SEARCH

SEARCH BY CITATION

Keywords:

  • dopamine transport;
  • mazindol;
  • sprouting;
  • synaptosomes;
  • ultrastructure;
  • voltammetry

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Lesioning
  5. DAT immunohistochemistry
  6. Preparation of SNpc for estimation of lesion size
  7. Estimation of SNpc lesion size
  8. Injection of tracer for ultrastructural examination of nigrostriatal synapses
  9. Preparation of tissue for analysis of nigrostriatal ultrastructure
  10. Rotational behaviour
  11. [3H]Mazindol binding to the dopamine transporter
  12. Measurement of [3H]Dopamine transport in synaptosomes
  13. Electrochemistry and microelectrodes
  14. In vivo electrochemistry
  15. In vitro electrochemistry
  16. Results
  17. Estimates of the number of neurones in the SNpc
  18. [3H]Dopamine transport in normal animals
  19. [3H]Dopamine transport in lesioned animals
  20. [3H]Mazindol binding to the DAT
  21. In vivo electrochemistry
  22. In vitro electrochemistry of dopamine release and clearance in synaptosomal preparations and the effects of quinpirole
  23. Ultrastructural changes to the DA synapse after lesions of the SNpc
  24. Rotational behaviour
  25. Discussion
  26. Acknowledgements
  27. References

Following partial substantia nigra lesions, remaining dopaminergic neurones sprout, returning terminal density in the dorsal striatum to normal by 16 weeks. This suggests regeneration and maintenance of terminal density is regulated to release appropriate levels of dopamine. This study examined the structure and function of these reinnervated terminals, defining characteristics of dopamine uptake and release, density and affinity of the dopamine transporter (DAT) and ultrastructural morphology of dopamine terminals in the reinnervated dorsal striatum. Finally, rotational behaviour of animals in response to amphetamine was examined 4 and 16 weeks after substantia nigra pars compacta (SNpc) lesions. Dopamine transport was markedly reduced 16 weeks after lesioning along with reduced density and affinity of DAT. Rate of dopamine release and peak concentration, measured electrochemically, was similar in lesioned and control animals, while clearance was prolonged after lesioning. Ultrastructurally, terminals after lesioning were morphologically distinct, having increased bouton size, vesicle number and mitochondria, and more proximal contacts on post-synaptic cells. After 4 weeks, tendency to rotate in response to amphetamine was proportional to lesion size. By 16 weeks, rotational behaviour returned to near normal in animals where lesions were less than 70%, although some animals demonstrated unusual rotational patterns at the beginning and end of the amphetamine effect. Together, these changes indicate that sprouted terminals are well compensated for dopamine release but that transport mechanisms are functionally impaired. We discuss these results in terms of implications for dyskinesia and other behavioural states.

Abbreviations used
anova

analysis of variance

AP

anteroposterior

Bmax

density of binding sites

CE

coefficient of error

CV

coefficient of variance

DA

dopamine

DAB

diaminobenzidine

DAT

dopamine transporter

D2R

D2 dopamine receptor

EM

electron microscope

HEPES

(N-[2-Hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid])

i.d.

internal diameter

i.p.

intraperitoneal

-ir

immunoreactive

Kd

dissociation constant

L

lateral

ms

milliseconds

nA

nanoamps

nr

neutral red

6-OHDA

6-hydroxydopamine

PBS

phosphate-buffered saline

PD

Parkinson's disease

R0-5

dopamine transport 0–5 min after assay initiation

S

saturation of dopamine transport

s.c.

subcutaneous

SD

standard deviation

SNpc

substantia nigra pars compacta

TH

tyrosine hydroxylase

T50

time for DA signal to decrease by 50% of peak amplitude

T80

time for DA signal to decrease by 80% of peak amplitude

Following partial substantia nigra pars compacta (SNpc) lesions, dopaminergic neurones sprout to reinnervate the dorsal striatum (Finkelstein et al. 2000; Parish et al. 2001). Reconstruction of individual axons show that by 4 months, terminal arbors of individual axons increase, commensurate with lesion size, to return terminal density to normal (Finkelstein et al. 2000). Terminal density appears to be regulated by the D2 dopamine (DA) receptor (D2R) suggesting that regeneration and maintenance of terminal density is controlled to maintain appropriate DA levels in the synaptic cleft (Parish et al. 2001). This implies that newly formed terminals have release and transport mechanisms for DA, capable of regulation. In normal nigrostriatal terminals, DA synthesis and release is highly regulated. Pre-synaptic D2R inhibits nerve terminal excitability (Bunney et al. 1973; Tepper et al. 1984) and reduces DA release (Ungerstedt et al. 1982; Bowyer and Weiner 1987), partially mediated via activation of K+ channels (Lacey et al. 1987; Cass and Zahniser 1990). Activation of D2R by DA reduces cAMP production and thereby reduces DA synthesis by adenylate cyclase-dependent phosphorylation of tyrosine hydroxylase (TH), the rate limiting enzyme in the DA synthesis pathway (el Mestikawy et al. 1986; Onali et al. 1988; Lindgren et al. 2001). As DA release is dependent on newly synthesized transmitter, this effect is likely to play a critical role in the control of striatal dopaminergic transmission. The D2 autoreceptor is tightly linked to the DA transporter (DAT), both anatomically (Hersch et al. 1997) and functionally (Kimmel et al. 2001; Robinson 2002). Thus return of regulated function of DA terminals following injury might be expected to include evidence of co-ordinated D2R and DAT interaction as evidenced by regulated DA release and turnover. Sprouting and formation of new terminals occurs following partial SNpc lesions induced by 6-hydroxydopamine (Pickel et al. 1992; Thomas et al. 1994; Blanchard et al. 1995; Anglade et al. 1996; Blanchard et al. 1996; Ingham et al. 1996, 1998; Finkelstein et al. 2000; Parish et al. 2001). Although only a portion of DA neurones are destroyed, it is likely that by 4 months after lesioning, most, if not all, DA terminals in the striatum are newly formed. One day after a lesion, nigrostriatal axons retract back to the level of the globus pallidus, and over the next 6 days, continue to retract progressively towards the SNpc (Rosenblad et al. 2000). We have observed that after SNpc lesioning regeneration has commenced by 4 weeks with the appearance of hypertrophic DAT immunoreactive (DAT-ir) terminals in the striatum (Stanic et al. 2002, 2003). Established synapses are present by 4 months, although regeneration continues for at least 7 months (Blanchard et al. 1996). The ultrastructure of regenerated SNpc terminals in the striatum is altered, suggesting they may produce, store, and release more DA than normal terminals (Finkelstein et al. 2000). In a medial forebrain bundle stimulation post-lesioning paradigm, DA terminals were shown to have increased DA release and retarded DA clearance (van Horne et al. 1992; Garris et al. 1997a, 1997b). This suggests that compensatory changes in the behaviour of the synapse may occur in response to lesioning that favours prolongation of DA half-life in the synaptic cleft. This study examined in detail the structure and function of nascent DA terminals. We made partial lesions to the SNpc of rats and examined DA release and transport by DA terminals following sprouting. We also examined the ultrastructural morphology of these terminals and correlated turning behaviour in response to amphetamine at 4 and 16 weeks after lesioning.

Adult male Wistar rats (Monash University) weighing between 250 and 350 g were used. All methods conformed to the Australian National Health and Medical Research Council published code of practice for the use of animal research and were approved by the Monash University Animal Ethics Committee. Throughout this study anovas with Tukey post-hoc tests were used with statistical differences set at the level of p ≤ 0.05. Where specified, significance levels were tested with an unpaired t-test set at p ≤ 0.05.

DAT immunohistochemistry

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Lesioning
  5. DAT immunohistochemistry
  6. Preparation of SNpc for estimation of lesion size
  7. Estimation of SNpc lesion size
  8. Injection of tracer for ultrastructural examination of nigrostriatal synapses
  9. Preparation of tissue for analysis of nigrostriatal ultrastructure
  10. Rotational behaviour
  11. [3H]Mazindol binding to the dopamine transporter
  12. Measurement of [3H]Dopamine transport in synaptosomes
  13. Electrochemistry and microelectrodes
  14. In vivo electrochemistry
  15. In vitro electrochemistry
  16. Results
  17. Estimates of the number of neurones in the SNpc
  18. [3H]Dopamine transport in normal animals
  19. [3H]Dopamine transport in lesioned animals
  20. [3H]Mazindol binding to the DAT
  21. In vivo electrochemistry
  22. In vitro electrochemistry of dopamine release and clearance in synaptosomal preparations and the effects of quinpirole
  23. Ultrastructural changes to the DA synapse after lesions of the SNpc
  24. Rotational behaviour
  25. Discussion
  26. Acknowledgements
  27. References

Rats were killed by an overdose of sodium pentobarbitone (100 mg/kg i.p., Rhone Merieux, Pinkenba, Australia) and perfused with 400 mL of heparinized (1 unit/mL, Fisons, Sydney, Australia) warmed (37°C) 0.1 m phosphate-buffered saline (PBS; pH 7.4), followed by 250 mL of chilled 4% paraformaldehyde (Sigma-Aldrich Pty Ltd, Castle Hill, Australia) in 0.1 m phosphate buffer and 0.2% picric acid (4°C; pH 7.4). Brains were removed and left overnight at 4°C in 30% sucrose and PBS solution. The following day, 20 µm-thick coronal sections were cut serially through the striatum with a cryostat (Leica CM 1850, Wetzlar, Germany) directly onto slides coated with 0.1% chrome alum (Ajax Chemicals, Sydney, Australia) and 1% gelatine (Sigma-Aldrich Pty Ltd) in distilled water. For DAT immunohistochemistry, sections were fixed to gelatinized slides with 10% neutral buffered formalin (15 s). Sections were then incubated for 30 min in blocking solution (0.1 m PBS, 0.3% Triton X-100; Sigma-Aldrich Pty Ltd; and 5.0% normal rabbit serum), and then for 48 h at 4°C in rat anti-DAT primary antibody (Chemicon, Temecula, CA, USA, 1 : 4000 in PBS, 0.3% Triton X-100 and 1.0% normal rabbit serum). This was followed by overnight incubation at 4°C in a biotinylated secondary antibody (rabbit, anti-rat IgG, 1 : 500, Vector, Burlingame, CA, USA) and for 2 h in 1 : 5000 avidin–peroxidase (Sigma-Aldrich Pty Ltd) with 0.75% Triton X-100. Sections were then reacted with cobalt and nickel-intensified diaminobenzidine (DAB, Sigma-Aldrich Pty Ltd) for 30 min. Hydrogen peroxide (3.33 µL/ml) was added to the DAB solution for a further 8 min. Rinses (4 × 10 min) in 0.1 m PBS were performed between each step. Sections were dehydrated in a series of graded ethanol solutions (50–100%), and cleared before being coverslipped with a polystyrene mounting medium.

Preparation of SNpc for estimation of lesion size

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Lesioning
  5. DAT immunohistochemistry
  6. Preparation of SNpc for estimation of lesion size
  7. Estimation of SNpc lesion size
  8. Injection of tracer for ultrastructural examination of nigrostriatal synapses
  9. Preparation of tissue for analysis of nigrostriatal ultrastructure
  10. Rotational behaviour
  11. [3H]Mazindol binding to the dopamine transporter
  12. Measurement of [3H]Dopamine transport in synaptosomes
  13. Electrochemistry and microelectrodes
  14. In vivo electrochemistry
  15. In vitro electrochemistry
  16. Results
  17. Estimates of the number of neurones in the SNpc
  18. [3H]Dopamine transport in normal animals
  19. [3H]Dopamine transport in lesioned animals
  20. [3H]Mazindol binding to the DAT
  21. In vivo electrochemistry
  22. In vitro electrochemistry of dopamine release and clearance in synaptosomal preparations and the effects of quinpirole
  23. Ultrastructural changes to the DA synapse after lesions of the SNpc
  24. Rotational behaviour
  25. Discussion
  26. Acknowledgements
  27. References

Rats used in synaptosome and membrane binding studies were decapitated and brains removed and dissected in a coronal plane 4.3 mm posterior to bregma (Paxinos and Watson 1998). The posterior portion containing the SNpc was placed into 10% formalin and stored at 4°C for 7 days (anterior portions, containing the striatum, were used for membrane binding and synaptosome preparations as described below). After 7 days, brains were placed in a solution containing 4% paraformaldehyde in 0.1 m phosphate buffer, 0.2% picric acid and 30% sucrose (4°C; pH 7.4) for 48 h and then sectioned on a cryostat. Coronal sections, each 45 µm thick, were serially cut (1 : 4) through the SNpc. On average, 12 of the 45-µm thick sections, each 180 µm apart, were stained with 1% neutral red (Merck, Darmstadt, Germany) for 3 min, washed in water, dehydrated in a series of graded ethanol solutions, cleared and coverslipped.

