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NGF treatment potentiates c-fos expression in the rat nucleus basalis upon excitotoxic lesion with quisqualic acid
Brain Research 853 Ž2000. 136–141 www.elsevier.comrlocaterbres
Research report
NGF treatment potentiates c-fos expression in the rat nucleus basalis upon excitotoxic lesion with quisqualic acid Lisa Giovannelli ) , Maria Veltri, Fiorella Casamenti, Giancarlo Pepeu Department of Preclinical and Clinical Pharmacology, UniÕersity of Florence, Viale Pieraccini 6, 50139 Florence, Italy Accepted 2 November 1999
Abstract The induction of the c-fos gene in the rat brain by NGF was studied in a model of acute cholinergic hypofunction, i.e., the lesion of the nucleus basalis magnocellularis ŽNBM. with quisqualic acid. Choline acetyltransferase and Fos immunoreactivity ŽIR. in the NBM were analyzed at different times after the excitotoxic lesion. NGF treatment induced a potentiation of Fos expression 4 and 24 h after lesion. The possibility is discussed that c-fos induction is one of the early mechanisms of the neuroprotective action of NGF. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Neurotrophin; Early gene; Cholinergic forebrain system
1. Introduction Neurotrophic factors are a family of polypeptides which play an important role in the development and maintenance of neurons in the CNS. NGF was the first neurotrophin to be discovered and since then the best characterized. Low concentrations of NGF are present in the brain and its distribution among the different cerebral areas is highly non-homogeneous, with the highest levels occurring in the hippocampus and cortex w14,26,10,16x. It has been shown that NGF is important for maintaining survival and function of the basal forebrain cholinergic neurons w2x. Most of these neurons express both the low affinity receptor Žp75 NG FR . and TrkA, a component of the NGF high affinity binding site w7x. The cholinergic neurons of the basal forebrain are mainly localized in the medial septal nucleus, in the diagonal band nuclei and in the complex of the nucleus basalis magnocellularis ŽNBM., which provide most of the cholinergic innervation to the cerebral cortex and hippocampus, and their degeneration is thought to be responsible for the decline of cognitive function associated with aging and neurodegenerative diseases such as Alzheimer’s w6x. The intracerebroventricular administration of NGF in experimental animals reduces the extent of cholinergic degeneration induced by different lesions, in-
)
Corresponding author. [email protected]
Fax: q0039-55-4271-280; e-mail:
cluding fimbria–fornix transection in both primate and rat brain w25,27x and neurotoxic injury w1x. Furthermore, NGF treatment improves learning and memory in animal models of cognitive impairment w5,3,22x. In view of these observations, NGF has been proposed as a possible therapeutic agent in neurodegenerative disorders involving cholinergic neuronal atrophy such as Alzheimer’s disease. The early gene c-fos, by means of its product, the nuclear protein Fos, participates in the transduction of extracellular stimuli by modulating the transcription of other genes, among which the NGF gene w11x. On the other hand, it has been demonstrated that NGF in turn can induce the c-fos gene both in vitro and in vivo w15,24,20x. In the central nervous system, the c-fos gene can be induced by a variety of experimental manipulations w12x, including toxic and traumatic stimuli. Thus, c-fos is induced by intracerebral infusion of excitotoxins w18x, immunolesioning of the basal forebrain cholinergic system w21x, cerebral trauma w24,4x, hypoxic–ischemic damage w13,17x. It has been hypothesized that the early and transient induction of c-fos might be part of a protective cellular response to stress, whereas delayed induction has been proposed to be related to programmed cell death Žapoptosis. w23x. We recently demonstrated that c-fos gene is rapidly induced by excitotoxic lesions of the rat NBM made by quisqualic acid, and hypothesized that this induction is related to the cellular ability to survive the toxic stimulus w9x.
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 2 3 1 2 - 4
L. GioÕannelli et al.r Brain Research 853 (2000) 136–141
From these observations, we speculate that the induction of c-fos might be one of the cellular mechanisms by which NGF exerts its trophic action on cholinergic neurons. Thus, the aim of the present study was to verify whether NGF treatment modified c-fos induction upon lesioning of the NBM.
