Lesion verification and behavioral measurements

The initial evaluation of the 6-OHDA lesion status was examined with drug-free cylinder and vibrissae tests. For both these tests, the Kruskal-Wallis one-way ANOVA on ranks revealed a main effect for the lesion (cylinder test: *H*_{(5)} = 55.513, *p* < 0.001; vibrissae test: *H*_{(5)} = 131.916, *p* < 0.001) and a significant deficit in the contralateral forepaw use for the 6-OHDA-lesioned rats in the cylinder test (2 weeks postlesion, *p* < 0.05; 3 weeks postlesion, *p* < 0.05) and in the vibrissae test (2 weeks postlesion, *p* < 0.05; 3 weeks postlesion, *p* < 0.05).

In the cylinder test, all rats used both forepaws equally to make contact with the cylinder wall before 6-OHDA surgery (contralateral forepaw contacts, 50.2 ± 0.01%; ipsilateral forepaw contacts, 49.8 ± 0.01%). Following the lesion, the parkinsonian rats showed a significant deficit in contralateral paw contacts at 2 weeks (24.2 ± 0.03%) and 3 weeks (21.7 ± 0.03%) postlesion compared with the contralateral paw contacts in the control group at 2 weeks (47.9 ± 0.03% contacts, *p* < 0.05) and 3 weeks (49.8 ± 0.02% contacts, *p* < 0.05) postlesion. The same comparisons were conducted with the vibrissae test. Before surgery, all rats showed a 100% tap response to vibrissae stimulation for both left and right forepaws (successful taps per 10 trials, 10 ± 0). Control rats maintained a 100% tap response for both forepaws. Rats receiving a 6-OHDA lesion showed a significant impairment on the side contralateral to the lesion compared with that of the ipsilateral side at 2 weeks (successful taps of the contralateral paw/10 trials, 1.60 ± 0.51, *p* < 0.05) and 3 weeks (successful taps of the contralateral paw/10 trials, 1.90 ± 0.40, *p* < 0.05) postlesion.

Final verification of lesion status was done by estimating the total number of TH^{+} neurons in the SNc using the optical fractionator (Stereo Investigator; MBF Bioscience) and by examining the pattern of TH^{+} fibers in the striatum. Rats with a 6-OHDA lesion lost >90% of all dopaminergic neurons from the SNc (one-way ANOVA *F*_{(3,17)} = 509.356, *p* < 0.001; ). There was no difference in the SNc TH^{+} neuron loss between the rat groups that received the 6-OHDA lesion (*p* > 0.05), and no difference in neuron loss between rats that were dyskinetic^{+} and those that were dyskinetic^{−} (*p* > 0.05, ). The striatum displayed a typical pattern of TH^{+} fiber loss, in that the dorsal striatum was completely devoid of fibers ().

| **Table 1.**Stereological estimates of the number of TH^{+} neurons in the SNc |

All

l-DOPA-treated, 6-OHDA-lesioned rats were rated for the severity of their AIMs. Rats with severe AIMs (dyskinesia

^{+}) displayed axial, orofacial, and limb AIM cumulative severity scores that ranged from 10 to 25. Rats with mild AIMs (dyskinesia

^{−}) had cumulative severity scores that ranged from 0 to 2. There was also a group of rats with moderate, cumulative severity scores that fell between the severe and mild scores. The latter animals displayed hyperkinetic but no dystonic movements. We eliminated the moderate group from further analysis and examined only the dyskinetic

^{+} and dyskinetic

^{−} groups. In anatomical studies, it is important to be able to relate an anatomical substrate to a specific behavioral outcome. Thus, the behavior must be distinct and not on a continuum. Such differences can only be detected by studying animals with fully separate score ranges on a given rating scale (

Meredith et al., 2000).

We measured the severity of AIMs and their severity over time (*A*; two-way repeated-measures ANOVA, main effect of AIMs severity: *F*_{(1,71)} = 245.97, *p* < 0.001; main effect of time: *F*_{(8,71)} = 8.972, *p* < 0.001; AIMs severity × time interaction: *F*_{(8,71)} = 5.969, *p* < 0.001). Dyskinetic^{+} animals showed significantly higher scores at each rating session and at each session over time compared with the dyskinetic^{−} group (*p* values <0.001; *A*). In addition, only the dyskinetic^{+} group showed that AIM scores were greater at all time points in sessions 2–9 than at the first rating session (*p* values <0.001). Thus, the AIMs increased in intensity over time for the dyskinetic^{+} group. In addition, there was no correlation between the severity of AIMs and the extent of the lesion (*B*).