Estimation of SNpc lesion size

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Lesioning
  5. DAT immunohistochemistry
  6. Preparation of SNpc for estimation of lesion size
  7. Estimation of SNpc lesion size
  8. Injection of tracer for ultrastructural examination of nigrostriatal synapses
  9. Preparation of tissue for analysis of nigrostriatal ultrastructure
  10. Rotational behaviour
  11. [3H]Mazindol binding to the dopamine transporter
  12. Measurement of [3H]Dopamine transport in synaptosomes
  13. Electrochemistry and microelectrodes
  14. In vivo electrochemistry
  15. In vitro electrochemistry
  16. Results
  17. Estimates of the number of neurones in the SNpc
  18. [3H]Dopamine transport in normal animals
  19. [3H]Dopamine transport in lesioned animals
  20. [3H]Mazindol binding to the DAT
  21. In vivo electrochemistry
  22. In vitro electrochemistry of dopamine release and clearance in synaptosomal preparations and the effects of quinpirole
  23. Ultrastructural changes to the DA synapse after lesions of the SNpc
  24. Rotational behaviour
  25. Discussion
  26. Acknowledgements
  27. References

The fractionator design for estimating the number of SNpc neurones were published in detail previously with the following modifications (Finkelstein et al. 2000; Parish et al. 2001). Using optical disector rules, counts of SNpc neurones, stained for neutral red, were made at regular pre-determined intervals (x = 125 µm, y = 200 µm) derived by means of a grid program (Stereo Investigator, MicroBrightField, VT, USA; viewed through a microscope, Leica DMLB) and a counting frame (40 µm × 27 µm = 1080 µm2). Therefore, the area sampling fraction is 1080/(125 × 200) = 0.043. In all animals, 45-µm thick sections through the SNpc, each 180 µm apart, were analysed, the fraction of sections sampled being 45/180 = 0.25. Lesion size was the number of SNpc neurones estimated in lesioned animals, expressed as a percent of the number in the normal SNpc.

Injection of tracer for ultrastructural examination of nigrostriatal synapses

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Lesioning
  5. DAT immunohistochemistry
  6. Preparation of SNpc for estimation of lesion size
  7. Estimation of SNpc lesion size
  8. Injection of tracer for ultrastructural examination of nigrostriatal synapses
  9. Preparation of tissue for analysis of nigrostriatal ultrastructure
  10. Rotational behaviour
  11. [3H]Mazindol binding to the dopamine transporter
  12. Measurement of [3H]Dopamine transport in synaptosomes
  13. Electrochemistry and microelectrodes
  14. In vivo electrochemistry
  15. In vitro electrochemistry
  16. Results
  17. Estimates of the number of neurones in the SNpc
  18. [3H]Dopamine transport in normal animals
  19. [3H]Dopamine transport in lesioned animals
  20. [3H]Mazindol binding to the DAT
  21. In vivo electrochemistry
  22. In vitro electrochemistry of dopamine release and clearance in synaptosomal preparations and the effects of quinpirole
  23. Ultrastructural changes to the DA synapse after lesions of the SNpc
  24. Rotational behaviour
  25. Discussion
  26. Acknowledgements
  27. References

Sixteen weeks after lesioning, two small injections (10–20 nL) of the anterograde neuronal tracer, 10% dextran biotin (Molecular Probes, Inc., Eugene, OR, USA) in 0.1 m phosphate buffer (pH 7.4), were made (Picospritzer II, General Valve Corporation, Fairfield, NJ, USA) into each SNpc (antero-posterior 5.2, 5.8 mm; lateral 2.1, 2.0 mm; dorso-ventral 7.8, 7.8 mm) to label SNpc axons and terminals for identification under electron microscope (EM). Following the injection, the micropipette was left in situ for approximately 10 min before slowly withdrawing (1 mm/min), to minimise spread of tracer along the needle track. Fourteen days after injection of tracer (to allow for transport of tracer), the rats were killed and perfused with warmed (37°C) PBS with heparin (1 unit/mL), followed by 500 mL of chilled 2.5% glutaraldehyde (Sigma-Aldrich Pty Ltd) and 1% paraformaldehyde in 0.1 m phosphate buffer (4°C, pH 7.4). The brains were then removed and left at 4°C in the same fixative for a period of 24 h.

Preparation of tissue for analysis of nigrostriatal ultrastructure

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Lesioning
  5. DAT immunohistochemistry
  6. Preparation of SNpc for estimation of lesion size
  7. Estimation of SNpc lesion size
  8. Injection of tracer for ultrastructural examination of nigrostriatal synapses
  9. Preparation of tissue for analysis of nigrostriatal ultrastructure
  10. Rotational behaviour
  11. [3H]Mazindol binding to the dopamine transporter
  12. Measurement of [3H]Dopamine transport in synaptosomes
  13. Electrochemistry and microelectrodes
  14. In vivo electrochemistry
  15. In vitro electrochemistry
  16. Results
  17. Estimates of the number of neurones in the SNpc
  18. [3H]Dopamine transport in normal animals
  19. [3H]Dopamine transport in lesioned animals
  20. [3H]Mazindol binding to the DAT
  21. In vivo electrochemistry
  22. In vitro electrochemistry of dopamine release and clearance in synaptosomal preparations and the effects of quinpirole
  23. Ultrastructural changes to the DA synapse after lesions of the SNpc
  24. Rotational behaviour
  25. Discussion
  26. Acknowledgements
  27. References

The striatum of each brain was cut into 150-µm coronal sections on a vibratome (Technical Products International, Inc., St Louis, MO, USA). All sections were subsequently incubated in avidin peroxidase (1 : 5000) and 0.015% Triton-X in PBS for 2 h. These sections were then washed three times in PBS (to remove excess unbound avidin peroxidase), incubated for 30 min in intensified cobalt-nickel DAB and finally hydrogen peroxide (0.01%) was added to this solution for a further 10 min (Adams 1981).

Sections from experimental and control animals which contained the striatum were examined under light microscopy (40× objective) and portions of the dorsal tier of the striatum containing labelling (Fallon and Moore 1978; Gerfen et al. 1987; Fallon and Loughlin 1995; Isacson and Deacon 1997) were cut from the sections using a blunt 14-gauge luer needle (i.d. 1.5 mm). These sections were then post-fixed in 1% osmium tetraoxide in 0.2 m cacodylate buffer for 1 h, dehydrated through a series of alcohol, rinsed in 1,2-epoxy-propane and finally embedded in pure epon-araldite. Ultrathin sections were cut from the resin blocks and mounted onto copper grids stained with 2% aqueous uranyl acetate and 2% lead citrate and viewed with a JEOL II electron microscope. Using EM, terminals emanating from SNpc cells were recognized by the presence of the DAB reaction product. Synaptic features, including the pre-synaptic terminal area, vesicle and mitochondria numbers, docking of vesicles at synaptic active zones and post-synaptic targets were examined directly from the electron micrographs. Kruskal–Wallis anova on Ranks (with Dunn's post-hoc test) as well as median, 2.5th and 97.5th percentiles were used for data without a normal distribution. anova's (with Tukey post-hoc tests), Chi square tests and mean ± standard deviation were used on normally distributed data. Statistical significance was recognised at the level of p < 0.05. The SNpc from these animals was cut and stained with neutral red, and lesion size estimated.

Rotational behaviour

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Lesioning
  5. DAT immunohistochemistry
  6. Preparation of SNpc for estimation of lesion size
  7. Estimation of SNpc lesion size
  8. Injection of tracer for ultrastructural examination of nigrostriatal synapses
  9. Preparation of tissue for analysis of nigrostriatal ultrastructure
  10. Rotational behaviour
  11. [3H]Mazindol binding to the dopamine transporter
  12. Measurement of [3H]Dopamine transport in synaptosomes
  13. Electrochemistry and microelectrodes
  14. In vivo electrochemistry
  15. In vitro electrochemistry
  16. Results
  17. Estimates of the number of neurones in the SNpc
  18. [3H]Dopamine transport in normal animals
  19. [3H]Dopamine transport in lesioned animals
  20. [3H]Mazindol binding to the DAT
  21. In vivo electrochemistry
  22. In vitro electrochemistry of dopamine release and clearance in synaptosomal preparations and the effects of quinpirole
  23. Ultrastructural changes to the DA synapse after lesions of the SNpc
  24. Rotational behaviour
  25. Discussion
  26. Acknowledgements
  27. References

Rotational behaviour was performed in a light- and soundproofed room to which the animals were habituated for 45 min. Animals were routinely tested between the hours of 09:00 and 12:00 h. The behaviour was filmed using a domestic video camera (Panasonic, Tokyo, Japan) and analysis performed post-hoc. Motor asymmetry of lesioned animals was quantified 4 and 16 weeks after SNpc lesions. Rotatory response to administration of amphetamine (5 mg/kg i.p., Sigma-Aldrich Pty Ltd) was measured by placing rats into a 45-cm diameter observation chamber. Observations were made for 30 min prior to amphetamine injection and for 2 h after injection. The net number of turns (right turns minus left) made in 5-min periods were recorded and divided by 5 to obtain the average number of rotations per minute.

Rats observed at 4 weeks were killed by an overdose of sodium pentobarbitone (100 mg/kg i.p.) and perfused, as described earlier (see DAT immunohistochemistry). Brains were removed and stored at 4°C in 30% sucrose and 4% paraformaldehyde in 0.1 m PB solution overnight. The following day, the SNpc was sectioned, stained for neutral red and lesion size estimated. Rats observed 16 weeks after lesioning were used in the synaptosome experiments (described below).

