Degeneration of rat cholinergic basal forebrain neurons and reactive changes in nerve growth factor expression after chronic neurotoxic injury—I. Degeneration and plastic response of basal forebrain neurons

Neuroscience Vol. 65, No. 3, pp. 633~645, 1995

~ ) Pergamon

0306-4522(94)00526-5

Elsevier ScienceLtd Copyright © 1995 IBRO Printed in Great Britain. All rights reserved 0306-4522/95 $9.50 + 0.00

DEGENERATION OF RAT CHOLINERGIC BASAL FOREBRAIN NEURONS A N D REACTIVE CHANGES IN NERVE GROWTH FACTOR EXPRESSION AFTER CHRONIC NEUROTOXIC INJURY--I. DEGENERATION A N D PLASTIC RESPONSE OF BASAL FOREBRAIN NEURONS T. A R E N D T , * t M. K. B R U C K N E R , t S. P A G L I U S I ~ and T. K R E L L t tDepartment of Neurochemistry, Paul Flechsig Institute of Brain Research, University of Leipzig, Jahnallee 59, 04109 Leipzig, Germany :[:GLAXO Institute of Molecular Biology, Geneva, Switzerland Abstract--The process of degeneration and dendritic reorganization of cholinergic neurons was investigated in the rat basal forebrain under the conditiofis of chronic neurotoxic injury induced by long-term consumption of ethanol. After 28 weeks of ethanol treatment (20% v/v), both the number of choline acetyltransferase-immunoreactive basal forebrain neurons and levels of biochemical measures of cholinergic neurons, such as the activity of choline acetyltransferase and the synthesis and content of acetylcholine, were decreased by about 60-80%. The number of cholinergic neurons showing a positive hybridization signal to choline acetyltransferase messenger RNA was decreased to a similar extent. On the contrary, the reduction in the number of neurons immunoreactive for nerve growth factor receptor p75, which in control brains is highly co-localized with the expression of choline acetyltransferase, was much less pronounced and reached only 20-30%. The loss of choline acetyltransferase expression was associated with a cellular hypertrophy. Neurons which had survived the neurotoxic damage, furthermore, showed a remodelling of the dendritic organization which was quantitatively investigated after Golgi impregnation. This process of dendritic reorganization was mainly characterized by an increase in number and length of terminal dendritic segments. The results indicate that under the conditions of the present paradigm of chronic neurodegeneration, a certain number of cholinergic neurons persists in a form where they lost their ability to express detectable amounts of choline acetyltransferase messenger RNA and the enzyme protein. Persisting neurons, however, show both expression of nerve growth factor receptor p75 and signs of perikaryal and dendritic growth. It might, therefore, be hypothesized that chronic degeneration of cholinergic basal forebrain neurons triggers reactive attempts of repair which involve the action of trophic factors such as nerve growth factor.

In Alzheimer's disease, in postalcoholic Wernicke-Korsakoff's disease and in a variety of other dementing disorders, the degeneration o f basal forebrain neurons giving rise to the cholinergic innervation of the entire cortical mantle is an early and constant feature and is most probably linked to the progressive deterioration of cognitive function. 2,5,9,16The development of these cholinergic neurons, as well as their maintenance in the adult nervous system, are critically dependent on trophic support provided by fl-nerve growth factor (NGF), 39'42'52 the prototype of a target-derived neurotrophic factor. 56 This has raised the hope that trophic factor therapies could eventually prove effective under the conditions of these

*To whom correspondence should be addressed. Abbreviations: ACh, acetylcholine; CHAT, choline acetyl-

transferase; DAB, 3,Y-diaminobenzidine; NGF, nerve growth factor; NGFR, nerve growth factor receptor; standard saline citrate.

