation task was shown to be sensitive to the effects of aging, i.e., the choice accuracy of 24-month-old rats was significantly less worse than either 3- or 9-month-old rats Ž P - 0.05., whereas the choice accuracy of 3- and 9-month-old rats did not differ significantly from each other. Expression of the two cholinergic markers used here was drastically reduced in those brain regions normally innervated by CBFNs as early as 10 days after the 192 IgG-saporin-mediated partial lesions. The decreased expression of ChAT and AChE activity in the target areas of the CBFNs is consistent with the loss of CBFN nerve terminals in the hippocampus, cortex, and olfactory bulb. Yu et al. w55x demonstrated that ChAT activity is progressively decreased in the septum, hippocampus, cortex and olfactory bulb during the first 11 days after immunotoxin injection and remained at those level for 5 months. There were no significant changes in ChAT activity in the cerebellum and a small decrease at 30 days in the striatum which are no or less p75 NTR positive cholinergic neurons. Other studies w18,39x have shown that several groups of non-cholinergic neurons in the basal forebrain ŽGABAergic septal neurons, Calbindin-D28K and NADPHd positive neurons. and one group of p75 NTR negative cholinergic neurons are not affected by the immunotoxin. The concomitant increase in NGF protein levels in these brain areas is evidence that the specific deafferentation of the CBFNs, in the absence of other direct interruptions of non-cholinergic neurotransmitter functions or direct lesions to any neurons in the hippocampus Žhippocampal neurons
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do not express p75 NTR ., triggers accumulation of NGF protein in those brain regions synthesizing NGF. Such an accumulation of NGF has been reported for total immunolesions as constant over time up to 5 months postlesion w55x. Given that cholinergic deafferentation alone has no effect on NGF mRNA levels in the hippocampus, cortex, and olfactory bulb and that the observed increases in NGF levels for a partial immunolesion are transient ŽFig. 2., it is likely that the return of NGF protein levels in target areas between 6 and 12 months postlesion reflect the restoration of retrograde transport of NGF by the residual CBFNs and therefore suggest that synaptic reorganization is carried out successfully after the deafferenting consequences of a partial immunolesion. Such an interpretation is consistent with the hypothesis that the lesion-induced increases in NGF suggest a role for NGF in postlesion remodeling. It is interesting that proximal sites witnessed a return to basal values earlier than sites more distant from the CBFNs. Reports of deafferentation associated with trauma, trauma alone, or deafferenting lesions that include a traumatic component, direct involvement of other neurotransmitter systems, or inflammatory responses have been shown to stimulate both NGF and BDNF mRNA and protein levels in contrast to the more restricted effects of cholinergic immunolesions on NGF accumulation but not synthesis, consistent with the hypothesis that the retrograde transport of NGF is largely responsible for cholinergic regulatory events and changes in basal levels of NGF synthesis may more directly reflect regulation of stress response gene products directly affecting neuronal commitment to apoptosis and inflammatory responses. It has been proposed that reinnervation by deafferented neurons after partial cholinergic lesions is due to lesion-induced increases in the release of NGF w13,40x. There was a transient and relatively smaller response in cholinergic marker activities in the basal forebrain after the partial immunolesions perhaps reflecting compensatory changes by the 50% residual CBFNs unaffected by the immunolesion ŽFig. 4.. There was a pronounced and very transient 150% increase in NGF protein levels that did not persist beyond 10 days postlesion ŽFig. 4. and could explain the delay in the decreases in ChAT activity in the CBFNs as compared to the more sudden decreases in cholinergic markers in the target areas for the CBFNs ŽFig. 2..
5. Conclusion In summary, partial immunolesions result in dose-dependent, region-specific and time-dependent losses in cholinergic activities in the innervation target areas of the CBFNs followed by long term recovery of activity over a 6 month to one year period. There are transient accumulations of NGF in the deafferented target regions consistent
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with impairment of retrograde transport of NGF to the basal forebrain and eventual recovery due to reinnervation of the target areas. The observed time course of changes in both the measured cholinergic markers and the levels of NGF protein are quite different for the CBFNs directly impacted by the 192 IgG-saporin injections and their projection areas in the hippocampus, cortex, and olfactory bulb. The differential time courses would suggest that different recuperative and regenerating processes are at work in the different regions. Furthermore, the responses to partial immunolesions would suggest that both ChAT and NGF levels respond to the state of cholinergic innervation of basal forebrain target areas and that there are recuperative processes in adult rats that might prove useful for the determination of the extent of plasticity of cholinergic function.