[3H]Mazindol binding to the dopamine transporter

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Lesioning
  5. DAT immunohistochemistry
  6. Preparation of SNpc for estimation of lesion size
  7. Estimation of SNpc lesion size
  8. Injection of tracer for ultrastructural examination of nigrostriatal synapses
  9. Preparation of tissue for analysis of nigrostriatal ultrastructure
  10. Rotational behaviour
  11. [3H]Mazindol binding to the dopamine transporter
  12. Measurement of [3H]Dopamine transport in synaptosomes
  13. Electrochemistry and microelectrodes
  14. In vivo electrochemistry
  15. In vitro electrochemistry
  16. Results
  17. Estimates of the number of neurones in the SNpc
  18. [3H]Dopamine transport in normal animals
  19. [3H]Dopamine transport in lesioned animals
  20. [3H]Mazindol binding to the DAT
  21. In vivo electrochemistry
  22. In vitro electrochemistry of dopamine release and clearance in synaptosomal preparations and the effects of quinpirole
  23. Ultrastructural changes to the DA synapse after lesions of the SNpc
  24. Rotational behaviour
  25. Discussion
  26. Acknowledgements
  27. References

[3H]Mazindol binding to the DAT was performed using methods described previously (Javitch et al. 1984). Sixteen weeks after lesioning, rats were decapitated, brains removed and cut in a coronal plane (4.3 mm posterior to bregma) to separate the striatum from the SNpc (Paxinos and Watson 1998). The dorsal striatum was dissected and homogenized in 5 mL of ice-cold assay buffer (50 mm Tris, 120 mm NaCl, 5 mm KCl, pH 7.9) then centrifuged at 48 000 g for 10 min at 4°C. The resulting pellets were resuspended in 5 mL of assay buffer and re-centrifuged under the same conditions. This process was repeated and the final pellet was weighed and then resuspended in assay buffer to a final concentration of 160 w/v. All radioligand binding assays were performed in a final volume of 250 µL and initiated by the addition of 100 µL membrane preparation to a mixture containing [3H]Mazindol (NEN Life Science Products, Boston, MA, USA) and assay buffer. Aliquots of membrane preparation were incubated in triplicate for 1 h at 4°C. Non-specific binding was defined as binding in the presence of 10 µm GBR-12935 (a competitive DAT antagonist, Sigma-Aldrich Pty Ltd). For all assays, binding was terminated by rapid filtration through glass-microfibre filters [GF/B (Whatman International Ltd, Maidstone, UK) soaked in 0.2% polyethylenimine (Sigma-Aldrich Pty Ltd) for 1 h] using a Brandel Cell Harvester. Filters were washed three times with 5 mL of ice-cold buffer, and radioactivity was measured by liquid scintillation spectometry. Saturation assays employed a range of concentrations of [3H]Mazindol (0.3–70 nm). Specific binding was calculated by subtracting the respective non-specific binding from total binding and was expressed as picomoles per milligram of tissue. Protein concentrations were determined as described previously (Lowry et al. 1951). All binding data were quantified using a computer software package, EBDA/LIGAND running on RADLIG40 [McPherson GA (1994) RADLIG (Version 4), Elsevier Biosoft, Cambridge, UK]. The affinity (Kd) and density (Bmax) of [3H]Mazindol binding sites in normal and lesioned rats was compared by unpaired t-tests p < 0.05. The brain posterior to the striatum was placed into a solution containing 10% formalin in 0.1 m PB, the SNpc sectioned and stained, and lesion size determined.

Measurement of [3H]Dopamine transport in synaptosomes

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Lesioning
  5. DAT immunohistochemistry
  6. Preparation of SNpc for estimation of lesion size
  7. Estimation of SNpc lesion size
  8. Injection of tracer for ultrastructural examination of nigrostriatal synapses
  9. Preparation of tissue for analysis of nigrostriatal ultrastructure
  10. Rotational behaviour
  11. [3H]Mazindol binding to the dopamine transporter
  12. Measurement of [3H]Dopamine transport in synaptosomes
  13. Electrochemistry and microelectrodes
  14. In vivo electrochemistry
  15. In vitro electrochemistry
  16. Results
  17. Estimates of the number of neurones in the SNpc
  18. [3H]Dopamine transport in normal animals
  19. [3H]Dopamine transport in lesioned animals
  20. [3H]Mazindol binding to the DAT
  21. In vivo electrochemistry
  22. In vitro electrochemistry of dopamine release and clearance in synaptosomal preparations and the effects of quinpirole
  23. Ultrastructural changes to the DA synapse after lesions of the SNpc
  24. Rotational behaviour
  25. Discussion
  26. Acknowledgements
  27. References

Sixteen weeks after lesioning, rats were decapitated, brains removed and cut in a coronal plane, as described above, to separate the striatum from the SNpc. The striatum was then hemisected and the dorsal striatum dissected and placed immediately in KRH buffer at 4°C. The KRH buffer consisted: NaCl 125 μm; K2HPO4 1.5 μm; MgSO4 1.5 μm; CaCl2 1.25 μm; d-glucose 10 μm; HEPES 25 μm; ascorbic acid 0.1 μm; pargyline 1 μm; and EDTA 0.1 μm, pH 7.4. The buffer was oxygenated with 95% O2 and 5% O2 for 10 min before use. The brain areas were homogenized in 25 mL of cold sucrose (0.32 m + 5 mm NaHCO3, pH 7.4) using a Teflon pestle with a clearance of 0.003–0.004 inches. The homogenates were centrifuged at 2000 g for 10 min at 4°C and the pellets discarded. The supernatant was centrifuged at 16 000 g for 15 min at 4°C. The resulting pellet remained on ice until it was resuspended for the transport assay. The pellets from the dorsal striatum were weighed and resuspended in KRH buffer so as to obtain a concentration of 1000 w/v. 980 µL of pellet suspension was added to each assay tube. The tubes were pre-incubated at 37°C for 3 min. The transport assay was initiated by the addition of 1 mL KRH buffer (37°C) containing 0.2 µm dopamine consisting of 0.25 µCi of [3H]DA (31.1 Ci/mmol; dihydroxyphenylethylamine 3,4-[Ring-2,5,6–3H]-, NEN Life Science Products) plus 20 µL of either KRH buffer or 1 mm Mazindol. Blanks consisted of samples of which the suspended pellets containing synaptosomes was substituted for 1 mL of KRH buffer. The volume of each assay was 2 mL. The assay continued for the length of time assigned (1, 2, 3, 4, 5, 7, 9, 10, 15, 20 or 30 min). After the designated time period, solutions were immediately filtered using a millipore filtration apparatus with glass-microfibre filters (GF/F, Whatman International Ltd, Maidstone, UK). After filtration, the filters were washed twice with 8 mL of cold KRH buffer. The filters were placed into scintillation vials, to which 3 mL of Biosafe scintillation fluid (Research Products Int., Mount Prospect, IL, USA) was added and the radioactivity was counted by liquid scintillation spectrometry. Protein was determined by the method of Lowry et al. (1951). Results are expressed as picomoles of DA taken up into the synaptosomes per milligram of protein. Statistical significance was determined with student's t-test and anova, p < 0.05. The brain posterior to the striatum was prepared for stereological assessment of SNpc lesion size. 2 mL of synaptosome preparation was used for electrochemical measurement of DA clearance and release (see in vitro electrochemistry).

Electrochemistry and microelectrodes

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Lesioning
  5. DAT immunohistochemistry
  6. Preparation of SNpc for estimation of lesion size
  7. Estimation of SNpc lesion size
  8. Injection of tracer for ultrastructural examination of nigrostriatal synapses
  9. Preparation of tissue for analysis of nigrostriatal ultrastructure
  10. Rotational behaviour
  11. [3H]Mazindol binding to the dopamine transporter
  12. Measurement of [3H]Dopamine transport in synaptosomes
  13. Electrochemistry and microelectrodes
  14. In vivo electrochemistry
  15. In vitro electrochemistry
  16. Results
  17. Estimates of the number of neurones in the SNpc
  18. [3H]Dopamine transport in normal animals
  19. [3H]Dopamine transport in lesioned animals
  20. [3H]Mazindol binding to the DAT
  21. In vivo electrochemistry
  22. In vitro electrochemistry of dopamine release and clearance in synaptosomal preparations and the effects of quinpirole
  23. Ultrastructural changes to the DA synapse after lesions of the SNpc
  24. Rotational behaviour
  25. Discussion
  26. Acknowledgements
  27. References

Thirty-micron thick carbon-fibre working microelectrodes (Textron Systems, Lowell, MA, USA) were coated with nafion (5% solution, Sigma-Aldrich Pty Ltd) and glued to a fused glass capillary (i.d. of 40 µm, SGE, Ringwood, Australia). The distance between the tips of the carbon-fibre electrode and capillary delivery tube was approximately 200 µm. Voltammetric measurements of extracellular DA concentration was performed using an axon gene clamp (Axon Instruments, Inc. Foster City, CA, USA). Voltammograms were simultaneously recorded at each carbon-fibre electrode at 10 Hz (potential + 550 mV, square-wave pulses). Potential was referenced to an Ag/AgCl electrode. Oxidative currents were calculated by integrating the area under the current curve. The linearity and sensitivity of all electrodes used in the experiments were determined by using DA (3-hydroxytyramine-hydrochloride, Sigma-Aldrich Pty Ltd) standard solutions in the range from 2 to 10 µm. All solutions were prepared in 0.1 m PBS, pH 7.4. Calibration curves for DA were determined for all electrodes prior to and after each experiment. Only electrodes exhibiting highly linear responses (r2 > 0.90) and selectivity to DA (> 100 : 1, compared with ascorbic acid) were used for the experiments. All signals were expressed as µm changes in DA by comparison to pre- and post-calibration curves. Average responses obtained with the Axon Scope Software (Axon Instruments, Inc. Foster City, CA, USA) were translated for further analysis using excel (Microsoft). These programs were used to translate electrical signals (nA/ms) into DA concentration (µm) and measure several parameters of the evoked responses, i.e. maximum amplitude of DA overflow, time to 50% and 80% decay after exogenous DA delivery or potassium stimulation. Differences in absolute values between normal and SNpc lesioned animals were analysed using an unpaired student's t-test with significance set at p < 0.05.

In vivo electrochemistry

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Lesioning
  5. DAT immunohistochemistry
  6. Preparation of SNpc for estimation of lesion size
  7. Estimation of SNpc lesion size
  8. Injection of tracer for ultrastructural examination of nigrostriatal synapses
  9. Preparation of tissue for analysis of nigrostriatal ultrastructure
  10. Rotational behaviour
  11. [3H]Mazindol binding to the dopamine transporter
  12. Measurement of [3H]Dopamine transport in synaptosomes
  13. Electrochemistry and microelectrodes
  14. In vivo electrochemistry
  15. In vitro electrochemistry
  16. Results
  17. Estimates of the number of neurones in the SNpc
  18. [3H]Dopamine transport in normal animals
  19. [3H]Dopamine transport in lesioned animals
  20. [3H]Mazindol binding to the DAT
  21. In vivo electrochemistry
  22. In vitro electrochemistry of dopamine release and clearance in synaptosomal preparations and the effects of quinpirole
  23. Ultrastructural changes to the DA synapse after lesions of the SNpc
  24. Rotational behaviour
  25. Discussion
  26. Acknowledgements
  27. References

Animals were anaesthetized with urethane (1.25 g/kg i.p., Sigma) 16 weeks after SNpc lesioning. Carbon-fibre electrodes were inserted into the dorsal striatum to electrochemically detect the presence of DA. The fused silica capillary was used to inject 325 ± 70 nL of 200 µm DA in experiments designed to measure clearance of DA in the striatum. Clearance was expressed in terms of the time (in seconds) for the DA electrochemical signal to decrease to 50% of peak amplitude (T50) and 80% of peak amplitude (T80), as shown in Fig. 1. This portion of the signal measures clearance independently of the rising phase of the signal and gives a clear indication of how quickly DA is cleared from tissue surrounding the tip of the electrode, allowing signals of similar amplitude to be compared directly (Fig. 1). The characteristics of DA release from the dorsal striatum was measured, using KCl (70 mm, 200 ± 50 nL) applied locally through the capillary. Statistical significance was determined using a Student's t-test, p < 0.05. The brain caudal to the striatum was removed and the SNpc serially sectioned and prepared for stereological analysis.

image

Figure 1. DA concentration in the dorsal striatum of normal and rats lesioned for 16 weeks after local application of exogenous DA or KCl. (a) an example of the measurements of DA concentration in the dorsal striatum, made before and after local application of 325 ± 70 nL of 200 µm DA in the vicinity of carbon-fibre recording electrodes. In normal animals (●), DA concentration rises rapidly to a peak and is also cleared promptly. Following a lesion (○), the time to peak DA concentration is significantly longer and clearance is greatly prolonged. (b) Peak DA concentration following injection of DA in the vicinity of the recording electrode. (c) T50 (d) T80 and (e) peak DA concentration following local application of KCl (70 mm, 200 ± 50 nL). (b–e) The mean (± SD) of 44 measurements from 24 animals (14 control, 10 lesioned). Peak DA concentration was similar in lesioned and non-lesioned animals following both injected DA and KCl however, clearance of DA was significantly prolonged in lesioned animals. □, normal animals; ▪, lesioned animals; T50, time for peak DA electrical signal to reduce by 50%; T80, time for peak DA electrical signal to reduce by 80%. No significant difference in peak dopamine levels, or in the re-uptake of DA as represented by T50 and T80 was observed between SNpc lesioned groups (small, medium or large).