Reference volumes do not differ between groups

We used the Cavalieri method to estimate the volume of the striatum (

Coggeshall, 1992). There was no difference in striatal volume between intact controls (saline or

l-DOPA treatments) and lesioned groups (one-way ANOVA,

*F*_{(4,25)} = 2.393

*p* = 0.08; ).

Corticostriatal and thalamostriatal synapses are differentially distributed in the control striatum, and l-DOPA treatment does not alter that distribution

We began the analysis by estimating the total number of VGluT1-immunopositive (VGluT1

^{+}), VGluT2-immunopositive (VGluT2

^{+}), and unlabeled asymmetric (presumed excitatory) synapses in the dorsal striatum of control rats (). The VGluT1

^{+} terminals comprised ~38% of all asymmetric contacts, and the VGluT2

^{+} synapses comprised ~20%, with the remainder being unlabeled. The VGluT1

^{+} and VGluT2

^{+} immunoreaction product (DAB) was found in terminals from the cortex and thalamus, respectively, and did not colocalize in the striatum (

Lacey et al., 2005). This indicates that ~42% of all asymmetric endings are VGluT1

^{−} or VGluT2

^{−}, as demonstrated by others (

Lacey et al., 2005), and could therefore originate from other regions, such as the serotonergic afferents from the raphé (

Soghomonian et al., 1989;

Descarries et al., 1992) or from cholinergic interneurons, both of which have been shown to express vesicular glutamate transporter 3 in the striatum (

Gras et al., 2002;

Fujiyama et al., 2004). We then examined the target specificity of asymmetric synapses (single synaptic contacts, not MSBs) in the control (sham/saline) striatum by quantifying the total number of VGluT1

^{+} (corticostriatal) and VGluT2

^{+} (thalamostriatal) terminals and the number contacting spines and dendrites (). We found that VGluT1

^{+} afferents form the great majority of their asymmetric contacts onto spines rather than dendritic shafts. In contrast, VGluT2

^{+} afferents also contact spines predominantly, but make more asymmetric contacts with dendritic shafts than VGluT1

^{+} synapses (). These data are very similar to earlier findings by others (

Lacey et al., 2005;

Moss and Bolam, 2008).

l-DOPA treatment does not affect corticostriatal or thalamostriatal synapses in intact animals, but when administered after the parkinsonian lesion l-DOPA dramatically increases corticostriatal synapses

It is important to control for the effects of the drug, since l-DOPA could be responsible for changes in synapse distribution or postsynaptic targets in intact animals or following the dopamine-depleting lesion in the dyskinetic^{−} group. Therefore, to determine whether l-DOPA alters synapses, we compared the effects of l-DOPA on the total number of corticostriatal and thalamostriatal synapses (two-way ANOVA; VGluT1^{+}: main effect of l-DOPA, *F*_{(1,22)} = 6.451, *p* = 0.019; main effect of lesion, *F*_{(1,22)} = 9.380, *p* = 0.006; no interaction between l-DOPA and lesion, *F*_{(1,22)} = 4.016, *p* > 0.05; VGluT2^{+}: no main effect of l-DOPA, *F*_{(1,22)} = 0.987, *p* > 0.05; no effect of lesion, *F*_{(1,22)} = 1.834, *p* > 0.05; no interaction between l-DOPA and lesion, *F*_{(1,22)} = 1.941, *p* > 0.05). There was no l-DOPA effect within the sham-lesioned groups for corticostriatal or thalamostriatal total synapses (sham/saline vs sham/l-DOPA: VGluT1^{+}, *p* > 0.05; VGluT2^{+}, *p* > 0.05; *A*). The parkinsonian lesion (6-OHDA/saline) significantly reduced the total number of corticostriatal synapses compared with controls (sham/saline, VGluT1^{+}, *p* = 0.003), but l-DOPA treatment of parkinsonian rats (6-OHDA/l-DOPA) greatly increased the total number of corticostriatal endings compared with those in the parkinsonian l-DOPA-naive group (6-OHDA/saline, VGluT1^{+}, *p* = 0.002; *A*). The parkinsonian lesion (6-OHDA/saline) did not alter the total number of thalamostriatal (VGluT2^{+}) synapses compared with controls. Moreover, l-DOPA treatment of parkinsonian rats (6-OHDA/l-DOPA) did not change the total number of thalamostriatal (VGluT2^{+}) endings compared with those in the parkinsonian l-DOPA-naive group (6-OHDA/saline). Overall, these data indicate that corticostriatal, but not thalamostriatal, synapses had been added by l-DOPA treatment after the lesion (*A*).

