The neural basis of positive reinforcement is often studied in the laboratory using intracranial self-stimulation (ICSS), a simple behavioral model in which subjects perform an action in order to obtain exogenous stimulation of a specific brain area. Recently we showed that activation of ventral tegmental area (VTA) dopamine neurons supports ICSS behavior, consistent with proposed roles of this neural population in reinforcement learning. However, VTA dopamine neurons make connections with diverse brain regions, and the specific efferent target(s) that mediate the ability of dopamine neuron activation to support ICSS have not been definitively demonstrated. Here, we examine in transgenic rats whether dopamine neuron-specific ICSS relies on the connection between the VTA and the nucleus accumbens (NAc), a brain region also implicated in positive reinforcement. We find that optogenetic activation of dopaminergic terminals innervating the NAc is sufficient to drive ICSS, and that ICSS driven by optical activation of dopamine neuron somata in the VTA is significantly attenuated by intra-NAc injections of D1 or D2 receptor antagonists. These data demonstrate that the NAc is a critical efferent target sustaining dopamine neuron-specific ICSS, identify receptor subtypes through which dopamine acts to promote this behavior, and ultimately help to refine our understanding of the neural circuitry mediating positive reinforcement. Actions that lead to beneficial outcomes are more likely to be repeated than those that do not. This process, whereby the probability of a behavioral response increases as a consequence of the outcome of that response, is referred to as positive reinforcement. ICSS is a simple behavioral model that distills positive reinforcement to its minimum neural elements. In ICSS paradigms, subjects make instrumental responses in order to deliver stimulation to a specific brain area. Sites containing dopamine neurons or their ascending projections are particularly effective in eliciting this behavior Importantly, VTA dopamine neurons send projections to many brain areas, and the specific efferent targets that support ICSS driven by optogenetic activation of dopamine neurons have not been demonstrated. Prior efforts to establish efferent targets that mediate ICSS employed electrical stimulation to reinforce operant responding We sought to determine whether VTA dopamine neuron-driven ICSS was mediated by the NAc and, if so, which dopamine receptors were involved. We relied on two complementary experimental approaches to address these questions. First, we optogenetically activated VTA dopamine neuron axon terminals innervating the NAc to determine if selective activation of this pathway would support ICSS. Next, we used targeted infusions of dopamine receptor antagonists into the NAc during ICSS behavior driven by optogenetic activation of dopaminergic somata in the VTA. We found that activation of the VTA dopamine neuron projection to the NAc was sufficient to support ICSS, and that ICSS behavior mediated by VTA dopamine neurons was significantly reduced by antagonism of either D1 or D2Rs in the NAc. Taken together, these results add to a growing body of evidence implicating dopaminergic transmission in the NAc as an important element of the neural circuitry mediating positive reinforcement.Th::Cre+, n = 19), allowing for selective targeting of dopamine neurons as described previously Th::Cre-, n = 10) were used as controls. All rats weighed >300 g at the time of surgery and were individually housed with free access to food and water. Animal care and all experimental procedures were in accordance with guidelines from the National Institutes of Health and approved in advance by the Gallo Center Institutional Animal Care and Use Committee. Surgical procedures were conducted under isoflurane anesthesia and all necessary precautions were taken to minimize animal suffering.29 male transgenic rats (on a Long-Evans background) were used in these studies. These rats expressed Cre recombinase under the control of the tyrosine hydroxylase promoter  and implant optical fibers. A total of 4 µL of virus was infused into the VTA at AP −5.4 and 6.2, ML ±0.7; at each AP site 1 µL virus was delivered (0.1 µL/min) at both DV −8.4 and −7.4. The infuser was left in place for an additional 10 min to allow for diffusion. An optical fiber  was chronically implanted dorsal to the NAc  ipsilateral to the virus infusion. All coordinates are in mm relative to bregma and skull surface.Standard stereotaxic surgical procedures were used to unilaterally infuse Cre-dependent virus  contained within sound-attenuating cubicles. Session start was indicated to the rat by the illumination of a chamber light and the onset of low-volume white noise (65 dB) to mask external sounds. Two nosepoke ports, designated “active” and “inactive”, were positioned on the left chamber wall; each had three LED lights at the rear. A response at the active nosepoke port resulted in optical stimulation  on a fixed-ratio 1 schedule, with the exception that a new stimulation train could not be earned until any ongoing train had finished. The LED lights in the recess of the active port were illuminated concurrent with stimulation. We chose to include a response-contingent cue in our experimental design because such cues have been show to facilitate