Download figure to PowerPoint

In vitro electrochemistry

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Lesioning
  5. DAT immunohistochemistry
  6. Preparation of SNpc for estimation of lesion size
  7. Estimation of SNpc lesion size
  8. Injection of tracer for ultrastructural examination of nigrostriatal synapses
  9. Preparation of tissue for analysis of nigrostriatal ultrastructure
  10. Rotational behaviour
  11. [3H]Mazindol binding to the dopamine transporter
  12. Measurement of [3H]Dopamine transport in synaptosomes
  13. Electrochemistry and microelectrodes
  14. In vivo electrochemistry
  15. In vitro electrochemistry
  16. Results
  17. Estimates of the number of neurones in the SNpc
  18. [3H]Dopamine transport in normal animals
  19. [3H]Dopamine transport in lesioned animals
  20. [3H]Mazindol binding to the DAT
  21. In vivo electrochemistry
  22. In vitro electrochemistry of dopamine release and clearance in synaptosomal preparations and the effects of quinpirole
  23. Ultrastructural changes to the DA synapse after lesions of the SNpc
  24. Rotational behaviour
  25. Discussion
  26. Acknowledgements
  27. References

The rate of DA clearance and release was also measured electrochemically (Fig. 2f) in synaptosome preparations isolated as described above. The effect of quinpirole (Sigma-Aldrich Pty Ltd), a selective D2 DA receptor agonist, on release and transport was also examined. One millilitre of synaptosome suspension was added to each well and pre-incubated at 37°C for 3 min. Recordings were made as described above for in vivo measurements. In preparations from normal and lesioned animals, 12 µL of 0.25 mm DA were injected by micropipette into the synaptosome suspension. At this concentration, clearance of DA into synaptosomes was too rapid in samples from normal animals to allow a meaningful comparison with clearance from lesioned animals (see Figs 2b and d). Therefore, for measurement of DA transport into synaptosomes from normal animals, the concentration of DA added to the preparation was subsequently increased to 0.5 mm. DA release from synaptosomes was examined by adding 50 µL 1 m KCl to the preparation once DA concentration in the well returned to a baseline level (i.e. when no further DA was being taken up into synaptosomes). The effect of D2R activation on uptake and release of DA was evaluated by adding 20 µL of 1 mm quinpirole to 1 mL of the synaptosome preparation for 10 min prior to injections of DA. Clearance was expressed as time (in seconds) for the DA electrochemical signal to decrease to 50% of peak amplitude (T50) and 80% of peak amplitude (T80).

image

Figure 2. In vitro recordings showing DA release and uptake in synaptosome preparations from the dorsal striatum of normal rats and those lesioned for 16 weeks, and the effects of quinpirole. (a) T50 and (c) T80 after application of 12 µL 0.5 mm DA showing that the rate of DA uptake increases in the presence of quinpirole. (b and d) Differences in the rate of DA uptake after addition of 12 µL 0.25 mm DA to striatal synaptosomes from normal and lesioned animals and synaptosomes from lesioned animals that were pretreated with quinpirole. Observe that DA uptake increases in synaptosomes pretreated with quinpirole. (e) DA release from striatal synaptosomes of normal and lesioned animals evoked by 50 µL 1 m KCl. The presence of quinpirole reduces peak DA concentration in samples from both normal and lesioned animals. Note there is no difference between normal and lesioned groups. (f) Electrochemical recordings of DA overflow in striatal synaptosomes from normal animals in vitro. The three peak DA concentrations are evoked by applying 12 µL 0.25 mm DA (top), 12 µL 0.5 mm DA (middle) and 50 µL 1 m KCl to synaptosome preparations, respectively. (a–c) The mean (± SEM). Scale bars: black (50 s, x-axis); grey (2 µm DA, y-axis). No significant difference in peak dopamine levels, or in the re-uptake of DA as represented by T50 and T80 was observed between SNpc lesioned groups (small, medium or large).

Download figure to PowerPoint

Estimates of the number of neurones in the SNpc

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Lesioning
  5. DAT immunohistochemistry
  6. Preparation of SNpc for estimation of lesion size
  7. Estimation of SNpc lesion size
  8. Injection of tracer for ultrastructural examination of nigrostriatal synapses
  9. Preparation of tissue for analysis of nigrostriatal ultrastructure
  10. Rotational behaviour
  11. [3H]Mazindol binding to the dopamine transporter
  12. Measurement of [3H]Dopamine transport in synaptosomes
  13. Electrochemistry and microelectrodes
  14. In vivo electrochemistry
  15. In vitro electrochemistry
  16. Results
  17. Estimates of the number of neurones in the SNpc
  18. [3H]Dopamine transport in normal animals
  19. [3H]Dopamine transport in lesioned animals
  20. [3H]Mazindol binding to the DAT
  21. In vivo electrochemistry
  22. In vitro electrochemistry of dopamine release and clearance in synaptosomal preparations and the effects of quinpirole
  23. Ultrastructural changes to the DA synapse after lesions of the SNpc
  24. Rotational behaviour
  25. Discussion
  26. Acknowledgements
  27. References

Using previously described methods (Finkelstein et al. 2000) we confirmed that there are 11 273 ± 827 (mean ± SD) SNpc neurones (stained for neutral red) in the normal rat (Table 1). Relative variations (CE = 0.074–0.083, CV = 0.073) were regarded as true interanimal differences and not a consequence of the stereological technique (Table 1; Finkelstein et al. 2000). For all experiments, SNpc lesions ranged from 3 to 96% (Table 1), and depended on the volume and concentration of toxin injected.

Table 1.  Number of neurones in the SNpc of normal and lesioned rats
 NormalEMBehaviour*[3H]Mazindol Binding[3H]DA Transport,In vivo electrochemistry
  • *

    Animals to whom behavioural tests were performed 4 weeks after SNpc lesion;

  • in vitro electrochemistry experiments (synaptosomes) were also performed from these animals;

  • behavioural tests were performed on these animals 16 weeks after lesioning.

n1071571810
Mean No. NR (SD)11 273 (827)6482 (1425)5556 (2145)4514 (2014)4651 (3356)3950 (2533)
Range SNpc lesion (%)022–5817–7522–863–9629–89
Mean percentage lesion (SD)041 (14)51 (19)60 (18)59 (29)65 (22)
CV0.0730.2190.3860.4460.7220.641
Mean CE (SD)0.079 (0.003)0.101 (0.045)0.14 (0.028)0.14 (0.033)0.196 (0.14)0.164 (0.066)

[3H]Dopamine transport in normal animals

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Lesioning
  5. DAT immunohistochemistry
  6. Preparation of SNpc for estimation of lesion size
  7. Estimation of SNpc lesion size
  8. Injection of tracer for ultrastructural examination of nigrostriatal synapses
  9. Preparation of tissue for analysis of nigrostriatal ultrastructure
  10. Rotational behaviour
  11. [3H]Mazindol binding to the dopamine transporter
  12. Measurement of [3H]Dopamine transport in synaptosomes
  13. Electrochemistry and microelectrodes
  14. In vivo electrochemistry
  15. In vitro electrochemistry
  16. Results
  17. Estimates of the number of neurones in the SNpc
  18. [3H]Dopamine transport in normal animals
  19. [3H]Dopamine transport in lesioned animals
  20. [3H]Mazindol binding to the DAT
  21. In vivo electrochemistry
  22. In vitro electrochemistry of dopamine release and clearance in synaptosomal preparations and the effects of quinpirole
  23. Ultrastructural changes to the DA synapse after lesions of the SNpc
  24. Rotational behaviour
  25. Discussion
  26. Acknowledgements
  27. References

Transport of [3H]DA was examined using synaptosomes from the dorsal striatum of normal rats (n = 8, Fig. 3a). [3H]DA transport into striatal DA synaptosomes was 0.22 ± 0.03 (mean ± SEM) fmol/mg protein at 5 min and saturated at 0.64 ± 0.15 fmol/mg protein, 30 min after the assay initiation (Fig. 3a). A transient drop in the rate, or ‘notch’ occurred between 7 and 10 min, suggesting a point of transition to a second lower affinity transporter that continued transporting for about 15 min. This second transport mechanism was not readily detected in the post-lesion synaptosome preparations. Thus, for the purpose of comparison with lesioned animals, we measured the line of best fit for the data points in the first 5 min after analysis (representing the rate of transport over this period, R0-5), and the level of saturation of transport (between 15 and 30 min, representing the saturation concentration, S). As judged by R0-5 and S, the transport of [3H]DA was the same in dorsal striatal synaptosomes from the side contralateral to the lesioned SNpc and normal animals (data not shown). A similar ‘notch’ was observed 7–10 min after start of the assay, suggesting that this was a consistent transition point between transporters (data not shown). Mazindol (100 µm), a DAT inhibitor, reduced the rate of [3H]DA transport (Fig. 3a). In the presence of mazindol, the S was only 0.01 ± 0.002 fmol/mg protein, compared to 0.62 ± 0.06 fmol/mg protein in the normal synaptosome (p ≤ 0.001, anova, Fig. 3a).

image

Figure 3. Binding and transport properties of the dopamine transporter on newly generated terminals in the dorsal striatum of rats 16 weeks after SNpc lesions. (a) [3H]DA transport into synaptosomes. The rate of DA transport over the first five minutes (R0–5) and the saturation concentration (S), was calculated. In the normal animal (bsl00046;), and those with small lesions (●), a transient drop in the rate, or ‘notch’ occurred between 7 and 10 min, suggesting a point of transition to a second lower affinity transporter that continued transporting till about 15 min. Mazindol (◊) reduced the rate of transport. In small lesions (0–30%, ●), R0-5 was near normal, but S was reduced to almost half of normal. Following medium-sized lesions (▪), both R0-5 and S were significantly reduced. In large lesions (> 70%, ▴), both R0-5 and S were greatly reduced. Mazindol reduce both R0-5 and S in all lesioned animals (◊). (b) Scatchard plots of [3H]Mazindol binding to the DAT in the dorsal striatum. (bi) normal animals (bii) lesioned animals (ipsilateral to SNpc lesion). The Kd and Bmax values for each plot are shown in Table 2. Data from normal animals required a two-line fit indicative of two distinct binding sites, one of high affinity and a second of lower affinity. Following partial SNpc lesions, the scatchard plots also required a two-line fit. Although the Kd of high-affinity sites in lesioned and unlesioned animals were similar, density was reduced by almost 40% in lesioned animals. In contrast, the low affinity site had a very high density. When animals with different sized lesions were compared, no significant trend in Kd and Bmax values were observed. Furthermore, no difference from normal was observed in the striatum contralateral to the SNpc lesion (refer Table 2).

