Axonal sprouting following lesions of the rat substantia nigra

Sprouting of substantia nigra neurons

Pergamon PII: S0306-4522(00)00009-9

Neuroscience Vol. 97, No. 1, pp. 99–112, 2000 99 Copyright q 2000 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00

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AXONAL SPROUTING FOLLOWING LESIONS OF THE RAT SUBSTANTIA NIGRA D. I. FINKELSTEIN,*† D. STANIC,*† C. L. PARISH,* D. TOMAS,* K. DICKSON‡ and M. K. HORNE*§¶ *Department of Medicine, Monash University, Clayton 3168, Victoria, Australia ‡School of Life Sciences and Technology, Victoria University, St Albans 3021, Victoria, Australia §Department of Neurology, Monash Medical Centre, Clayton Road, Clayton 3168, Victoria, Australia

Abstract—Parkinson’s disease is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Symptoms do not appear until most nigral neurons are lost, implying that compensatory mechanisms are present. Sprouting has been proposed as one of these mechanisms. This study quantified the extent of compensatory axonal sprouting following injury of dopaminergic neurons within the substantia nigra pars compacta. Specifically, the extent of the axonal arbour and axonal varicosity morphology was measured after partial destruction (with 6-hydroxydopamine) of the substantia nigra of the adult male rat. Four months later, the substantia nigra was injected with the anterograde neuronal tracer dextran–biotin to trace the full extent of individual axons. An unbiased estimate of neuron number was performed in each animal. This demonstrated nigral neuronal loss ranging from 10 to 90% on the side that received the injection whilst a 7% reduction was observed in the side contralateral to the lesion. Coincident with this loss, some nigral neurons lose tyrosine hydroxylase expression. Vigorous axonal sprouting was observed in the terminal arbours of lesioned animals and was associated with an increased axonal varicosity size. Axonal varicosities and branching points were primarily confined to the dorsal 1.5 mm of the caudate–putamen, an area predominantly innervated by nigral neurons. It appears that dopaminergic neurons were responsible for this sprouting because the density of dopamine transporter immunoreactive varicosities in the caudate–putamen was maintained until about a 70% loss of neurons. It was concluded that substantial compensation in the form of sprouting and new dopaminergic synapse formation occurs following lesions of the substantia nigra pars compacta. q 2000 IBRO. Published by Elsevier Science Ltd. Key words: Parkinson’s disease, ultrastructure, stereology, compensation, axonal reconstruction, dopamine transporter.

Symptoms of Parkinson’s disease (PD) do not appear until most substantia nigra pars compacta (SNpc) neurons are lost, implying that the remaining dopaminergic neurons may compensate for this neuronal loss. 1,27,31,41,47,54,55 Neurotoxins [6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine] have been administered to the rat to injure SNpc neurons and model this compensatory response. 1,5,6,26,28,33,38,43,44,46 Axonal sprouting has been proposed as the compensatory mechanism responsible for forming additional collateral branches and new synapses.3,16,19,22,29,30,34,40,43,48 Sprouting occurs in the caudate–putamen (CPu) following 6OHDA lesions of the SNpc. 5,6,35 Four to seven months after lesioning, the density of tyrosine hydroxylase-immunoreactive (TH-IR) fibers in the CPu were increased, suggesting sprouting of axons from spared nigrostriatal neurons. In addition, electron microscopic examination of the CPu revealed axonal sprouts, larger than normal axonal varicosities and immunoreactive growth cone-like structures. 5 In this study, we have quantified the extent of sprouting by SNpc neurons in response to varying degrees of denervation of the CPu. The extent of sprouting of individual surviving axons was related to the degree of cell loss in the SNpc. Analyses of the ultrastructural morphology of varicosities in

the CPu showed that newly formed profiles were larger and had an increased number of vesicles than those from control rats. The density of dopamine transporter immunoreactive (DAT-IR) varicosities in the CPu was also estimated and remained unchanged from control values until SNpc cell loss was more than 70%. This suggests that compensatory sprouting by dopaminergic (DA) SNpc neurons had occurred. EXPERIMENTAL PROCEDURES

All methods conformed with the Australian National Health and Medical Research Council published code of practice for the use of animals in research and were approved by the Monash University Animal Ethics Committee. Forty-eight adult male outbred Wistar rats (Monash University, Clayton, Victoria, Australia) weighing 250–350 g were used for these experiments. Ten were used in the ultrastructure study, 26 for DAT studies, eight in the reconstruction studies and four for TH and glutamate (Glut) immunohistochemistry. Throughout this study significance levels were tested with unpaired ttest and set at P # 0.05. Lesioning The same method of lesioning was used in all of the study groups. Twenty-eight rats were lesioned. The remaining animals were left intact and used as age matched controls. Each of these animals were anaesthetized with sodium pentobarbitone (60 mg/kg, i.p.), treated with atropine (0.24 mg/kg, i.p.) and placed in a stereotaxic head frame. Anaesthesia was maintained with a mixture of ketamine (28 mg/kg, i.m.) and xylazine (2.4 mg/kg, i.m.) at a level where a paw pinch elicited a weak withdrawal reflex. The skull was exposed and a small hole drilled through the right side using a site and method previously described. 38 A 10-ml Hamilton syringe (with a 26-gauge needle) mounted in a syringe pump (Cole-Parmer, Vernon Hills, IL, U.S.A.) was used to inject the 6-OHDA (Sigma, St Louis, MO, U.S.A.) into the SNpc. The extent of SNpc cell loss was varied by altering the amount of 6-OHDA injected (12–80 mg). The skin was sutured,

†Dr Finkelstein and Dr Stanic contributed equally to this work and are joint first authors. ¶To whom correspondence should be addressed. Abbreviations: CE, coefficient of error; CPu, caudate–putamen; CV, coefficient of variance; DA, dopaminergic; DAB, diaminobenzidine; DAT, dopamine transporter; DB, dextran–biotin; Glut, glutamate; IR, immunoreactive; 6-OHDA, 6-hydroxydopamine; PBS, phosphatebuffered saline; PD, Parkinson’s disease; SNpc, substantia nigra pars compacta; SNpr, substantia nigra pars reticulata; TH, tyrosine hydroxylase; VTA, ventral tegmental area. 99