2. Materials and methods Male Wistar rats ŽHarlan Nossan, Milan, Italy. of 3 months of age were used. The rats were individually housed in macrolon cages with ad lib food and water and maintained on a 12-h lightrdark cycle, at 238C room temperature ŽRT.. All experiments were carried out according to the guidelines of the European Community’s Council for Animal Experiments. A unilateral Žright side. injection into the right NBM was performed under chloral hydrate Ž400 mgrkg i.p.. anesthesia by means of a stereotaxic apparatus Žcoordinates: AP s y0.5; L s y2.8 from bregma; H s y7.0 from the dura w19x.. A volume of 0.5 ml of either 0.12 M quisqualic acid ŽSigma, Dorset, UK. or saline solution was injected with a Hamilton syringe. The injection lasted 3 min and the needle was kept in place for 5 min before withdrawing. Four to six animals were prepared for each experimental group. After surgery, the animals were returned to their home cages, and sacrificed at different times. To investigate the early changes in gene expression and morphology, the times of 4 and 24 h were chosen. Longer time points of 1 and 3 weeks were investigated to assay long-term changes and plastic responses. NGF Ž2.5 S, Fidia Res. Laboratories, Abano Terme, Italy. was administered to both lesioned and unlesioned animals bi-weekly by i.c.v. injection using a polyethylene cannula ŽPE 10 tubing. implanted in the right ventricle on the day of lesioning. Treatment started immediately after quisqualic acid injection in the NBM. Experimental animals received 10 mg of NGF dissolved in 10 ml of vehicle Žsaline solution. and control animals 10 ml of saline in a single i.c.v. injection. Animals were subsequently sacrificed at different times after NGF injection Ž4 and 24 h, 1 and 3 weeks.. At the time of sacrifice, the rats were deeply anaestethized with ketamine Ž100 mgrkg i.p.. and perfused through the ascending aorta with 50 ml of saline solution followed by 200–300 ml of ice-cold paraformaldehyde solution Ž2% in phosphate-buffer, pH 7.4.. The brains were removed, postfixed in the same fixative for 2 h, cryoprotected in sucrose solution Ž18% in water. for at least 24 h at 48C, and cut in a cryostat from the level of the caudal septum to the level of the caudal caudate–putamen in 20-mm thick coronal sections. The sections were mounted on gelatin-coated slides, let dry at RT and stored at y208C. Sections throughout the extension of the in-
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jected area were stained with Richardson’s solution according to the Nissl method, in order to check the position of the injection and the general morphology of the area. Rats that did not bear a correctly placed lesion Žabout 5%. were discarded. C-fos and ChAT immunoreactivity ŽIR. were visualized by means of two polyclonal antibodies Žanti-c-fos made in sheep from Genosys, Northwick, UK, and anti-ChAT made in rabbit from Chemicon, Temecula, CA. used at a 1:1000 dilution in phosphate-buffered saline ŽPBS. and localized with the same procedure. After a pre-incubation of 1 h at RT in 2% bovine serum albumin ŽBSA. in PBS with the addition of 0.3% Triton-X-100 ŽTX., the slides were incubated overnight at RT with the primary antibody in the presence of 0.5% BSA and TX. On the following day, the sections were washed in PBS and incubated for 1 h at RT with the secondary antibody ŽVector Laboratories, Burlingame, CA. diluted 1:1000 in PBS–0.5% BSA. After a further PBS wash, incubation with the avidin–biotin– peroxidase complex ŽVector kit. followed for 1 h at RT. The primary antibody was finally localized by the diaminobenzidine ŽDAB. –H 2 O 2 –peroxidase color reaction, using NiCl 2 as intensifier. The reaction was usually completed in 8 min, at the end of which the slides were washed, dehydrated and mounted. Colocalization of the Fos protein and ChAT was carried out as described w9x at the 4-h time point after lesion. Briefly, immunohistochemistry for Fos was performed first as described above, then after PBS washes the slides were further incubated with the anti-ChAT antibody overnight Ž1:200.. A fluorescein-labelled anti-rabbit antibody ŽVector. was then used at 1:100 dilution to visualize ChATpositive neurons, and the slides were mounted in aqueous medium. Controls for the immunocytochemical procedures were obtained running some slides through the entire procedure with the omission of the primary or secondary antibodies. No staining was observed in these control slides. For microscopic analysis and photography, a Nikon Labophot-2 microscope equipped for epifluorescence was used. Cell counting was performed with the aid of a computerized image analysis system using the Image-Pro Plus software. NBM ChAT-immunoreactive cells were identified as purple–black bodies with neuronal shape located in the ventral pallidum and the adjoining internal capsule. Five sections per animal were analyzed between bregma y0.3 mm and bregma y1.3 mm w19x. The total number of ChAT-positive cells in the lesioned NBM was compared to that in the contralateral unlesioned side of the same section. Counting of c-fos-positive nuclei was performed similarly in the area surrounding the injection site. Three fields of 0.5 mm2 each were randomly chosen in the NBM area and the number of nuclei averaged among the three selected fields. Five sections per animal were counted and
L. GioÕannelli et al.r Brain Research 853 (2000) 136–141
138
averaged. Final data were expressed as density of immunoreactive nuclei per area unit Žmm2 .. Statistical analysis was carried out by means of the paired Student’s t-test for ChAT-immunoreactive neuronal counts, and by the unpaired Student’s t-test for c-fos-positive nuclei counts.