Download figure to PowerPoint

[3H]Dopamine transport in lesioned animals

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Lesioning
  5. DAT immunohistochemistry
  6. Preparation of SNpc for estimation of lesion size
  7. Estimation of SNpc lesion size
  8. Injection of tracer for ultrastructural examination of nigrostriatal synapses
  9. Preparation of tissue for analysis of nigrostriatal ultrastructure
  10. Rotational behaviour
  11. [3H]Mazindol binding to the dopamine transporter
  12. Measurement of [3H]Dopamine transport in synaptosomes
  13. Electrochemistry and microelectrodes
  14. In vivo electrochemistry
  15. In vitro electrochemistry
  16. Results
  17. Estimates of the number of neurones in the SNpc
  18. [3H]Dopamine transport in normal animals
  19. [3H]Dopamine transport in lesioned animals
  20. [3H]Mazindol binding to the DAT
  21. In vivo electrochemistry
  22. In vitro electrochemistry of dopamine release and clearance in synaptosomal preparations and the effects of quinpirole
  23. Ultrastructural changes to the DA synapse after lesions of the SNpc
  24. Rotational behaviour
  25. Discussion
  26. Acknowledgements
  27. References

[3H]DA transport was measured in synaptosomes from the dorsal striatum of rats with lesions of the SNpc and compared with those from normal animals. In the initial analysis, data from all animals (n = 18), regardless of lesion size were pooled. Both R0-5 and S were significantly different (p ≤ 0.001, Student's t-test), with S being reduced by 79 ± 2%. Following Mazindol administration, there was no perceivable R0-5, and the value of S was 86 ± 4% less than the S measured from lesioned synaptosomes (p = 0.005, Student's t-test). However, as the density of terminals in the striatum (and possibly function) is related to the size of the SNpc lesion (Finkelstein et al. 2000), we re-analysed the data after sorting animals into three groups based on lesion size. Transport of [3H]DA into striatal synaptosomes was compared in these three groups. A small lesion (n = 3) resulted in a non-significant reduction in R0-5 of 16 ± 6% compared with normal synaptosomes, but S was reduced by 31 ± 2% (p ≤ 0.001, anova, Fig. 3a). In synaptosomes from animals with medium-sized (n = 6) SNpc lesions, both R0-5 and S were significantly less than normal (71 ± 5%, p ≤0.001–0.007 and 79 ± 2%, p ≤ 0.001, respectively, Student's t-test, Fig. 3a). When lesions were large (n = 9), both R0-5 and S were also significantly less than normal (85 ± 3% and 96 ± 0.4%, respectively, p ≤ 0.001–0.004 and p ≤ 0.001, Student's t-test), with transport after 5 min being almost indistinguishable from S. Mazindol was found to statistically reduce both R0-5 and S into synaptosomes from all animals regardless of lesion size (p ≤ 0.001, anova). Furthermore, the absolute level to which mazindol reduced R0-5 and S was similar in all groups.

[3H]Mazindol binding to the DAT

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Lesioning
  5. DAT immunohistochemistry
  6. Preparation of SNpc for estimation of lesion size
  7. Estimation of SNpc lesion size
  8. Injection of tracer for ultrastructural examination of nigrostriatal synapses
  9. Preparation of tissue for analysis of nigrostriatal ultrastructure
  10. Rotational behaviour
  11. [3H]Mazindol binding to the dopamine transporter
  12. Measurement of [3H]Dopamine transport in synaptosomes
  13. Electrochemistry and microelectrodes
  14. In vivo electrochemistry
  15. In vitro electrochemistry
  16. Results
  17. Estimates of the number of neurones in the SNpc
  18. [3H]Dopamine transport in normal animals
  19. [3H]Dopamine transport in lesioned animals
  20. [3H]Mazindol binding to the DAT
  21. In vivo electrochemistry
  22. In vitro electrochemistry of dopamine release and clearance in synaptosomal preparations and the effects of quinpirole
  23. Ultrastructural changes to the DA synapse after lesions of the SNpc
  24. Rotational behaviour
  25. Discussion
  26. Acknowledgements
  27. References

The DA transport studies imply that lesioning alters the affinity of DA for DAT. The binding properties of the DAT in the rat dorsal striatum were measured using [3H]Mazindol, a selective, high-affinity DAT inhibitor. Properties observed in membrane preparations from normal animals (n = 5) were compared with those of animals with partial SNpc lesions (n = 7). A 10-point saturation binding curve (5–70 nm[3H]Mazindol) confirmed that specific binding to membranes of the dorsal striatum was saturable with a plateau at 50 nm[3H]Mazindol in normal and lesioned animals (data not shown). In normal animals, a Scatchard plot of [3H]Mazindol binding to DAT required a two-site fit, indicative of two distinct binding sites, one of high affinity and a second of lower affinity (Fig. 3bi, Table 2). Following partial SNpc lesions, scatchard analysis of [3H]Mazindol binding to DAT also revealed a two-site fit (Fig. 3bii, Table 2). Although the Kd value of the high-affinity site was similar to that observed in normal animals, the density (Bmax) was reduced by almost 40%, p < 0.05). In contrast, lesions reduced the affinity of the second binding site, concurrent with a fivefold increase in the density of the lower-affinity binding site (p < 0.05, Table 2). [3H]Mazindol binding to DAT in the dorsal striatum contralateral to the lesioned SNpc was not different to normal, as indicated by Kd and Bmax values (Table 2). Furthermore, no correlation was observed between lesion size and changes in [3H]Mazindol binding to the DAT in SNpc lesioned animals.

Table 2.  Scatchard analysis of [3H]Mazindol binding to DA transporter in the dorsal striatum of normal and SNpc lesioned animals 16 weeks after injury (mean ± SD)
Dorsal striatumKd* (nm)Bmax1 (fmol/mg protein)Kd (nm)Bmax2 (fmol/mg protein)
  1. *High-affinity binding site for [3H]Mazindol to the DA transporter; †low-affinity binding site for [3H]Mazindol to the DA transporter.

Normal7.68 ± 2.21441 ± 483306 ± 1824284 ± 1631
(n = 5)(n = 5)(n = 5)(n = 4)
Contralateral9.87 ± 2.71517 ± 439126 ± 624688 ± 2308
(n = 7)(n = 8)(n = 7)(n = 7)
Lesioned9.6 ± 3.1954 ± 2818349 ± 693822739 ± 11465
(n = 7)(n = 7)(n = 7)(n = 6)

In vivo electrochemistry

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Lesioning
  5. DAT immunohistochemistry
  6. Preparation of SNpc for estimation of lesion size
  7. Estimation of SNpc lesion size
  8. Injection of tracer for ultrastructural examination of nigrostriatal synapses
  9. Preparation of tissue for analysis of nigrostriatal ultrastructure
  10. Rotational behaviour
  11. [3H]Mazindol binding to the dopamine transporter
  12. Measurement of [3H]Dopamine transport in synaptosomes
  13. Electrochemistry and microelectrodes
  14. In vivo electrochemistry
  15. In vitro electrochemistry
  16. Results
  17. Estimates of the number of neurones in the SNpc
  18. [3H]Dopamine transport in normal animals
  19. [3H]Dopamine transport in lesioned animals
  20. [3H]Mazindol binding to the DAT
  21. In vivo electrochemistry
  22. In vitro electrochemistry of dopamine release and clearance in synaptosomal preparations and the effects of quinpirole
  23. Ultrastructural changes to the DA synapse after lesions of the SNpc
  24. Rotational behaviour
  25. Discussion
  26. Acknowledgements
  27. References

The membrane and synaptosome studies suggested that clearance of DA would be prolonged because of altered uptake through the DAT. Clearance of DA was measured by injecting known amounts of DA. Recordings were made from the dorsal striatum of normal and SNpc lesioned rats. Local application of DA in the vicinity of the recording electrode resulted in reproducible and stable electrochemical signals (Fig. 1a). In most animals, three measurements of clearance (i.e. three separate applications of DA) were performed in each striata. In total, 44 clearance measurements, obtained from 24 rats (control n = 14, lesioned n = 10) were performed. Clearance times (T50 and T80) were obtained for each measurement and grouped as either controls or lesioned and then averaged (Figs 1c and d). Peak amplitudes of DA concentration were similar in lesioned and control animals (Figs 1a and b), but clearance of DA was significantly prolonged in lesioned animals (p ≤ 0.001), with T50 and T80 being about twice those from normal animals (Figs 1c and d).

In order to address the question of whether differences in clearance were localized to specific regions with the dorsal striatum, measurements in all animals were taken at three stereotaxically determined sites within the lesioned and control dorsal striatum. The co-ordinates were 1.0–1.8 mm anterior and 2.0–4.0 mm lateral to bregma and at depths of 4.5–5.5 mm below the dural surface. DA clearance was consistently prolonged at each site (data not shown). Peak DA concentration evoked by KCl was the same in both normal and lesioned rats (p = 0.074, Fig. 1e).

In vitro electrochemistry of dopamine release and clearance in synaptosomal preparations and the effects of quinpirole

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Lesioning
  5. DAT immunohistochemistry
  6. Preparation of SNpc for estimation of lesion size
  7. Estimation of SNpc lesion size
  8. Injection of tracer for ultrastructural examination of nigrostriatal synapses
  9. Preparation of tissue for analysis of nigrostriatal ultrastructure
  10. Rotational behaviour
  11. [3H]Mazindol binding to the dopamine transporter
  12. Measurement of [3H]Dopamine transport in synaptosomes
  13. Electrochemistry and microelectrodes
  14. In vivo electrochemistry
  15. In vitro electrochemistry
  16. Results
  17. Estimates of the number of neurones in the SNpc
  18. [3H]Dopamine transport in normal animals
  19. [3H]Dopamine transport in lesioned animals
  20. [3H]Mazindol binding to the DAT
  21. In vivo electrochemistry
  22. In vitro electrochemistry of dopamine release and clearance in synaptosomal preparations and the effects of quinpirole
  23. Ultrastructural changes to the DA synapse after lesions of the SNpc
  24. Rotational behaviour
  25. Discussion
  26. Acknowledgements
  27. References

Clearance of DA into synaptosomes from the dorsal striatum was measured electrochemically by directly injecting known volumes and concentrations of DA into the synaptosome preparation. Rate of clearance of DA (T50 and T80) was measured from synaptosome preparations extracted from the dorsal striatum of normal rats (n = 6) and rats with unilateral 6-OHDA lesions of the SNpc (n = 12; Figs 2a–d). In synaptosomes from normal animals, the rate of DA clearance was increased in the presence of quinpirole (i.e. reduced T50, p < 0.05, Figs 2a and c). However, clearance of DA by synaptosomes from lesioned animals was markedly prolonged and following the addition of 0.5 nm DA, a meaningful measure of T50 or T80 could not be obtained. Consequently clearance of DA by synaptosomes from lesioned animals was measured following the addition of a more dilute DA concentration (0.25 nm DA; Figs 2b and d). The rate of DA uptake into synaptosomes decreased in lesioned animals. As for normal synaptosomes, quinpirole enhanced the clearance of DA (p ≤ 0.001). Release of DA from synaptosomes was evoked by KCl. Peak amplitudes of DA concentration evoked by KCl were the same in both normal and lesioned rats (Fig. 2e). In the presence of quinpirole, DA release in response to KCl was reduced to a similar extent in both normal and lesioned animals (p ≤ 0.001).

Ultrastructural changes to the DA synapse after lesions of the SNpc

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Lesioning
  5. DAT immunohistochemistry
  6. Preparation of SNpc for estimation of lesion size
  7. Estimation of SNpc lesion size
  8. Injection of tracer for ultrastructural examination of nigrostriatal synapses
  9. Preparation of tissue for analysis of nigrostriatal ultrastructure
  10. Rotational behaviour
  11. [3H]Mazindol binding to the dopamine transporter
  12. Measurement of [3H]Dopamine transport in synaptosomes
  13. Electrochemistry and microelectrodes
  14. In vivo electrochemistry
  15. In vitro electrochemistry
  16. Results
  17. Estimates of the number of neurones in the SNpc
  18. [3H]Dopamine transport in normal animals
  19. [3H]Dopamine transport in lesioned animals
  20. [3H]Mazindol binding to the DAT
  21. In vivo electrochemistry
  22. In vitro electrochemistry of dopamine release and clearance in synaptosomal preparations and the effects of quinpirole
  23. Ultrastructural changes to the DA synapse after lesions of the SNpc
  24. Rotational behaviour
  25. Discussion
  26. Acknowledgements
  27. References