Acknowledgements This study was supported in part by NINDS Grant NS 33288 to JR P-P.
References w1x J. Andra, ¨ Z. Lojda, A histochemical method for the demonstration of acetylcholinesterase activity using semipermeable membranes, Histochemistry 84 Ž1986. 575–579. w2x C.E. Bandtlow, R. Heumann, M.E. Schwab, H. Thoenen, Cellular localization of nerve growth factor synthesis by in situ hybridization, EMBO J. 6 Ž1987. 891–899. w3x A.J. Bean, Z. Xu, S.Y. Chai, S. Brimijoin, T. Hokfelt, Effect of intracerebral injection of monoclonal acetylcholinesterase antibodies on cholinergic nerve terminals in the rat central nervous system, Neurosci. Lett. 133 Ž1991. 145–149. w4x V. Bigl, N.J. Woolf, L.L. Butcher, Cholinergic projections from the basal forebrain to frontal, parietal, temporal, occipital, and cingulate cortices: a combined fluorescent tracer and acetylcholinesterase analysis, Brain Res. Bull. 8 Ž1982. 727–749. w5x A.A. Book, R.G. Wiley, J.B. Schweitzei, Specificity of 192 IgGsaporin for NGF receptor-positive cholinergic basal forebrain neurons in the rat, Brain Res. 590 Ž1992. 350–355. w6x D.M. Bowen, C.B. Smith, P. White, A.N. Davison, Neurotransmitter-related enzymes and indices of hypoxia in senile dementia and other abiotrophies, Brain 99 Ž1976. 459–496. w7x P. Davies, Neurotransmitter-related enzymes in senile dementia of the Alzheimer’s type, Brain Res. 171 Ž1979. 319–327. w8x A.J. Dekker, D.J. Conner, L.J. Thal, The role of cholinergic projections from the nucleus basalis in memory, Neurosci. Biobehav. Rev. 15 Ž1991. 299–317. w9x S.B. Dunnett, B.J. Everitt, T.W. Robbins, The basal forebrain-cortical cholinergic systems: interpreting the functional consequences of excitotoxic lesions, Trend. Neurosci. 14 Ž1991. 494–501. w10x G.L. Ellman, K.D. Courtney, V. Andres Jr., R.M. Featherstone, A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol. 7 Ž1961. 88–95. w11x A. Fisher, I. Hanin, Potential animal models for senile dementia of Alzheimer’s type, with emphasis on AF64A induced cholinotoxicity, Ann. Rev. Pharmacol. Toxicol. 26 Ž1986. 161–181.
196
Z. Gu et al.r Brain Research 801 (1998) 190–197
w12x F. Fonnum, A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem. 24 Ž1975. 407–409. w13x F.H. Gage, A. Bjorklund, U. Stenevi, Reinnervation of the partially deafferented hippocampus by compensatory collateral sprouting from spared cholinergic and noradrenergic afferents, Brain Res. 268 Ž1983. 39–47. w14x R.B. Gibbs, J.T. McCabe, C.R. Buck, M.V. Chao, D.M. Pfaff, Expression of NGF receptor in the rat forebrain detected with in situ hybridization and immunohistochemistry, Mol. Brain Res. 6 Ž1989. 275–287. w15x R.B. Gibbs, D.W. Pfaff, In situ hybridization detection of trkA mRNA in brain; distribution, colocalization with p75 NG FR and up regulation by nerve growth factor, J. Comp. Neurol. 341 Ž1994. 324–339. w16x H. Gnahn, F. Hefti, R. Heumann, M.E. Schwab, H. Thoenen, NGF-mediated increase of choline acetyltransferase ŽChAT. in the neonatal rat forebrain: evidence for a physiological role of NGF in the brain, Dev. Brain Res. 9 Ž1983. 45–52. w17x I. Hanin, The AF64A model of cholinergic hypofunction: an update, Life Sci. 58 Ž1996. 1955–1964. w18x S. Heckers, T. Ohtake, R.G. Wiley, D.A. Lappi, C. Geula, M.-M. Mesulam, Complete and selective cholinergic denervation of rat neocortex and hippocampus but not amygdala by an immunotoxin against the p75 NGF receptor, J Neurosci. 14 Ž1994. 1271–1289. w19x F. Hefti, A. Dravid, J. Hartikka, Chronic intraventricular injections of nerve growth factor elevate hippocampal choline acetyltransferase activity in adult rats with partial septo-hippocampal lesions, Brain Res. 293 Ž1984. 