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antiseptic (1% w/w iodine, Betadine, Faulding and Company, Salisbury, South Australia) applied to the wound and the rats recovered in a warmed cage. Paracetamol (100 mg/l) was added to the animal’s water for two days after surgery. Injection of tracers Reconstruction of axons and ultrastructural studies required injection of an anterograde tracer, dextran–biotin (DB) into the SNpc. Four months after injection of 6-OHDA, each animal (normal and lesioned) was deeply anaesthetized (as described above) and the anterograde neuronal tracer, dextran–biotin (2.5% for light microscopy, 10% for electron microscopy, Molecular Probes, Eugene, OR, U.S.A.) in 0.1 M phosphate buffer (pH 7.4), was injected into the SNpc. Two small volumes of the tracer (approx. 10–20 nl) were pressure injected from a micropipette into the SNpc (antero-posterior 5.2 mm, 5.8 mm; lateral 2.1 mm, 2.0 mm; dorsoventral 7.8 mm, 7.8 mm, with respect to bregma 37) with a Picospritzer II (General Valve, NJ, U.S.A.). Normal rats received a single injection at the first set of co-ordinates listed above. To minimize backfilling along the injection tract, the micropipette was left in situ for 5 min before slowly withdrawing it. Preparation of tissue for reconstruction of axons Fourteen days after the tracer injections, each rat was killed with sodium pentobarbitone (100 mg/kg, i.p.) and perfused. The perfusate consisted of 400 ml of warmed (378C) 0.1 M phosphate-buffered saline (PBS; pH 7.4) with 1 unit of heparin/ml, followed by 400 ml of chilled 4% paraformaldehyde (Sigma, St Louis, MO, U.S.A.) in 0.1 M phosphate buffer (48C, pH 7.4). The brain was removed and left at 48C in 20% sucrose and 4% paraformaldehyde solution overnight. Fiftymicrometer-thick serial frozen sagittal sections were cut through the CPu and SNpc. The sections were washed three times for 10 min in 0.1 M PBS and then incubated for 2 h in 1:5000 avidin–peroxidase (Sigma, St Louis, MO, U.S.A.) with 0.75% Triton X-100 (Sigma, St Louis, MO, U.S.A.). This was followed by four 10 min washes (to remove unbound avidin–peroxidase) and 20 min of exposure to cobalt and nickel-intensified diaminobenzidine (DAB) (Sigma, St Louis, MO, U.S.A.). Hydrogen peroxide (3.33 ml/ml) was added to the DAB solution for a further 10 min. After a final wash (3 × 10 min), the sections were mounted on microscope slides with a 0.5% gelatine solution (w/v H2O). The mounted sections were air dried, counterstained with 1% Neutral Red (3 min), dehydrated in a series of graded ethanol solutions and then cleared before coverslips were applied with a mounting medium (DPX). Reconstructed axons The axons were reconstructed using methods previously described. 2 Axons were only reconstructed when the injection site was centred on the SNpc and there was no tracer spread to the ventral tegmental area (VTA). Reconstructions were acceptable for inclusion in this study when there was confidence that all axonal branches were filled to their terminal varicosities and the axon could be traced back through the internal capsule close to, or within the SNpc. Single labelled SNpc axons were reconstructed using a drawing tube attached to a light microscope (Leitz GmbH, Wetzlar, Germany). Their axons, branches and terminal varicosities (identified as a swelling of the axon, usually circular or oval in shape) were traced through serial sections under high power ( × 100 oil objective and × 10 ocular). Surrounding blood vessels and other labelled axons were used to reliably follow the selected neuron from section to section under lower power ( × 10 objective). The morphology of each axon was quantified by counting the number of branching points and varicosities. The data obtained from the normal and lesioned animals were compared. Preparation of tissue for ultrastructural studies Fourteen days after the tracer injections, each rat was perfused with PBS, followed by 500 ml of chilled 2.5% glutaraldehyde (Sigma, St Louis, MO, U.S.A.) and 1% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The following day, 100–150 mm coronal sections were cut on a Vibratome (Technical Products International, St Louis, MO, U.S.A.). DB was conjugated to avidin–peroxidase and reacted with DAB and hydrogen peroxide, as previously described (with the exception that the concentration of Triton X-100 was 0.015%).

Sections were then viewed under a dissecting microscope ( × 40 objective) in search of axonal varicosities (i.e. circular or oval-shaped swellings on an axon). Segments of the dorsal 1.5 mm of the CPu that contained varicosities were removed with a blunt 14-gauge luer needle (approximate i.d. 1.5 mm). These punch biopsies were postfixed in 1.0% osmium tetroxide (60 min), dehydrated in ethanol, washed in epoxy propane and flat embedded in Epon Araldite. Ultrathin sections were cut, mounted on grids, stained with 2% aqueous uranyl acetate and 2% lead citrate and examined with a Jeol 100S electron microscope. Axonal varicosities emanating from cell bodies in the SNpc were recognized by the presence of DAB reaction product (Fig. 10). An axonal varicosity was defined as one that contained at least three vesicles (identified by the absence of DAB reaction product within them and appearing as small white circular structures). In this study we made no attempt to distinguish between synapses (containing a postsynaptic density) and varicosities. Varicosities were photographed, their area calculated with an image analysis program (Sigma Scan, 1997, Jandel, Chicago, IL, U.S.A.) and number of vesicles noted. Dopamine transporter immunohistochemistry DAT immunohistochemistry was used to label DA SNpc terminals in the CPu. Twenty-six rats were used in this study. Four months after lesioning, each rat was perfused with PBS (400 ml), followed by 150 ml of chilled 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) and 0.2% picric acid. The brains were removed and snap frozen in isopentane (21208C) cooled in liquid nitrogen. The CPu was serially sectioned at 20 mm and 10 of these sections, each 600 mm apart, were stained for DAT and used for stereology. The sections were fixed to gelatinized slides with 10% neutral buffered formalin (30 s) and rinsed in PBS (3 × 10 min). They were then incubated for 15 min in a blocking solution (PBS, 0.3% Triton X-100 and 5.0% normal rabbit serum), and overnight at 48C in rat anti-DAT primary antibody (Chemicon, Temecula, CA, U.S.A., 1:3000 in PBS, 0.3% Triton X-100 and 1.0% normal rabbit serum). This was followed by incubations at room temperature in a biotinylated secondary antibody (Rabbit anti-rat IgG, 1:300, Vector, Burlingame, CA, U.S.A.) for 1 h and in avidin–peroxidase (1:5000) for 1.5 h. Sections were then reacted with cobalt and nickel-intensified DAB. PBS rinses (4 × 10 min) were performed between each step. Sections were dehydrated and coverslipped as described previously. Tyrosine hydroxylase and glutamate immunohistochemistry Glut and TH immunohistochemistry and Neutral Red counterstaining was performed. Neutral Red staining and TH immunoreactivity were compared as methods for evaluating the lesion size induced by 6-OHDA. Four months after lesioning with 6-OHDA, the SNpc of four rats were serially sectioned at 50 mm and Glut and TH immunohistochemistry was performed on the first and second of every four sections. Sections were incubated in a blocking solution (PBS, 0.3% Triton X100 and 3.0% normal goat serum) for 10 min and then overnight in primary antibody (rabbit, anti-glutamate, Chemicon, Temecula, CA, U.S.A., 1:500; mouse, anti-tyrosine hydroxylase, Boehringer– Mannheim, Castle Hill, Australia, 1:1000, respectively, in PBS, 0.3% Triton X-100 and 1.0% normal serum) at 48C. This was followed by incubations at room temperature in a biotinylated secondary antibody (goat, anti-rabbit IgG, 1:1000, Sigma, St Louis, MO, U.S.A.; sheep, anti-mouse IgG, 1:300, Silenus, Hawthorn, Australia) for 2 h at room temperature, then in avidin–peroxidase (1:5000) for 2 h. Sections were then reacted with cobalt and nickel-intensified DAB. PBS rinses (3 × 10 min) were performed between each step. Sections were mounted, stained with 1% Neutral Red, dehydrated and coverslipped. Fractionator sampling scheme for estimating total numbers of substantia nigra pars compacta neurons and dopamine transporter labelled varicosities The number of neurons in the SNpc and density of DAT-IR varicosities in the CPu were estimated using a fractionator sampling design. 24,51 Staining with Neutral Red delineated the area of the SNpc in each section. The SNpc was recognized as the sheet of densely packed neurons of approximately 11 × 20 mm in soma size. At the ventral margin of the SNpc, the substantia nigra pars reticulata (SNpr) neurons were recognized because their soma were larger

