Changes in function and ultrastructure of striatal dopaminergic

terminals that regenerate following partial lesions of the SNpc

D. Stanic,*,1 C. L. Parish,*,1 W. M. Zhu,* E. V. Krstew,� A. J. Lawrence,� J. Drago,�D. I. Finkelstein� and M. K. Horne�

*Department of Medicine, Monash University, Monash Medical Centre, Clayton, Australia

�Howard Florey Institute of Experimental Physiology and Medicine, The University of Melbourne, Victoria, Australia

Abstract

Following partial substantia nigra lesions, remaining dopamin-

ergic 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 demon-

strated 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.

Keywords: dopamine transport, mazindol, sprouting, syna-

ptosomes, ultrastructure, voltammetry.

J. Neurochem. (2003) 86, 329–343.

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 excita-

bility (Bunney et al. 1973; Tepper et al. 1984) and reduces

DA release (Ungerstedt et al. 1982; Bowyer and Weiner

Received December 13, 2002; revised manuscript received April 1,

2003; accepted April 2, 2003.

Address correspondence and reprint requests to Professor Malcolm K.

Horne, Howard Florey Institute of Experimental Physiology and

Medicine, The University of Melbourne Victoria, 3010, Australia.

E-mail: m.horne@hfi.unimelb.edu.au1Stanic and Parish contributed equally and should be considered equal

first authors.

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; HE-

PES, (N-[2-Hydroxyethyl]piperazine-N¢-[2-ethanesulfonic acid]); i.d.,

internal diameter; i.p., intraperitoneal; -ir, immunoreactive; Kd, dissoci-

ation 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 initi-

ation; S, saturation of dopamine transport; s.c., subcutaneous; SD, standard

deviation; SNpc, substantia nigra pars compacta; TH, tyrosine hydroxy-

lase; T50, time for DA signal to decrease by 50% of peak amplitude; T80,

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

Journal of Neurochemistry, 2003, 86, 329–343 doi:10.1046/j.1471-4159.2003.01843.x

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 329–343 329

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 phosphoryla-

tion 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 hypertroph-

ic 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 amphetam-

ine at 4 and 16 weeks after lesioning.

Materials and methods

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.

Lesioning

Partial SNpc lesions ranging from 3 to 96% loss of SNpc neurones

were produced by 6-OHDA injections into the SNpc. Lesioning

methods have been described in detail previously (Perese et al.

1989; Finkelstein et al. 2000). With respect to lambda, the

co-ordinates for the first injection of 6-OHDA were AP 3.7 mm,

L 1.7 mm at a depth of 8.1 mm and for the second AP 3.7 mm,

L 2.1 mm at a depth of 7.5 mm (Paxinos and Watson 1998). Lesion

size was estimated by counting the number of nissl stained (neutral

red) neurones in the SNpc.

DAT immunohistochemistry

Rats were killed by an overdose of sodiumpentobarbitone (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 lm-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 Chem-

icals, 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 lL/ml) wasadded 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

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

330 D. Stanic et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 329–343

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 lm thick,

were serially cut (1 : 4) through the SNpc. On average, 12 of the

45-lm thick sections, each 180 lm 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

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 ¼ 125 lm, y ¼ 200 lm)derived by means of a grid program (Stereo Investigator, Micro-

BrightField, VT, USA; viewed through a microscope, Leica DMLB)

and a counting frame (40 lm · 27 lm ¼ 1080 lm2). Therefore,

the area sampling fraction is 1080/(125 · 200) ¼ 0.043. In all

animals, 45-lm thick sections through the SNpc, each 180 lmapart, were analysed, the fraction of sections sampled being 45/

180 ¼ 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

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�Cin the same fixative for a period of 24 h.

Preparation of tissue for analysis of nigrostriatal ultrastructure

The striatum of each brain was cut into 150-lm coronal sections on

a vibratome (Technical Products International, Inc., St Louis, MO,

USA). All sections were subsequently incubated in avidin peroxi-

dase (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

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

[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 lL and initiated by the addition of 100 lL membrane

preparation to a mixture containing [3H]Mazindol (NEN Life

Function and ultrastructure of SNpc terminals after lesioning 331

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 329–343

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 lM 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

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 lM; K2HPO4 1.5 lM;MgSO4 1.5 lM; CaCl2 1.25 lM; D-glucose 10 lM; HEPES 25 lM;ascorbic acid 0.1 lM; pargyline 1 lM; and EDTA 0.1 lM, 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. Theresulting 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 lL 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 lM dopamine consisting of 0.25 lCi of [3H]DA(31.1 Ci/mmol; dihydroxyphenylethylamine 3,4-[Ring-2,5,6–3H]-,

NEN Life Science Products) plus 20 lL 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, Maid-

stone, 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 measure-

ment of DA clearance and release (see in vitro electrochemistry).