3. Results Table 1 shows that treatment for 3 weeks with NGF according to the above described schedule induces a recovery of the number of ChAT-positive neurons in the lesioned nucleus basalis. A 68% decrease in the number of immunoreactive neurons was found in the right nucleus basalis of the animals treated with saline post-lesion, whereas no statistically significant decrease was detected in the animals treated with NGF. This effect of NGF was not present after 1 week of treatment, when the number of ChAT-positive neurons in the lesioned nucleus basalis was still strongly reduced Žy60%. as compared to the unlesioned contralateral side ŽFig. 2.. For c-fos studies, we initially investigated whether NGF induces the expression of c-fos per se. The results of this group of experiments are shown in Fig. 1, where the effects of NGF and saline in unlesioned animals are compared 4 h post-injection. A single i.c.v. injection of NGF induced a moderate expression of the c-fos gene in the ipsilateral cerebral cortex, in the thalamus Žparaventricular nucleus, central lateral nucleus and central medial nucleus. and in the hypothalamus. At longer times Ž24 h, 1 and 3 weeks. no induction of c-fos by NGF or saline was detected. A second group of experiments was aimed to verify whether the treatment with NGF modifies lesion-induced c-fos expression. Table 2 shows that a strong ipsilateral
Table 1 Effect of NGF treatment on the number of ChAT-immunoreactive neurons in the nucleus basalis Animals were lesioned with quisqualic acid and treated i.c.v. with either saline solution Žsaline. or NGF Ž10 mg. twice a week for 7 or 21 days starting the day of surgery. Data are the mean"S.E. of four animals per group and are expressed as the total number of neurons counted in five coronal sections per rat in the nucleus basalis of both sides. In the far right column, the percent change in the lesioned right ŽDX. side as compared to the unlesioned left ŽSX. side is shown. Post-lesion treatment
Treatment duration Ždays.
Unlesioned side
Lesioned side
% change ŽDXrSX.
Saline NGF Saline NGF
7 7 21 21
233"27.3 241"29.3 211"19.3 229"23.4
103"22.2U 96"19.5U 67"11.4U 208"25.2
y56 y60 y68 y9
U
p- 0.05 statistically significant difference from the corresponding unlesioned side Žpaired Student’s t-test..
Fig. 1. Schematic drawing showing the distribution of Fos-immunoreactive neurons in coronal rat brain sections. Left: 4 h after i.c.v. injection with saline; right: 4 h after i.c.v. injection with NGF Ž10 mg.. Three different coronal levels are shown Ža, b, c. whose corresponding bregma values in millimeters are: Ža. y0.30, Žb. y0.80, Žc. y1.30. Each dot represents one Fos-positive nucleus.
induction of c-fos was rapidly elicited in animals lesioned with quisqualic acid and treated i.c.v. with saline, and that such induction was much reduced 24 h post-lesion, thus, confirming our previous results w9x. Furthermore, post-lesion treatment with NGF potentiated the induction of c-fos both 4 ŽFig. 2. and 24 h after quisqualic acid administration. In fact, a single injection of NGF induced an increase in the density of Fos-positive nuclei in the lesioned nucleus basalis as compared to the animals injected with saline post-lesion Žq44% at 4 h and q263% at 24 h.. Analysis of counterstained sections showed that c-fos expression was exclusively neuronal Ždata not shown.. Colocalization experiments showed that the Fos protein and Table 2 Effect of NGF treatment on the density of c-fos-positive nuclei in the rat nucleus basalis upon quisqualic acid local injection Animals were lesioned with quisqualic acid, treated i.c.v. with either saline solution Žsaline. or NGF Ž10 mg. immediately after surgery and sacrificed 4 or 24 h later. Data are the mean"S.E. of four animals per group and are expressed as mean density of labeled nuclei per area unit Žmm2 . in the right nucleus basalis. Five coronal sections per rat were analyzed. Post-lesion treatment
Time after lesion Žh.
Number of c-fospositive nucleirmm2
Saline NGF Saline NGF
4 4 24 24
79.8"12.91 114.9"16.45U 5.7"0.1 20.7"3.10U
U
p- 0.05 statistically significant difference from saline ŽStudent’s t-test..
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Fig. 2. Photomicrographs showing coronal sections of the rat NBM immunohistochemically stained for ChAT Ža, b., Fos Žc, d., ChAT and Fos Že, f.. Ža. ChAT-immunoreactive neurons in the unlesioned left NBM after 1 week of i.c.v. NGF treatment; Žb. loss of ChAT-immunoreactive neurons in the lesioned right NBM after 1 week of i.c.v. NGF treatment; gp s globus pallidus; ic s internal capsule; Žc. Fos expression in the right NBM 4 h after lesion; Žd. Fos expression 4 h after lesion and i.c.v. injection of NGF; Že. ChAT immunofluorescence in the right NBM 4 h after lesion and i.c.v. injection of NGF; Žf. same field shown in Že., viewed under normal light to detect Fos-immunoreactive nuclei; the position occupied by the cholinergic neurons is indicated by open triangles. Note the lack of colocalization of the two antigens. Magnification: =125 Ža, b., =190 Žc, d., =340 Že, f.. Scale bar s 40 m.
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L. GioÕannelli et al.r Brain Research 853 (2000) 136–141
ChAT did not colocalize in NGF-treated animals 4 h after lesion ŽFig. 2.. Finally, we investigated whether NGF potentiates the induction of the c-fos gene in lesioned animals also at longer times post-lesion. Thus, we have analyzed two further groups of animals lesioned with quisqualic acid in the right nucleus basalis and treated i.c.v. with NGF for 1 week Žtotal of two NGF injections. or for 3 weeks Žtotal of six NGF injections.. We have found no c-fos expression in any brain area in either group.