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Fig. 1. Axon N-2. The normal axon illustrated in A originated from the injection site presented in C (scale bar ˆ 500 mm). It divided into two main branches in the CPu. Each of these primary branches diverged to form a number of secondary branches as they passed further through the CPu. “En passant” varicosities were seen throughout the length of each branch. In total, the length of this axon from its entry into the CPu was 6074 mm. The axons entry into the CPu is marked by the letter E. A line delineates the area of the CPu and nucleus accumbens. A.C. indicates the location of the anterior commisure. Scale bars of the reconstructed axon represent a length of 500 mm in rostrocaudal and dorsoventral planes. Branches and axonal varicosities are shown in photomicrographs D and F, whose locations correspond to the letters in the main diagram (scale bar ˆ 20 mm). Arrows in the photomicrographs point to axonal varicosities while arrowheads indicate branching points. The inset (B) is of three sagittal sections through the rat brain with a superimposed 3-dimensional representation of the axon to help visualize the orientation and scale of the reconstruction (scale bar ˆ 2 mm). The number below each section corresponds to the distance of the section from the midline. The medial/lateral (L) orientation and nomenclature is adapted from previous work. 37 Note that the axon was reconstructed beyond the border of the CPu down to the SNpc, even though the course of the axon outside the CPu is not shown in this figure.

(approximately 20–45 mm) and less densely packed than those in the SNpc. The VTA had smaller and less densely packed cells than the SNpc, making the rostromedial border of the SNpc easy to delineate. Caudally, the medial border of the SNpc excluded the loosely scattered neurons in the medial lemniscus. Both SNpc nuclei from normal and from lesioned animals were examined. In each of the sections sampled, counts of SNpc neurons were made using optical disector rules 24 and the nuclei of stained SNpc cells were the counting unit. For TH and Glut immunohistochemistry, labelled profiles were counted only if the first recognizable profile of the cell came into focus within the counting frame. 51 Counts of SNpc neurons were made at regular predetermined intervals (x ˆ 250 mm, y ˆ 125 mm for sections cut sagitally to accommodate axonal tracing, and x ˆ 150 mm, y ˆ 200 mm for sections cut coronally for DAT, Glut and TH immunohistochemistry analysis). These counts were derived by means of a grid program (Olympus BX50, DK A/S C.A.S.T.—Grid version 1.10, Glostrup, Denmark), through which a systematic sample of the area occupied by the SNpc was made from a random

starting point. An unbiased counting frame of known area (39.2 × 27.2 mm ˆ 1067 mm 2) was superimposed on the image of the tissue sections viewed under a × 100, N.A. 1.30 oil immersion objective. After all sections from each SNpc were analysed, the fraction of the area of the sections sampled was calculated, as previously described. 50,51 This fraction, the area sampling fraction, is obtained by dividing the area of the counting frame by the area of the distance between sampling regions, i.e. the x and y intervals. As detailed above, the x and y intervals in sections cut sagitally were 250 mm and 125 mm and the area of the counting frame was 1067 mm 2. Therefore, the area sampling fraction is 1067/(250 × 125) ˆ 0.0341. In animals used for axonal reconstructions all saggital sections containing the SNpc were analysed and consequently, the fraction of sections sampled was 1. Every 5 th section through the SNpc was examined in rats used for DAT immunohistochemistry. Therefore the sampling fraction was 1/5. For TH and Glut immunohistochemistry, every 4 th section through the SNpc was assessed, the section sampling fraction being 1/4.

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Fig. 2. Axon N-3. The normal axon illustrated in A originated from the injection site presented in C (scale bar ˆ 500 mm). It divided into three main branches in the CPu. Each of these primary branches diverged to form a number of secondary branches as they passed further through the CPu. “En passant” varicosities were seen throughout the length of each branch. The total length of this axons terminal arborization from its entry into the CPu was 11,516 mm. The axons entry into the CPu is marked by the letter E. A line delineates the area of the CPu and nucleus accumbens. The letters A.C. mark the location of the anterior commisure. Scale bars of the reconstructed axon represent a length of 500 mm in rostrocaudal and dorsoventral planes. Branches and axonal varicosities are shown in photomicrographs D and F, whose locations correspond to the letters in the main diagram (scale bar ˆ 20 mm, arrows indicate axonal varicosities; arrowheads indicate branching points). The inset (B) is of three sagittal sections through the rat brain with a superimposed 3-dimensional representation of the axon to help visualize the orientation and scale of the reconstruction (scale bar ˆ 2 mm). The number below each section corresponds to the distance of the section from the midline. The medial/lateral (L) orientation and nomenclature is adapted from previous work. 37 Note that the axon was reconstructed beyond the border of the CPu down to the SNpc, even though the course of the axon outside the CPu is not shown in this figure.