Electrochemistry and microelectrodes

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 lm, SGE, Ringwood, Australia). The distance between the tipsof the carbon-fibre electrode and capillary delivery tube was

approximately 200 lm. Voltammetric measurements of extracellularDA 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 lM. All solutionswere 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

lM 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 (lM)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

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 lM 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 character-

istics of DA release from the dorsal striatum was measured, using

KCl (70 mM, 200 ± 50 nL) applied locally through the capillary.

332 D. Stanic et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 329–343

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.

In vitro electrochemistry

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 lL 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 lL 1 M KCl to the preparation once DA

Fig. 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 lM DA in the vicinity of carbon-fibre recording electrodes. In

normal animals (d), DA concentration rises rapidly to a peak and is

also cleared promptly. Following a lesion (s), the time to peak DA

concentration is significantly longer and clearance is greatly pro-

longed. (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 how-

ever, clearance of DA was significantly prolonged in lesioned animals.

h, normal animals; j, 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).

Fig. 2 In vitro recordings showing DA release and uptake in syna-

ptosome 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 lL 0.5 mM DA showing that the rate of

DA uptake increases in the presence of quinpirole. (b and d) Differ-

ences in the rate of DA uptake after addition of 12 lL 0.25 mM DA to

striatal synaptosomes from normal and lesioned animals and syna-

ptosomes 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 lL 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 lL 0.25 mM DA (top), 12 lL

0.5 mM DA (middle) and 50 lL 1 M KCl to synaptosome preparations,

respectively. (a–c) The mean (± SEM). Scale bars: black (50 s,

x-axis); grey (2 lM DA, y-axis). No significant difference in peak dop-

amine levels, or in the re-uptake of DA as represented by T50 and T80

was observed between SNpc lesioned groups (small, medium or

large).

Function and ultrastructure of SNpc terminals after lesioning 333

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 329–343

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 lL 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).

Results

Estimates of the number of neurones in the SNpc

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 ¼ 0.074–0.083, CV ¼ 0.073) were

regarded as true interanimal differences and not a conse-

quence 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.

[3H]Dopamine transport in normal animals

Transport of [3H]DA was examined using synaptosomes

from the dorsal striatum of normal rats (n ¼ 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 lM), aDAT 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).

[3H]Dopamine transport in lesioned animals

[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 ¼ 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 ¼ 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 ¼ 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 ¼ 6) SNpc lesions, both R0-5and 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 ¼ 9), both R0-5and 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,

Table 1 Number of neurones in the SNpc of normal and lesioned rats

Normal EM Behaviour* [3H]Mazindol Binding [3H]DA Transport�,� In vivo electrochemistry

n 10 7 15 7 18 10

Mean No. NR (SD) 11 273 (827) 6482 (1425) 5556 (2145) 4514 (2014) 4651 (3356) 3950 (2533)

Range SNpc lesion (%) 0 22–58 17–75 22–86 3–96 29–89

Mean percentage lesion (SD) 0 41 (14) 51 (19) 60 (18) 59 (29) 65 (22)

CV 0.073 0.219 0.386 0.446 0.722 0.641

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)

*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.

334 D. Stanic et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 329–343

the absolute level to which mazindol reduced R0-5 and S was

similar in all groups.

[3H]Mazindol binding to the DAT

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 ¼ 5) were

compared with those of animals with partial SNpc lesions

(n ¼ 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.

In vivo electrochemistry

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 measure-

ments, obtained from 24 rats (control n ¼ 14, lesioned

n ¼ 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

Fig. 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 concen-

tration (S), was calculated. In the normal animal (r), and those with

small lesions (d), 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. Mazin-

dol (e) reduced the rate of transport. In small lesions (0–30%, d), R0-5

was near normal, but S was reduced to almost half of normal. Following

medium-sized lesions (j), both R0-5 and S were significantly reduced. In

large lesions (> 70%, m), both R0-5 and S were greatly reduced.

Mazindol reduce both R0-5 and S in all lesioned animals (e). (b) Scat-

chard 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).

Function and ultrastructure of SNpc terminals after lesioning 335

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 329–343

DA concentration evoked by KCl was the same in both

normal and lesioned rats (p ¼ 0.074, Fig. 1e).

