ANALYSIS OF DOPAMINE TRANSPORTER-EXPRESSING NEURON-SPECIFIC TYROSINE HYDROXYLASE KNOCKOUT MICE
Authors: Fujita *Masayo , Ide Soichiro , Okitsu Masato , Ikeda Kazutaka 
Published: February 2025

Abstract

Background
Tyrosine hydroxylase (TH), an enzyme synthesizing L-dopa from tyrosine, expresses dopaminergic and noradrenergic neurons. Dopamine transporter (DAT) expresses specifically in synaptic terminals of dopaminergic neurons and re-uptakes extracellular dopamine. Although DAT is a specific dopaminergic marker, it has been suggested that dopaminergic neurons that express no or very slight levels of DAT (DAT-negative dopaminergic neurons) may also exist in the midbrain. However, it is not known whether DAT negative neurons really exist and the role of DAT negative neurons.

Aims & Objectives
We generated DAT-expressing neurons-specific TH knockout mice to evaluate the existence and the role of DAT-negative dopaminergic neurons.

Method
We generated TH-floxed mice by inserting loxP sequence in front of and behind exon 3 of TH gene. TH-floxed mice were crossed with transgenic (Tg) mice which express tamoxifen inducible cre recombinase under the control of DAT promoter (DAT-ER Cre Tg mice). Generated TH-floxed/DAT-ER- Cre Tg mice received 100 mg/kg tamoxifen for continuous 5 days after they became 8 weeks old. Motor function was analyzed by rotarod test, catalepsy test and open field test more than 4 weeks after the last day of tamoxifen injection. Brain samples were collected after motor function tests to evaluate TH expression levels.

Results
TH-floxed/DAT-ER-Cre Tg mice received 100 mg/kg tamoxifen for continuous 5 days did not show motor impairment. Although TH immunoreactivity was slightly decreased in the striatum, many TH- positive neurons were observed in the substantia nigra and the ventral tegmental area. These results indicate that 1 cycle of tamoxifen treatment was not enough to completely knock out TH-expressing DAT-positive neurons. Therefore, we conducted 2 more cycles of tamoxifen injection. TH-floxed/DAT- ER-Cre Tg mice that received 3 cycles of tamoxifen injection showed significant motor function impairment. In addition, few TH-positive neurons were observed in the substantia nigra. These results indicate that the mice that received 3 cycles of tamoxifen injection can be considered as DAT-THKO mice. Interestingly, TH-positive fibers and cells were observed in the prefrontal cortex, the medial nucleus accumbens shell, and the ventral tegmental area. These fibers and cells were DAT and dopamine beta hydroxylase negative, indicating that remaining TH-positive fibers and cells were not derived from remaining DAT-positive TH neurons or noradrenergic neurons.

Discussion & Conclusion
We could generate DAT-THKO mice by 3 cycles of tamoxifen injection and showed existence of DAT-negative dopaminergic neurons. DAT-THKO mice showed significant motor impairment, suggesting DAT-positive dopaminergic neurons regulate motor function. DAT-negative dopaminergic neurons were observed in the ventral tegmental area. These neurons may innervate specifically to the prefrontal cortex and the nucleus accumbens shell and regulate functions other than motor control.