As described above, DAT-IR varicosities in the dorsal 1.5 mm of the CPu were counted from 20 mm-thick serial sections, each 600 mm apart. This area was selected because we found that sprouting occurred in this region and because it receives the SNpc projection. 4,20,23 The section sampling fraction was therefore 1/30. Counts of DAT-IR varicosities were made at regular predetermined intervals (x ˆ 400 mm, y ˆ 400 mm). An unbiased counting frame of known area (7.8 × 5.5 mm ˆ 43 mm 2) was superimposed on the image of the tissue sections viewed under a × 100, N.A. 1.30 oil immersion objective. DAT-IR varicosities were identified as being a swollen part of an axon (usually circular or oval in shape) and was only included in the count if it was attached to an axon. The fraction of the area of the sections sampled was calculated, as described above. The entire z-dimension of each section was sampled, the section thickness sampling fraction being 1. The total number of neurons in the SNpc was estimated by multiplying the number of neurons counted within the sampled regions with the reciprocals of the fraction of the sectional area sampled and the fraction of the section thickness sampled. 17,51 Similarly, the total number and density of DAT-IR varicosities in the CPu was estimated. Additionally, coefficients of error (CE) and coefficients of variance (CV) were calculated as estimates of precision. 9,49,51

RESULTS

Axonal reconstructions Four axons were reconstructed from normal animals and six were reconstructed from lesioned animals. The number of branching points and the number of axonal varicosities of each axon was counted. Reconstructions commenced by selecting a branch of an axon in the CPu and tracing it back through the internal capsule towards the SNpc. Each parent axon ascended rostrally from the SNpc, through the subthalamic nucleus, internal capsule and globus pallidus and entered the CPu close to the anterior commissure. These axons branched in the striatal terminal field (Figs 1 and 2). With one exception, (Fig. 2) every axon branched close to the rostral border of the CPu (Figs 1, 3 and 4). While there was individual variation in the pattern and orientation of branching, two to three main branches innervating a distinct strip of the CPu was usual.

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Fig. 3. Axon L-1. This axon (A) was reconstructed from a rat with a 25% lesion of the SNpc. The injection sites are illustrated in C (scale bar ˆ 500 mm). Two primary branches emerge from the parent axon as the rostral border of the CPu is approached. Each of these branches ascend dorsally parallel to the rostral border of the CPu on their way to their point of termination. A large number of higher order terminal branches and varicosities were seen throughout the course of each branch. The length of this axons terminal arborization was 17,567 mm. The letter E marks the axons entry into the nucleus accumbens. The area of the CPu and nucleus accumbens is delineated by a line. A.C. indicates the location of the anterior commisure. Scale bars of the reconstructed axon represent a length of 500 mm in rostrocaudal and dorsoventral planes. Branches and axonal varicosities are shown in photomicrograph D, whose location corresponds to the letter in the main diagram (scale bar ˆ 20 mm, arrows indicate axonal varicosities; arrowheads indicate branching points). The inset (B) is of three sagittal sections through the rat brain with a superimposed 3-dimensional representation of the axon to help visualize the orientation and scale of the reconstruction (scale bar ˆ 2 mm). The number below each section corresponds to the distance of the section from the midline. The medial/lateral (L) orientation and nomenclature is adapted from previous work. 37 Note that the axon was reconstructed beyond the border of the nucleus accumbens down to the SNpc, but is not shown in this figure.

The distribution of varicosities in a dorsoventral plane was graphed (Fig. 5), demonstrating that 94% of the varicosities were confined to the dorsal 1.5 mm of the CPu even after lesioning. The remainder (6%) were located 0.1–0.5 mm below this point. This shows that lesions of the SNpc resulted in an increase in the number of axonal varicosities and branch points that was proportional to the size of the lesion. Interestingly, sprouting of SNpc neurons was principally confined to the dorsal 1.5 mm of the CPu (Figs 3–5). The large number of small branches and axonal varicosities appeared to form in clusters within this confined space and may have corresponded to areas of the CPu which were denervated as a result of the lesion of SNpc neurons. Stereology of the substantia nigra pars compacta The normal SNpc contained a mean of 11,239 ^ 314 (S.D., n ˆ 11) neurons (Table 1). There was no difference between the numbers in the left and the right SNpc nuclei. The number of neurons in SNpc lesioned by 6-OHDA administration ranged from 9 to 92% (all lesioned animals), depending on

the extent of injury (Tables 2, 3). Note that the maximum lesion in the axonal reconstruction studies was 55%. There was also a significant (7%, P , 0.001) reduction in the number of neurons in the contralateral (non-lesioned) SNpc (mean ˆ 10,431 ^ 1016), a measure not considered in previous studies where the contralateral nigrostriatal projection is often used as a control. The coefficient of error (CE) (Table 1) of the estimate of N for every normal SNpc nuclei ranged from 0.022 to 0.057, indicating that the sampling protocol was precise. The coefficient of variance (CV) was 0.028. Therefore observed relative variations of SNpc cell numbers among the normal animals were regarded as true inter-animal differences and not as a consequence of the stereological techniques, (Table 1). In a separate set of animals (n ˆ 4), we made a formal comparison of TH immunohistochemistry and Neutral Red staining as methods for estimating the number of neurons in the SNpc following 6-OHDA injections (Fig. 6). In this study, sections were stained with Neutral Red and for TH so that Neutral Red counts indicated the number of cells that were not TH-IR. As expected, most neurons in the non-lesioned

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Fig. 4. Axon L-4. The most extensive arborization of a reconstructed SNpc axon is illustrated in A. This axon originated from the injection sites illustrated in C (scale bar ˆ 500 mm). It had 1324 varicosities and 275 branching points, the most recorded from all axons reconstructed. Interestingly this is also the axon, which was reconstructed from the animal with the largest lesion of SNpc cells (55%). This axon divided into three main branches at the rostral border of the CPu. All three branches were directed dorsally and later curved caudally. Branch B1, the most dorsally oriented, terminated 150 mm lateral to the axons entry into the CPu. Branch B2 was the most rostroventrally oriented and its terminal point was located 1300 mm lateral to the point at which the parent axon entered the CPu. Branch B3 was the most caudally directed. Its final terminal point was 1350 mm lateral to the axons entry into the CPu. In total, the length of this axons terminal arborization was 26,229 mm. The axons entry into the CPu is marked by the letter E. The area of the CPu and nucleus accumbens is delineated by a line. A.C. indicates the location of the anterior commisure. The scale bars of the reconstructed axons represent a length of 500 mm in rostrocaudal and dorsoventral planes. Branches and axonal varicosities are shown in photomicrograph D, whose location corresponds to the letter in the main diagram (scale bar ˆ 20 mm, arrows indicate axonal varicosities; arrowheads indicate branching points). The inset (B) is of three sagittal sections through the rat brain with a superimposed 3-dimensional representation of the axon to help visualize the orientation and scale of the reconstruction (scale bar ˆ 2 mm). The number below each section corresponds to the distance of the section from the midline. The medial/lateral (L) orientation and nomenclature is adapted from previous work. 37 Note that the axon was reconstructed beyond the border of the CPu down to the SNpc, even though the course of the axon outside the CPu is not shown in this figure.