Within the dorsal striatum, nigrostriatal terminals were recognised by the presence of the anterogradely transported dextran biotin as indicated by dense DAB reaction product. Terminals with at least three synaptic vesicles, a widened synaptic cleft, parallel pre- and post-synaptic membranes and, a post-synaptic density were defined as ‘synaptic terminals’ (e.g. Figs 4a and c–h). Labelled terminals with at least three vesicles present in the pre-synaptic terminal but with no clear active zone (i.e. no synaptic cleft, parallel membranes or a post-synaptic density) were referred to as ‘varicosities’ (Fig. 4b). Collectively, all labelled terminals containing at least three vesicles (with or without an active zone) were referred to as ‘boutons’. Previous studies have drawn implications about the function of synapses by assessing the symmetry of synaptic densities and vesicle shape according to Gray's classification (Eccles 1964; Gray 1969). However, the complete filling of the pre-synaptic terminals by dense DAB reaction product prevented visualisation of the pre-synaptic density and hence determination of the symmetry (and consequently the use of Gray's criteria). The post-synaptic targets for synaptic terminals were identified in the following manner. Proximal dendrites were identified by their larger size, the number of mitochondria and the presence of granular endoplasmic reticulum (Fig. 4e). Distal dendrites were smaller and had fewer mitochondria and minimal amounts of ribosomal matter (Fig. 4f). The dendritic spines were recognised by their narrow neck and a bulb, with a spine apparatus commonly present but no mitochondria or tubules (Peters et al. 1991; Fig. 4g). A docked vesicle was classified as a vesicle present in the synaptic terminal in close association with the active membrane and directly opposite to the post-synaptic density. Perforated synapses were recognised as having a discontinuous post-synaptic density (Ingham et al. 1998; Fig. 4d, white arrows). In total, 1017 boutons were studied of which 736 were classified as varicosities and 281 classified as synaptic terminals.

image

Figure 4. Electron micrographs of nigrostriatal terminals of normal and rats 16 weeks after partial SNpc lesions. All panels are synaptic terminals in the dorsal striatum of lesioned animals except for panel (c), which illustrates a synaptic terminal from a control animal. (a) An example of a labelled nigrostriatal synaptic terminal (greater than three vesicles and has an active zone). This is a typical regenerated synapse showing a large pre-synaptic terminal area (0.246 µm2) and several vesicles (49). The black arrowheads in this and subsequent panels, indicate the active zone of the synapse (where the post-synaptic density and synaptic cleft are visible). (b) An example of a varicosity (greater than three vesicles but with no active zone). (c) A synaptic terminal from a control animal (small area, 0.103 µm2 and few vesicles, 13) making contact onto a proximal dendrite. (d) A terminal making greater than one synaptic contact. White arrowheads point to a synapse formed with a proximal dendrite of a striatal cell and black arrowheads indicate the second synapse formed with a distal dendrite of a striatal cell. This micrograph also illustrates an example of a perforated post-synaptic density (two white arrowheads). (e, f and g) Examples of the post-synaptic targets of synapses from lesioned animals. (e) A synaptic contact onto a proximal dendrite where numerous mitochondria can be seen in the post-synaptic target cell. (f) A distal dendrite contact, showing two mitochondria and little granular material present and (g) a dendritic spine, recognized by their narrow neck and a bulb. (h) An example of a bouton from a lesioned animal showing five mitochondria (*) present in the pre-synaptic element. Scale = 0.5 µm. All animals (except for one) in the ultrastructural studies had medium (30–75%) sized SNpc lesions.

Download figure to PowerPoint

The median area of nigrostriatal synaptic terminals in the dorsal striatum of lesioned animals (n = 7) were 84% larger than those in control animals and varicosities from the lesioned dorsal striatum were 93% larger than those from control animals (n = 5, Figs 5a and b). Synaptic terminals from the dorsal striatum of lesioned animals contained 129% more vesicles than those from control animals (Figs 5c and d). Similarly, varicosities from lesioned animals contained significantly more vesicles than those from control animals.

image

Figure 5. Morphological changes of nigrostriatal terminals following SNpc lesions. (a and b) Area of the pre-synaptic element; (c and d) the number of vesicles in nigrostriatal synaptic terminals (▪) and varicosities (grey bars). The vertical lines indicate the median value for the area of the varicosities. Regenerated terminals had significantly larger pre-synaptic areas and vesicle numbers than controls, clearly indicated by the median values (anova, Tukey's post-hoc, p < 0.05). (e) Regression plots of the area of the pre-synaptic terminal (µm2) plotted against the number of vesicles present in the pre-synaptic terminal (with 95% confidence intervals for the lesioned groups verses the control group). The black lines show the correlation between area and vesicle number for lesioned animals whereas the grey line is the regression line for normal animals. For any pre-synaptic terminal area, there were more vesicles in terminals of lesioned animals than in a similar sized terminal from a normal animal. Note also that there is minimal overlap of the confidence lines, suggesting two distinct populations. (f) Histogram showing proportions of post-synaptic targets of nigrostriatal terminals in control and lesioned animals. Note increased proportion of proximal dendritic contacts in lesioned animals compared with control (chi-square test, p < 0.05). (g) Histogram showing number of mitochondria present in synaptic terminals of control and SNpc lesioned groups. There was significantly more terminals containing multiple mitochondria in lesioned groups than in controls (χ2 test, p < 0.05). In the lesioned group, 23% of synaptic terminals contained one or two mitochondria per bouton, with up to five mitochondria present in some terminals (see h). mt, mitochondria. All animals (except for one) in this set of experiments had medium (30–75%) sized SNpc lesions.

Download figure to PowerPoint

In both control and lesioned animals, the area of SNpc boutons was correlated with the number of vesicles within the bouton, demonstrating that vesicle number is proportional to the area of the pre-synaptic elements (Fig. 5e). Regression lines with 95% confidence intervals were fitted to these data (Fig. 5e). Comparisons of regression lines and 95% confidence interval lines show minimal overlap, suggesting that the two populations are distinct.

Twenty-eight per cent of the boutons examined were synaptic terminals, the majority of which made only one synaptic contact with the post-synaptic striatal cells. Following a lesion, a small but significant increase (p < 0.05) was seen in the number of synaptic terminals making greater than one synaptic contact (Fig. 4d). After lesioning, a greater number of synaptic terminals made contacts with proximal dendrites and dendritic spines (control, 13%; lesioned, 28%, χ2 test, p < 0.05) although, distal dendrites remained the predominant post-synaptic target for these synaptic terminals (Fig. 5f). There was an increase in the number of perforated synapses (defined by the presence of a discontinuous specialization) after lesioning (control, 9%; lesioned, 14%; χ2 test, p < 0.05). Lesioning also resulted in a small increase in the number of synaptic terminals containing mitochondria. However, when mitochondria were present in terminals from the lesioned animals, they were significantly more likely to be multiple. In the lesioned group, 23% of the synaptic terminals contained more than one mitochondria compared with only 4% in the controls (χ2 test, p < 0.05, Fig. 5g). There were also a greater number of docked vesicles per unit of active zone after lesioning (9 docked vesicles/µm in the control and 15/µm in the lesioned ipsilateral group; χ2 test, p < 0.05).

Rotational behaviour

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Lesioning
  5. DAT immunohistochemistry
  6. Preparation of SNpc for estimation of lesion size
  7. Estimation of SNpc lesion size
  8. Injection of tracer for ultrastructural examination of nigrostriatal synapses
  9. Preparation of tissue for analysis of nigrostriatal ultrastructure
  10. Rotational behaviour
  11. [3H]Mazindol binding to the dopamine transporter
  12. Measurement of [3H]Dopamine transport in synaptosomes
  13. Electrochemistry and microelectrodes
  14. In vivo electrochemistry
  15. In vitro electrochemistry
  16. Results
  17. Estimates of the number of neurones in the SNpc
  18. [3H]Dopamine transport in normal animals
  19. [3H]Dopamine transport in lesioned animals
  20. [3H]Mazindol binding to the DAT
  21. In vivo electrochemistry
  22. In vitro electrochemistry of dopamine release and clearance in synaptosomal preparations and the effects of quinpirole
  23. Ultrastructural changes to the DA synapse after lesions of the SNpc
  24. Rotational behaviour
  25. Discussion
  26. Acknowledgements
  27. References

Rotational behaviour was assessed following amphetamine administration at 4 (n = 15) and 16 (n = 12) weeks after SNpc lesions and compared with age-matched control animals (n = 7). Without amphetamine treatment, neither lesioned nor control animals had a propensity to rotate (Figs 6a, ci and di). In control animals, amphetamine treatment induced a persistent but modest bias toward leftward rotation that persisted for 2 h after injection (Fig. 6a). The effect of amphetamine on rotational behavior at 4 and 16 weeks was complex and examples are shown in Figs 6(cii, ciii, dii and diii). For a more comprehensive examination of the effects of lesion size and time after lesioning, an estimate of the area under the curve was made. The area was obtained by adding each data point for 140 min after amphetamine administration (allowing for the arithmetic sign, i.e. left or right turning). The area was then plotted against lesion size (Fig. 6b). This demonstrated that at 4 weeks, lesion size was proportional to the extent of right turning bias. However, by 16 weeks, animals with lesions of less than 70% recovered to the point of having a modest, even normal propensity to turn to the left. Lesions larger than 70% still showed a right ward bias, but this was much less marked than in the 16 week test.

image

Figure 6. Rotational behavior of individual animals in response to administration of amphetamine (5 mg/kg i.p). In these graphs, each symbol represents the net rotation (right turns minus left turns) made in a 5-min period divided by 5 to obtain the average number of turns per minute in that interval. (a) Turning behaviour of a normal animal. ●, Behaviour before amphetamine; ○, behaviour after amphetamine; V, amphetamine injection. (b) From the plot of each animal's rotational behaviour, an estimate of the area under the curve was made by adding each data point for 140 min after amphetamine administration. Animals were grouped according to lesion size and the mean area (± SE) for each group was plotted. The small black square shows the normal unlesioned animals rate of turns to the left following amphetamine. The black bars are from animals 4 weeks postlesion and white bars are from animals 16 weeks post-lesion. At 4 weeks, lesion size was proportional to the extent of right turning bias but by 16 weeks, animals whose lesions were less than 70% had a near normal propensity to turn to the left. Animals with lesions larger than 70% still showed a right ward bias, but this was much less marked than in the 4 week animals. (c) Behaviour of animals 4 weeks after a lesion. (ci) Averaged response of all animals prior to amphetamine administration (n = 15). (cii) Response of an animal with a 40% lesion. (ciii) Turning response of an animal with a 68% lesion. (d) Behaviour of animals 16 weeks after a lesion. (di) Averaged response of all animals prior to amphetamine administration (n = 12). (dii) Response of an animal with a 44% lesion. (diii) Turning response of an animal with a 65% lesion. In the absence of amphetamine, animals did not tend to turn in either direction (a, ci and di) although amphetamine treatment in normal animals induced a persistent but modest bias toward leftward rotation that persisted for 2 h after injection (a). (cii) Shows that even with moderate lesions animals tended to turn toward the right whereas by 16 weeks turning behaviour had tended toward the left, even after large lesions. Nevertheless, rotational responses were often complex at 16 weeks (diii). On the y-axis, positive numbers indicate right turns and negative numbers indicate left turns.

