Figure 1.
Dopamine uptake between striatum and substantia nigra.
The inherent differences in DA uptake between striatum and substantia nigra in non-lesioned tissue, as per our synaptosome preparation and uptake protocol are illustrated. Our results reflect the previous observations that DAT expression is significantly less in SN than in striatum and in vivo assessments of DA clearance also show less DA uptake in the SN compared to striatum. Statistics, **p = 0.001, two-tailed paired t-test of 16 matched observations in synaptosomes prepared from striatum and substantia nigra dissected contralateral to medial forebrain bundle 6-OHDA lesion.
Figure 2.
Dopamine uptake profiles per equal synaptosomal protein related to percent loss of tyrosine hydroxylase. A.
DA uptake with TH loss at 30–60%. Statistics, p = 0.055, one-tailed paired t-test of 6 matched observations in synaptosomes prepared from striatum ∼9 days following mfb 6-OHDA lesion. TH loss was confirmed in a tissue fraction during synaptosome preparation. B. DA uptake with TH loss at 30–60% at 70–99% loss. Statistics, p<0.05, one-tailed paired t-test of 26 matched observations in synaptosomes prepared from striatum ∼9 days following mfb 6-OHDA lesion. TH loss was confirmed in a tissue fraction during synaptosome preparation. C. Representative western blot depicting TH loss. TH loss by the 6-OHDA lesion (L) is shown versus quantity in contralateral striatum (C) and interpolation by accompanying standard curve of TH protein (0.5 to 2.0 ng TH). Associated Ponceau stain (below TH bands) on same blot before TH antibody blotting demonstrates similar striatal protein loading.
Figure 3.
Dopamine uptake per remaining DAT protein. A.
DA uptake in synaptosomes normalized to remaining DAT protein recovered in the synaptosome fraction with confirmed DAT loss of at least 70%. Statistics, **p<0.01, t = 3.49, two-tailed paired t-test of 19 matched observations in synaptosomes prepared from striatum ∼9 days following mfb 6-OHDA lesion. B. Significant correlation of DA uptake with the severity of DAT loss. Statistics, p = 0.015, Spearman correlation analysis of DA uptake versus %DAT protein loss, r = 0.55, n = 19 pairs. C. Inherent DAT immmunoreactivity in synaptosome aliquots of equivalent total protein quantity from the 6-OHDA-lesioned (L) and contralateral striatum (C).
Figure 4.
Establishment of uptake competition differences between the endogenous monoamines of striatum in naïve tissue.
The percentage of DA uptake was assessed in the presence of varying concentrations of NE, DA and 5HT in the naïve striatum. The concentration of 1 uM of NE, DA and 5HT differentially inhibited uptake of labeled DA, and was thus chosen for discerning involvement of other monoamine transporters in the paradoxical increase of DA uptake per remaining DAT (as seen in Fig. 3).
Figure 5.
Dopamine uptake profiles with monoamine competition in 6-OHDA-lesioned striatum versus intact striatum. A.
Dopamine. 1 µM DA was added in striatal synaptosomes prepared from at least 70% lesioned striatum and from the operationally-matched contralateral control. After 5 min preincubation period, 500 nM [7-, 8- 3H-DA] was added and uptake was determined for 2 min. In the lesioned striatal synaptosomes, DA was significantly less effective (30% less inhibition than in control) effective to inhibit DA uptake, as compared to the control. Statistics: *p<0.05, t = 3.47, two-tailed Student’s paired t-test, n = 6 paired observations. B. Norepinephrine 1 µM NE was added in striatal synaptosomes prepared from at least 70% lesioned striatum and from the operationally-matched contralateral control. After 5 min preincubation period, 500 nM [7-, 8- 3H-DA] was added and uptake was determined for 2 min. In the lesioned striatal synaptosomes, NE was significantly more effective (38% greater inhibition than in control) to inhibit DA uptake, as compared to the control. Statistics: *p<0.05, t = 2.59, two-tailed Student’s paired t-test, n = 6 paired observations. C. Serotonin 1 µM 5-HT was added in striatal synaptosomes prepared from at least 70% lesioned striatum and from the operationally-matched contralateral control. After 5 min preincubation period, 500 nM [7-, 8- 3H-DA] was added and uptake was determined for 2 min. There was no significant difference in the ability of 5-HT to inhibit DA uptake in lesioned striatal synaptosomes, as compared to the control, n = 6 paired observations.
Figure 6.
Norepinephrine Transporter (NET) striatal protein expression and function in 6-OHDA lesioned rats at >70% TH protein loss. A
. NET protein expression. Quantification of NET showed that NET was significantly increased in lesioned striatum compared to its matched non-lesioned control. Statsitics: NET, *p<.05, t = 3.251, n = 4 paired observations. B. Representative western blot of NET expression. Relative expression of NET (∼80 kDa band) between contralateral, control, striatum (C) and 6-OHDA-lesioned striatum (L). C. Increased NET function in 6-OHDA lesioned striatum. Desipramine (DMI)-mediated inhibition of [3H] NE uptake between contralateral (control) and lesioned striatum (mean TH loss = 66%). Statistics p<0.05, t = 3.127, n = 3 observations.
Figure 7.
Monoamine content in operationally matched 6-OHDA lesioned rats at >70% TH protein loss.
Representation of relative impact of our 6-OHDA lesion protocol on monoamine tissue content (expressed as ng monoamine per mg protein) in striatum with at least 70% confirmed loss of dopamine (top panel). As shown in the middle panel, there was no loss of NE in the 6-OHDA lesion method employed, but there was a trend toward a decrease in 5-HT (bottom panel). Statistics: DA, ***p<0.0001, t = 10.87. n = 7 paired observations for all monoamines.
Figure 8.
Impact of L-DOPA on monoamine uptake A. Dopamine uptake in lesioned striatum in presence of L-DOPA.
1 µM L-DOPA was added in striatal synaptosomes prepared from at least 70% lesioned striatum and from the operationally-matched contralateral control. After 5 min preincubation period, 500 nM [7-, 8- 3H-DA] was added and uptake was determined for 2 min. In the lesioned striatal synaptosomes, L-DOPA was significantly more (77% above inhibition in control) effective to inhibit DA uptake. Statistics: *p<0.05, t = 3.31, two-tailed Student’s paired t-test, n = 5 paired observations. B. Impact of L-DOPA on DA versus NE uptake. 1 µM L-DOPA was added to naïve (unlesioned) striatal synaptosomes and after 5 min preincubation, either 500 nM 3H-DA or 250 nM [3H] NE was added and uptake was determined for 2 min. Statistics (*p<0.001, t = 12.75, unpaired two-tailed Student’s t-test, n = 4 for NE, 5 for DA).