SNpc were TH-IR (9138 ^ 483) but there were a number of neurons stained only by the Neutral Red (1657 ^ 433) (Fig. 6B). This was statistically the same as the number (1125 ^ 159) of Glut-IR cells in adjacent sections (Fig. 6B).

There was also no difference between the number of Glut-IR (1125 ^ 159) cells in the contralateral SNpc and in lesioned SNpc (1293 ^ 80) (Fig. 6B and D). In the lesioned SNpc, the number of TH-IR neurons was reduced to 3063 ^ 866 while

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Table 1. Estimates of total number of substantia nigra pars compacta neurons of each normal rat Animal No. N-1* N-2* N-3* N-4* N-5 N-6 N-7 N-8 N-9 N-10 N-11 Mean n (S.D.) Mean n LHS and RHS (CE) S.D. (n ˆ 8) CV (LHS and RHS)

LHS

RHS

11,611 (0.039) 10,728 (0.031) 11,187 (0.032) 11,041 (0.027) 11,106 (0.050) 10,965 (0.056) 11,106 (0.057) 11,949 (0.030) 10,965 (0.045) 11,387 (0.039) 10,965 (0.045) 11,183 (345) 11,239 (0.040) 314 0.028

11,715 (0.022) 11,442 (0.032) 11,162 (0.031) 11,071 (0.038) 10,965 (0.052) 11,387 (0.045) 10,965 (0.033) 11,809 (0.039) 11,246 (0.043) 11,106 (0.052) 11,387 (0.048) 11,296 (283)

Cell estimates were made from both SNpc from 11 rats. The coefficient of error (CE) is listed in brackets unless otherwise stated. *Rats used for axonal reconstructions. DAT immunohistochemistry was performed on all other rats listed in this table.

SNpc (Fig. 6A, C and E), the estimate of cells loss is between 65–83% (74 ^ 8%) (Fig. 6E and F). However if the calculations are done on the total number of cells (TH-IR cells plus Neutral Red only cells), a different sized lesion is estimated. This results in a 29–56% (37 ^ 14%) reduction in the number of neurons in the lesioned SNpc (Fig. 6E and F). This is consistent with previous studies. 8 Because of this finding, estimates of lesion size were obtained from counts of Neutral Red-stained neurons throughout this study. Estimates of sprouting

Fig. 5. Distribution of varicosities in the CPu. This figure illustrates the number of varicosities counted on a single reconstructed axonal arborization plotted against their depth below the dorsal surface of the CPu. (A) Plots the average number of varicosities counted from the four normal reconstructions plotted against their depth beneath the dorsal surface of the CPu. (B–G) are from lesioned animals, B and G representing axons shown in Figs 3 and 4. Note that 94% of all observed varicosities were found in the dorsal tier (no more than 1.5 mm below the dorsal surface of the CPu), with the exception of B, F and G where 6% were seen 0.1– 0.5 mm below this point. Note also the vastly greater number of varicosities found on axons from lesioned animals. (A), distribution of mean varicosities from reconstructions from normal rats (n ˆ 4); (B), axon L-1 (25% SNpc lesion); (C), axon L-2a (45% SNpc lesion); (D), axon L-2b (45% SNpc lesion); (E), axon L-3a (50% SNpc lesion); (F), axon L-3b (50% SNpc lesion); (G), axon L-4 (55% SNpc lesion).

the number of neurons stained only by Neutral Red increased to 4076 ^ 842 (Fig. 6D). The number of Neutral Red-stained neurons was significantly more (P ˆ 0.012) than the number of Glut-IR labelled neurons (Fig. 6D). In summary, there was an increase in the absolute numbers of Neutral Red-stained cells at the expense of TH-IR neurons in the lesioned SNpc. When, as is conventionally done, the number of TH-IR neurons in the lesioned SNpc is expressed as a percentage of the number of TH-IR neurons in the non-lesioned

Two measures of sprouting were considered: the number of axonal varicosities and axonal branching points of reconstructed SNpc neurons and the density of DAT-labelled varicosities in the CPu. The number of varicosities and branching points of axons from a lesioned SNpc were estimated and compared with those from axons of normal animals (Table 2, Fig. 7A, B). The average number of varicosities on the four reconstructed SNpc axons from normal rats was 132 and the average number of branching points was 23 (Table 2). Table 2 and Fig. 7A and B show that the number of branching points and varicosities increase with the size of lesion. The estimated number of SNpc varicosities within the normal CPu was 1.5 × 10 6, which was obtained by multiplying the number of varicosities per neuron by the number of neurons in the normal SNpc (Table 2, Fig. 7C). Similarly, an estimate of varicosities in the CPu of lesioned animals was obtained (Table 2, Fig. 7C). The number of varicosities on reconstructed axons of lesioned animals was multiplied by the number of cells counted in the relevant SNpc (Table 2). These estimates suggest that the number of varicosities in the CPu on lesioned axons increase as the degree of cell loss in the SNpc increases. Twenty-six animals were processed for DAT immunohistochemistry. Eleven were normal controls, and the remainder had lesions of the SNpc ranging from 9 to 92% (Table 3). The total number of varicosities was estimated using the stereological techniques described in the methods. The effect of lesioning was assessed by examining the