4. Discussion In the present report, we show that post-lesion NGF treatment for 3 weeks reduces the loss of ChAT-immunopositive neurons induced by excitotoxic lesions of the NBM, in agreement with previous reports w1x. Furthermore, we demonstrate that NGF treatment induces in lesioned animals an increase in c-fos induction as compared to the lesioned animals treated with saline. This effect of NGF is evident both at 4 and 24 h after lesion, and not at later times. A relationship between NGF and the c-fos gene has been repeatedly suggested. In our experimental conditions, NGF treatment per se rapidly induces the expression of c-fos in some cerebral areas, but not in the NBM. Previous reports have shown small increases in Fos IR in this area upon NGF administration w20,8x. These discrepancies are probably accounted for by differences in the technical procedures. Our present data show that NGF treatment increases the number of neurons expressing Fos upon lesioning of the NBM, and we have previously hypothesized that early c-fos induction by NBM excitotoxic lesions might be a protective cellular response w9x. In view of these results, it appears feasible that one of the mechanisms of the neuroprotective action of NGF is the potentiation of c-fos induction. However, the potentiation of c-fos expression takes place within 24 h after lesion, whereas 3 weeks of NGF treatment are necessary to achieve a significant recovery of ChAT IR, and we have not detected further Fos protein induction later than 24 h post-lesion, although we cannot exclude that Fos might be expressed at intermediate time points that we have not investigated. This seems to indicate that c-fos induction is not sufficient for mediating the trophic effects of NGF on cholinergic neurons, and that other NGF-induced events need to occur at later times for the recovery to be completed. Whether these later events require early Fos protein expression or are independent from the c-fos gene needs to be elucidated. It has been reported that NGF can prevent delayed apoptotic cell death in vivo w27x. Thus, it can be speculated that NGF exerts both a fast, Fos-mediated action that protects the cells from rapid excitotoxic death, and, a later, possibly Fos-indepen-
dent action which supports cell repair and prevents damaged neurons from undergoing apoptosis. We have previously shown that the early induction of c-fos by excitotoxic lesions in the NBM does not occur in cholinergic neurons, but in other neuronal cells of the NBM itself and in the surrounding areas w9x. In the present study, a further increase in the number of Fos-positive neurons in the NBM was found upon NGF administration to lesioned animals. Thus, we performed ChAT and Fos protein colocalization experiments in order to verify whether the NGF-induced increase in the number of Fospositive nuclei was due to c-fos induction in cholinergic neurons of the NBM. These experiments have shown that in lesioned animals the cholinergic neurons do not express the Fos protein 4 h after NGF treatment, indicating that at this time point the increase in the number of Fos-positive nuclei induced by NGF is due to Fos expression in noncholinergic NBM neurons. However, we were not able to perform colocalization experiments at 24 h post-lesion, in view of the fact that at this time point cholinergic neurons have already lost ChAT IR and are no longer detectable w9x. Thus, the possibility cannot be ruled out that NGF can induce a slower expression of the c-fos gene in the damaged cholinergic neurons, and that this might contribute to the significant increase in the number of Fos-positive nuclei that we found at 24 h upon NGF treatment. Furthermore, it is possible that cholinergic neurons do express Fos at later times during the process of recovery. In conclusion, the experimental model of the excitotoxic lesion of the rat NBM has allowed us to study the role of the c-fos gene in the molecular mechanisms through which NGF supports the recovery of lesioned cholinergic neurons. The hypothesis can be put forward that c-fos induction is one of the early events that contribute to the trophic actions of NGF.
Acknowledgements This investigation was supported by a research contract with NE.FA.C., Pomezia, Italy ŽMURST Neurobiological Systems National Research Plan.. We thank FIDIA Research Laboratories ŽAbano Terme, Italy. for providing NGF.
