Date of Award

2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Molecular and Systems Pharmacology

Research Advisor

Fu-Ming Zhou, PhD

Committee

Francesca-Fang Liao, PhD; Il Hwan Kim, PhD; Kafait U. Malik, PhD; Max Fletcher, PhD; Wenlin Sun, PhD

Keywords

c-Fos, Dopamine, Motor cortex, Parkinson's Disease, Spike activity, Striatum

Abstract

Purpose. The striatum receives excitatory glutamatergic input from nearly all parts of the cerebral cortex, and its output is delivered by direct pathway medium spiny neurons (dMSNs) expressing D1 type dopamine (DA) receptors (D1Rs) and indirect pathway MSNs (iMSNs) expressing D2 DA receptors (D2Rs). Loss of DA results in the imbalance between the activities of dMSNs and iMSNs in Parkinson’s disease (PD), leading to ab-normal interactions between the cerebral cortex and striatum. Thus, understanding the inter-actions between the cortex and striatum is important. The monosynaptic corticostriatal cir-cuit has been extensively studied. In contrast, due to the poly-synaptic nature of the connec-tions between the striatum and the cortex, how the striatum and striatal dopaminergic activi-ty each influences or feedbacks the cerebral cortex is more difficult to study and thus poorly understood. Our study is designed to fill this knowledge gap.

Methods. Tyrosine hydroxylase knockout (TH KO) mice were used as the DA-depleted mouse model, while wild-type (WT) C57Bl/6 mice were used as the normal control mice. In TH KO mice, the TH gene is selectively deleted in DA neurons (TH is intact in epineph-rine and norepinephrine neurons), leading to a total lack of DA in the striatum, mimicking the severe DA loss in late-stage PD. TH KO and WT were crossed with D2-GFP mice to generate D2-GFP TH KO and D2-GFP WT, respectively, for identifying D1-MSNs and D2-MSNs in the striatum. Four principal methods were used as follows: First, intracranial microinjection was used to deliver drugs directly into the striatum and motor cortex. A guide cannula was implanted into the right striatum/motor cortex of each TH KO mouse, and a single microinjection of L-dopa/ D1 agonist SKF81297/ D2 agonist quinpirole/ saline into the right striatum/right motor cortex was performed 75 min before mouse perfusion. Second, c-Fos mapping was used to evaluate neuronal activity. C-Fos is an immediate-early gene with activity-dependent protein expression that can be detected by immunostaining. We observed neuronal activity in the motor cortex, striatum, motor thalamus, external seg-ment of globus pallidus (GPe), subthalamic nucleus (STN), and substantia nigra pars retic-ulata (SNr) by c-Fos immunostaining at baseline and upon activation by treatments with L-dopa and DA agonists, separately. Third, in vivo unit spike recording was used to monitor the spike firing activity of cortical neurons in freely moving mice. A micro-drivable tetrode array was implanted in the motor cortex and connected to a headstage to detect the cortical spike activity using a 16-channel spike data acquisition system. The firing activity of neu-rons was recorded for 30 minutes as baseline and 60 minutes after drug injection. Fourth, behavioral videotaping was used to record the behavioral response to the drug treatments during the spike recording sessions.

Results. The basal c-Fos expression in the striatum, motor cortex, motor thalamus, and STN was much lower in TH KO mice compared with WT mice. 10mg/kg L-dopa and 3mg/kg benserazide systemic administration significantly increased the c-Fos expression in the striatum, the entire cerebral cortex including the motor cortex, and the key brain areas in striatal-thalamocortical circuit (motor thalamus, GPe, STN, and SNr) in TH KO mice. In the neuronal spike recording, the mean firing rates of cortical pyramidal neurons (PNs) were 1.0±0.1Hz in TH KO mice and 1.8±0.2Hz in WT mice, respectively. The mean firing rates of cortical interneurons (INs) were 1.6±0.3Hz and 2.7±0.8Hz in TH KO and WT mice, separately. Both cortical PNs and INs in TH KO mice had significantly lower firing activities than those in WT mice. Moreover, with L-dopa IP injection, the firing rate of 61% PNs (N=83/135) and 66% of INs (N=25/38) was significantly increased by 80% and 150%, respectively. 24% of PNs (N=32/135) and 16% of INs (N = 6/38) had correspond-ing significant decrease of 50% and 45% in firing rates after L-dopa IP injection. L-dopa 4μg microinjection in the unilateral striatum triggered a striking increase of c-Fos expression in the injected striatum and ipsilateral wide-spreading cortex as well as ipsilat-eral STN and motor thalamus but in contralateral SNr. 92.9% of the triggered striatal c-Fos protein was expressed in D1 MSN, and the c-fos expression in the motor cortex was in all cortical layers 1-6. In addition, the firing rate of 60% PNs (N=34/57) in the motor cortex was significantly increased by 275%, and 26% PNs (N=14/57) had a 62% decrease in fir-ing rates after striatal L-dopa microinjection. However, the effect of striatal L-dopa injection to spike activity of cortical INs was detected as statistically insignificant due to the insuffi-cient number of IN recorded (N=11). Furthermore, unilateral striatal L-dopa microinjection induced consistent contralateral. Both 0.4μg SKF81297 and 0.4μg quinpirole microinjection into the right striatum triggered significantly increased c-Fos expression in the injected striatum and ipsilateral cortex, GPe, STN, and motor thalamus. But they decreased c-Fos expression in the ipsilateral SNr in TH KO mice. Striatal 0.4μg SKF81297 microinjection excited 65% PNs (N=34/52) by an in-crease of 100% and inhibited 14% PNs (N=7/52) by a decrease of 50% in the motor cor-tex. However, the cortical INs didn’t have a statistically significant response to striatal SKF81297 microinjection because of the insufficient number of IN recorded (N=14). Stria-tal 0.4μg quinpirole microinjection significantly increased the firing rate of 66% PNs (N=23/35) and 71% INs (N=17/24). Unilateral striatal SKF81297 and quinpirole mi-croinjection induced consistent contralateral rotations, respectively.

Conclusions. In Parkinson’s disease, loss of DA innervation and DA receptor activity in the striatum reduces and impairs neuronal activity in the thalamus and cerebral cortex. Stria-tal D1R activation enhances the neuronal activity in the thalamocortical circuits by activating striatal D1-MSNs; striatal D2R activation induces D2-MSN inhibition and also enhances the neuronal activity in the thalamocortical circuits in PD. Thus, striatal DA receptor activity is required for the normal circuitry operation of the cerebral cortex and brain’s motor and cognitive functions.

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

0009-0004-8558-6879

DOI

10.21007/etd.cghs.2024.0675

Additional Files
2024-014-Wang-DOA.pdf (420 kB)
Declaration of Authorship
Download

Available for download on Thursday, July 23, 2026

Share

COinS