Histological Correlates of Neuroanatomical Changes in a Rat Model of Levodopa-Induced Dyskinesia Based on Voxel-Based Morphometry

Long-term therapy with levodopa (L-DOPA) in patients with Parkinson’s disease (PD) often triggers motor complications termed as L-DOPA-induced dyskinesia (LID). However, few studies have explored the pathogenesis of LID from the perspective of neuroanatomy. This study aimed to investigate macroscopic structural changes in a rat model of LID and the underlying histological mechanisms. First, we established the hemiparkinsonism rat model through stereotaxic injection of 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle, followed by administration of saline (PD) or L-DOPA to induce LID. Magnetic resonance imaging (MRI) and behavioral evaluations were performed at different time points. Histological analysis was conducted to assess the correlations between MRI signal changes and cellular contributors. Voxel-based morphometry (VBM) analysis revealed progressive bilateral volume reduction in the cortical and subcortical areas in PD rats compared with the sham rats. These changes were partially reversed by chronic L-DOPA administration; moreover, there was a significant volume increase mainly in the dorsolateral striatum, substantia nigra, and piriform cortex of the lesioned side compared with that of PD rats. At the striatal cellular level, glial fibrillary acidic protein-positive (GFAP+) astrocytes were significantly increased in the lesioned dorsolateral striatum of PD rats compared with the intact side and the sham group. Prolonged L-DOPA treatment further increased GFAP levels. Neither 6-OHDA damage nor L-DOPA treatment influenced the striatal expression of vascular endothelial growth factor (VEGF). Additionally, there was a considerable increase in synapse-associated proteins (SYP, PSD95, and SAP97) in the lesioned striatum of LID rats relative to the PD rats. Golgi-Cox staining analysis of the dendritic spine morphology revealed an increased density of dendritic spines after chronic L-DOPA treatment. Taken together, our findings suggest that striatal volume changes in LID rats involve astrocyte activation, enrichment of synaptic ultrastructure and signaling proteins in the ipsilateral striatum. Meanwhile, the data highlight the enormous potential of structural MRI, especially VBM analysis, in determining the morphological phenotype of rodent models of LID.

Altered Functional Segregated Sensorimotor, Associative, and Limbic Cortical-Striatal Connections in Parkinson's Disease: An fMRI Investigation

Multiple studies have identified segregated functional territories in the basal ganglia for the control of goal-directed and habitual actions. It has been suggested that in PD, preferential loss of dopamine in the posterior putamen may cause a major deficit in habitual control (mediated by the sensorimotor cortical-striatal loop), and the patients may therefore be forced into a progressive reliance on the goal-directed behavior (regulated by the associative cortical-striatal loop). Functional evidence supporting this point is scarce at present. This study aims to verify the functional connectivity changes within the sensorimotor, associative, and limbic cortical-striatal loops in PD. Resting-state fMRI of 70 PD patients and 30 controls were collected. Bilateral tripartite functional territories of basal ganglia and their associated cortical structures were chosen as regions of interest, including ventral striatum and ventromedial prefrontal cortex for limbic loop; dorsomedial striatum and dorsolateral prefrontal cortex for associative loop; dorsolateral striatum and sensorimotor cortex for sensorimotor loop. Pearson's correlation coefficients for each seed pair were calculated to obtain the functional connectivity. The relationships between functional connectivity and disease severity were further investigated. Functional connectivity between dorsolateral striatum and sensorimotor cortex is decreased in PD patients, and negatively correlated with disease duration; whereas functional connectivity between dorsomedial striatum and dorsolateral prefrontal cortex is also decreased but postitively correlated with disease duration. The functional connectivity within the sensorimotor loop is pathologically decreased in PD, while the altered connectivity within the associative loop may indicate a failed attempt to compensate for the loss of connectivity within the sensorimotor loop.

Aging reduces calreticulin expression and alters spontaneous calcium signals in astrocytic endfeet of the mouse dorsolateral striatum

Aging-related impairment of the blood brain barrier (BBB) and neurovascular unit (NVU) increases risk for neurodegeneration. Among the various cells participating in BBB and NVU function, spontaneous Ca2+ signals in astrocytic endfeet are crucial for maintaining BBB and NVU integrity. To assess if aging is associated with changes in spontaneous Ca2+ signals within astrocytic endfeet of the dorsolateral striatum (DLS), we expressed a genetically encoded Ca2+ indicator, Lck-GCaMP6f in DLS astrocytes of young (3-4 month) and aging (20-24 month) mice. Compared to young mice, endfeet in the DLS of aging mice demonstrated a decrease in calreticulin (CALR) expression, and dramatic alterations in the dynamics of endfoot membrane-associated and mitochondrial Ca2+ signals. While young mice required both extracellular and endoplasmic reticulum (ER) Ca2+ sources for generating endfoot Ca2+ signals, aging mice showed exclusive dependence on ER Ca2+. These data suggest that aging is associated with significant changes in Ca2+ buffers and Ca2+ signals within astrocytic endfeet, which has important implications for understanding mechanisms involved in aging-related impairment of the BBB and NVU.

Opposing roles for striatonigral and striatopallidal neurons in dorsolateral striatum in consolidating new instrumental actions

Comparatively little is known about how new instrumental actions are encoded in the brain. Using whole-brain c-Fos mapping, we show that neural activity is increased in the anterior dorsolateral striatum (aDLS) of mice that successfully learn a new lever-press response to earn food rewards. Post-learning chemogenetic inhibition of aDLS disrupts consolidation of the new instrumental response. Similarly, post-learning infusion of the protein synthesis inhibitor anisomycin into the aDLS disrupts consolidation of the new response. Activity of D1 receptor-expressing medium spiny neurons (D1-MSNs) increases and D2-MSNs activity decreases in the aDLS during consolidation. Chemogenetic inhibition of D1-MSNs in aDLS disrupts the consolidation process whereas D2-MSN inhibition strengthens consolidation but blocks the expression of previously learned habit-like responses. These findings suggest that D1-MSNs in the aDLS encode new instrumental actions whereas D2-MSNs oppose this new learning and instead promote expression of habitual actions.

Activity in the dorsomedial striatum decreases with improvement in motor coordination

It is widely thought that during early stages of motor learning, the dorsomedial striatum facilitates the learning of goal-directed actions, and at later stages, the learned actions are transferred to the dorsolateral striatum, which enables motor actions to become a skill or habit. It is however unknown if these striatal regions are simultaneously active as expertise is acquired during practice. To address this question, we developed a treadmill task to track changes in mouse locomotor coordination during practice running at a range of speeds. We analyzed body position and paw movement to evaluate changes in motor coordination over practice using DeepLabCut and custom-built code. By simultaneous evaluation of motor coordination improvements and fiber photometry recordings of neuronal calcium activity during training, we found that direct pathway dorsomedial striatum neurons exhibited reduced activity as the mouse became proficient at running on the treadmill. In contrast, direct pathway activity in dorsolateral striatum was similar throughout training and did not correlate with increased skill proficiency. These results provide new tools to measure changes in fine motor skills during simultaneous recordings of brain activity, revealing fundamental features of the neural substrates of motor learning.