Download figure to PowerPoint

Animals were grouped into three lesion sizes to allow comparison with the membrane studies already described. This approach however, failed to reveal the complex patterns of turning observed in individual lesioned animals. Examination of the response of individual animals that demonstrated complex patterns of turning was quite informative. An example is shown in Figs 6(diii), and demonstrates a biphasic pattern, with an initial tendency to turn to the right followed by a relative normal leftward bias, followed again by a propensity to turn to the right. On other occasions the pattern was changed with the right bias only emerging at about 60 min, being preceded and followed by turning to the left. These tendencies were most prominent after 16 weeks when SNpc lesions were between 60 and 75% (four of five animals tested within this lesion range) and were not seen when the SNpc lesion was less than 60%.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Lesioning
  5. DAT immunohistochemistry
  6. Preparation of SNpc for estimation of lesion size
  7. Estimation of SNpc lesion size
  8. Injection of tracer for ultrastructural examination of nigrostriatal synapses
  9. Preparation of tissue for analysis of nigrostriatal ultrastructure
  10. Rotational behaviour
  11. [3H]Mazindol binding to the dopamine transporter
  12. Measurement of [3H]Dopamine transport in synaptosomes
  13. Electrochemistry and microelectrodes
  14. In vivo electrochemistry
  15. In vitro electrochemistry
  16. Results
  17. Estimates of the number of neurones in the SNpc
  18. [3H]Dopamine transport in normal animals
  19. [3H]Dopamine transport in lesioned animals
  20. [3H]Mazindol binding to the DAT
  21. In vivo electrochemistry
  22. In vitro electrochemistry of dopamine release and clearance in synaptosomal preparations and the effects of quinpirole
  23. Ultrastructural changes to the DA synapse after lesions of the SNpc
  24. Rotational behaviour
  25. Discussion
  26. Acknowledgements
  27. References

These studies show that sprouting of DA neurones following partial SNpc lesioning results in altered structure and function of DA terminals in the dorsal striatum. It is likely that most DA terminals in the dorsal striatum are newly formed. At 4 weeks we found very few DAT-ir terminals in the dorsal striatum yet by 16 weeks, DA terminal density was normal (Fig. 7c). Many studies have described a decrease in dopaminergic innervation of the striatum soon after 6-OHDA lesioning, indicating a significant or total loss of innervation (Perese et al. 1989; Thomas et al. 1994; Blanchard et al. 1995, 1996). Taken together with previous studies these results point to a near total retraction of the dopaminergic terminal tree (Stanic et al. 2002, 2003). However, regulatory mechanisms are in place that seem to ensure that the density of dopaminergic terminals return to near normal levels unless the lesion size exceeds 70% (Finkelstein et al. 2000). These observations are important for interpretation of ultrastructural studies because they imply that at the time of our EM studies (16 weeks after lesioning) the majority of terminals had been generated de novo. These terminal changes (increased bouton size, increased number of vesicles, contacts onto more proximal targets, increased numbers of mitochondria) followed a medium sized lesion (on average, 40% loss of SNpc neurones). It seems unlikely that these observations could be explained by absence of DAT-ir expression in axons that have survived. Furthermore, there are numerous reports of large numbers of TH and DAT-ir neurites entering the striatum following injury indicating substantial reinnervation and presumably new synapse formation (Blanchard et al. 1995, 1996; Finkelstein et al. 2000; Parish et al. 2001).

image

Figure 7. Photomicrographs of DAT-ir terminals and fibres in the dorsal striatum of normal and SNpc lesioned rats. (a) Normal animal. (b) 4 weeks after SNpc lesion (54% SNpc lesion). (c) 16 weeks after SNpc lesion (47% SNpc lesion). Note reduced density and hypertrophy of DAT-ir fibres 4 weeks after SNpc lesion, indicative of growing fibres. Also observe the density of DAT-ir terminals and fibres has returned to normal levels 16 weeks after the SNpc lesion. Scale bar = 50 µm.

Download figure to PowerPoint

Previous studies of regenerating neuromuscular junctions suggest that following retraction of the terminal arbour, new terminals reform at pre-existing post-synaptic sites (McMahan and Wallace 1989). However, a significant proportion of terminals we observed made contact more proximally onto dendrites and spines, arguing for new terminal formation rather than simply re-establishment of synaptic contacts at pre-existing sites. Figure 5 also demonstrates that the synapses observed are at least remodelled even if some are synapses that were present before the lesion. Although these morphological changes could result from increased activity they would be expected to also result in increased synaptic efficiency and therefore constitute an appropriate compensatory response to injury. Our current findings indicate that these new terminals appear to have diminished transport of DA into the terminals, although the amount of DA released is normal. Given the observed increase in vesicle number per terminal, we suggest that there have been compensatory changes in DA release.

The membrane binding studies indicate that in normal animals, there is a large density of high-affinity DAT sites and a much lower density of low-affinity sites. Following a lesion there is a very large increase in the density of the low affinity sites, with an even further reduction in their affinity. The net effect of these changes would be reduced transport of DA into postlesion regenerated terminals, an observation confirmed by the synaptosome study in which saturation was shown to be proportional to lesion size. The findings with the synaptosome preparation also suggest that the high affinity transport site is substantially reduced after lesioning, especially if the lesion is greater than 30%. The electrochemistry studies also confirm abnormal uptake of DA with clearance rates doubling. Even though peak DA concentration is similar in normal and lesioned animals, time to peak is longer in lesioned animals, suggesting compromised release. The synaptosome study shows that after large lesions, rate of DA transport is only slightly greater than blockade with Mazindol, whereas small lesions (< 30%) result in approximately 50% reduction in transport. Therefore although lesions of less than 70% appear to have established normal terminal density within the dorsal striatum after 16 weeks (Finkelstein et al. 2000), our findings suggest that synaptic function is not normal.

It is likely that the turnover and functionality of DAT protein is regulated through D2 autoreceptors (Hersch et al. 1997; Kimmel et al. 2001; Robinson 2002). Normal synaptosomes exposed to quinpirole demonstrated that activation of the D2R reduces uptake of DA (presumably through the transporter). A similar reduction is seen in synaptosomal preparations from lesioned animals suggesting that the D2R/DAT molecular interaction is preserved in new synapses.

Interestingly, release of DA is normal after lesioning as measured by the peak DA concentration produced by KCl injection. The EM appearance of postlesion terminals with the larger number and larger size of vesicles would intuitively suggest that these terminals are capable of delivering larger amounts of DA into the cleft. Although larger vesicle numbers and size suggest increased capacity for DA release, it may also reflect increased demand for synthesis in lieu of the impaired transport. Although the peak DA concentration obtained is comparable in lesioned animals, the time to reach the peak is significantly longer, suggesting that rate of release in lesioned animals is less than normal (Garris et al. 1997a). However, other studies have found that release of dopamine in the partially denervated striatum was similar to that in the intact striatum (Robinson and Whishaw 1988).

The studies of rotational behaviour confirm that in normal animals amphetamine administration is followed by a propensity to rotate left (Jerussi and Glick 1974; Pycock 1980), whereas 4 weeks after lesioning, amphetamine induces turning toward the side of lesion (Ungerstedt and Arbuthnott 1970; Pycock 1980; Dravid et al. 1984), with this effect being proportional to lesion size. At 16 weeks, by which time animals with small and medium lesions (< 70%) have established a normal density of terminals in the striatum, the pattern of turning is substantially altered. Most animals with small lesions and many with intermediate lesions turn left or have only a modest tendency to turn toward the side of the lesion. Only animals with large lesions persist in turning toward the lesioned side. These results provide a functional measure of the degree to which regenerated DA terminals can release DA.

Previously we reported that sprouting of DA neurones that follows partial SNpc lesions is regulated, with the effect that normal terminal density is maintained until lesions became particularly large (> 70%; Finkelstein et al. 2000; Parish et al. 2001). We have proposed that the D2 autoreceptor regulates the size of the terminal arbour of DA neurones, and this receptor is well placed to monitor and thereby respond to levels of DA within the synaptic cleft (Parish et al. 2001). On the basis of findings reported here, we speculate that as synaptic contacts are re-established, underexpression of high-affinity DAT acts to maintain DA concentrations in the synaptic cleft. Although peak delivery is normal, the altered transport is likely to lead to prolonged stimulation of the D2R and thus reduce the demand for further sprouting. It may also lead to altered, even augmented patterns of post-synaptic activation (discussed below). With time, and as the number of contacts normalise, normal transport may also be restored. This however, requires a lengthy process and would not be completed in animals with extensive lesions, even after 16 weeks. It is interesting to note that Blanchard et al. (1996) observed growth cones entering the striatum 7 months after partial lesions, suggesting that 12 months or more may be required for normalization of synaptic function.