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w15x W. Kruijer, D. Schubert, I.M. Verma, Induction of the proto-oncogene c-fos by nerve growth factor, Proc. Natl. Acad. Sci. U.S.A. 82 Ž1985. 7330–7334. w16x E.J. Mufson, J.M. Conner, S. Varon, J.H. Kordower, Nerve growth factor-like immunoreactive profiles in the primate basal forebrain and hippocampal formation, J. Comp. Neurol. 341 Ž1994. 507–519. w17x H. Onodera, K. Kogure, Y. Ono, K. Igarashi, Y. Kiyota, A. Nagaoka, Proto-oncogene c-fos is transiently induced in the rat cerebral cortex after forebrain ischemia, Neurosci. Lett. 98 Ž1989. 101–104. w18x K.J. Page, A. Saha, B.J. Everitt, Differential activation and survival of basal forebrain neurons following infusions of excitatory amino acids: studies with the immediate early gene c-fos, Exp. Brain Res. 93 Ž1993. 412–422. w19x G. Paxinos, G. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, New York. w20x Z.C. Peng, S. Chen, M. Fusco, G. Vantini, M. Bentivoglio, Fos induction by nerve growth factor in the adult rat brain, Brain Res. 632 Ž1993. 57–67. w21x S. Roßner, J.R. Perez-Polo, R.G. Wiley, R. Schliebs, 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. w22x C. Scali, F. Casamenti, M. Pazzagli, L. Bartolini, G. Pepeu, Nerve growth factor increases extracellular acetylcholine levels in the parietal cortex and hippocampus of aged rats and restores object recognition, Neurosci. Lett. 170 Ž1994. 117–120. w23x S.S. Schreiber, M. Baudry, Selective neuronal vulnerability in the hippocampus — a role for gene expression?, Trends Neurosci. 18 Ž1995. 446–451. w24x F.R. Sharp, M.F. Gonzales, K. Hisanaga, W.C. Mobley, S.M. Sagar, Induction of the c-fos gene product in rat forebrain following cortical lesions and NGF injections, Neurosci. Lett. 100 Ž1989. 117–122. w25x M.H. Tuszynski, U.H. Sang, D.G. Amaral, F.H. Gage, Nerve growth factor infusion in the primate brain reduces lesion-induced cholinergic neuronal degeneration, J. Neurosci. 10 Ž1990. 3604–3614. w26x S.R. Whittemore, A. Sieger, The expression, localization and functional significance of b-nerve growth factor messenger RNA and protein in the central nervous system, Brain Res. Rev. 12 Ž1987. 439–464. w27x B.J. Wilcox, M.D. Applegate, C. Portera-Cailliau, V.E. Koliatsos, Nerve growth factor prevents apoptotic cell death in injured central cholinergic neurons, J. Comp. Neurol. 359 Ž1995. 573–585.
Research report
NGF treatment potentiates c-fos expression in the rat nucleus basalis upon excitotoxic lesion with quisqualic acid Lisa Giovannelli ) , Maria Veltri, Fiorella Casamenti, Giancarlo Pepeu Department of Preclinical and Clinical Pharmacology, UniÕersity of Florence, Viale Pieraccini 6, 50139 Florence, Italy Accepted 2 November 1999
Abstract The induction of the c-fos gene in the rat brain by NGF was studied in a model of acute cholinergic hypofunction, i.e., the lesion of the nucleus basalis magnocellularis ŽNBM. with quisqualic acid. Choline acetyltransferase and Fos immunoreactivity ŽIR. in the NBM were analyzed at different times after the excitotoxic lesion. NGF treatment induced a potentiation of Fos expression 4 and 24 h after lesion. The possibility is discussed that c-fos induction is one of the early mechanisms of the neuroprotective action of NGF. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Neurotrophin; Early gene; Cholinergic forebrain system
1. Introduction Neurotrophic factors are a family of polypeptides which play an important role in the development and maintenance of neurons in the CNS. NGF was the first neurotrophin to be discovered and since then the best characterized. Low concentrations of NGF are present in the brain and its distribution among the different cerebral areas is highly non-homogeneous, with the highest levels occurring in the hippocampus and cortex w14,26,10,16x. It has been shown that NGF is important for maintaining survival and function of the basal forebrain cholinergic neurons w2x. Most of these neurons express both the low affinity receptor Žp75 NG FR . and TrkA, a component of the NGF high affinity binding site w7x. The cholinergic neurons of the basal forebrain are mainly localized in the medial septal nucleus, in the diagonal band nuclei and in the complex of the nucleus basalis magnocellularis ŽNBM., which provide most of the cholinergic innervation to the cerebral cortex and hippocampus, and their degeneration is thought to be responsible for the decline of cognitive function associated with aging and neurodegenerative diseases such as Alzheimer’s w6x. The intracerebroventricular administration of NGF in experimental animals reduces the extent of cholinergic degeneration induced by different lesions, in-
)
Corresponding author. [email protected]
Fax: q0039-55-4271-280; e-mail:
cluding fimbria–fornix transection in both primate and rat brain w25,27x and neurotoxic injury w1x. Furthermore, NGF treatment improves learning and memory in animal models of cognitive impairment w5,3,22x. In view of these observations, NGF has been proposed as a possible therapeutic agent in neurodegenerative disorders involving cholinergic neuronal atrophy such as Alzheimer’s disease. The early gene c-fos, by means of its product, the nuclear protein Fos, participates in the transduction of extracellular stimuli by modulating the transcription of other genes, among which the NGF gene w11x. On the other hand, it has been demonstrated that NGF in turn can induce the c-fos gene both in vitro and in vivo w15,24,20x. In the central nervous system, the c-fos gene can be induced by a variety of experimental manipulations w12x, including toxic and traumatic stimuli. Thus, c-fos is induced by intracerebral infusion of excitotoxins w18x, immunolesioning of the basal forebrain cholinergic system w21x, cerebral trauma w24,4x, hypoxic–ischemic damage w13,17x. It has been hypothesized that the early and transient induction of c-fos might be part of a protective cellular response to stress, whereas delayed induction has been proposed to be related to programmed cell death Žapoptosis. w23x. We recently demonstrated that c-fos gene is rapidly induced by excitotoxic lesions of the rat NBM made by quisqualic acid, and hypothesized that this induction is related to the cellular ability to survive the toxic stimulus w9x.