We next determined the effects of l-DOPA on corticostriatal and thalamostriatal axospinous synapses (two-way ANOVA; VGluT1^{+}: no effect of l-DOPA, *F*_{(1,22)} = 3.961, *p* > 0.05; main effect of lesion, *F*_{(1,22)} = 7.991, *p* = 0.010; no interaction between l-DOPA and lesion, *F*_{(1,22)} = 2.922, *p* > 0.05; VGluT2^{+}: no effect of l-DOPA, *F*_{(1,22)} = 0.987, *p* > 0.05; no effect of lesion, *F*_{(1,22)} = 1.834, *p* > 0.05; no interaction between l-DOPA and lesion, *F*_{(1,22)} = 1.941, *p* > 0.05) and axodendritic synapses (two-way ANOVA; VGluT1^{+}: main effect of l-DOPA, *F*_{(1,22)} = 10.105, *p* = 0.004; no effect of lesion, *F*_{(1,22)} = 0.913, *p* > 0.05; no interaction between l-DOPA and lesion, *F*_{(1,22)} = 0.474, *p* > 0.05; VGluT2^{+}: no effect of l-DOPA, *F*_{(1,22)} = 0.809, *p* > 0.05; no effect of lesion, *F*_{(1,22)} = 0.265, *p* > 0.05; significant interaction between l-DOPA and lesion, *F*_{(1,22)} = 5.196, *p* = 0.033). There was no l-DOPA effect within the sham-lesioned control groups for corticostriatal or thalamostriatal axospinous or axodendritic synapses (sham/saline vs sham/l-DOPA: VGluT1^{+}, *p* > 0.05; VGluT2^{+}, *p* > 0.05; *A*). There was, however, a significant difference between the parkinsonian groups (6-OHDA/saline vs 6-OHDA/bpl-DOPA; *A*). The lesioned l-DOPA-naive group (6-OHDA/saline) had significantly reduced the total number of corticostriatal synapses onto spines (VGluT1^{+}, *p* = 0.01) but not onto dendrites (VGluT1^{+}, *p* > 0.05) compared with controls (sham/saline), and l-DOPA treatment of the parkinsonian rats (6-OHDA/l-DOPA) significantly increased the number of VGluT1^{+} endings onto spines compared with that in the parkinsonian l-DOPA-naive group (6-OHDA/saline, VGluT1^{+}, *p* = 0.009), restoring the total axospinous endings to the level of controls (*A*). In addition, l-DOPA treatment significantly increased the number of corticostriatal synapses onto dendrites compared with that in the parkinsonian l-DOPA-naive group (VGluT1^{+}, *p* = 0.007). While thalamostriatal axospinous and axodendritic synapses were not lost after the 6-OHDA lesion (VGluT2^{+}, p > 0.05), l-DOPA treatment did significantly increase the number of contacts onto dendrites compared with that in the parkinsonian l-DOPA-naive group (VGluT2^{+}, *p* = 0.023; *A*).

It then became important to establish whether these synaptic changes following the l-DOPA treatment are due to the development of dyskinesias per se. Therefore, we compared the dyskinetic^{+} and dyskinetic^{−} groups separately to parkinsonian l-DOPA-naive (6-OHDA/saline) and control (sham/saline) groups (*B*,*C*). Both the dyskinetic^{+} and the dyskinetic^{−} rats showed a significant increase in the total number of corticostriatal synapses compared with the parkinsonian l-DOPA-naive group [one-way ANOVA; VGluT1^{+}: *F*_{(2,13)} = 16.680, *p* < 0.001; dyskinetic^{+} group significantly increased compared with the parkinsonian l-DOPA-naive group (6-OHDA/saline), *p* < 0.001; dyskinetic^{−} group significantly increased compared with the 6-OHDA/saline group, *p* = 0.027]. Neither the dyskinetic^{+} nor the dyskinetic^{−} rats showed a change in the total number of thalamostriatal synapses compared with the parkinsonian l-DOPA-naive group (one-way ANOVA; VGluT2^{+}, *F*_{(2,13)} = 2.068, *p* = 0.16).