SSC,

disorders. Recent studies, however, have demonstrated that degeneration in the cholinergic basal forebrain in Alzheimer's disease is associated with a plastic dendritic growth response of surviving neurons, 6'9'1! as well as with an increase of N G F 1'25 and a number of other growth factors. 7'17'4°'78'79 The progression of the disease despite supranormal N G F levels might, therefore, indicate that neurodegeneration in Alzheimer's disease is due to a defective utilization rather than to a lack of N G F . A number of experimental lesion paradigms has demonstrated a reactive increase in the endogenous content of N G F , 3s'59'6°'68-7°'8~85 brain-derived neurot r o p h i c f a c t o r 14'29'47'57'58'74'87 and other polypeptide growth-promoting factors, such as glia maturation factor, 64 ciliary neurotrophic factor, 66 basic fibroblast growth factor, 32'5~ acidic fibroblast growth factor 64 or interleukin-1. 65 A cholinergic deafferentation of the hippocampus and neocortex in rat brain by transecting the fimbria-fornix or by placing a lesion to basal 633

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forebrain neurons results in an increase of N G F , both at the site of neuronal death and in loci deafferented by the lesion. 3s'53,59,6s's3's4 Moreover, an increase in N G F has been described in the septum and in the cerebral cortex of aged rats which show a severe impairment of learning and memory. 43,7° Altogether, these experimental data provide evidence that endogenous mechanisms can maintain N G F content at supranormal levels under conditions where the nervous system is undergoing a progressive degeneration, a process which might be of importance for the stimulation of compensatory functional changes and repair mechanisms. The question, however, has not been addressed yet whether the same holds true under chronic degenerative conditions which more closely mimic the situation observed in Alzheimer's disease. As chronic intake of ethanol both in human and rat has previously been shown to induce a chronic degeneration of cholinergic basal forebrain neurons comparable to those observed in Alzheimer's disease,2 5.15this experimental paradigm of chronic n e u r otoxic damage was used in the present investigation. The aim of the present study was to investigate whether cholinergic basal forebrain neurons under conditions of chronic injury show any signs of repair attempting to counteract degenerative processes. In subsequent experiments, which are described in the accompanying paper, ~° we have investigated whether the processes of neurodegeneration and plastic adaptive response of cholinergic neurons might be associated with changes in the expression of N G F in their microenvironment, which can provide the trophic stimuli for mechanisms of compensation and repair. EXPERIMENTAL PROCEDURES

Treatment of animals Adult male Sprague Dawley rats (Charles River) weighing between 180 and 250 g were maintained on an artificial 12:12 h light/dark cycle under conditions of constant temperature (22°C) and relative humidity (50%). Ethanol was administered in the drinking water (20% v/v) as the only source of fluid. Daily intake of ethanol was 9-10 g/kg body weight, resulting in blood alcohol levels between 40 (daytime) and 120 mg/100 ml (darkness). After different periods of time (four, eight, 18 and 28 weeks of ethanol treatment), animals were slowly withdrawn from ethanol over two weeks and then maintained on water for another four weeks before killing. No seizures were noted during withdrawal, and no animals were lost during any stage of the experiment. Animals maintained throughout on tap water served as controls. Assays of acetylcholine content, acetylcholine synthesis and choline acetyltransferase activity Animals were killed by decapitation [for determination of choline acetyltransferase (CHAT) activity, acetylcholine (ACh) synthesis] or focused microwave radiation (for determination of ACh content) between 10.00 and 12.00 a.m. Samples were dissected at 4°C from the septum and from the substantia innominata ventral to the anterior commissure. Tissue samples were weighed and homogenized in ice-cold 0.25 M sucrose containing 0.2% Triton X-100 (CHAT) or in trichloroacetic acid (ACh). Determination of ACh synthesis

was performed on tissue prisms (side 0.12mm) prepared with a tissue chopper. ACh was assayed by a chemiluminescent method. 49 Synthesis of [t4C]ACh from [14C]choline was measured at intervals of 10 min up to 1 h in tissue minces by extraction with tetraphenylboron followed by thin-layer chromatography. 73 Activity of ChAT was determined as described by Fonnum. 36

