The VTA in horizontal midbrain slices is identified and recognized as the area medial to the substantia nigra compacta and medial to terminal nucleus of the accessory optic tract. Additionally, a clear hyperpolarization-activated cation current (Ih) emerges after hyperpolarizing the VTA DA neuron from -70 to -150 mv, in 10 mv step size, immediately after break-in was observed in each recorded neuron. Ih was shown to be a reliable marker for VTA DA neurons [32–34]. A recent report has questioned the identification of VTA DA neurons using Ih. However, in previous studies [6, 7, 34, 36] and in the present study, this criterion was sufficient to provide necessary identification.
The disruption between PFC and VTA was observed at the time the brain was removed from the skull. Once the brain has been removed from the skull, the area immediately caudal to PFC has been observed to be cut. This indicates the PFC has completely lost its connection with the rest of the brain.
Previous studies had reported that single nicotine injection could enhance the peak ratio of VTA DA neurons AMPA/NMDA receptors responses within 24 hours, even after nicotine metabolized . Moreover, Mansvelder and McGehee reported brief nicotine application rapidly induced LTP and maintained for more than one hour in VTA slice . Additionally, the positive correlation between glutamatergic synaptic enhancement and behavioral locomotion were well described . In vivo experiments also showed that following nicotine application, dopamine release to NAc and the locomotion activity of rats both would peak within one hour . These evidences led us to predict that, within only one hour after single nicotine injection, the postsynaptic response will be changed. Our hypotheses are supported by the results, since we found that even within one hour after a single nicotine injection, the peak ratio of AMPA/NMDA on VTA DA neurons could be enhanced. However, the peak ratio increase observed within one hour was lower in comparison to 24 hours after a single nicotine injection . This suggests that, after one hour of a single systemic nicotine injection, not all subset of synapses were potentiated.
This finding was also supported by previous reports that the AMPA/NMDA ratio enhancement was observed two hours after a single cocaine injection  and observed in another study two hours after a single amphetamine injection .
The PFC is a key structure for executive functions of the brain [38, 39] and has been shown to regulate the firing pattern of dopamine (DA) neurons in the VTA. Gao et al  stated that there is an indirect coupling between PFC and VTA. Thus, PFC stimulation increases burst firing in DA neurons, whereas PFC inactivation produces the opposite effect [40–44].
Glutamate transmission from PFC to VTA is important in controlling VTA DA neurons firing activities and animal behavior [11–15]. Treatments of most drugs of abuse has been reported to increase excitatory inputs to the midbrain, which is thought to contribute, impartially, to the enhancement of VTA DA neurons AMPA/NMDA ratio. All the evidences suggested it is a common mechanism of addiction through neural adaptation [6, 7].
The disconnection of the functional pathway between PFC and VTA could significantly reduce the effects of drugs of addiction, including nicotine [9, 28, 45]. We recently have demonstrated in in vivo experiments that the responses of main VTA DA neurons to acute nicotine injection are greatly changed after the PFC transaction [23, 24]. Based on these, in this study, we transected the PFC and examined the changes of AMPA/NMDA peak ratio of VTA DA neurons. Interestingly, without the intact input from PFC, the AMPA/NMDA ratio was still enhanced by nicotine injection.
Like LTP, AMPA/NMDA ratio alteration reflects the plasticity change in synapse. Normally these changes are caused by either increase in excitatory input or decrease in inhibitory input. In VTA, DA neurons receive excitatory inputs from PFC and the inhibitory inputs from GABAergic interneuron in VTA and/or NAc, which also should have functional coupling with PFC. The openings of GluR were changed after nicotine treatments, via the regulation from PFC to VTA DA neurons, that induced EPSCs. After PFC transection, the signals induced by nicotine could not be transferred from PFC to VTA adequately, it may be the result of the different alteration to GluR on VTA DA neurons. Measurement of AMPA/NMDA peak ratio only takes into account the maximum GluR response. However, the AMPA and NMDA curves represent the whole GluR response with respect to time. Therefore, we estimated the AMPA/NMDA area ratio and KL divergence to better understand the dynamics of AMPA and NMDA signals since they took into consideration the whole current response rather than just the peak response. With these two methods, we found that there is statistical significance between PFC intact and PFC transected rats with saline treatments as seen in Figures 4C and 5C. This is not observed when measuring the AMPA/NMDA peak ratio. One possible reason for the observed differences may be due to the loss of glutamatergic inputs from PFC, which is one of the most important factors that contribute to the ratio level .
The use of traditional analysis method of AMPA/NMDA peak ratio suggests the PFC is not a "must" area and the ratio enhancement could occur locally in VTA. Previous studies showed that in vitro exposure of VTA slices to amphetamine did not enhance AMPA/NMDA ratio . However, local injection of amphetamine to the VTA in vivo triggered sensitization [47, 48]. This suggests that the enhancement effects should be triggered in VTA and need other areas to provide functional feedback, increase the excitatory input and/or reduce the inhibitory input.
However, the use of different analysis methods based on AMPA/NMDA area ratio and KL divergence, show that PFC is suggested to play an important role in affecting the VTA DA neurons. These two methods took into account the dynamics of AMPA and NMDA signals of the complete response and not just the maximum, making them more suitable in understanding the effects caused by nicotine and PFC transection.