Histology and behaviour
As expected, the 6-hydroxydopamine lesions of the medial forebrain bundle produced a ~95–97% loss of tyrosine hydroxylase-positive dopamine neurons in the striatum and SNC (A and D). Electrode tracks were histologically confirmed in the SNR and subthalamic nucleus (H and I).
Figure 1 Histology. (A) Sample tyrosine hydroxylase stained section of hemiparkinsonian rat showing lesioned left striatum. (B) Sample tyrosine hydroxylase stained section of transplanted rat showing lesioned left striatum with surviving tyrosine hydroxylase-positive (more ...)
Nine transplanted rats showed definite surviving striatal grafts with tyrosine hydroxylase-positive neurons. B and C shows an example striatal graft. Each identified graft site in each animal had healthy appearing tyrosine hydroxylase-positive neurons and graft derived tyrosine hydroxylase-positive neurites around the graft that appeared to innervate the surrounding denervated striatum. Graft-derived tyrosine hydroxylase-positive cells were also dopamine transporter-positive (E–G), confirming their dopaminergic status. Supplementary Fig. 2
shows additional views of the graft cells.
The nine rats with surviving grafts fell naturally into two categories: five showed large, robust grafts with many surviving cells, and four showed small grafts with few surviving cells. The discrimination between the large graft and small graft groups was based on stereological cell counts, using an arbitrary threshold of 4000 cells. The mean count of tyrosine hydroxylase-positive grafted neurons in the large graft group was 8975
2386 (SEM), with coefficient of error 0.18
0.01. The mean in the small graft group was 1471
328 neurons with coefficient of error 0.29
0.03. Although this range of surviving graft size was not the intended result of our transplantation paradigm, it gave us the opportunity to analyse the electrophysiology from these two groups separately to evaluate whether the size of the striatal graft had a differential effect on the downstream firing rates and patterns.
As shown in , both the ‘large graft’ and ‘small graft’ groups showed apomorphine-induced rotation improvement at months 1, 2 and 3 compared to pre-transplant (P
0.05), but only the large graft group showed improvements in the extended body axis bias test (P
0.05). The correlation of cell count against behavioural improvement index was significant (P
0.01), with an R2
value of 0.8055. We did not observe graft-induced dyskinesias in any of the rats.
Figure 2 Behavioural tests. (A) Apomorphine-induced rotations showing behavioural improvement at 1, 2 and 3 months after transplant (*P<0.05, **P<0.01 compared to pre-transplant). (B) Extended Body Axis Bias Test showing reduction (more ...)
The remaining 17 transplanted rats were excluded from the analysis for one of the following reasons: (i) the perfusion or tyrosine hydroxylase histology was problematic allowing no firm conclusion to be made about graft survival (10 rats); (ii) surviving cells were seen but were all misplaced outside the boundary of the striatum (one rat); or (iii) no surviving tyrosine hydroxylase-positive graft cells were seen but not enough total neurons were recorded to make a valid ‘no-graft-seen’ control group to compare with the other groups (six rats).
shows a sample neural recording, including a transition from slow-wave activity to global activation states. lists the numbers of neuronal spike trains and local field potential waveforms recorded in the different groups and brain activation states. When possible, neurons were recorded from both slow-wave activity and global activation states, but these transitions were deliberately kept spontaneous rather than manipulated (e.g. by paw pinch or thermal stimuli, cf
. Mallet et al., 2008b
), causing some unevenness of sampling among the different activation states.
Figure 3 Sample waveforms. These recordings are from a subthalamic nucleus neuron in a rat from the large graft group. A transition is visible between slow-wave activity (left) to global activation (right), with characteristic EEG spectra changes between the two (more ...)
Firing rates in the slow-wave activity state were significantly different between groups in the SNR (P
0.05) but not in the subthalamic nucleus (A). Post hoc
testing showed that the only significant difference in the SNR was that the large graft group had lower firing rates than the small graft group.
Firing patterns in the slow-wave activity state in both SNR and subthalamic nucleus showed increased burstiness in the hemiparkinsonian state and a restoration of burstiness towards more normal levels in the large graft group (B–G). In the SNR this trend was significant in the coefficient of variance (P
0.001), proportion of spikes in bursts (P
0.001), and density discharge histogram range (P
0.001). In each of these measures, post hoc
tests showed that the hemiparkinsonian group and the small graft group were burstier than the normal group, and the large graft group was restored to a level not statistically different from the normal group. Additionally, the sample entropy was reduced in the hemiparkinsonian and small graft groups (P
0.001) restored to normal levels in the large graft group. The proportion of SNR cells classified as bursty by the Poisson density discharge histogram comparison increased in the hemiparkinsonian group, but the trend did not reach statistical significance (Fisher's exact two-sided test, P
In the subthalamic nucleus, the same pattern was seen of increased burstiness in the hemiparkinsonian group and restoration to normal levels in the large graft group. This trend was significant in the Poisson density discharge histogram comparison (Fisher's exact two-sided test; P
0.025 between normal and hemiparkinsonian groups; P
0.0046 between hemiparkinsonian and large graft groups) and proportion of spikes in bursts (P
0.05). Post hoc
tests showed that the large graft group was significantly less bursty than the hemiparkinsonian control group in the coefficient of variance, burst index, density discharge histogram range, and sample entropy metrics.