robust operant responding over long periods in drug self-administration studies Subjects in experiment 2 received surgery as described above except that the optical fiber was targeted dorsal to the VTA  instead of the NAc. Additionally, 26 gauge cannulae (Plastics1) were implanted bilaterally dorsal to the NAc . Infusers extended 2 mm past the end of the guide cannula, for a final DV of −6.8.Subjects in experiment 2 underwent ICSS training as described above except that instead of single daily 120 min sessions, subjects were given a 60 min baseline session, removed from the chamber for drug infusions, and then returned for a further 60 min test session to assess drug effects on behavior. This was done because while ICSS responding was stable within a single day, the absolute magnitude of behavior emitted was variable across multiple days, even with extended training.Subjects in experiment 2 received targeted intracranial drug infusions into the NAc once ICSS behavior was established (at least 4 training sessions prior to drug administration). The following drugs were used: (1) flupenthixol, a non-selective dopamine receptor antagonist dissolved in water ; (2) SCH23390, a D1R-selective antagonist dissolved in saline ; (3) Raclopride, a D2R-selective antagonist dissolved in saline  or (4) saline control. Drug doses were chosen based on studies that have previously demonstrated a lack of non-specific locomotor impairments 2 . Light power was measured before and after every behavioral session to ensure that all equipment was functioning properly.Prior to all behavioral sessions, rats were gently attached to custom-made optical cables  encased in durable metal covering . The cables were secured to the rat's implanted optical fiber with a ceramic sleeve  and attached at the other end to an optical commutator (Doric Lenses). The commutator was mounted on a counterbalanced lever arm to facilitate unhindered behavioral responding, and connected via a second cable to a 100–150 mW DPSS laser (OEM Laser Systems). Light output during individual light pulses was estimated to be ∼2 mW at the tip of the intracranial fiber. This value was derived by measuring the average light power when the laser was pulsed at the parameters used for our experiments  and then correcting for the duty cycle . Based on this value we estimate that light power density at the tip of the fiber was ∼7 mW/mmTh::Cre+ and Th::Cre- groups. In experiment 2, the effect of drug infusions on active nosepoke responding was assessed by expressing post-drug responding as a percentage of a pre-drug baseline value, and the numbers of c-Fos+ cells were compared between Th::Cre+ and Th::Cre- groups. Parametric (one- or two-way ANOVA followed by post-hoc Student-Newman-Keuls tests) or non-parametric  tests were used where appropriate.In experiment 1, the total number of active and inactive nosepoke responses made across multiple training days was compared within and between Immunohistochemical detection of TH and YFP was performed in all subjects used in Experiments 1 and 2 as described previously Th::Cre+ rats were imaged using identical magnification and exposure settings on a confocal microscope. The portion of the image containing the VTA was manually isolated as a region of interest and fluorescence intensity was calculated in this area using imageJ software.For the quantification of ChR2-YFP expression (measured as fluorescence intensity) in experiment 1, VTA sections from Th) promoter (Th::Cre rats) in order to gain selective control over dopamine neuron activity Th::Cre+ rats (Th::Cre+ rats or their wild-type (Th::Cre-) littermates were chronically implanted with an optical fiber targeting the NAc ; however, variability in virus expression may account for the lack of a between-group effect. In support of this, ChR2 expression strength in the VTA of Th::Cre+ rats was significantly correlated with total responses made at the active port (2 = 0.482), and the Th::Cre+ rats that displayed above-average expression of ChR2 (n = 4) performed significantly more active nosepokes than Th::Cre- rats on day 4 . Thus, optical activation of the dopaminergic projection to the NAc is sufficient to support ICSS, confirming an important role for this pathway in the neural basis of positive reinforcement.We initially set out to determine whether selective activation of dopaminergic axon terminals innervating the NAc would be sufficient to support ICSS. We performed our experiments in a recently developed transgenic rat line where Cre recombinase expression is driven by the tyrosine hydroxylase  = 6.771, p = 0.343, 6,34 = 6.414, p<0.001, 2(6) = 5.829, p = 0.443; Next, we combined our optogenetic approach with pharmacological tools that allowed us to assess the contribution of dopamine acting on specific dopamine receptor subtypes to ICSS behavior. tructure , 4A. Addtructure , 4A. Afttructure . Drug efInterestingly, subsequent analyses demonstrated that ipsilateral or contralateral dopamine antagonist infusions  caused similar decreases in ICSS behavior . This finding was surprising, since the dopaminergic projection from the VTA to the NAc is thought to be almost exclusively unilateral Th::Cre+ rats (n = 4) and Th::Cre- rats (n = 3) performed a mean ± SEM of 8063±151 and 6±4 active nosepokes, respectively; the number of c-Fos+ cells in the NAc and VTA was counted by