Immunohistochemistry Tissue was cryoprotected in 30o sucrose and cut (30 #m) on a freezing microtome. Endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide in methanol and non-specific binding sites were blocked with 0.3% non-fat dried milk (Sigma) and 0.1% gelatine in 0.1 M phosphate-buffered saline. Free-floating sections were incubated overnight (4°C) with mouse monoclonal antiChAT (1:2000; Chemicon) or anti-NGF receptor p75 (NGFR; clone 192; 1:2000; Oncogene Science) and further processed with the biotin-avidin system (biotinylated sheep anti-mouse immunoglobulin, 1:300, Amersham, Extravidin-peroxidase conjugate, 1:300; Sigma), and 0.04% 3,Y-diaminobenzidine (DAB)/0.015% H202 as chromogen. Primary antibodies were omitted in control incubations. The extent to which ChAT and NGFR co-localize was visualized by a double immunoperoxidase procedure with the NGFR antigen localized first using the DAB reaction (brown reaction product). Sections were subsequently processed for anti-ChAT applying a metallic intensification of the DAB reaction [0.4% (NH4)2Ni(SO4)2/0.02% DAB/0.015% H202 in 0.05 M Tris-HCl (pH 8.0); dark blue reaction product]. In situ hybridization

Construction of probes. A 43-mer oligonucleotide complementary to nucleotides 1818-1860 of the sequence of rat ChA'I"48 was synthesized on an Applied Biosystems 380A. Specificity of the probe has been established previously) 3 The oligonucleotides were labelled at the 3' end with [~-35S]dATP, using terminal deoxynucleotidyl transferase (Gibco) to a specific activity of approximately 2 x 107 c.p.m./pmol. The probes were purified on a Bio-Spin P-6 polyacrylamide gel column (Bio-Rad). Hybridization protocol. Brains were removed from the cranium and cut on a freezing microtome at a thickness of 16/~m. Sections were mounted onto RNAase-free polylysine-coated slides and fixed in 4% paraformaldehyde in phosphate-buffered saline. Sections were dehydrated in a graded ethanol series, air dried and incubated overnight at 42°C with 100/21 hybridization buffer per section86containing labelled probe at a concentration of 10,000 c.p.m./#l. Thereafter, slides were washed in 2 x SSC (300 mM sodium chloride, 30 mM sodium citrate, pH 7.0) supplemented with 0.025% sodium pyrophosphate/0.02% 2-mercaptoethanol at room temperature for 20 min, followed by three subsequent washes for 20min each in 1 × SSC at 55°C, 0.1 × SSC at 55°C and 0.1 × SSC at room temperature. Tissue sections were dehydrated by immersing the slides for a few seconds in serial ethanol solutions (70%, 90%, 100%) and air dried. For appreciation of hybridization signals, slides were exposed to X-ray film (Kodak XAR 5) for one week. For autoradiography with cellular resolution, slides were dipped in silver emulsion (Kodak NTB3) diluted 1:I in water and exposed at 4°C for six weeks. Sections were counterstained by Cresyl Violet. Golgi impregnation Animals were transcardially perfused with a solution of 5% glutaraldehyde/4% potassium dichromate. The brains were removed and coronal slabs of about 5 mm thickness were cut. Tissue was postfixed in the perfusate for 12 h and transferred into silver nitrate (0.75%) for 48h. Thereafter, the tissue slabs were treated again with potassium dichromate (3.6%) for 12-24h, followed by silver

Degeneration and plastic response of cholinergic neurons nitrate (0.75%) for 24h. Sections were cut on a.freezing microtome at a thickness of 150 #m and mounted in neutral balsam.