It is interesting to speculate further on the implications these findings may have for Parkinson's disease and drug induced dyskinesia. Examination of the response of individual lesioned animals demonstrated complex patterns of turning that were reminiscent of peak dose and biphasic dyskinesia of Parkinson's disease (Poewe 1994). It is conceivable that dysregulated terminals with prolonged reuptake of DA from arbours that stretch throughout much of the striatum could result in complex patterns of DA release. Some of these animals turned to the left (non-lesioned side) more frenetically than normals and approached the rate seen when turning towards the lesion. We speculate that sprouting of axons, whether drug induced (by D2R antagonists like haloperidol) or as a response to lesioning, will result in abnormal DA delivery. This abnormal delivery will be to unusually large regions as a consequence of both the large terminal arbours of individual axons and because of impaired synaptic clearance and reduced function of DAT. We hypothesize that these factors and the altered synaptic contact form a common basis for both the dyskinesia of Parkinson's disease and tardive dyskinesia. The altered uptake is likely to lead to more prolonged stimulation of post-synaptic receptors with altered, even augmented patterns of post-synaptic activation leading to altered patterns of motor activation. As previously noted, nigrostriatal synaptic terminals most commonly form contacts with dendritic spines and shafts, and less commonly with the somata of striatal neurones (Freund et al. 1984; Zahm 1992; Groves et al. 1994; Anglade et al. 1996; Descarries et al. 1996; Hanley and Bolam 1997; Ingham et al. 1998). Following lesioning, the number of distal dendrite and spine contacts decrease and consequently there is a greater proportion of more proximal dendrite and somal contacts (Ingham et al. 1996; Ingham et al. 1998). Recently, Reynolds et al. (2001) described how stimulation of the SNpc induced potentiation of the glutamatergic synapses between the cortex and the striatum that was dependent on activation of dopamine receptors. The cortico-striatal glutamatergic fibres synapse onto the ends of dendritic spines of the striatal neurones whereas the SNpc terminals normally synapse onto the shaft. As more proximal synapses are believed to elicit greater physiological changes in the target neurones than distal synapses (Pickel et al. 1992), the more proximal site of termination of the reinnervated DA terminals could enhance the efficiency of DA augmentation of glutamatergic transmission. Indeed, Picconi (2001) described that plasticity at the cortical projection onto spiny neurones was altered by selective DA receptor blockade and following dopamine denervation but restored by L-DOPA therapy (Calabresi et al. 2000; Centonze et al. 2001; Picconi 2001). Others have noted that following neuroleptic treatment, there is persistent alteration in dendrites and spines, especially in the ventral striatum. As lesioning and haloperidol therapy both produce sprouting (Parish et al. 2001), it is possible that this sprouting provides the drive for the synaptic remodelling described here and elsewhere (Meshul and Tan 1994; Meredith et al. 2000; Meshul and Allen 2000). We speculate that the altered morphology and function of these newly formed terminals not only reflect mechanisms that may compensate for the loss of nigral neurones but may also be important in understanding the molecular processes underlying the dyskinesias of Parkinson's disease and neuroleptic treatment.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Lesioning
  5. DAT immunohistochemistry
  6. Preparation of SNpc for estimation of lesion size
  7. Estimation of SNpc lesion size
  8. Injection of tracer for ultrastructural examination of nigrostriatal synapses
  9. Preparation of tissue for analysis of nigrostriatal ultrastructure
  10. Rotational behaviour
  11. [3H]Mazindol binding to the dopamine transporter
  12. Measurement of [3H]Dopamine transport in synaptosomes
  13. Electrochemistry and microelectrodes
  14. In vivo electrochemistry
  15. In vitro electrochemistry
  16. Results
  17. Estimates of the number of neurones in the SNpc
  18. [3H]Dopamine transport in normal animals
  19. [3H]Dopamine transport in lesioned animals
  20. [3H]Mazindol binding to the DAT
  21. In vivo electrochemistry
  22. In vitro electrochemistry of dopamine release and clearance in synaptosomal preparations and the effects of quinpirole
  23. Ultrastructural changes to the DA synapse after lesions of the SNpc
  24. Rotational behaviour
  25. Discussion
  26. Acknowledgements
  27. References
  • Adams J. C. (1981) Heavy metal intensification of DAB-based HRP reaction product. J. Histochem. Cytochem. 29, 775.
  • Anglade P., Mouatt-Prigent A., Agid Y. and Hirsch E. (1996) Synaptic plasticity in the caudate nucleus of patients with Parkinson's disease. Neurodegeneration 5, 121128.
  • Blanchard V., Chritin M., Vyas S., Savasta M., Feuerstein C., Agid Y., Javoy-Agid F. and Raisman-Vozari R. (1995) Long-term induction of tyrosine hydroxylase expression: compensatory response to partial degeneration of the dopaminergic nigrostriatal system in the rat brain. J. Neurochem. 64, 16691679.
  • Blanchard V., Anglade P., Dziewczapolski G., Savasta M., Agid Y. and Raisman-Vozari R. (1996) Dopaminergic sprouting in the rat striatum after partial lesion of the substantia nigra. Brain Res. 709, 319325.
  • Bowyer J. F. and Weiner N. (1987) Modulation of the Ca2+-evoked release of [3H]dopamine from striatal synaptosomes by dopamine (D2) agonists and antagonists. J. Pharmacol. Exp. Ther. 241, 2733.
  • Bunney B. S., Walters J. R., Roth R. H. and Aghajanian G. K. (1973) Dopaminergic neurons: effect of antipsychotic drugs and amphetamine on single cell activity. J. Pharmacol. Exp. Ther. 185, 560571.
  • Calabresi P., Giacomini P., Centonze D. and Bernardi G. (2000) Levodopa-induced dyskinesia: a pathological form of striatal synaptic plasticity? Ann. Neurol. 47, S60S68; discussion S68–69.
  • Cass W. A. and Zahniser N. R. (1990) Inhibition of striatal dopamine release by the selective D-2 dopamine receptor agonist N-0437 is blocked by quinine. Synapse 5, 336337.
  • Centonze D., Picconi B., Gubellini P., Bernardi G. and Calabresi P. (2001) Dopaminergic control of synaptic plasticity in the dorsal striatum. Eur. J. Neurosci. 13, 10711077.
  • Descarries L., Watkins K. C., Garcia S., Bosler O. and Doucet G. (1996) Dual character, asynaptic and synaptic, of the dopamine innervation in adult rat neostriatum: a quantitative autoradiographic and immunocytochemical analysis. J. Comp Neurol. 375, 167186.
  • Dravid A., Jaton A. L., Enz A. and Frei P. (1984) Spontaneous recovery from motor asymmetry in adult rats with 6-hydroxydopamine-induced partial lesions of the substantia nigra. Brain Res. 311, 361365.
  • Eccles J. C. (1964) The Physiology of Synapses, pp. 1126. Springer-Verlag, Berlin.
  • Fallon J. H. and Loughlin S. E. (1995) Substantia nigra. In: The Rat Nervous System (PaxinosG., ed.), pp. 215237. Academic Press, San Diego.
  • Fallon J. H. and Moore R. Y. (1978) Catecholamine innervation of the basal forebrain. IV. Topography of the dopamine projection to the basal forebrain and neostriatum. J. Comp. Neurol. 180, 545580.
  • Finkelstein D. I., Stanic D., Parish C. L., Tomas D., Dickson K. and Horne M. K. (2000) Axonal sprouting following lesions of the rat substantia nigra. Neuroscience 97, 99112.
  • Freund T. F., Powell J. F. and Smith A. D. (1984) Tyrosine hydroxylase-immunoreactive boutons in synaptic contact with identified striatonigral neurons, with particular reference to dendritic spines. Neuroscience 13, 11891215.
  • Garris P. A., Walker Q. D. and Wightman R. M. (1997a) Dopamine release and uptake rates both decrease in the partially denervated striatum in proportion to the loss of dopamine terminals. Brain Res. 753, 225234.
  • Garris P. A., Christensen J. R., Rebec G. V. and Wightman R. M. (1997b) Real-time measurement of electrically evoked extracellular dopamine in the striatum of freely moving rats. J. Neurochem. 68, 152161.
  • Gerfen C. R., Herkenham M. and Thibault J. (1987) The neostriatal mosaic. II. Patch- and matrix-directed mesostriatal dopaminergic and non-dopaminergic systems. J. Neurosci. 7, 39153934.
  • Gray E. G. (1969) Electron microscopy of excitatory and inhibitory synapses. Prog. Brain Res. 31, 141155.
  • Groves P. M., Linder J. C. and Young S. J. (1994) 5-hydroxydopamine-labeled dopaminergic axons: three-dimensional reconstructions of axons, synapses and postsynaptic targets in rat neostriatum. Neuroscience 58, 593604.
  • Hanley J. J. and Bolam J. P. (1997) Synaptology of the nigrostriatal projection in relation to the compartmental organization of the neostriatum in the rat. Neuroscience 81, 353370.
  • Hersch S. M., Yi H., Heilman C. J., Edwards R. H. and Levey A. I. (1997) Subcellular localization and molecular topology of the dopamine transporter in the striatum and substantia nigra. J. Comp. Neurol. 388, 211227.
  • Van Horne C., Hoffer B. J., Stromberg I. and Gerhardt G. A. (1992) Clearance and diffusion of locally applied dopamine in normal and 6-hydroxydopamine-lesioned rat striatum. J. Pharmacol. Exp. Ther. 263, 12851292.
  • Ingham C. A., Hood S. H., Taggart P. and Arbuthnott G. W. (1996) Synaptic plasticity in the rat neostriatum after unilateral 6-hydroxydopaminelesion of the nigrostriatal dopaminergic pathway. In: The Basal Ganglia (OhyeC., KimuraM. and McKenzieJ. S., eds), pp. 157164. Plenum Press, New York.
  • Ingham C. A., Hood S. H., Taggart P. and Arbuthnott G. W. (1998) Plasticity of synapses in the rat neostriatum after unilateral lesion of the nigrostriatal dopaminergic pathway. J. Neurosci. 18, 47324743.
  • Isacson O. and Deacon T. (1997) Neural transplantation studies reveal the brain's capacity for continuous reconstruction. Trends Neurosci. 20, 477482.
  • Javitch J. A., Blaustein R. O. and Snyder S. H. (1984) [3H]mazindol binding associated with neuronal dopamine and norepinephrine uptake sites. Mol. Pharmacol. 26, 3544.
  • Jerussi T. P. and Glick S. D. (1974) Amphetamine-induced rotation in rats without lesions. Neuropharmacology. 13, 283286.
  • Kimmel H. L., Joyce A. R., Carroll F. I. and Kuhar M. J. (2001) Dopamine D1 and D2 receptors influence dopamine transporter synthesis and degradation in the rat. J. Pharmacol. Exp. Ther. 298, 129140.
  • Lacey M. G., Mercuri N. B. and North R. A. (1987) Dopamine acts on D2 receptors to increase potassium conductance in neurones of the rat substantia nigra zona compacta. J. Physiol. 392, 397416.
  • Lindgren N., Xu Z. Q., Herrera-Marschitz M., Haycock J., Hokfelt T. and Fisone G. (2001) Dopamine D(2) receptors regulate tyrosine hydroxylase activity and phosphorylation at Ser40 in rat striatum. Eur. J. Neurosci. 13, 773780.
  • Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275.
  • McMahan U. J. and Wallace B. G. (1989) Molecules in basal lamina that direct the formation of synaptic specializations at neuromuscular junctions. Dev. Neurosci. 11, 227247.
  • Meredith G. E., De Souza I. E., Hyde T. M., Tipper G., Wong M. L. and Egan M. F. (2000) Persistent alterations in dendrites, spines, and dynorphinergic synapses in the nucleus accumbens shell of rats with neuroleptic- induced dyskinesias. J. Neurosci. 20, 77987806.
  • Meshul C. K. and Allen C. (2000) Haloperidol reverses the changes in striatal glutamatergic immunolabeling following a 6-OHDA lesion. Synapse 36, 129142.
  • Meshul C. K. and Tan S. E. (1994) Haloperidol-induced morphological alterations are associated with changes in calcium/calmodulin kinase II activity and glutamate immunoreactivity. Synapse 18, 205217.
  • El Mestikawy S., Glowinski J. and Hamon M. (1986) Presynaptic dopamine autoreceptors control tyrosine hydroxylase activation in depolarized striatal dopaminergic terminals. J. Neurochem. 46, 1222.
  • Onali P., Olianas M. C. and Bunse B. (1988) Evidence that adenosine A2 and dopamine autoreceptors antagonistically regulate tyrosine hydroxylase activity in rat striatal synaptosomes. Brain Res. 456, 302309.
  • Parish C. L., Finkelstein D. I., Drago J., Borrelli E. and Horne M. K. (2001) The role of dopamine receptors in regulating the size of axonal arbors. J. Neurosci. 21, 51475157.
  • Paxinos G. and Watson C. (1998) The Rat Brain in Stereotaxic Co-Ordinates, 3rd edn. Academic Press, Sydney.
  • Perese D. A., Ulman J., Viola J., Ewing S. E. and Bankiewicz K. S. (1989) A 6-hydroxydopamine-induced selective parkinsonian rat model. Brain Res. 494, 285293.
  • Peters D. A., Palay S. L. and Webster H. (1991) The Fine Structure of the Nervous System, pp. 138211. Oxford University Press, New York.
  • Picconi B. (2001) Effects of dopamine denervation and chronic levodopa treatment of synaptic plasticity and spontaneous synaptic activity of stratal spiny neurones. Soc. Neurosci. Abstract 27, 292.218.
  • Pickel V. M., Johnson E., Carson M. and Chan J. (1992) Ultrastructure of spared dopamine terminals in caudate-putamen nuclei of adult rats neonatally treated with intranigral 6-hydroxydopamine. Brain Res. Dev Brain Res. 70, 7586.
  • Poewe W. H. (1994) Clinical aspects of motor fluctuations in Parkinson's disease. Neurology 44, S6S9.
  • Pycock C. J. (1980) Turning behaviour in animals. Neuroscience 5, 461514.
  • Reynolds J. N., Hyland B. I. and Wickens J. R. (2001) A cellular mechanism of reward-related learning. Nature 413, 6770.
  • Robinson M. B. (2002) Regulated trafficking of neurotransmitter transporters: common notes but different melodies. J. Neurochem. 80, 111.
  • Robinson T. E. and Whishaw I. Q. (1988) Normalization of extracellular dopamine in striatum following recovery from a partial unilateral 6-OHDA lesion of the substantia nigra: a microdialysis study in freely moving rats. Brain Res. 450, 209224.
  • Rosenblad C., Kirik D. and Bjorklund A. (2000) Sequential administration of GDNF into the substantia nigra and striatum promotes dopamine neuron survival and axonal sprouting but not striatal reinnervation or functional recovery in the partial 6-OHDA lesion model. Exp Neurol. 161, 503516.
  • Stanic D., Finkelstein D. I., Parish C. L., Zhu W., Krstew E., Drago J., Lawrence A. J. and Horne M. K. (2002) Changes in function and ultrastructure of striatal dopaminergic terminals following partial lesions of the SNpc. FENS 1, A186.116.
  • Stanic D., Finkelstein D. I., Bourke D. W., Drago J. and Horne M. K. (2003) Timecourse of striatal reinnervation following lesions of dopaminergic SNpc neurons of the rat. Eur. J. Neurosci. in press.
  • Tepper J. M., Nakamura S., Young S. J. and Groves P. M. (1984) Autoreceptor-mediated changes in dopaminergic terminal excitability: effects of striatal drug infusions. Brain Res. 309, 317333.
  • Thomas J., Wang J., Takubo H., Sheng J., De Jesus S. and Bankiewicz K. S. (1994) A 6-hydroxydopamine-induced selective parkinsonian rat model: further biochemical and behavioral characterization. Exp Neurol. 126, 159167.
  • Ungerstedt U. and Arbuthnott G. W. (1970) Quantitative recording of rotational behavior in rats after 6-hydroxy-dopamine lesions of the nigrostriatal dopamine system. Brain Res. 24, 485493.
  • Ungerstedt U., Herrera-Marschitz M., Stahle L. and Zetterstrom T. (1982) Models for studying the synaptic mechanisms: correlative measurements of transmitter release and drug altered behaviour. In: Behavioural Models and the Analysis of Drug Action (SpiegelsteinM. Y. and LevyA., eds), pp. 5770. Elsevier, Amsterdam.
  • Zahm D. S. (1992) An electron microscopic morphometric comparison of tyrosine hydroxylase immunoreactive innervation in the neostriatum and the nucleus accumbens core and shell. Brain Res. 575, 341346.