For corticostriatal synapses, the dyskinetic^{+}, but not the dyskinetic^{−} rats, showed a significant increase in the total number of axospinous synapses (one-way ANOVA; VGluT1^{+}, main effect of dyskinesia status, *F*_{(2,13)} = 12.74, *p* < 0.001) and axodendritic terminals (one-way ANOVA; VGluT1^{+}, main effect of dyskinesia status, *F*_{(2,13)} = 16.93, *p* < 0.001) compared with the parkinsonian l-DOPA-naive group (6-OHDA/saline; *B*). When the thalamostriatal synapses were examined, neither the dyskinetic^{+} or dyskinetic^{−} animals showed any change in the number of axospinous synapses (one-way ANOVA; VGluT2^{+}, *F*_{(2,13)} = 1.736, *p* > 0.05) or axodendritic contacts (one-way ANOVA; VGluT2^{+}, *F*_{(2,13)} = 3.248, *p* = 0.07) compared with the parkinsonian l-DOPA-naive (6-OHDA/saline) group (*B*).

The greatest corticostriatal synaptic changes occurred in the parkinsonian rats that were dyskinetic^{+} (*C*). There was a significant increase in the total number of corticostriatal synapses [one-way ANOVA; VGluT1^{+} main effect of dyskinesia status, *F*_{(2,13)} = 4.378, *p* = 0.035; dyskinetic^{+} significantly greater than dyskinetic^{−}, *p* = 0.036; but no difference from controls (sham/saline), *p* > 0.05], axospinous synapses [one-way ANOVA; VGluT1^{+}, main effect of dyskinesia status, *F*_{(2,13)} = 4.364, *p* = 0.035; dyskinetic^{+} significantly greater than dyskinetic^{−}, *p* = 0.042; but no difference from controls (sham/saline) *p* > 0.05], and axodendritic synapses [main effect of dyskinesia status, *F*_{(2,13)} = 11.417, *p* = 0.001; dyskinetic^{+} significantly greater than dyskinetic^{−}, *p* = 0.003, and significantly greater than controls (sham/saline) *p* = 0.004] for the dyskinetic^{+} group. For the thalamostriatal synapses, there was no difference among the dyskinetic^{+}, dyskinetic^{−}, and control groups in the total number of contacts (one-way ANOVA; VGluT2^{+}, no effect of dyskinesia status, *F*_{(2,13)} = 0.119, *p* > 0.05), the number of axospinous contacts (VGluT2^{+}, no effect of dyskinesia status, *F*_{(2,13)} = 0.343, *p* > 0.05), or the number of axodendritic contacts (VGluT2^{+}, no effect of dyskinesia status, *H*_{(2)} = 0.647, *p* > 0.05; *C*).

Multisynaptic boutons are lost in parkinsonian rats with or without dyskinesias

Significant changes in the number of MSBs are an important indication of circuit modifications, such as remodeling (

Rademacher et al., 2010). In the control group (sham/saline), VGluT1

^{+} and VGluT2

^{+} terminals occasionally contacted more than one target, documenting a small population of MSBs in control brains. These MSBs generally contacted two distinct spines, especially for the VGluT1

^{+} afferents. However, a few MSBs that were VGluT1

^{+} or VGluT2

^{+} contacted two dendritic shafts or a shaft and a spine (see also

Moss and Bolam, 2008). An insufficient number of MSBs was detected among the thalamostriatal synapses to test for differences. Among corticostriatal MSBs and under all conditions of striatal dopamine depletion, regardless of dyskinesia status, the total number of corticostriatal (VGluT1

^{+}) MSBs decreased significantly (two-way ANOVA; no effect of

l-DOPA,

*F*_{(1,22)} = 0.068,

*p* > 0.05; main effect of lesion,

*F*_{(1,22)} = 24.094,

*p* = 0.003; and no interaction between

l-DOPA and lesion

*F*_{(1,22)} = 3.561,

*p* > 0.05). Thus, regardless of treatment, the 6-OHDA lesion significantly reduced the total number of corticostriatal MSBs (

*p* < 0.001).

