This outcome is consistent with increased D2 receptor modulation of corticostriatal synapses one month after 3-NP (fig. not change on the same post 3-NP time points. These data show even brief exposure to 3-NP can have lasting behavioral effects mediated by changes in the way DA and glutamate synapses interact. analysis two weeks (24 hour post 3-NP group) or 6 weeks (one month post 3-NP group) after the 6-OHDA lesions. Corticostriatal long-term synaptic plasticity was compared between slices taken ipsilateral and contralateral to the unilateral 6-OHDA lesion to demonstrate the DA- dependence of the unique 3-NP-induced changes in synaptic plasticity seen at 24 hours and one month after the 3-NP injection. Each animal was tested three days prior to becoming injected with 3-NP for rotational behavior induced by apomorphine injection (0.5 mg/kg). Animals showing 10 rotations per minute or more were included in the study. Data on synaptic plasticity were from striatal neurons located ipsilateral and contralateral to the lesion. The mesencephalic block containing both the lesioned and the intact substantia nigra was then fixed in 4% paraformaldehyde and processed for tyrosine hydroxylase (TH) immunocytochemistry. The TH immunocytochemistry was performed on 10C15 mesencephalic sections per animal, using the commercially available antibody from Chemicon (Temicula, CA). Immunopositive cells were counted in every additional section through the rostral to caudal degree of the substantia nigra. Having a section thickness of 60 mm, we had approximately 15C20 sections per animal. 6-OHDA lesions produced an 85% or higher loss of TH positive neurons ipsilateral to the lesion as compared to the contralateral part (mean 24 hour post 3-NP: 90%, n=3; mean 1-month post 3-NP group: 91%, for 20 min. The supernatant was discarded and the pellet washed twice under the conditions explained above. The final pellet was suspended in ~10 quantities of buffer, pH 7.4. Protein concentrations for the final pellet were identified using the Bio-Rad Protein Assay with BSA as the standard. Striatal homogenates were assayed in duplicate in test tubes comprising 5 nM [ 3H] (+)-5-methyl-10,11-dihydro-516; aCSF + SCH 23390, 8) (Fig. 3). analysis of post-tetanic plasticity (average 0C3 min post-tetanus plasticity) also exposed a difference ( test), and a similar trend was seen for long-term plasticity (average 15C20 min post-tetanus; CD3G 0.09, unpaired test) when comparing slices from rats injected 24 Dihydroartemisinin hours earlier with 3-NP that were bathed in saline or SCH 23390. The DA-dependence of the improved manifestation of corticostriatal LTD seen one month after 3-NP exposure was also examined using the same strategy of showing 1) the DA requirement through 6-OHDA-mediated DA depletion and 2) the DA receptor centered pharmacology for the induction of the enhanced LTD seen 1 month after the 3-NP injection. Corticostriatal synapses examined on the side ipsilateral to the 6-OHDA lesion did not display Dihydroartemisinin the exaggerated synaptic major depression seen in the side contralateral to the 6-OHDA lesion one month after the 3-NP injection (Fig. 4) (p 0.03 d.f., 1, 8; F=8.07 repeated measures ANOVA) (ipsilateral = 5, contralateral = 5). Post-hoc assessment of post-tetanic plasticity (average 0C3 min post-tetanus plasticity) showed no difference, but long-term plasticity approached was different (average 15C20 min post-tetanus) (p 0.05, unpaired t-test) (Fig. 4). Corticostriatal LTD is definitely D2 DA receptor dependent in normal rodents (for review observe Calabresi et al, 2007), and we tested whether the 3-NP induced increase in corticostriatal LTD seen Dihydroartemisinin one month after 3-NP exposure was D2 receptor dependent as well by bathing mind slices from3-NP injected rats in the selective D2 receptor antagonist l-sulpiride (10 M). Dihydroartemisinin Assessment between neurons from slices taken from rats injected one month earlier with 3-NP and bathed in aCSF only (16) with those bathed in aCSF + l-sulpiride (8) during the tetanic induction exposed a significant difference across the entire post-tetanus sampling period (panalysis of post-tetanic plasticity (average 0C3 min post- tetanus plasticity) exposed a difference (ptest) as did long-term plasticity (average 15C20 min post-tetanus; 0.04, unpaired test). 3-NP and [3H] MK-801 binding in the dorsomedial striatum [3H]MK-801 binding measured in dorsomedial striatal homogenates did not reveal variations between 3-NP and saline injected rats at any of the post injection time points measured (24 hours to 3 months) (Fig. 5)(3-NP n=6, saline n=6). These data show 3-NP did not alter the number of NMDA receptors at any recovery time point. Open in a separate window.
- Wash the cells once briefly with PBS, then incubate for 15 minutes in blocking solution made up of 4% normal goat serum and 0
- When gated about viable immune cells in the tumor, the annexin V+ single-positive cells are distinct from caspase-3/7+ or apoptotic cells (Figure 1B)
- Miller 1995b)
- Each dot represents one patient, and bars indicate mean with SEM