an experimenter blind to the subject's genotype. We observed significantly more c-Fos+ cells in the NAc of Th::Cre+ rats as compared to Th::Cre- controls , although overall c-Fos expression was higher ipsilaterally in Th::Cre+ rats . In the VTA, we observed a trend towards increased c-Fos expression in Th::Cre+ rats . C-Fos+ cells in the VTA often co-expressed TH and ChR2-YFP , these effects were statistically indistinguishable when the data were considered collectively. This similarity in magnitude is intriguing given clear inter-hemispheric differences in ChR2 and c-Fos expression. Critically, the pharmacological actions of dopamine antagonists reported here would be expected to block all effects of dopamine, whether released by optical stimulation or endogenous neural processing. It is possible that endogenous dopamine release must be intact in both hemispheres to permit normal ICSS behavior, although this idea is not supported by prior work which has demonstrated that ICSS behavior for an electrical stimulation reinforcer is minimally affected by unilateral lesions of ascending dopaminergic projections Our results are in accord with a rich literature implicating VTA dopamine neurons, and their major efferent projection to the NAc, in reward-related behaviors Th::Cre+ mice; it is not clear which procedural, or other, variables account for this difference. However, all of the above mentioned efforts have focused on the behavioral effects of manipulating a mixed population of dopamine neurons with diverse projection targets. In contrast, the experiments described here were designed to isolate the contribution of a specific dopaminergic projection (VTA to NAc) to behavior. Because dopamine neurons are embedded in a complex and multifunctional circuitry, such pathway-specific approaches are essential in developing a detailed understanding of the ways in which this important neural population contributes to behavior.Other recent studies have also used optogenetics to examine the contributions of midbrain dopamine neurons to positive reinforcement and learning Midbrain dopamine neurons are known to co-release other neurotransmitters and peptides in addition to dopamine, and these molecules may be important mediators of the signals relayed by dopamine neurons to the rest of the brain The present findings indicate that the VTA to NAc projection is positively reinforcing in that it can support acquisition and performance of ICSS; these studies do not determine the distinct behavioral mechanisms that may contribute to this effect. The behavioral procedure we used in the present study was designed such that each nosepoke that resulted in dopamine neuron stimulation also resulted in simultaneous presentation of a visual cue within the nosepoke operandum. Thus, it remains to be determined whether the optical stimulation reinforced the instrumental action, or via association of the stimulation with the cue, allowed the response-paired cue to act as a conditioned reinforcer. Of note, we recently showed that sucrose reward-paired dopamine neuron stimulation can promote conditioned responding to reward cues, in agreement with a role for dopamine as a reward prediction error signal in temporal difference learning (TDL) models


PubChem is an open repository for molecular structures, their properties and biological activities . The num


Then the newly developed GP-GRNN approach further reduces the RMS to 0.31 kcal mol−1. Thus, the GP-GRNN correction on top of B3LYP/6-31G (d) can improve the accuracy of calculating the homolysis BDE in quantum chemistry and can predict homolysis BDE which cannot be obtained experimentally.We propose a generalized regression neural network (GRNN) approach based on grey relational analysis (GRA) and principal component analysis (PCA) (GP-GRNN) to improve the accuracy of density functional theory (DFT) calculation for homolysis bond dissociation energies (BDE) of Y-NO bond. As a demonstration, this combined quantum chemistry calculation with the GP-GRNN approach has been applied to evaluate the homolysis BDE of 92 Y-NO organic molecules. The results show that the ull-descriptor GRNN without GRA and PCA (F-GRNN) and with GRA (G-GRNN) approaches reduce the root-mean-square (RMS) of the calculated homolysis BDE of 92 organic molecules from 5.31 to 0.49 and 0.39 kcal mol NO haet al. proposed the concept of NO affinity which can be defined as the measure of the strength for a receptor (X) to bind with the NO group m*m. Therein,(b) Calculate the correlation coefficient λi  of related matrix R and then, arrange the eigenvalue λi according to the descending order λ1 ≥ λ2 ≥ λ3 ≥ … ≥ λm ≥ 0. iZ, i.e., the weight of the principal component iZ.