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 2 3 1 2 - 4
L. GioÕannelli et al.r Brain Research 853 (2000) 136–141
From these observations, we speculate that the induction of c-fos might be one of the cellular mechanisms by which NGF exerts its trophic action on cholinergic neurons. Thus, the aim of the present study was to verify whether NGF treatment modified c-fos induction upon lesioning of the NBM.
2. Materials and methods Male Wistar rats ŽHarlan Nossan, Milan, Italy. of 3 months of age were used. The rats were individually housed in macrolon cages with ad lib food and water and maintained on a 12-h lightrdark cycle, at 238C room temperature ŽRT.. All experiments were carried out according to the guidelines of the European Community’s Council for Animal Experiments. A unilateral Žright side. injection into the right NBM was performed under chloral hydrate Ž400 mgrkg i.p.. anesthesia by means of a stereotaxic apparatus Žcoordinates: AP s y0.5; L s y2.8 from bregma; H s y7.0 from the dura w19x.. A volume of 0.5 ml of either 0.12 M quisqualic acid ŽSigma, Dorset, UK. or saline solution was injected with a Hamilton syringe. The injection lasted 3 min and the needle was kept in place for 5 min before withdrawing. Four to six animals were prepared for each experimental group. After surgery, the animals were returned to their home cages, and sacrificed at different times. To investigate the early changes in gene expression and morphology, the times of 4 and 24 h were chosen. Longer time points of 1 and 3 weeks were investigated to assay long-term changes and plastic responses. NGF Ž2.5 S, Fidia Res. Laboratories, Abano Terme, Italy. was administered to both lesioned and unlesioned animals bi-weekly by i.c.v. injection using a polyethylene cannula ŽPE 10 tubing. implanted in the right ventricle on the day of lesioning. Treatment started immediately after quisqualic acid injection in the NBM. Experimental animals received 10 mg of NGF dissolved in 10 ml of vehicle Žsaline solution. and control animals 10 ml of saline in a single i.c.v. injection. Animals were subsequently sacrificed at different times after NGF injection Ž4 and 24 h, 1 and 3 weeks.. At the time of sacrifice, the rats were deeply anaestethized with ketamine Ž100 mgrkg i.p.. and perfused through the ascending aorta with 50 ml of saline solution followed by 200–300 ml of ice-cold paraformaldehyde solution Ž2% in phosphate-buffer, pH 7.4.. The brains were removed, postfixed in the same fixative for 2 h, cryoprotected in sucrose solution Ž18% in water. for at least 24 h at 48C, and cut in a cryostat from the level of the caudal septum to the level of the caudal caudate–putamen in 20-mm thick coronal sections. The sections were mounted on gelatin-coated slides, let dry at RT and stored at y208C. Sections throughout the extension of the in-
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jected area were stained with Richardson’s solution according to the Nissl method, in order to check the position of the injection and the general morphology of the area. Rats that did not bear a correctly placed lesion Žabout 5%. were discarded. C-fos and ChAT immunoreactivity ŽIR. were visualized by means of two polyclonal antibodies Žanti-c-fos made in sheep from Genosys, Northwick, UK, and anti-ChAT made in rabbit from Chemicon, Temecula, CA. used at a 1:1000 dilution in phosphate-buffered saline ŽPBS. and localized with the same procedure. After a pre-incubation of 1 h at RT in 2% bovine serum albumin ŽBSA. in PBS with the addition of 0.3% Triton-X-100 ŽTX., the slides were incubated overnight at RT with the primary antibody in the presence of 0.5% BSA and TX. On the following day, the sections were washed in PBS and incubated for 1 h at RT with the secondary antibody ŽVector Laboratories, Burlingame, CA. diluted 1:1000 in PBS–0.5% BSA. After a further PBS wash, incubation with the avidin–biotin– peroxidase complex ŽVector kit. followed for 1 h at RT. The primary antibody was finally localized by the diaminobenzidine ŽDAB. –H 2 O 2 –peroxidase color reaction, using NiCl 2 as intensifier. The reaction was usually completed in 8 min, at the end of which the slides were washed, dehydrated and mounted. Colocalization of the Fos protein and ChAT was carried out as described w9x at the 4-h time point after lesion. Briefly, immunohistochemistry for Fos was performed first as described above, then after PBS washes the slides were further incubated with the anti-ChAT antibody overnight Ž1:200.. A fluorescein-labelled anti-rabbit antibody ŽVector. was then used at 1:100 dilution to visualize ChATpositive neurons, and the slides were mounted in aqueous medium. Controls for the immunocytochemical procedures were obtained running some slides through the entire procedure with the omission of the primary or secondary antibodies. No staining was observed in these control slides. For microscopic analysis and photography, a Nikon Labophot-2 microscope equipped for epifluorescence was used. Cell counting was performed with the aid of a computerized image analysis system using the Image-Pro Plus software. NBM ChAT-immunoreactive cells were identified as purple–black bodies with neuronal shape located in the ventral pallidum and the adjoining internal capsule. Five sections per animal were analyzed between bregma y0.3 mm and bregma y1.3 mm w19x. The total number of ChAT-positive cells in the lesioned NBM was compared to that in the contralateral unlesioned side of the same section. Counting of c-fos-positive nuclei was performed similarly in the area surrounding the injection site. Three fields of 0.5 mm2 each were randomly chosen in the NBM area and the number of nuclei averaged among the three selected fields. Five sections per animal were counted and
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averaged. Final data were expressed as density of immunoreactive nuclei per area unit Žmm2 .. Statistical analysis was carried out by means of the paired Student’s t-test for ChAT-immunoreactive neuronal counts, and by the unpaired Student’s t-test for c-fos-positive nuclei counts.