Aberrant morphological plasticity of MSNs in dyskinetic^{+} animals

The restoration of axospinous and axodendritic corticostriatal synapses in the dyskinetic

^{+} animals led us to question how the substrate (MSNs) adapted to this change in wiring. Reconstructions of Golgi-impregnated neurons revealed typical MSN morphology. Specifically, there were from four to eight primary dendrites on each MSN, and proximal dendrites emerging from the soma were initially aspiny, with spines increasing in frequency from proximal to distal branch orders. The MSNs in the control striatum had an overall mean spine density of (8.5/10 μm), and distal branches were more densely covered in spines (8.9/10 μm) compared with proximal branches (7.9/10 μm). As expected and, in support of the corticostriatal spine and synaptic loss seen in the 6- OHDA-lesioned animals previously (

Ingham et al., 1989,

1998), there was a significant loss of spines for the parkinsonian rats (6-OHDA), regardless of their dyskinetic state (one-way ANOVA;

*F*_{(2,31)} = 10.42,

*p* < 0.001; parkinsonian significantly reduced compared with controls,

*p* < 0.001; and dyskinetic

^{+} significantly reduced compared with controls,

*p* = 0.008;

*A*). For the proximal segments of the dendrites, there was a significant loss of spines for the parkinsonian rat, regardless of dyskinetic status (one-way ANOVA;

*F*_{(2,31)} = 8.60,

*p* = 0.001). However, for the dyskinetic

^{+} rats, the spine losses were confined to the proximal segments of MSNs compared with controls (dyskinetic

^{+} significantly reduced compared with controls,

*p* = 0.012;

*A*), but for the parkinsonian rats, both proximal (6-OHDA significantly reduced compared with controls,

*p* = 0.002) and distal spines were lost compared with controls (one-way ANOVA;

*F*_{(2,31)} = 4.982,

*p* = 0.013; 6-OHDA group significantly reduced compared with controls,

*p* = 0.11;

*A*). The distal spine density of dyskinetic

^{+} rats did not differ significantly from controls (dyskinetic

^{+} was not significantly different compared with controls,

*p* > 0.05) and showed no difference with the parkinsonian (6-OHDA) group [trend for dyskinetic

^{+} to have an increase in spine density compared with parkinsonian (6-OHDA) group,

*p* = 0.1]. These data suggest a restoration of spines in the distal branch orders for the dyskinetic

^{+} group only. Finally, the density of mushroom, but not branched or thin, spines (

*B*,

*D*) was significantly greater for the dyskinetic

^{+} rats compared with the parkinsonian group (6-OHDA;

*H*_{(2)} = 21.703,

*p* < 0.001; dyskinetic

^{+} significantly greater than 6-OHDA group,

*p* < 0.05) and control group (dyskinetic

^{+} significantly more than controls,

*p* < 0.05). Neither the length of dendrites (

*C*) nor their surface areas differed between groups (not illustrated).

The ultrastructural analysis of spines showed that multisynaptic spines were increased in the dyskinetic^{+} rats compared with controls and parkinsonian (6-OHDA) rats (). These differences were significant (one-way ANOVA; main effect of dyskinesia status, *F*_{(2,15)} = 8.969, *p* = 0.004), suggesting that the restoration of VGluT1^{+} axospinous synapses was accommodated, in part, by multiple excitatory inputs onto individual spines. There was no difference in the number of multisynaptic spines between dyskinetic^{−} and parkinsonian l-DOPA-naive (6-OHDA) rats (*p* > 0.05). Furthermore, we estimate that the total number of asymmetric (excitatory) synapses in the control striatum (immunohistochemically labeled and unlabeled) is ~21.6 billion; of these, only 35 million single spines receive more than one contact. The total number of asymmetric (excitatory) synapses in the dyskinetic^{+} striatum (immunohistochemically labeled and unlabeled) is ~15 billion, but, in contrast, 118 million of these single spines receive at least two of these contacts.

| **Table 3.**Stereological estimates of spines |