(c) Calculate the eigenvalue 2.3.GRNN was proposed by the American scholar D. F. Specht . The appX = T. The number of neurons in the input layer is equal to the dimensions of input vector p in the study sample. The number of neurons in pattern layer is equal to the number of study sample n. The transfer function of neuron n isX is the network’s input variable and δ is the smoothing factor which determines the shape of function. The larger the value is, the smoother the function is. iX is the corresponding study sample of neuron i. Each unit in the pattern layer corresponds to a training sample and the Gaussian function is treated as the activation of kernel function. Two types of neurons are used for summation in the summation layer, one isiy of the output sample in the pattern layer in which the weighted sum is adopted to work out the summation of the output of corresponding neurons. The output of neurons in the output layer isi.e., the output of network. In our paper, The output is Y which corresponds to the net input 3.3.1.N-nitrosamine compounds, O-nitrite, C-nitroso compounds, S-nitrosylation compounds (their molecular structures are shown in Ninety-two (92) important organic carrier molecules containing NO are studied in this work. They are the four typical NO carrier molecules in the acetonitrile solution: 3.2.All of the calculations were done using Gaussian03 . The geoH298–0 is the standard temperature correction term including Hvib, Hrot and Htrans.The zero point energy correction was taken into account in the calculation. ΔHhomo), the net charge (QY) on the Y atom which is bonded with the NO, the net charge (QN) and (QO) on the atoms N, O in the NO molecule fragment, the number of electrons (NX) on the fragment X , the molecule dipole moment (μ) and the molecule polarizability (α), the highest occupied molecular orbital and the lowest unoccupied molecular orbital energy (EHOMO) and (ELUMO), the second highest occupied molecular orbital and the second lowest unoccupied molecular orbital energy (EHOMO−1) and (ELUMO+1), the energy gap (ΔE) between EHOMO and ELUMO. The molecular descriptors can reflect covalent and ionic interactions in a molecule bond.A set of molecular descriptors can be obtained from the geometry optimization and frequency calculations. The molecular descriptors include the calculated homolysis BDE value  bond, the B3LYP function is used to optimize the geometry at 6-31G (d) basis set level and the frequency is calculated to confirm the stable structure. Finally, the homolysis BDE of Y-NO  bond and relevant molecular descriptors are obtained and shown in vice versa. Lower homolysis BDE indicates that the carrier can serve as a good NO releasing agent. The four types of carrier molecules studied in this paper have lower homolysis BDE and are excellent NO radical carriers in the body. From −1, 32.5–38.6 kcal mol−1, 17.2–29.2 kcal mol−1 and 27.5–31.4 kcal mol−1 respectively. The carrier molecule containing the S-NO bond has the lowest homolysis BDE which indicates that the carrier containing the S-NO bond is the best free NO radical carrier among the four compounds studied. The theoretical calculated homolysis BDE levels for the four compounds are at 12.63–35.57 kcal mol−1, 29.80–40.39 kcal mol−1, 17.49–27.96 kcal mol−1 and 17.69–24.62 kcal mol−1 respectively. Compared with the experimental values, the calculated value for the molecule carrier containing the N-NO bond is close to its experimental value with some underestimations and the calculated energy for the molecule carrier containing the O-NO and S-NO bonds is very close to their experimental values. The calculated energy for the molecule carrier containing the C-NO bond is lower than its experimental value.In addition, 4.2.vice versa.The experimenatal value of homolysis BDE of the 92 carrier molecules are used as the reference array. The 12 computed molecular descriptors are used as the contrast array. The closer the relation is to 1, the more relations the two arrays have and Hhomo, QY, QN, QO, NX, µ, α, EHOMO−1, EHOMO, ELUMO, ELUMO+1, ΔE are 0.8902, 0.6137, 0.7946, 0.8626, 0.8079, 0.7899, 0.8096, 0.8889, 0.8925, 0.8359, 0.7020, 0.8827 respectively. The relational coefficients of the 12 parameters on the experimental values in decreasing order are EHOMO, ΔHhomo, EHOMO−1, ΔE, QO, ELUMO, α, NX, QN, μ, ELUMO+1, QY. Clearly, the EHOMO and EHOMO−1 have a big impact on the homolysis BDE. The ELUMO has a certain influence on the homolysis BDE. The ELUMO+1 has little impact on the homolysis BDE. The relational coefficient is 0.8902 which indicates a good match between the theoretical calculations and the experimental values. The difference between the EHOMO energy level and ELUMO energy level ΔE can measure the stability of the molecules and has a large impact on the homolysis BDE. In the NO molecular fragments, the oxygen’s electronegativity is greater than that of nitrogen and the net charge of oxygen has a larger impact than that of the nitrogen on the homolysis. The net charge on the Y atoms which are connected to NO has the minimal impact on the homolysis which implies that the ionicity of chemical bonds is smaller. The polarization rate can measure the molecular deformation and affect the homolysis BDE. The number of electrons on the molecular fragment X which is connected to NO has a certain impact on the homolysis. The dipole moment is a vector that has little impact on the homolysis which indicates that the Y-NO direction has little impact on the molecular dipole moment.When the distinguishing coefficient is set to 0.5, Δ4.3.NX is as high as 0.9331. It is inevitable that increasing the complexity of data analysis, if these eight selected molecular descriptors are taken as the final attribute characteristics directly, there would be some problems such as instable calculation and ill-conditioned matrix which are caused by superposed information existing in the above eight molecular descriptors. Nevertheless, these problems can be avoided through PCA that can also make