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Morphometric methods Neuronal cell counts. The number of neurons immunereactive for ChAT and NGFR, respectively, or neurons showing a hybridization signal to ChAT mRNA, was counted at a magnification of × 250 in every third section throughout the entire length of basal forebrain nuclei. Sections were subsequently Nissl stained and the total number of nucleolated neurons was determined. Analysis of Golgi-impregnated neurons. A three-dimensional morphometric analysis of Golgi-impregnated neurons was performed on a semiautomatic image analysing system (SIS, Miinster) as described. 8 Golgi-impregnated neurons were excluded from the analysis if either: (i) dendrites were so obscured by other elements that optical tracing was precluded; or (ii) dendrites trailed off as a series of dots, indicating poor impregnation. Neurons with cut dendritic segments were reconstructed from serial sections. From the digitized image, topological and metric parameters were calculated to characterize the degree of dendritic branching and the size of soma and dendrites. Data were averaged for each animal to determine an individual mean for each' parameter measured. These individual means were averaged to obtain group means, whose differences were analysed with parametric statistical tests (programme SPSS/PC+; SPCC Inc., Chicago). For the topological characterization of the dendritic tree, the number of dendrites (d) and the degree of dendritic arborization of the cell (At), defined as the total number of dendritic endings per cell, were determined. The degree of arborization of a single dendrite (Ae) was defined as Ae = Add. The number and length of dendritic segments of each segment order were determined using the centrifugal ordering system.l~ The total length of all dendritic elements per neuron (La) was determined by considering actual three-dimensional trajectories. The neuronal soma size was defined as the cross-sectional area of the two-dimensional projection of the soma (So). RESULTS

The chronic neurotoxic damage o f cholinergic basal forebrain neurons Long-term treatment of rat with a 20% (v/v) solution of ethanol in the drinking water results in a severe degeneration of neurons of basal forebrain nuclei giving rise to the cholinergic innervation of the cerebral cortex, i.e. the medial septal nucleus, diagonal band nucleus and basal nucleus (Figs 1 and 2; Table 1). After 28 weeks Of treatment, both the number of ChAT-immunoreactive neurons and the levels of biochemical measures of cholinergic neurons, such as activity of C h A T and synthesis and content of ACh, were decreased by about 60-80% (Fig. 1). The extent of this degeneration within the basal forebrain shows a gradient throughout its rostrocaudal extension, thereby affecting the cholinergic septohippocampal system more severely than. the basalocortical system (Table 1). The loss of CHATimmunoreactive neurons appeared to be rather selective, since changes in the total neuronal number were much less pronounced and reached only 20-30%. Moreover, the number of neurons showing immunoreactivity of N G F R , which in control brain is

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Ethanol intake (weeks) Fig. 1. Progressive degeneration of cholinergic basal forebrain neurons in relation to the duration of ethanol intake. (A) Changes in biochemical measures of cholinergic neurons in the basal forebrain. (B) Changes in neuronal number. Data are mean values (+S.E.M.; group size n = 5 animals) averaged over the medial septal nucleus, the vertical and horizontal limbs of the diagonal band nucleus and the substantia innominata/nucleus basalis complex (for biochemical measures: averaged for the septum and substantia innominata). Control values (+ S.E.M.) are: ACh synthesis (conversion of [14C]choline into [14C]ACh): 4 5 + 6 % ; ChAT activity: 108 __+3.5 nmol/mg h; ACh content: 31.5 _+ 1.2 pmol/mg. Differences between experimental and control animals are significant for *P < 0.05, **P < 0.01 (Student's t-test). highly co-localized with C h A T immunoreactivity, was decreased by only 30-40% (Table 1). The discrepancy between changes in the number of CHATimmunoreactive and NGFR-immunoreactive neurons (Fig. 2) might indicate that a certain number of cholinergic neurons persists in a form where they lost their ability to express immunocytochemically detectable amounts of CHAT. These neurons, which still express the N G F R but fail to express CHAT, can be directly visualized by a double immunoreaction (Fig. 3). To further characterize these neurons with an altered C h A T content, expression of the C h A T gene was investigated at a single cell level by in situ