305–311. w20x F. Hefti, B. Knusel, P.A. Lapchak, Protective effects of nerve growth factor and brain-derived neurotrophic factor on basal forebrain cholinergic neurons in adult rats with partial fimbrial transections, Prog. Brain Res. 98 Ž1993. 257–263. w21x Z. Henderson, Responses of basal forebrain cholinergic neurons to damage in the adult brain, Prog. Neurobiol. 48 Ž1996. 219–254. w22x D.M. Holtzman, Y. Li, L.F. Parada, S. Kinsma, C.-K. Chen, J.S. Valleta, J. Zhou, J.B. Long, W.C. Mobley, p140 trk mRNA marks NGF-responsive forebrain neurons: evidence that trk gene expression in induced by NGF, Neuron 9 Ž1992. 465–478. w23x E.M. Johnson, M. Taniuchi, Nerve growth factor ŽNGF. receptors in the central nervous system, Biochem. Pharmacol. 36 Ž1987. 4189– 4195. w24x E.M. Johnson, M. Taniuchi, H.B. Calrk, J.E. Springer, S. Koh, M. Tayrien, R. Loy, Demonstration of the retrograde transport of nerve growth factor in the peripheral and central nervous system, J. Neurosci. 7 Ž1987. 923–928. w25x S. Korsching, The role of nerve growth factor in the CNS, Trends Neurosci. 11 Ž12. Ž1986. 570–573. w26x G. Leanza, O.G. Nilsson, R.G. Wiley, A. Bjorklund, Selective lesioning of the basal forebrain cholinergic system by intraventricular 192 IgG-saporin: behavioral, biochemical and stereological studies in the rat, Eur. J. Neurosci. 7 Ž1995. 329–343. w27x C.A. Lucidi-Phillipi, F.H. Gage, The neurotrophin hypothesis and the cholinergic basal forebrain projection, Prog. Brain Res. 98 Ž1993. 241–256. w28x D.C. Mash, D.D. Flynn, L.T. Potter, Loss of M2 muscarine receptors in the cerebral cortex in Alzheimer’s disease and experimental cholinergic denervation, Science 228 Ž1985. 1115–1117. w29x H. Michalek, S. Fortuna, A. Pintor, Age-related differences in brain cholinesterase and muscarinic receptor sites in two strains of rats, Neurobiol. Aging 10 Ž1989. 143–148. w30x D. Neary, J.S. Snowden, D.M. Mann, D.M. Bowen, N.R. Sims, B. Northen, P.O. Yates, A.N. Davison, Alzheimer’s disease: a correlative study, J. Neurol. Neurosurg. Psychiatr. 49 Ž1986. 229–237. w31x J. Pascual, A. Fontan, J.J. Zarranz, J. Berciano, J. Florez, A. Pazos, High-affinity choline uptake carrier in Alzheimer’s disease: implications for the cholinergic hypothesis of dementia, Brain Res. 552 Ž1991. 170–174.
w32x G. Paxinos, C. Watson, The rat brainatlas in stereotaxic coordinates, 2nd edn., Academic Press, Sydney, 1986. w33x J.R. Perez-Polo, Roles of trophic factors in neuronal aging, Adv. Exp. Med. Biol. 265 Ž1990. 101–116. w34x E.K. Perry, B.E. Tomlinson, G. Blessed, K. Bergmann, P.H. Gilbson, R.H. Perry, Correlation of cholinergic abnormalities with senile plaques and mental test scores in senile dementia, Br. Med. J. 2 Ž1978. 1457–1459. w35x D.L. Price, V.E. Koliatsos, R.C. Clatterbuck, Cholinergic systems: human diseases, animal models, and prospects for therapy, Prog. Brain Res. 98 Ž1993. 51–60. w36x Z. Rakonczay, P. Hammond, S. Brimijoin, Lesion of central cholinergic system by systemically administered acetylcholinesterase antibodies in newborn rats, Neuroscience 54 Ž1993. 225–238. w37x J.A. Richter, E.K. Perry, B.E. Tomlinson, Acetylcholine and choline levels in post mortem human brain tissue: preliminary observations in Alzheimer’s disease, Life Sci. 26 Ž1980. 1683–1689. w38x S. Roßner, J.R. Perez-Polo, R.G. Wiley, V. Bigl, Differential expression of immediate early genes in distinct layers of rat cerebral cortex after selective immunolesion of the forebrain cholinergic system, J. Neurosci. Res. 38 Ž1994. 