the weight distribution of the molecular descriptors more reasonable, avoid the redundant information, and eliminate the not useful information.According to the results of GRA, the molecular descriptors that have a relation of greater than 0.8 are selected. The first eight molecular descriptors are used as the basic characteristics. A correlation matrix is generated after the correlation analysis on the eight molecular descriptors . It can The PCA is performed on the eight selected molecular descriptors to obtain the eigenvalues, variance and cumulative variance contribution rate for each principle component . It can EHOMO−1 and ELUMO have a higher load in the first principal component, the ΔHhomo and ΔE on the second principal component have a higher load, the NX and α on the third principal component have a higher load, the ΔHhomo on the fourth principal component has a higher load, the QO on the fifth principal component has a higher load and the EHOMO on the sixth principal component has a higher load. Thus the assignment to the weights has the theoretical basis. Meanwhile, the variance between either two of six principal components is 0 which means that the six principal components are unrelated to each other. Therefore, the instability and ill-conditioned matrix problems can be avoided in the computation.The weights of the first six principal components and the corresponding molecular descriptors are shown in After the GRA and PCA approaches are performed for the molecular descriptors selection and optimization, the first six principal components are used as the final GRNN inputs. To assess the GP-GRNN approach’s effect on calculating the homolysis BDE of 92 organic molecules, we compare the GP-GRNN correction results with the B3LYP/6-31G (d) correction results, the correction results of the full-descriptor GRNN without GRA and PCA (F-GRNN) and with GRA (G-GRNN), respectively. The differences between the experimental and calculated homolysis BDE for the F-GRNN, G-GRNN and GP-GRNN correction results are tabulated in δ. In the training process, the learning algorithm is to adjust the transfer function of each unit to acquire the best results of regression estimation by changing δ, not by adjusting the connection weight between neurons.For GRNN, the initialization is to determine the study process of training samples. Then, the connection weight between the network structure and each neuron is determined after the determination of the study samples. The training process of network is just the process of determining δ is increased progressively from 0.02 to 1 by the constant of the variation of 0.02. The optimal output of neural network can be decided in the process of the variation of δ. For F-GRNN, G-GRNN and GP-GRNN approaches, when the values of δ are respectively 0.18, 0.08 and 0.10, the best results of regression estimation appear. For the training test, the RMS before correction is 5.40 kcal mol−1, and the RMS of F-GRNN and G-GRNN after correction is 0.48 and 0.38 kcal mol−1; however the RMS of GP-GRNN is 0.30 kcal mol−1. For the test set, the RMS respectively decreases from 4.69 to 0.55, 0.46 and 0.39 kcal mol−1  approaches the mean of experimental value of homolysis BDE in all samples. On the contrary, when the smoothing factor δ tends to 0, Y is close to training sample. When the points which need predicating are included in the training sample, the predictive value of the experimental value of homolysis BDE calculated by using δ is not the larger the better, nor the smaller the better. When the value of δ is moderate, all the experimental value of homolysis BDE in the training sample are taken into account and the experimenatal value of homolysis BDE corresponding to the sample points close to the predictive points add more weight. Hence, the value of δ is selected in this paper after many experiments with the aim to acquire the best network output. (2) Sample data is few in the training set and the features cannot be extracted in the training procedure of neural network. The prediction accuracy of GP-GRNN approach can be further improved as more and better experimental data are available. The consistency between the training and test set implied that the GP-GRNN results could indeed predict the homolysis BDE with higher accuracy than F-GRNN and G-GRNN.In the transfer function, the value of al mol−1 . The GP-−1. Upon the traditional F-GRNN correction approach, the RMS of the calculated homolysis BDE of the 92 organic molecules is reduced from 5.31 to 0.49 kcal mol−1 for the B3LYP/6-31G (d) calculation. With the G-GRNN and GP-GRNN correction, the RMS is reduced from 5.31 to 0.39 and 0.31 kcal mol−1, respectively (The B3LYP/6-31G (d) calculations are carried out to evaluate the homolysis BDE of the 92 organic molecules, and their overall resulting RMS from the experimental data is 5.31 kcal molectively .From 5.−1. Compared with the F-GRNN and G-GRNN, GP-GRNN is more feasible and effective. Further, the GP-GRNN correction on top of the B3LYP/6-31G (d) results is a better approach to correct homolysis BDE and can be used as the approximation of experimental results, when the experimental results are limited to measurement, with very high accuracy. GP-GRNN approach extends the B3LYP/6-31G (d)’s feasibility and applicability. The more experimental data the training set has, the more accurate the GP-GRNN approach will be. GP-GRNN approach can be not only used to calculate the homolysis BDE, but also it can be applied to calculate the heterolysis BDE, absorption energy, ionization energy, formation heat and so on. In summary, the GP-GRNN approach is an effective and predictive tool that can be used in the study of physical and chemical properties at the molecular level.GP-GRNN approach was successfully used to improve the homolysis BDE calculation’s accuracy. GRA is used to select the appropriate molecular descriptors. PCA is used to optimize the selection of molecular descriptors. GRNN is used to establish non-linear model. The GP-GRNN approach reduced the calculated RMS of 92 organic molecules from 5.31 to 0.31 kcal mol