636

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Fig. 2. Degeneration of cholinergic neurons in the substantia innominata/nucleus basalis complex after chronic treatment with ethanol as demonstrated by anti-ChAT (A, B) and anti-NGFR immunocytochemistry (C-F). (A, C, E)Control. (B, D, F) Ethanol treatment for 28 weeks. Note that the extent of neuronal loss and the presence of atrophic neurons (arrows) are more pronounced for the anti-ChAT (B) than for the anti-NGFR-preparation (D). Ethanol-induced degeneration is accompanied by a rarefication of the NGFR-immunoreactive neuropil (D, asterisk) and a hypertrophy of NGFR-immunoreactive neurons (compare E and F). Scale bars = 50 #m (A-D); 10/~m (E, F).

hybridization using a [35S]ChAT mRNA-antisense oligonucleotide probe. In the basal forebrain of control animals, labelled° cells were associated prominently with the medial septal nucleus, the vertical and horizontal limbs of the diagonal band nucleus, the magnocellular preoptic nucleus and the substantia innominata/nucleus basalis complex. Autoradiographic grains were preferentially found over the large fusiform, round and polygonal hyperchromic

neurons (Fig. 4). In the medial septal nucleus, the vertical limb of the diagonal band nucleus and the substantia innominata/nucleus basalis complex of control animals, neurons were showing a ChAT m R N A hybridization signal with frequencies of about 30-50%, 50-70% and 85-95%, respectively (Table 2). The distribution of individual neurons throughout basal forebrain nuclei revealing a hybridization signal to ChAT m R N A was identical to those

Degeneration and plastic response of cholinergic neurons

637

Fig. 3. Double immunoreaction for the presence of ChAT and NGFR in the substantia innominata/nucleus basalis complex in control animal (A) and after ethanol treatment for 28 weeks (B). CHAT, dark blue reaction product of DAB/nickel intensification; NGFR, brown DAB reaction product. While in controls, ChAT and NGFR immunoreaction is highly co-localized (A), an increasing number of NGFR-immunoreactive neurons fails to express ChAT immunoreactivity (arrow) after chronic ethanol treatment (B). Scale bar = 20 #m. showing ChAT immunoreactivity, although the latter appeared to be slightly more sensitive (cf. Tables 1 and 2). In experimental animals exposed to ethanol for 28 weeks, expression of the ChAT gene in the basal forebrain was severely affected. Both the number of neurons labelled by the [35S]ChAT mRNA-antisense oligonucleotide probe and the intensity of labelling of individual neurons were significantly decreased as compared to controls (Table 2; Fig. 4). The number of neurons showing a ChAT mRNA hybridization signal was slightly more reduced than the number of ChAT-immunoreactive neurons (see Tables 1 and 2). Intending to characterize the fraction of neurons which lose the ability to express the ChAT gene, the cell size distribution of labelled and unlabelled neurons was comparatively analysed under both normal and experimental conditions. The results clearly show that the loss of ChAT expression after chronic neurotoxic damage is associated with a perikaryal and nuclear hypertrophy (Fig. 5, Table 3; cf. also Fig. 4). The cross-sectional cell area of unlabelled neurons was increased in experimental animals by about 60-80% as compared to controls, while neurons showing a hybridization signal to ChAT mRNA did

not differ in size. These changes in size of unlabelled neurons result in a small, but significant shift in the size distribution of both the total neuronal population and the population of neurons which are N G F R immunoreactive (Figs 4, 5, Table 3; see also Fig. 7).

Dendritic reorganization of basal forebrain neurons Changes in the dendritic organization of basal forebrain neurons induced by chronic neurotoxic damage were investigated on Golgi-impregnated material. Impregnated neurons showed a fusiform, round or polygonal soma shape and a sparsely ramified dendritic tree. Varicose dendrites were frequently encountered (Fig. 6). Performing a threedimensional quantitative analysis of the dendritic arborization of Golgi-impregnated neurons in the basal nucleus, a remodelling of the dendritic organization could be established for those neurons which had survived the neurotoxic damage (Fig. 7). This process of dendritic reorganization was associated with an increase in both the length and arborization of dendrites. An increase in soma size was observed, as previously noted on Nissl preparations and after N G F R immunocytochemistry (cf. Fig. 5 and