3. Results Table 1 shows that treatment for 3 weeks with NGF according to the above described schedule induces a recovery of the number of ChAT-positive neurons in the lesioned nucleus basalis. A 68% decrease in the number of immunoreactive neurons was found in the right nucleus basalis of the animals treated with saline post-lesion, whereas no statistically significant decrease was detected in the animals treated with NGF. This effect of NGF was not present after 1 week of treatment, when the number of ChAT-positive neurons in the lesioned nucleus basalis was still strongly reduced Žy60%. as compared to the unlesioned contralateral side ŽFig. 2.. For c-fos studies, we initially investigated whether NGF induces the expression of c-fos per se. The results of this group of experiments are shown in Fig. 1, where the effects of NGF and saline in unlesioned animals are compared 4 h post-injection. A single i.c.v. injection of NGF induced a moderate expression of the c-fos gene in the ipsilateral cerebral cortex, in the thalamus Žparaventricular nucleus, central lateral nucleus and central medial nucleus. and in the hypothalamus. At longer times Ž24 h, 1 and 3 weeks. no induction of c-fos by NGF or saline was detected. A second group of experiments was aimed to verify whether the treatment with NGF modifies lesion-induced c-fos expression. Table 2 shows that a strong ipsilateral
Table 1 Effect of NGF treatment on the number of ChAT-immunoreactive neurons in the nucleus basalis Animals were lesioned with quisqualic acid and treated i.c.v. with either saline solution Žsaline. or NGF Ž10 mg. twice a week for 7 or 21 days starting the day of surgery. Data are the mean"S.E. of four animals per group and are expressed as the total number of neurons counted in five coronal sections per rat in the nucleus basalis of both sides. In the far right column, the percent change in the lesioned right ŽDX. side as compared to the unlesioned left ŽSX. side is shown. Post-lesion treatment
Treatment duration Ždays.
Unlesioned side
Lesioned side
% change ŽDXrSX.
Saline NGF Saline NGF
7 7 21 21
233"27.3 241"29.3 211"19.3 229"23.4
103"22.2U 96"19.5U 67"11.4U 208"25.2
y56 y60 y68 y9
U
p- 0.05 statistically significant difference from the corresponding unlesioned side Žpaired Student’s t-test..
Fig. 1. Schematic drawing showing the distribution of Fos-immunoreactive neurons in coronal rat brain sections. Left: 4 h after i.c.v. injection with saline; right: 4 h after i.c.v. injection with NGF Ž10 mg.. Three different coronal levels are shown Ža, b, c. whose corresponding bregma values in millimeters are: Ža. y0.30, Žb. y0.80, Žc. y1.30. Each dot represents one Fos-positive nucleus.
induction of c-fos was rapidly elicited in animals lesioned with quisqualic acid and treated i.c.v. with saline, and that such induction was much reduced 24 h post-lesion, thus, confirming our previous results w9x. Furthermore, post-lesion treatment with NGF potentiated the induction of c-fos both 4 ŽFig. 2. and 24 h after quisqualic acid administration. In fact, a single injection of NGF induced an increase in the density of Fos-positive nuclei in the lesioned nucleus basalis as compared to the animals injected with saline post-lesion Žq44% at 4 h and q263% at 24 h.. Analysis of counterstained sections showed that c-fos expression was exclusively neuronal Ždata not shown.. Colocalization experiments showed that the Fos protein and Table 2 Effect of NGF treatment on the density of c-fos-positive nuclei in the rat nucleus basalis upon quisqualic acid local injection Animals were lesioned with quisqualic acid, treated i.c.v. with either saline solution Žsaline. or NGF Ž10 mg. immediately after surgery and sacrificed 4 or 24 h later. Data are the mean"S.E. of four animals per group and are expressed as mean density of labeled nuclei per area unit Žmm2 . in the right nucleus basalis. Five coronal sections per rat were analyzed. Post-lesion treatment
Time after lesion Žh.
Number of c-fospositive nucleirmm2
Saline NGF Saline NGF
4 4 24 24
79.8"12.91 114.9"16.45U 5.7"0.1 20.7"3.10U
U
p- 0.05 statistically significant difference from saline ŽStudent’s t-test..