The small graft group showed mixed results in both SNR and subthalamic nucleus, with burstiness levels closer to the normal group in some measures, and closer to the hemiparkinsonian group in other measures. The majority of measures showed the burstiness of the small graft group falling between that of the hemiparkinsonian group and the large graft group.
The percentage of oscillatory SNR neurons was significantly higher in the hemiparkinsonian group compared to the normal group (P
0.022). In the small graft group, the percentage of oscillatory SNR neurons was even higher than in the hemiparkinsonian group (P
0.0025, between hemiparkinsonian and small graft groups). The percentage of oscillatory SNR neurons in the large graft group was restored to normal levels. The subthalamic nucleus showed a similar trend. The hemiparkinsonian oscillatory percentage was not significantly increased from normal, but the small graft group had more oscillatory neurons than the normal group (P
In the global activation state, firing rates did not significantly differ between groups in either the SNR or subthalamic nucleus (A). Firing patterns in the global activation state were less bursty overall than in the slow-wave activity state in both SNR and subthalamic nucleus, and smaller differences were seen between the groups than in the slow-wave activity state. In the SNR, the density discharge histogram range showed an increase in burstiness in the hemiparkinsonian group (P
0.01). The sample entropy showed a significant reduction in the hemiparkinsonian group (P
0.05) and the percentage of oscillatory neurons was higher in the hemiparkinsonian group than in the normal group (Fisher's exact two-sided test; P
0.022). In contrast, the small graft and large graft groups did not show any significant differences in firing pattern from the normal group.
In the slow-wave activity state, the mean EEG spectra showed a slight increase in power
Hz in the large graft group (). This increase was also seen in the SNR local field potentials and in the EEG-local field potential coherence, especially in the 30–55
Hz band. Quantification of this effect by summing the power in the 30–55
Hz coherence band showed a significant increase in the large graft group (P
0.05). However, this peak was seen primarily in only two out of the five rats, from which a large number of recordings were obtained (Supplementary Fig. 1
). To visualize the coherence results in another way that de-emphasizes the variability of contributions from rats with greater or lesser numbers of recordings, the mean of each rat's mean coherences was also computed (, inset
). This mean showed smaller differences between the groups (statistics not performed because of small sample size). The subthalamic nucleus in the slow-wave activity state showed an increase in the 10–15
Hz band in the hemiparkinsonian and large graft groups (P
0.05). However, while the differences between groups were significant in the 10–15
Hz band in the SNR and subthalamic nucleus in the slow-wave activity state, these peaks did not cross the coherence significance level, and were thus not considered significant in summary . All rat groups showed a significant coherence peak in the 30–55
Hz band, with no significant differences seen between groups in this band.
Figure 6 Spectral measures. (Top row) Mean EEG log-spectra (bold lines) and SEM (thin lines) across all recordings in each group. Power units are arbitrary. (Second row) Mean local field potential log-spectra (bold lines) and SEM (thin lines) across all recordings (more ...)
Summary of electrophysiological changes in the large graft group compared to hemiparkinsonian control group
In the global activation state, a significant peak was seen in the 10–15
Hz band in the hemiparkinsonian group in both the SNR and subthalamic nucleus. This peak was significantly reduced in the small graft group SNR and in the large graft group SNR and subthalamic nucleus. However, these peaks were seen primarily in one rat in each group, and the average of each rat's mean coherences (, inset
) showed smaller differences between the groups in this band. The hemiparkinsonian SNR and the small graft group subthalamic nucleus also showed significantly increased coherence in the 30–55
Hz band compared to the normal group.
In the slow-wave activity state, spike triggered averages showed a trend toward increased post-spike amplitude in the hemiparkinsonian and small graft groups from both the local field potential and EEG waveforms (). This trend was significant in the EEG (P
0.05, post hoc
rank-sum tests on peak-to-peak amplitudes). Additionally, the local field potential showed significant modulation in the small graft SNR and the hemiparkinsonian subthalamic nucleus, but no significant modulation in either SNR or subthalamic nucleus of the normal and large graft groups.
Figure 7 Spike-triggered waveform averages. In each box, the top four rows show the mean spike triggered averages for each group (bold lines) with the SEM (thin lines). Dotted lines show the maximum and minimum of the mean randomized spike triggered averages. (more ...)
In the global activation state, the majority of groups showed significant spike triggered average modulation, but no significant differences between groups were observed.