Pharmacogenetic Activation Of Midbrain Dopamine Neurons Produces Hyperactivity Of DAT-Cre Mouse

[Objective]Dopamine (dopamine, DA) is a catecholamine neurotransmitter, DA has an extremelyimportant role in the central nervous system, dopamine neurons regulate and control manyimportant behavioral processes, including motor activity, cognition, reward, emotion,consciousness and memory. Consistently, Regulation of brain dopamine concentration canchange the level of animal activity. Dopamine in the brain is mainly synthesized by the threeDA neuron nuclei: the ventral tegmental area (ventral tegmental area, the VTA, there areabout25,000DA neurons), substantia nigra compacta (zona compacta of the substantia nigra,the SNc)(10000DA neurons) and hypothalamus and olfactory bulb. Middle undeterminedwith other DA neurons located in the hypothalamus (medial zona incerta)(about900DAneurons). Previous studies mainly through the dopamine transporter in the area of themidbrain VTA/SNc dopamine neurons as well as drug and can not be directly confirmedmidbrain dopamine neuron activity and high animal activity. The study DREADD (DesignerReceptors Exclusively Activated by Designer Drugs DREADD,) technology selectivelyactivate the midbrain dopamine neurons.This study is induced through the stereotacticinjection Cre recombinase adeno-associated virus (AAV), the modified M3muscarinicreceptor (hM3Dq) expression in DAT-Cre mice VTA/SNc, and DA neurons activated byintraperitoneal injection hM3Dq ligand with clozapine,-N-oxide (CNO). Acute activation ofVTA/SNc DA neurons can immediately produce high activity. Further confirmed that thedopamine system in the brain and regulation of animal activity level of direct contact.[Methods]1. Stereotactic injection： AAV viral were bilaterally injected into the VTA/SNC areas ofDAT-Cre mice to express the evolved human muscarinic receptor hM3Dq.2. Recordings: To examine whether CNO can excite midbrain dopamine neurons, wefirst carried out target recordings from hM3Dq/mCherry+dopamine neurons from brain slices(n=6cells). We further tested whether CNO could activate neurons in the VTA/SNc in vivoby using c-Fos expression as the indicator of neuronal activation.3. Immunostaining: By using the bicistronically expressed mCherry as the marker of hM3Dq expression and TH-immunoreactivity as the marker of dopamine neurons.4. Open-field test:We analyzed the dose dependency of the CNO induced hyperactivity.Mice expressing hM3Dq/mCherry or mCherry were given CNO at the doses of0,0.5,1.0and2.0mg/kg in consecutive days. Thirty minutes after injection, animal activity levels in anopen field were measured for15min.5.24-hour motor activity monitoring: Mice expressing hM3Dq/mCherry or mCherrywere injected with either saline or CNO (1mg/kg, i.p.) at the onset of dark phase and theirlocomotor activity in a test arena was video-recorded and online analyzed for24h.[Results]1. Recordings: we first carried out target recordings from hM3Dq/mCherry+dopamineneurons from brain slices (n=6cells). Cell-attached recordings showed that pressureinjection of CNO (10μmol/L) elicited vigorous firing of action potentials (Fig.2A).Whole-cell current-clamp recordings demonstrated that CNO depolarized these neurons andevoked action-potential firing. As a control, CNO did not evoke responses from DAT-Cremice injected with AAV DIO mCherry virusImmunostaining detection of c-Fos signal, CNO induced c-Fos signals in many neuronsin the VTA/SNc area of DAT-Cre mice injected with hM3Dq/mCherry vectors but not in thoseinjected with mCherry vectors.2. Immunostaining: we found that~78%of dopamine neurons were infected with AAVvirus and produced clear mCherry signals(425±75mCherry+neurons out of540±75TH+neurons from one coronal section of each of seven mice examined), a vast majority ofmCherry+neurons (>95%) were also TH-immunopositive.3. Open-field test:The activity levels of hM3Dq mice were further enhanced by injecting1.0mg/kg CNO and reached plateau at the CNO dose of2.0mg/kg. the ratio of time in centralexploration was not affected by hM3Dq expression. CNO at the dose of0.5mg/Kgsignificantly increased the activity levels of hM3Dq-expressing mice (P <0.001; paired t-testbetween saline and CNO; n=12mice) but not mCherry-expressing mice (P=0.59; pairedt-test between saline and CNO; n=12mCherry-expressing mice)4.24-hour motor activity monitoring: An over eight-fold increase was produced duringthe initial hour of drug injection. In contrast, CNO did not produce any clear effect on mice injected with mCherry control vectors. The statistical differences were significant.[Conclusions]This study demonstrates that the use of the Cre-induced the AAV virus hM3Dq/mCherrycan be expressed in midbrain dopamine neurons. Pharmacological method to selectivelyactivate the midbrain dopamine neurons can lead to DAT-Cre mice significantly high activity.Further confirmed that the dopamine system in the brain and regulation of animal activitylevel of direct contact.