Role of vesicle pools in action potential pattern-dependent dopamine overflow in rat striatum in vivo.

Action potential (AP) patterns and dopamine (DA) release are known to correlate with rewarding behaviors, but how codes of AP bursts translate into DA release in vivo remains elusive. Here, a given AP pattern was defined by four codes, termed total AP number, frequency, number of AP bursts, and interburst time [N, f, b, i].. The 'burst effect' was calculated by the ratio (γ) of DA overflow by multiple bursts to that of a single burst when total AP number was fixed. By stimulating the medial forebrain bundle using AP codes at either physiological (20 Hz) or supraphysiological (80 Hz) frequencies, we found that DA was released from two kinetically distinct vesicle pools, the fast-releasable pool (FRP) and prolonged-releasable pool (PRP), in striatal dopaminergic terminals in vivo. We examined the effects of vesicle pools on AP-pattern dependent DA overflow and found, with given 'burst codes' [b=8, i=0.5 s], a large total AP number [N = 768, f = 80 Hz] produced a facilitating burst-effect (γ[b8/b1] = 126 ± 3%), while a small total AP number [N=96, 80 Hz] triggered a depressing-burst-effect (γ[b8/b1] = 29 ± 4%). Furthermore, we found that the PRP (but not the FRP) predominantly contributed to the facilitating-burst-effect and the FRP played an important role in the depressing-burst effect. Thus, our results suggest that striatal DA release captures pre-synaptic AP pattern information through different releasable pools.

A quantitative DNA methylation assay using mismatch hybridization and chemiluminescence.

OBJECTIVE: To develop a quantitative method for methylation analysis of the p16 gene based on mismatch hybridization and chemiluminescence. METHODS: Genomic DNA was modified by sodium bisulfite to convert all unmethylated but not methylated cytosines to uracil, and subsequently a pair of primer having no CpG sites was designed for amplification target DNA containing methylated or unmethylated CpG sites. The PCR product spanning CpG sites were hybridized with two oligonucleotide probes which perfectly matched the methylated and unmethylated CpG sequences respectively, and the hybrids were detected by chemiluminescent method. The percentage of methylated target sequences could be estimated by calculating the ratio of signals obtained with two probes. RESULTS: The percentage of methylation of artificial mixtures DNA showed a linear relation. There was a negative correlation between the methyaltion index with p16 transcriptional mRNA of p16 gene in tumor cell lines. CONCLUSION: Compared with existing methods, this assay is nonisotopic, rapid, simple, and can be widely applied to the study of DNA methylation.

Impulse-dependent extracellular resting dopamine concentration in rat striatum in vivo.

The ambient resting dopamine (DA) concentration in brain regulates cognition and motivation. Despite its importance, resting DA level in vivo remains elusive. Here, by high-frequency stimulation of the medial forebrain bundle and immediately following the stimulus-induced DA overflow, we recorded a DA "undershoot" which is a temporal reduction of DA concentration to a level below the baseline. Based on the DA undershoot, we predicted a resting DA concentration of ∼73nM in rat striatum in vivo. Simulation studies suggested that removing basal DA by DAT during the post-stimulation inhibition of tonic DA release caused the DA undershoot, and the resting concentration of DA modulated the kinetics of the evoked DA transient. The DA undershoot was eliminated by either blocking D2 receptors with haloperidol or blocking the DA transporter (DAT) with cocaine. Therefore, the impulse-dependent resting DA concentration is in the tens of nanomolar range and is modulated by the presynaptic D2 receptors and the DAT in vivo.

Nicotine dynamically modulates dopamine clearance in rat striatum in vivo.

Nicotine binds to nicotinic acetylcholine receptors on dopaminergic terminals to evoke dopamine (DA) release. The clearance of released DA occurs rapidly through reuptake into nerve terminals through the DA transporter (DAT). However, whether nicotine modulates DAT function in vivo is still not well understood. In the present study, we determined the effect of nicotine on DA clearance using in vivo amperometric recording in the striatum of urethane-anesthetized rats. Stable DA release was evoked by electrical stimulation of the medial forebrain bundle (MFB). Subsequently, nicotine or saline was administered with MFB stimulation at 10-min intervals for 60 min. Kinetic analysis revealed that nicotine decreased the amplitude of DA overflow and the maximal DA clearance rate (V(max)) in response to stimulation of 96 pulses at 80 Hz. Surprisingly, nicotine enhanced the maximal DA clearance rate (V(max)) by stimulation of 768 pulses at 80 Hz. Furthermore, we found that this paradoxical effect of nicotine on V(max) depended on the stimulation pattern. These results suggest that nicotine may exert its addictive role by dynamically modulating DAT function in vivo.