Table 1. Total number of basal forebrain neurons and number of neurons immunoreactive for choline acetyltransferase or nerve growth factor receptor in control animals and after ethanol treatment (28 weeks) ChAT-immunoreactive neurons

NGFR-immunoreactive neurons

Number

Frequency (%)

Medial septal nucleus control 9844.6+ 836.2 ethanol 7708.2__+332.6 (-22%)*

4430.2 ± 410.5 784.0 + 56.8 (-82%)*

45 21

4682.6 __+362.1 2802.4 + 162.6 (-40%)*

47 36

Diagonal band nucleus (vertical limb) control 15,721.2± 1089.8 ethanol 11,795.1_+776.3(-25%)*

9747.2 ± 715.3 2319.1 ±263.7(-76%)*

62 26

9953.6 -+ 683.3, 6270.5 + 573.7 (-37"/o)*

63

Substantia innominata/nucleus basalis complex control 11,223.1_+936.4 10,213.2 ± 913.7 ethanol 8333.5± 548.2 (-26%)* 4702.3 + 428.3 (-54%)*

91 62

10,428.3 ___845.4 7645.7 ± 689.2 (-27%)*

93 92

Brain area

Total number of neurons

Number

Frequency (%)

Data are mean values for both hemispheres (_S.D.), group size n = 5 animals; *differences from control values are significant at a level of P < 0.01 (Student's t-test).

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Table 2. Number of choline acetyltransferase mRNA-positive basal forebrain neurons in control and after chronic ethanol treatment (28 weeks) ChAT mRNA-positive neurons Frequency (%)

Mean grain density

Medial septal nucleus control 3852.3 + 328.2 ethanol 616.5 + 56.8 (-84%)*

39 16

18.2 + 0.8 4.2 _+0.5 (-77%)*

Diagonal band nucleus (vertical limb) control 9262.6 + 862.5 ethanol 1946.5 _+ 367.8 ( - 79%)*

59 22

20.3 _+ 1.3 3.6 + 0.6 ( - 82%)*

Substantia innominata/nucleus basalis complex control 9906.4 + 728.4 88 ethanol 4160.7 + 526.3 ( - 58%)* 55

19.8 _+ 1.1 4.6 + 0.4 ( - 77%)*

Brain area

Total number

Data are mean values for both hemispheres (+S.D.), group size n = 5 animals; The mean grain density was determined as the ratio of grain density over neurons/grain density of background; *differences from control values are significant for P < 0.01 (Student's t-test). Table 3). The overall increase in dendritic branching was mainly due to an increase in the number and length of higher order branches, while low order branches tended to be unaffected or even showed an atrophy (Fig. 8). DISCUSSION In the present study, chronic degeneration and cell death of cholinergic basal forebrain neurons induced by long-term application of ethanol were found to be associated with a reactive dendritic growth response o f surviving neurons. A number of neurons surviving

chronic neurotoxic damage failed to express the C h A T gene, while the expression of N G F R was largely unaltered.

Degeneration o f cholinergic neurons Chronic intake of ethanol has previously been shown to result in a degeneration of cholinergic basal forebrain neurons both in human 2'5 and rodent 4'~5. The present study extends these findings and shows that a prominent feature of this degenerative process is the decrease in the ability of cholinergic neurons to express the ACh-synthesizing enzyme CHAT. Therefore, the synthesis of A C h in basal forebrain nuclei is

Fig. 4. Autoradiographic localization of hybridization of the [35S]ChAT oligonucleotide probe in the substantia innominata/nucleus basalis complex, counterstained by Cresyl Violet. In control animals (A), hybridization to ChAT mRNA is localized over large fusiform (arrow) or round (arrowhead) neurons. Only a small percentage of neurons is unlabelled (double arrow). After chronic treatment (28 weeks) with ethanol (B), the number of strongly labelled neurons (arrowhead) is decreased, while unlabelled hypertrophic neurons are more abundant (double arrow). Scale bar = 15/~m.