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Fig. 6. This figure compares the Neutral Red staining and TH immunohistochemistry methods for estimating SNpc lesions induced by 6-OHDA. In this figure, Neutral Red counts indicated the number of cells that were not TH-IR. (A) Counts of neurons stained by Neutral Red only (black bars) and TH-IR neurons (white bars) in the contralateral (non-lesioned) SNpc, four months after 6-OHDA injections. (B) The average number of neurons in the non-lesioned SNpc of rats TH1–TH4 which stained for Neutral Red only (black bar) and the average of those that were TH-IR (white bar). The average number of Glut-IR neurons in the non-lesioned SNpc of these same rats is represented by the grey bar. Note that the number of Glut-IR neurons is statistically the same as the number of Neutral Red-stained neurons. (C) Counts of neurons stained by Neutral Red only (black bars) and TH-IR neurons (white bars) in the 6-OHDA-lesioned SNpc. Compared with non-lesioned SNpc (6A), TH-IR neurons are decreased and Neutral Red-stained neurons are increased in the lesioned SNpc. (D) The average number of neurons in the lesioned SNpc of rats TH1–TH4 which stained for Neutral Red only (black bar) and the average of those that were TH-IR (white bar). The average number of Glut-IR neurons in the lesioned SNpc of these same rats is represented by the grey bar. Note that the number of Glut-IR neurons is unchanged from B but significantly less than the number of Neutral Red-stained neurons in the lesioned SNpc. E compares the two methods of estimating lesion size. For each animal, the total number of neurons in the lesioned SNpc (TH-IR neurons plus Neutral Red-labelled neurons in C) is expressed as a percentage of the total number of neurons in the non-lesioned SNpc (TH-IR neurons plus Neutral Red-labelled neurons in A). Lesion size is greater when estimated by only counting TH-IR neurons than by counting total number of neurons. (F) The average % SNpc lesion estimated with Neutral Red (black bar with error bars) and with TH immunohistochemistry (white bar and error bars). Lesion size is 37% greater when estimated by only counting TH-IR neurons than by counting total number of neurons.

density of varicosities (varicosities/mm 3) in the lesioned CPu, expressed as a percentage of the ipsilateral side (Fig. 8). Density of varicosities was used because we have no clearly defined or recognizable boundary of the area of the CPu innervated by the SNpc. The lesioned side was compared with the contralateral side because there was variability in immunohistochemical labelling between animals (see Discussion). Figure 9 provides examples of DAT-IR varicosities. These were from the dorsal CPu of a normal rat (A) and a rat with a 65% lesioned SNpc (B). The data show that the density of DAT-labelled varicosities in the denervated CPu remained normal until approximately 75% of the SNpc neurons were destroyed, at which point the density of

terminals decreased rapidly (Table 3, Fig. 8, sigmoidal line fit r ˆ 0.934, f ˆ a/(1 1 exp(2(x 2 x0)/b)), Sigmaplot, 1997, Jandel, Chicago, IL, U.S.A.). This corresponds closely to the relation between axonal sprouting and size of the lesion (Fig. 7). Electron microscopic analyses of terminals Samples from the CPu from 10 animals were processed for examination under the electron microscope. A 40% lesion (range 33–66%, established using methods described above) was produced by a 56 mg injection of 6-OHDA into the SNpc.

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non-lesioned animals were smaller and had fewer vesicles than the profiles from animals with SNpc lesions (Table 4, Fig. 10). DISCUSSION

Fig. 7. Extent of sprouting in SNpc neurons following partial lesions of the SNpc. (A) The number of axonal varicosities counted on a single reconstructed axonal arborisation plotted against the lesion size. (B) The number of branching points observed on a single reconstructed axon plotted against lesion size. The lesion size is the average number of cells in the normal SNpc minus the number of cells in the SNpc of the lesioned animal from which the reconstruction was made (expressed as a percent of the average number of normal SNpc cells). All lesions were less than 60%. Note that the number of varicosities per axon appears to rise sharply in proportion to the size of the lesion. (C) An estimate of the total number of varicosities in the CPu of normal and lesioned animals. This estimate was obtained by multiplying the number of varicosities observed on the corresponding reconstruction by the number of SNpc neurons in the respective animals.

Nigrostriatal axonal varicosities were recognised by anterograde filling and subsequent reaction of biotin in the presence of DAB. One hundred and twenty axonal varicosities from four CPu nuclei of normal animals and 190 axonal varicosities from four CPu nuclei ipsilateral to the lesion were examined. Varicosities of SNpc axons in the CPu of normal

This study demonstrates that following a partial lesion of the SNpc, surviving neurons sprout extensively within the dorsal tier of the CPu. The increased terminal arborization is manifested by a substantial increase in collateral branching (increased branching points) and axonal varicosities; both of which correlated with the size of the SNpc lesion. The morphology of these new synaptic varicosities was altered, being larger and containing more vesicles than normal profiles. Although there may be a small proportion of nondopaminergic neurons in the SNpc, the reconstructed axons demonstrating vigorous sprouting were most likely DA neurons. We argue this because firstly, it seems unlikely that our entire sample was inadvertently from the small proportion of non-dopaminergic neurons in the SNpc. Even after a 50% lesion of the SNpc, 80% of the remaining neurons would be DA. Secondly, the observation that the density of DAT-IR varicosities remains constant until the lesion is large, supports the contention that DA neurons have mounted a compensatory response for the loss of SNpc neurons. When the lesion exceeded 75%, remaining SNpc neurons seemed unable to increase the size of the axonal arbour to compensate for the missing neurons and varicosities. Up to that point, the degree of sprouting appeared to be regulated to match the extent of SNpc neuronal loss. A variety of counting methods have been used to determine the effects of 6-OHDA. These include cumulative cell counts on every third or fourth TH-IR section through the SNpc 10,32,42 and qualitative estimation of lesion size by examining single TH-IR stained sections of the SNpc. 5,26,38 Our method of lesioning with 6-OHDA was modelled on a previous study. 38 They, and others, 5,10,26,32,42 report that injection of 6-OHDA caused loss of more than 90% of SNpc DA neurons. We also found that if TH-IR neurons were counted, 6-OHDA injections into the SNpc caused an almost complete lesion of DA neurons (74 ^ 8%) whereas the lesion was estimated to be much smaller (37 ^ 14%) if Neutral Red-stained neurons were counted (Fig. 6). This discrepancy has also been reported by others. 8 The differences between the two techniques in estimating the lesion size may have been greater if normal rats rather than the contralateral SNpc were used as a control group for this aspect of the study. It is not clear whether Neutral Red or TH staining of SNpc neurons is the better measure of lesion size. On one hand, the presence of TH is an indication of DA production and it could be argued that the total number of neurons is not as relevant as the number of DA synthesizing neurons. The results illustrated in Fig. 6 suggest that the ability to express TH is lost by some DA cells following exposure to 6-OHDA. It is possible that, with time most, if not all, neurons will recover the ability to express TH 8 and so the total number of surviving neurons would be important. If this is the case, studies performed 10–14 days after the administration of 6-OHDA, and using TH as the measure of cell loss, could substantially over-estimated the extent of neuronal damage. The use of Neutral Red (or similar stains) in the absence of functional markers such as TH, has the potential to create difficulty in delineating the borders of the SNpc, especially

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D. I. Finkelstein et al. Table 2. Analysis and morphology of substantia nigra pars compacta axons reconstructed from normal (N-1–N-4) and lesioned animals (L-1–L-4) Axon No.