For a long time, the prediction of molecular crystal structures from first principles has been one of the most challenging computational problems. Because of the large number of degrees of freedom, methods with low computational effort have to be used. The price for this efficiency is, however, a loss of accuracy. In recent years, important progress has been made in the development of dispersion corrections -3, whichBased on these developments, the software package GRACE  was the In the most recent, 5th blind test, organized in 2010, the excellent agreement between experimental and predicted structures for small, neutral molecules was confirmed. However, it turned out that the crystal structure prediction of molecular salts and hydrates is still a challenging task. The inability of DFT without exact exchange to describe anions properly, as recently published by Jensen , suggest


However, the underlying neuronal mechanisms are unclear. Here we present an overview of some of the recent research on this topic. Electrophysiological studies reveal that DA neurons in the pVTA are a target of salsolinol. In acute brain slices from rats, salsolinol increases the excitability and accelerates the ongoing firing of dopamine neurons in the pVTA. Intriguingly, this action of salsolinol involves multiple pre- and post-synaptic mechanisms, including: (1) depolarizing dopamine neurons; (2) by activating μ opioid receptors on the GABAergic inputs to dopamine neurons – which decreases GABAergic activity – dopamine neurons are disinhibited; and (3) enhancing presynaptic glutamatergic transmission onto dopamine neurons via activation of dopamine type 1 receptors, probably situated on the glutamatergic terminals. These novel mechanisms may contribute to the rewarding/reinforcing properties of salsolinol observed in vivo.Salsolinol, a tetrahydroisoquinoline present in the human and rat brains, is the condensation product of dopamine and acetaldehyde, the first metabolite of ethanol. Previous evidence obtained  Alcohol/Ethanol is one of the most widely used drugs and the global burden of alcoholism is immense, with an estimated 3.8%  has been extensively studied as a target for the central effects of alcohol  as the MOR agonist DAMGO and MOR antagonist naltrexone significantly attenuated the increase in firing induced by ethanol and even altered the basal firing rate of the DA neurons, indicating ongoing opioid modulation   and D2-like  families. Both D1R and the D2R family (D2R in particular) have been implicated in the mechanisms of drug dependence and abuse  in the VTA was eliminated when dopamine was depleted by pretreatment with reserpine (Deng et al., in vitro findings in combination with in vivo experiments by other groups—reviewed here—identify cellular mechanisms underlying SAL's psychoactive effects on the mesolimbic dopamine system. SAL's stimulating action in the pVTA involves modulation of synaptic inputs and intrinsic properties of DA neurons (schematically depicted in Figure in vivo. Understanding how SAL affects the activity of VTA dopamine neurons could have profound implications for the prevention and treatment of alcoholism.Our The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


PPP1R1B  and whether the genetic effect may differ between age groups. We observed larger P2 amplitudes in individuals carrying the genotype associated with higher dopamine receptor efficacy, i.e., a allele homozygotes of a single nucleotide polymorphism (rs907094) of the PPP1R1B gene. Moreover, this effect was more pronounced in children and older adults in comparison to adolescents and younger adults. Together, our findings indicate that polymorphic variations in a dopamine relevant gene are associated with individual differences in brain-evoked potentials of outcome updating and hint at the possibility that genotype effects on neurocognitive phenotypes may vary as a function of brain maturation and aging.Maximizing gains during probabilistic reinforcement learning requires the updating of choice – outcome expectations at the time when the feedback about a specific choice or action is given. Extant theories and evidence suggest that dopaminergic modulation plays a crucial role in reinforcement learning and the updating of choice – outcome expectations. Furthermore, recently a positive component of the event-related potential about 200 ms (P2) after feedback has been suggested to reflect such updating. The efficacy of dopaminergic modulation changes across the life span. However, to date investigations of age-related differences in feedback-related P2 during reinforcement learning