In vitro electrochemistry of dopamine release

and clearance in synaptosomal preparations and the

effects of quinpirole

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 ¼ 6) and rats with unilateral

6-OHDA lesions of the SNpc (n ¼ 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 pro-

longed and following the addition of 0.5 nM DA, a

meaningful measure of T50 or T80 could not be obtained.

Consequently clearance ofDAby 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 clear-

ance 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

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 visual-

isation 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 mitochon-

dria 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 discontinu-

ous 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.

The median area of nigrostriatal synaptic terminals in the

dorsal striatum of lesioned animals (n ¼ 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 ¼ 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 con-

tained significantly more vesicles than those from control

animals.

In both control and lesioned animals, the area of SNpc

boutons was correlated with the number of vesicles within

Table 2 Scatchard analysis of [3H]Mazin-

dol binding to DA transporter in the dorsal

striatum of normal and SNpc lesioned ani-

mals 16 weeks after injury (mean ± SD)

Dorsal striatum Kd* (nM) Bmax1 (fmol/mg protein) Kd� (nM) Bmax2 (fmol/mg protein)

Normal 7.68 ± 2.2 1441 ± 483 306 ± 182 4284 ± 1631

(n ¼ 5) (n ¼ 5) (n ¼ 5) (n ¼ 4)

Contralateral 9.87 ± 2.7 1517 ± 439 126 ± 62 4688 ± 2308

(n ¼ 7) (n ¼ 8) (n ¼ 7) (n ¼ 7)

Lesioned 9.6 ± 3.1 954 ± 281 8349 ± 6938 22739 ± 11465

(n ¼ 7) (n ¼ 7) (n ¼ 7) (n ¼ 6)

*High-affinity binding site for [3H]Mazindol to the DA transporter; �low-affinity binding site for

[3H]Mazindol to the DA transporter.

336 D. Stanic et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 329–343

the bouton, demonstrating that vesicle number is propor-

tional 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

Fig. 4 Electron micrographs of nigrostriatal terminals of normal and

rats 16 weeks after partial SNpc lesions. All panels are synaptic ter-

minals 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 lm2) 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 lm2 and

few vesicles, 13) making contact onto a proximal dendrite. (d) A ter-

minal 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 arrow-

heads). (e, f and g) Examples of the post-synaptic targets of synap-

ses 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 mito-

chondria 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 ¼ 0.5 lm. All animals (except for

one) in the ultrastructural studies had medium (30–75%) sized SNpc

lesions.

Function and ultrastructure of SNpc terminals after lesioning 337

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 329–343

synaptic contact with the post-synaptic striatal cells. Follow-

ing 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%,

v2 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%;

v2 test, p < 0.05). Lesioning also resulted in a small increasein 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 (v2 test, p < 0.05, Fig. 5g). There werealso a greater number of docked vesicles per unit of active

zone after lesioning (9 docked vesicles/lm in the control and

15/lm in the lesioned ipsilateral group; v2 test, p < 0.05).

Rotational behaviour

Rotational behaviour was assessed following amphetamine

administration at 4 (n ¼ 15) and 16 (n ¼ 12) weeks after

SNpc lesions and compared with age-matched control

animals (n ¼ 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 arith-

metic 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.

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. Exam-

ination of the response of individual animals that demon-

strated 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

(a) (c)

(b) (d)

(e)

(f) (g)

Fig. 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 (j) and varico-

sities (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 indica-

ted by the median values (ANOVA, Tukey’s post-hoc, p < 0.05). (e)

Regression plots of the area of the pre-synaptic terminal (lm2) 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 pop-

ulations. (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 (v2 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.

338 D. Stanic et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 329–343

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

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

(a)

(b)

(c)

Fig. 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 hypertro-

phy 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 ¼ 50 lm.

Fig. 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.

d, Behaviour before amphetamine; s, 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 amphet-

amine. 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 pro-

pensity 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 ¼ 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 ¼ 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.

Function and ultrastructure of SNpc terminals after lesioning 339

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 329–343

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).

Previous studies of regenerating neuromuscular junctions

suggest that following retraction of the terminal arbour, new

terminals reform at pre-existing post-synaptic sites (McMa-

han 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 compensa-

tory 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, espe-

cially 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 Mazin-

dol, 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 syna-

ptosomes 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.

340 D. Stanic et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 329–343

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 autore-

ceptor 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 augmen-

ted 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, suggest-

ing that 12 months or more may be required for normaliza-

tion 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 individ-

ual 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 pat-

terns 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 trans-

mission. 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 under-

lying the dyskinesias of Parkinson’s disease and neuroleptic

treatment.

Acknowledgements

This research was supported by grants from the Australian National

Health and Medical Research Council. John Drago is a Logan

Fellow at Monash University

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