Estimated cell numbers in SNpc

% SNpc degeneration

No. of axonal varicosities

No. of branching points

N-1 N-2 N-3 N-4 L-1 L-2a* L-2b* L-3a† L-3b† L-4

11,611 11,442 11,187 11,071 8347 (0.037) 6297 (0.038)

0 0 0 0 25 45 45 50 50 55

71 96 151 208 384 983 918 835 1065 1324

19 20 21 40 95 143 147 200 227 275

5623 (0.041) 4950 (0.021)

No. of varicosities in the CPu ( × 10 6)

1.5‡ 3.2 6.0§ 5.5§ 6.6

Estimates of SNpc neurons are listed with coefficients of error in brackets. Per cent of SNpc degeneration was expressed as the difference between the average of the total number of neurons in the normal SNpc (Table 1) and the lesioned SNpc. Coefficients of error of the estimates for each lesioned animal are listed in brackets. *Axons L-2a and L-2b were reconstructed from the same SNpc. †Axons L-3a and L-3b were reconstructed from the same SNpc. ‡This estimate obtained by multiplying the average number of neurons in the SNpc (11,239, n ˆ 11, Table 1) by the average number of varicosities (132) observed on reconstructed axons, N-1–N-4. §These estimates obtained by multiplying the number of neurons in the SNpc by the average of the number of varicosities observed on the axons with similar sized lesions of the SNpc. Table 3. Analysis of dopamine transporter immunohistochemistry in the dorsal 1.5 mm of the CPu of normal (N-5–N-15) and lesioned rats (L-5–L-19) Animal No.

Total No. of neurons (CE)

N-5–N-15 L-5 L-6 L-7 L-8 L-9 L-10 L-11 L-12 L-13 L-14 L-15 L-16 L-17 L-18 L-19

11,239 (0.028)* 10,262 (0.060) 8856 (0.076) 8716 (0.065) 8013 (0.068) 7450 (0.047) 6522 (0.158) 4948 (0.109) 4948 (0.128) 4076 (0.082) 3823 (0.052) 3823 (0.113) 2811 (0.110) 2474 (0.085) 1237 (0.148) 937 (0.126)

% SNpc lesion 0 9 21 22 29 34 42 56 56 64 66 66 75 78 89 92

% Difference in No. of DAT varicosities

% Difference in density of DAT varicosities/mm 3

4.96 (4.59)† 213 21 21 7 4 45 214 14 6 11 26 28 86 99 99

6.03 (2.19)‡ 220 1 213 2 220 31 21 8 26 22 26 17 83 99 99

Estimates of SNpc neurons are listed with coefficients of error in brackets. Per cent SNpc lesion as in Table 2. Per cent difference in the total number and density of DAT-IR varicosities is the total number and density on the denervated CPu expressed as a percentage of the non-denervated CPu. *Average number of neurons in normal SNpc with coefficient of variance in brackets. †Mean difference in DAT varicosity numbers among normal rats with S.D. in brackets. ‡Mean difference in DAT density found in normal rats with S.D. in brackets.

with the SNpr. In practice, the borders were consistently detected, as confirmed by the low CE and recounting, which produced similar estimates of lesion size. The use of pre-lesion retrograde labelling with FluoroGold has been used to circumvent this problem. 8 This was not possible in this study because the mechanical injury of the CPu resulting from the number of injections of the FluoroGold required to completely fill the dorsal tier of the CPu may have inadvertently stimulated sprouting as a result of local trauma. Interestingly, there was a small (9 ^ 7%) but significant (P ˆ 0.010) reduction of neurons in the contralateral (nonlesioned) SNpc (mean ˆ 10,431 ^ 1016). While it has been

suggested the 6-OHDA injected unilaterally may leak to the contralateral SNpc, 45 it is common practice to use the contralateral SNpc as a normal control. Our findings suggest that untreated animals are the preferred control group because the effect of 6-OHDA may be underestimated by about 10% when the control is the contralateral SNpc. We estimated the average number of neurons in the normal rat SNpc to be 11,239 (Table 1), in contrast to a recent study that reported an average of 7200 neurons. 36 We included the dense aggregation of neurons at the caudal boundary of the SNpc that have been described as part of the SNpr anatomically 21,25 but are functionally part of the

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Fig. 8. Differences in the density of DAT varicosities between the left and right CPu of normal and rats with unilateral partial lesions of the SNpc. Density was measured in the dorsal 1.5 mm of each CPu and the differences between hemispheres is expressed as a percentage and plotted against lesion size. These data demonstrate that the density of DAT-labelled varicosities in the CPu ipsilateral to the lesioned SNpc remained normal until approximately 75% of its neurons were destroyed (r ˆ 0.9342, f ˆ a/(1 1 exp(2(x 2 x0)/b))). When the lesion exceeded 75%, remaining SNpc neurons appeared unable to increase the size of the axonal arbour to compensate for the missing varicosities/neurons resulting in a rapid decrease in DAT varicosity density in the dorsal CPu.

DA SNpc output. 23 Because we were investigating the sprouting response of DA neurons, we have included them as part of the SNpc. The spread of tracer is an important methodological consideration. Animals were used only if the centre of the injection was located in the SNpc. It is unlikely that a VTA cell was mistakenly reconstructed in the present study as no tracer from the injection site diffused into this area (Figs 1–4) and all arborizations only innervated the dorsal tier of the CPu. The ventrolateral layers of the VTA, situated medial to the SNpc, have projections that innervate the ventral tier of the CPu. 4,21,43 Although the tracer spread into the adjacent SNpr and occasionally the subthalamic nucleus, these nuclei do not project to the CPu. 21 Because of these precautions and the care taken to trace axons through the internal capsule to the SNpc, we think it is likely that only cells arising from the SNpc were studied. Even so, a proportion of axons reconstructed and axonal varicosities examined ultrastructurally may be non-dopaminergic because a proportion (10%) of SNpc neurons were glutamatergic. 21 We accept that for a 50% lesion of the SNpc, as many as 20% of axonal reconstructions and varicosities studied ultrastructurally may be glutamatergic and the risk of this contamination is proportional to the size of the lesion. However, this does not detract from the main thrust of this study, which is to demonstrate that the nigrostriatal DA system is capable of mounting a vigorous sprouting response. This opinion is supported by the results of our DAT-IR study. This vigorous sprouting response observed may explain the spontaneous improvements in motor function 18,26,43,44,53 and the increased density of TH-IR fibres that occurs four months after a SNpc lesion. 5–7,35 Neurons that survive the SNpc injury sprout, possibly to compensate for denervation and to reinnervate postsynaptic receptors in the CPu left vacant by the