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Fig. 2. Photomicrographs showing coronal sections of the rat NBM immunohistochemically stained for ChAT Ža, b., Fos Žc, d., ChAT and Fos Že, f.. Ža. ChAT-immunoreactive neurons in the unlesioned left NBM after 1 week of i.c.v. NGF treatment; Žb. loss of ChAT-immunoreactive neurons in the lesioned right NBM after 1 week of i.c.v. NGF treatment; gp s globus pallidus; ic s internal capsule; Žc. Fos expression in the right NBM 4 h after lesion; Žd. Fos expression 4 h after lesion and i.c.v. injection of NGF; Že. ChAT immunofluorescence in the right NBM 4 h after lesion and i.c.v. injection of NGF; Žf. same field shown in Že., viewed under normal light to detect Fos-immunoreactive nuclei; the position occupied by the cholinergic neurons is indicated by open triangles. Note the lack of colocalization of the two antigens. Magnification: =125 Ža, b., =190 Žc, d., =340 Že, f.. Scale bar s 40 m.
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ChAT did not colocalize in NGF-treated animals 4 h after lesion ŽFig. 2.. Finally, we investigated whether NGF potentiates the induction of the c-fos gene in lesioned animals also at longer times post-lesion. Thus, we have analyzed two further groups of animals lesioned with quisqualic acid in the right nucleus basalis and treated i.c.v. with NGF for 1 week Žtotal of two NGF injections. or for 3 weeks Žtotal of six NGF injections.. We have found no c-fos expression in any brain area in either group.
4. Discussion In the present report, we show that post-lesion NGF treatment for 3 weeks reduces the loss of ChAT-immunopositive neurons induced by excitotoxic lesions of the NBM, in agreement with previous reports w1x. Furthermore, we demonstrate that NGF treatment induces in lesioned animals an increase in c-fos induction as compared to the lesioned animals treated with saline. This effect of NGF is evident both at 4 and 24 h after lesion, and not at later times. A relationship between NGF and the c-fos gene has been repeatedly suggested. In our experimental conditions, NGF treatment per se rapidly induces the expression of c-fos in some cerebral areas, but not in the NBM. Previous reports have shown small increases in Fos IR in this area upon NGF administration w20,8x. These discrepancies are probably accounted for by differences in the technical procedures. Our present data show that NGF treatment increases the number of neurons expressing Fos upon lesioning of the NBM, and we have previously hypothesized that early c-fos induction by NBM excitotoxic lesions might be a protective cellular response w9x. In view of these results, it appears feasible that one of the mechanisms of the neuroprotective action of NGF is the potentiation of c-fos induction. However, the potentiation of c-fos expression takes place within 24 h after lesion, whereas 3 weeks of NGF treatment are necessary to achieve a significant recovery of ChAT IR, and we have not detected further Fos protein induction later than 24 h post-lesion, although we cannot exclude that Fos might be expressed at intermediate time points that we have not investigated. This seems to indicate that c-fos induction is not sufficient for mediating the trophic effects of NGF on cholinergic neurons, and that other NGF-induced events need to occur at later times for the recovery to be completed. Whether these later events require early Fos protein expression or are independent from the c-fos gene needs to be elucidated. It has been reported that NGF can prevent delayed apoptotic cell death in vivo w27x. Thus, it can be speculated that NGF exerts both a fast, Fos-mediated action that protects the cells from rapid excitotoxic death, and, a later, possibly Fos-indepen-
dent action which supports cell repair and prevents damaged neurons from undergoing apoptosis. We have previously shown that the early induction of c-fos by excitotoxic lesions in the NBM does not occur in cholinergic neurons, but in other neuronal cells of the NBM itself and in the surrounding areas w9x. In the present study, a further increase in the number of Fos-positive neurons in the NBM was found upon NGF administration to lesioned animals. Thus, we performed ChAT and Fos protein colocalization experiments in order to verify whether the NGF-induced increase in the number of Fospositive nuclei was due to c-fos induction in cholinergic neurons of the NBM. These experiments have shown that in lesioned animals the cholinergic neurons do not express the Fos protein 4 h after NGF treatment, indicating that at this time point the increase in the number of Fos-positive nuclei induced by NGF is due to Fos expression in noncholinergic NBM neurons. However, we were not able to perform colocalization experiments at 24 h post-lesion, in view of the fact that at this time point cholinergic neurons have already lost ChAT IR and are no longer detectable w9x. Thus, the possibility cannot be ruled out that NGF can induce a slower expression of the c-fos gene in the damaged cholinergic neurons, and that this might contribute to the significant increase in the number of Fos-positive nuclei that we found at 24 h upon NGF treatment. Furthermore, it is possible that cholinergic neurons do express Fos at later times during the process of recovery. In conclusion, the experimental model of the excitotoxic lesion of the rat NBM has allowed us to study the role of the c-fos gene in the molecular mechanisms through which NGF supports the recovery of lesioned cholinergic neurons. The hypothesis can be put forward that c-fos induction is one of the early events that contribute to the trophic actions of NGF.
Acknowledgements This investigation was supported by a research contract with NE.FA.C., Pomezia, Italy ŽMURST Neurobiological Systems National Research Plan.. We thank FIDIA Research Laboratories ŽAbano Terme, Italy. for providing NGF.
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