282–293. w39x S. Roßner, R. Schliebs, J.R. Perez-Polo, R.G. Wiley, V. Bigl, Differential changes of cholinergic markers from selected brain regions after specific immunolesion of the rat cholinergic basal forebrain system, J. Neurosci. Res. 40 Ž1995. 31–43. w40x S. Roßner, J. Yu, D. Pizzo, K. Werrbach-Perez, R. Schliebs, V. Bigl, J.R. Perez-Polo, Effects of intraventricular transplantation of NGFsecreting cells on cholinergic basal forebrain neurons after partial immunolesion, J. Neurosci. Res. 45 Ž1996. 40–56. w41x R.J. Rylett, M.J. Ball, E.H. Colhoun, Evidence for high affinity choline transport in synaptosomes prepared from hippocampus and neocortex of patients with Alzheimer’s disease, Brain Res. 289 Ž1983. 169–175. w42x T.L. Steininger, B.H. Wainer, R. Klein, M. Barbacid, H.C. Palfrey, High-affinity nerve growth factor receptor ŽTrk. immunoreactivity is localized in cholinergic neurons of the basal forebrain and striatum in the adult rat brain, Brain Res. 62 Ž1993. 330–335. w43x E.M. Torres, T.A. Perry, A. Blokland, L.S. Wilkinson, R.G. Wiley, D.A. Lappi, S.B. Dunnett, Behavioral, histochemical and biochemical consequences of selective immunolesions in discrete regions of the basal forebrain cholinergic system, Neuroscience 63 Ž1994. 95–122. w44x H. Thoenen, C. Bandtlow, R. Heumann, The physiological function of nerve growth factor in the central nervous system: comparison with the peripheral, Rev. Physiol. Biochem. Pharmacol. 109 Ž1987. 145–178. w45x J.J. Waite, M.L. Wardlow, A.C. Chen, D.A. Lappi, R.G. Wiley, Time course of cholinergic and monoaminergic changes in rat brain after immunolesioning with 192 IgG-saporin, Neurosci. Lett. 169 Ž1994. 154–158. w46x T.J. Walsh, C.D. Herzog, C. Gandhi, R.W. Stachman, R.G. Wiley, Injection of IgG-saporin into the medial septum produces cholinergic hypofunction and dose-dependent working memory deficits, Brain Res. 726 Ž1996. 69–79. w47x G.L. Wenk, J.D. Stoehr, G. Quintana, S. Mobley, R.G. Wiley, Behavioral, biochemical, histological, and electrophysiological effects of 192 IgG-saporin injections in the basal forebrain of rats, J. Neurosci. 14 Ž1994. 5986–5995. w48x G.L. Wenk, J.D. Stoehe, S.L. Mobley, J. Gurney, R.J. Morris, Age-related decrease in vulnerability to excitatory amino acids in the Nucleus Basalis, Neurobiol. Aging 17 Ž1996. 1–7. w49x G.L. Wenk, The nucleus basalis magnocellularis cholinergic system: one hundred years of progress, Neurobiol. Learn. Mem. 67 Ž1997. 85–95. w50x P.J. Whitehouse, D.L. Price, R.G. Struble, A.W. Clark, J.T. Coyle, M.R. DeLong, Alzheimer’s disease and senile dementia—loss of neurons in the basal forebrain, Science 215 Ž1982. 1237–1239.
Z. Gu et al.r Brain Research 801 (1998) 190–197
˚ Seiger, The expression, localization and funcw51x S.R. Whittemore, A. tional significance of b-nerve growth factor in the central nervous system, Brain Res. Rev. 12 Ž1987. 439–464. w52x R.G. Wiley, Neural lesioning with ribosome-inactivating proteins: suicide transport and immunolesioning, Trends Neurosci. 15 Ž1992. 285–290. w53x G. Wortwein, J. Yu, T. Toliver-Kinsky, J.R. Perez-Polo, Responses ¨ of young and aged rat CNS to partial cholinergic immunolesions and NGF treatment, J. Neurosci. Res. 52 Ž1998. 322–333. w54x J. Yu, D.P. Pizzo, L.A. Hutton, J.R. Perez-Polo, Role of the
197
cholinergic system in the regulation of neurotrophin synthesis, Brain Res. 705 Ž1995. 247–254. w55x J. Yu, R.G. Wiley, J.R. Perez-Polo, Altered NGF protein levels in different brain areas after immunolesion, J. Neurosci. Res. 43 Ž1996. 213–223. w56x G.S. Zubenko, J. Moossy, A.J. Martinez, G.R. Rao, U. Kopp, I. Hanin, A brain regional analysis of morphologic and cholinergic abnormalities in Alzheimer’s disease, Arch. Neurol. 46 Ž1989. 634– 638.