damage. Because the SNpc lesion was only partial, the denervation of the dorsal tier of the CPu may have been patchy. Indeed, many small branches and axonal varicosities appeared to form in clusters that may have corresponded to these areas in the CPu (Figs 3 and 4). The SNpc supplies the dorsal tier of the CPu and sprouting we observed remained confined to this region (Fig. 5). 4,20,23 This suggests that sprouting was a response to local denervation and the total number of axonal varicosities and branching points increased in relation to the size of lesion (Table 2, Fig. 7). The increase in the size of the terminal arbour of a single axon that follows SNpc injury can extend over the whole dorsal tier of the CPu. The consequence of this extensive arborization will mean that remaining neurons can no longer have local influence on specific sub-regions of the CPu. Removal of DA afferents by 6-OHDA lesions may enhance the production of neurotrophic factors. 39 Growth promoting activity in the CPu increases in response to loss of DA or pharmacological blockade of DA receptors. 11,12,52 The growth of cultured mesencephalic DA neurons was enhanced by extracts from the CPu of PD patients or rats with 6-OHDA mesencephalic lesions. 14,52 Chronic administration of levodopa to animals with mesencephalic lesions decreased the striatal-derived growth promoting activity in a dose-dependent fashion 13 and the arrival of DA and other catecholamines in growing animals decreased the production of growth factors. Our findings support the proposal that DA neurotransmission regulates the production of target derived neurotrophic factors and that denervation would result in an increase in this neurotrophic activity and stimulate sprouting of residual DA neurons. 11,14,39 The density of axonal varicosities in the dorsal tier of the CPu was estimated by counting the DAT-IR varicosities in this region (Fig. 9) with the contralateral CPu used as the

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control. The lesioned side was compared with the contralateral side because there was variability in immunohistochemical labelling between animals. Many factors could have contributed to this variation, including fixation time, penetration of the antibodies and density of the reaction product. While it

Fig. 10. Electron micrographs of nigrostriatal axonal varicosities. The dense reaction product formed by the DAB reaction can clearly be seen within the varicosity profile. (A) Axonal varicosities from a lesioned rat. Forty-nine vesicles are present and the area of the profile is 0.246 mm 2. (B) The axonal varicosity illustrated is from a normal rat. It contains eight vesicles and its area is 0.103 mm 2. Scale bar ˆ 0.35 mm. Table 4. Morphology of nigrostriatal axonal varicosities in the normal and denervated caudate–putamen Animal type Normal Lesioned

Median area of profiles

Number of vesicles

0.18 (0.04, 0.80) 0.25 (0.07, 0.67)

7 (3, 40) 16 (3, 49)

Figures in brackets represent the 2.5 percentile and the 97.5 percentile values. Normal and lesioned animals are significantly different (P # 0.05, Kruskal–Wallis, ANOVA post hoc Dunn’s). Non-parametric statistics were used as the data was not normally distributed (Sigma Stat, 1997, Jandel, Chicago, IL, U.S.A.).

Fig. 9. DAT-IR varicosities in the dorsal 1.5 mm of the rat CPu. (A) Normal CPu. (B) CPu ipsilateral to a SNpc with a 65% lesion (see rat L-13, Table 3). Observe that DA sprouting has occurred in the CPu shown in (B). This is demonstrated by the relatively equal distribution of DAT-IR varicosities between the two CPu nuclei despite the difference in the number of SNpc neurons. Arrows point to DAT varicosities; Scale bar ˆ 20 mm. (C) The counting frame in A has been magnified and a cartoon of the varicosities and axons within close proximity has been drawn. The eight filled varicosities were counted. The unfilled varicosity marked with a letter x was not counted because it fell on the exclusion line of the counting frame. The varicosity marked with an asterisk was counted because it was attached to an axon and it lay on the inclusion line of the counting frame.

Sprouting of substantia nigra neurons

was possible to obtain sufficient normal (unlesioned) animals to overcome this variability, it was not possible to produce sufficient numbers of each lesion size. As we have shown, the contralateral size may also be affected (by about 10%), and so there may be some underestimate of the change on the ipsilateral side. The number of axonal varicosities in the dorsal tier of the CPu was estimated by multiplying the number of SNpc neurons by the number of varicosities on the corresponding axonal reconstruction. Because there was often only one axon representing a particular size of lesion, errors might be expected. However the points in Fig. 7C seem to fall on a straight line, suggesting that these were not substantial. Presumably, as the lesion approaches 100%, sprouting and other compensatory responses will no longer be capable of replacing denervated varicosities and the total number will fall below the normal level and approach zero. This was demonstrated in the DAT studies where the density of DAT-IR varicosities fell sharply when the size of the lesion exceeded 70% (Fig. 8 and Table 3). Explanations for the late onset of symptoms in PD include compensatory mechanisms, including increased synthesis,

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metabolism and release of DA in the remaining SNpc neurons and terminals, the upregulation of DA receptors and a prolonged clearance time of extracellular DA. 1,27,41,54,55 The large arborizations of each residual DA SNpc neuron are capable of influencing a much larger postsynaptic field than normal and similarly, individual varicosities are also larger and contain more vesicles (Fig. 10 and Table 4). The altered morphology of varicosities suggests to us that synaptic mechanisms such as DA re-uptake channels and auto receptors could also be altered. The large arborisations and large terminals could deliver large boluses of DA to the CPu especially if the presynaptic DA re-uptake mechanisms and auto receptors are disrupted. 15,47 Delivery of boluses of DA in a mass action without regional control or regulation of delivery and uptake could provide an explanation for many features associated with PD and dyskinesia. Acknowledgements—This research was supported by grants from the Bethlehem Griffith Research Foundation and the Australian National Health and Medical Research Council. We are grateful for the skilled technical assistance of Anthony Natoli and Avril Horne, and to Nigel Wreford for his contribution during a stereological discussion.

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