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Fig. 5. Size distribution of the total population of basal forebrain neurons and of basal forebrain neurons showing immunoreactivity for NGFR and a hybridization signal for ChAT mRNA, respectively, in control animals and after 28 weeks of ethanol treatment. (A) Vertical limb of the diagonal band nucleus. (B) Substantia innominata/nucleus basalis complex.

reduced to a much larger extent than would be expected from the extent of neuronal loss alone. Biochemical measures of A C h synthesis, i.e. the conversion of [J4C]choline into [14C]ACh and the levels of ACh, were reduced by the same order of magnitude as was the expression of C h A T m R N A determined by quantitative in situ hybridization. In agreement with a previous report, 67 in control animals, the distribution of basal forebrain neurons showing a hybridization signal to C h A T m R N A was largely identical to those of anti-ChAT-immunoreactive neurons. The number of basal forebrain neurons labelled after in situ hybridization, however, tended to be slightly less than those that were immunoreactive for CHAT. This could be attributed to differences in the turnover and stability of m R N A

and the corresponding protein or simply to differences in the detection limits of the two methods.

Plastic changes o f basal forebrain neurons The loss of the ability of neurons to express the C h A T gene was associated with a cellular and nuclear hypertrophy. Plastic changes, furthermore, were observed in the dendritic organization of surviving neurons. Morphological features of Golgi-impregnated basal forebrain neurons with respect to soma shape and characteristics of dendritic arborization were in agreement with those of previous reports on rat 18,28 and human basal forebrain neurons. 8 Dendritic varicosities, a typical, even though not entirely specific feature of cholinergic basal forebrain neurons, TM were constantly observed. Dendritic plasticity

Table 3. Nuclear and cellular size of nerve growth factor receptor-immunoreactive basal forebrain neurons and of neurons showing either a hybridization signal or no hybridization to choline acetyltransferase mRNA in control animals and after ethanol treatment (28 weeks) NGFR-immunoreactive neurons

ChAT mRNA-positive neurons

ChAT mRNA-negative neurons

cross-sectional area (,um2) soma nucleus

cross-sectional area (~m 2) soma nucleus

cross-sectional area (#m 2) soma nucleus

Diagonal band nucleus (vertical limb) control 284.7 + 18.3 77.2 __+6.2 ethanol 313.3 + 20.2* 89.8 + 7.2*

279.3 ___23.1 287.4 __+16.2

75.6 + 5.3 78.1 + 6.1

281.6 + 21.2 492.3 + 52.8**

77.2 + 6.1 123.2 _+ 5.3**

Substantia inn0minata/nucleus basalis complex control 309.2 _ 26.2 106.8 _+ 8.2 ethanol 367.7 + 29.6* 128.2 + 9.2**

310.6 ___28.2 309.2 __+21.6

98.5 __+8.2 104.2 _ 9.7

312.7+ 22.8 502.3+ 61.2"*

102.6 + 9.2 138.7 + 9.6**

Data are mean values (+ S.D.), group size n = 350 neurons (five animals); differences between the groups of neurons are significant at levels of *P < 0.05; **P < 0.01 (Student's t-test).

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Fig. 7. Topological and metric parameters of Golgi-impregnated neurons of the substantia innominata/nucleus basalis complex in control animals and after 28 weeks of ethanol treatment. Data are mean values (+S.E.M.), group size n = 20 animals (1005 neurons); differences are statistically significant for • P < 0.05; **P < 0.01 (Student's t-test). was largely confined to the distal parts of the dendritic tree, indicating a regrowth of terminal and preterminal dendritic segments, while proximal segments tended to be unaffected or even showed an atrophy. Structural equivalents of dendritic growth in the adult brain have been observed under various neurodegenerative conditions, suggesting that regenerative attempts occur in parallel with degenerative processes. A hypertrophy of basal forebrain neurons associated with a process of dendritic growth similar to those shown in the present study has also been described for the human brain during normal ageing, 27'33 in Alzheimer's disease9,11 Parkinson's disease 9 and Wernicke-Korsakoff's disease. 9 Dendritic growth, furthermore, has been observed in the animal and human brain both under the conditions of ageing and alcoholism on a variety of other neurons. 23'3°'5°'54'6j Proliferative processes on dendrites in the adult brain, however, appear to be restricted to certain brain regions and/or neuronal types. 19'22"24'26'31'34'35'44'55'75'8°'81A substantial increase in the dendritic extent in the normal human ageing brain has clearly been demonstrated on layer II pyramidal neurons of the parahippocampal gyrus. 19 On the contrary, continuing dendritic regression has been established for pyramidal cells of the human motor cortex. 63 Studies of age-related changes on granule cells of the human hippocampal dentate gyrus provided further evidence that the original finding of continuing net dendritic growth in the normal human ageing brain 19 is not universal to all cell types and brain regions. Plastic changes of the dendritic organization during ageing and a variety of other degenerative conditions appear to affect largely

the same neuronal population, which is particularly vulnerable to degeneration and cell death. Chronic treatment with ethanol has been reported to induce dendritic growth on cortical and hippocampal pyramidal neurons 2°'54'61 and on dentate gyrus granule cells.3°'5°'7j These neurons are, at the same time, particularly prone to alcohol:induced degeneration and cell death. 21'82 Although alcohol-induced changes in dendritic organization follow a rather complex pattern of remodelling during alcohol exposure and subsequent withdrawal, they have been reported to be largely restricted to terminal dendritic segments. 4t'61 This observation was confirmed in the present study. These alterations in number and length of dendrites, particularly the terminal branches, agree with current theories on dendritic plasticity both during development and repair. 37'72'77In the ageing nervous system, neuronal hypertrophy accompanied by an increase in the spatial extension of the dendritic tree has been regarded as a sign of a compensatory mechanism attempting to counteract for the loss of neighbouring neurons. 44'76 As integration of synaptic inputs from different dendritic locations strongly depends on the geometry of the dendritic tree, minor changes in length and branching pattern of dendrites affect neuronal function: 5 The process of dendritic reorganization not only changes the weighting of synaptic inputs, thereby altering synaptic integration, it may also shift synapses closer to optimal effectiveness. If the length of terminating dendrites increases, then the synapses that used to be the most distal could increase in effectiveness.46,62 Dendritic growth, furthermore, may increase synaptic effectiveness by providing an increased capacity, resulting in a smaller

Fig. 6. Golgi-impregnated neurons of the substantia innominata/nucleus basalis complex. (A) Photomicrograph of sparsely ramified neurons with ovoid and round cell bodies. (B) Computer-generated image, corresponding to upper panel (reconstructed from serial sections). (C, D) Dendritic varicosities (arrows) are typical morphological features of basal forebrain neurons. (E) Basal forebrain neuron with polygonal cell body. Scale bars = 30#m (A, B); 20 #m (C-E).

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5

6

1

7

Order of segments

2

3

4

5

~ 6

7

Order of segments

Fig. 8. Dendritic changes of Golgi-impregnated neurons of the substantia innominata/nucleus basalis complex after 28 weeks of ethanol treatment depicted as a function of somatofugal branch order. (A) Frequency distribution of dendritic segments. (B) Distribution of segment length. Data are mean values (±S.E.M.), group size n = 20 animals (1005 neurons).

voltage change at the synapse. 45 It might therefore be assumed that a physiological process of dendritic remodelling in the adult brain subserves a compensatory function counteracting regressive changes in electrotonic membrane properties, resulting in alterations of conduction velocities as described for cholinergic rat basal forebrain neurons during ageing. 12

pression of N G F R , despite the dramatic reduction of C h A T expression. Therefore, it remains to be determined whether these plastic changes involve alterations in the expression of N G F in the microenvironment of cholinergic neurons, which might provide the stimulus for reparative attempts. This question is addressed in the accompanying paper. 1°

Dendritic reorganization of the NGF-responsive cholinergic basal forebrain neurons in the present study was associated with a largely unaltered ex-

Acknowledgement--This study was supported by the Bundesministerium ffir Forschung und Technik (0316914A).

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