are still scarce. The present study thus aims to investigate whether individual differences in the feedback-related P2 would be associated with polymorphic variations in a dopamine relevant gene  Flexible control of behavior requires the representation, maintenance, and updating of goal-relevant context information. For instance, in real-life situations of choosing between stocks, even though fluctuations in the markets are highly unpredictable, the profit histories, and risk profiles of different stocks are common context information that are considered when making choices. In laboratories, maximizing gains during probabilistic reinforcement learning requires the updating of choice–outcome expectations at the time when feedback about a specific choice is given. Studies using electroencephalography (EEG) have identified event-related potentials that are related to reinforcement learning  as well as subcortical and midbrain regions is less effective during childhood and old age in comparison to other life periods. For instance, there is ample evidence for aging-related declines of pre- and postsynaptic markers of the dopamine system  to investigate whether polymorphic variations in a dopamine relevant gene may contribute to individual differences in the feedback-related P2 amplitude; (ii) to investigate whether dopamine relevant genotype effects on the feedback-related P2 may interact with age, given that the efficacy of dopamine modulation changes across the lifespan.PPP1R1B, protein phosphatase 1, regulatory inhibitor subunit 1B), which is richly expressed in the striatum. The DARPP-32 protein is phosphorylated by dopamine D1 receptor stimulation, and dephosphorylated by D2 receptor stimulation  in the PPP1R1B gene affects the mRNA expression of the protein. A homozygosity of the PPP1R1B gene is associated with higher mRNA expression and higher dopamine receptor efficacy. Furthermore, individuals carrying haplotypes of the DARPP-32 gene associated with greater mRNA expression show greater changes in BOLD signal in the striatum, greater frontal-striatal connectivity during cognitive performance, as well as better cognitive performance on executive functions . DNA was extracted from saliva using standard techniques. TaqMan probes for the single nucleotide polymorphism (SNP) genotyping were designed and synthesized by Applied BioSystems . The SNP rs907904 for the PPP1R1B gene was selected based on previous studies  in our sample and were in the Hardy–Weinberg equilibrium, χ2(1) = 3.39, p > 0.05. Given that the frequency of the reference allele (G) is low, similar to previous reports  to AG and GG carriers combined .Saliva was collected from the participants using the OrageneDuring EEG recordings, participants were comfortably seated in an electrically and acoustically shielded room. The distance to the computer screen was 80 cm. In a reinforcement learning task, participants were presented with pairs of Japanese characters that were each associated with probabilistic gains and losses. Feedback was presented 500 ms after the choice. However, within each pair, one symbol had a higher probability of leading to a gain than the other symbol , using BrainVision Recorder. The sampling rate was 1000 Hz with a bandpass filter applied in the range of 0.01–250 Hz. EEG recordings were referenced online to the right mastoid. The ground was positioned above the forehead. Impedances were kept below 5 kΩ. Vertical and horizontal electrooculograms were recorded next to each eye and below the left eye.http://www.ru.nl/fcdonders/fieldtrip), the data were then segmented into epochs of 2 s before and 2.5 s after the onset of the feedback. Epochs or channels with severe muscular artifacts or saturated recordings were excluded manually. The prepared data were subjected to an ICA decomposition using EEGLAB  and SAS 9.1 for Windows . Given that age groups differ in the amount of intragroup between-person variance, comparisons across age groups were done using the PROC MIXED procedure, which is robust to variance heterogeneity between age groups. The intraclass correlation coefficient (ICC) was calculated as the effect size indicator for ANOVA. Cohen’s PPP1R1B “any G” and AA genotypes, whereas Figure Figure PPP1R1B, F = 4.72, p < 0.05, ICC = 0.39. As evident in Figure F = 13.20, p < 0.05, ICC = 0.84, reflecting larger P2 amplitudes in children and adolescents . In addition, P2 amplitudes were reliably higher in the gain as compared to the loss condition, F = 5.25, p < 0.05, ICC = 0.44. The overall two-way age × PPP1R1B interaction effect was not reliable, F = 1.23, p = 0.33, likely due to limited power.Indeed, in a three-way ANOVA with the factor gain-loss as within-subject factor, and age and genotype as between-subject factors, we observed a reliable main effect of PPP1R1B effect across the lifespan was performed  × PPP1R1B (AA – any G) 1 −1 −1 1 −1 1 1 −1; t = 1.65, p = 0.05, d = 0.37). As can also be seen in Figure t-tests comparing the PPP1R1B subgroups within age groups confirmed the specificity of this effect for children and older adults: .As a next step, we applied planned contrasts to test our second hypothesis of an amplification of the genetic effect in age groups with reduced dopamine levels. Specifically, a contrast testing for a U-shaped pattern of the beta = −0.04, ns; Any G carrier after negative feedback: beta = −0.03, ns; AA carrier after positive feedback: beta = 0.01, ns; Any G carrier after positive feedback: beta = 0.19, ns). This might be due to the fact that the updating processes in the P2 only reflect flexibility with respect to new information and is neutral with respect to the appropriate use of the new information. However, a relation to behavioral parameters might be expected when a change in the flexibility is behaviorally adaptive. In the present probabilistic reinforcement learning task, it is, for instance, adaptive to reduce the updating based on loss feedback in later stages of learning. Once the better option is identified, the infrequent, improbable losses (in this task about 25%) on this option should not be considered much during updating. In line with this reasoning, we observed a positive relationship of the decrease in P2 amplitude to losses after reaching the learning criterion (about 75% correct choices) with the mean accuracy . Subjects with higher mean accuracy exhibited a reduced updating response to negative feedback after reaching the learning criterion. Of particular interest, this relationship was stronger in subjects with the AA genotype, suggesting that higher dopamine receptor efficiency supports the flexibility in the updating process .Similar to prior studies , who presumably are associated with higher frontostriatal function, show larger P2 responses to feedbacks during reinforcement learning. Furthermore, this observed genotype effect is stronger in individuals with lower efficacy of dopamine signaling, i.e., in children and older adults.The present study provides evidence for genotype effects on the P2 amplitude during outcome updating. Homozygotes of the A allele of the O-methyltransferase (COMT), an enzyme that degrades dopamine in the PFC has been found to affect the amplitude of the error-related positivity, which is assumed to reflect the expectedness of an outcome , with their presumably higher dopamine levels in the PFC, showed a larger error-related positivity than the Val carriers  was associated with a higher decrease of P2 amplitude after learning for losses, but not for gains. Moreover, this effect was stronger for subjects with the AA genotype, which again hints at the role of dopamine signaling in a flexible updating process. This finding, however, needs to be considered in light of other, related studies which did not observe a relationship between P2 amplitude and accuracy  on the P2 amplitude hint at a link between dopamine modulation and outcome updating during reinforcement learning. Individuals carrying the genotype associated with higher striatal dopamine receptor efficacy and fronto-striatal connectivity showed higher P2 responses to feedback during reinforcement learning. Furthermore, this observed genotype effect is stronger in individuals with lower subcortical and/or frontal dopamine levels, i.e., in children and older adults. We hope that these results encourage further research on the link between frontostriatal dopaminergic modulation and functional differences in ERPs and behavior during reinforcement learning. At the same time this suggests a need to consider lifespan age differences in brain functions when investigating genotype-phenotype relations.Our findings of a dopamine related genotype effect  between dopamine genotype effect and ERP components of reinforcement learning are less direct than often assumed and may be mediated by interactions between transmitter systems. For instance, it has been suggested that a co-release of glutamate from midbrain dopaminergic neurons might underlie midbrain-prefrontal interactions in the sub-second range as the effects of dopamine release in the PFC unfold too slowly (in the time range of seconds to minutes) to provide an effective updating of prediction errors (see Lapish et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The GLP-1 receptor agonist, exenatide, has previously been shown to improve insulin secretion, protect beta cells from apoptosis, and promote beta cell regeneration. We propose that pretreatment with exenatide will promote islet graft survival and improve graft function. Pancreatectomized cynomolgus monkeys underwent islet allotransplantation and were treated with exenatide beginning on day 0 or day −2. A third group of animals was treated with an immunosuppressive regimen while a fourth group remained untreated. Fasting blood glucose (FBG) was used to evaluate graft function along with intravenous glucose tolerance tests (IVGTTs) performed at study endpoint (day 10 for untreated and posttransplant exenatide or day 90 for pretreatment exenatide and immunosuppression). The average FBG for pre-treated animals day 5 following transplant was 52.7 ± 14.8 mg/dl, compared to 154.3 ± 105.5 mg/dl for animals treated only following transplant, 59.4 mg/dl ±12.1 for animals treated with immunosuppression, and 265.5 ± 172.3 mg/dl for untreated animals. IVGTTs performed at study endpoint showed normal glucose and insulin curves in the pre-treated exenatide and immunosuppression groups only, with beta cell function actually improving after transplant in the pre-treated group. We conclude, therefore, that exenatide pre-treatment can successfully maintain islet graft survival in nonhuman primates. Traditionally, type 1 diabetes has been treated by either life-long insulin therapy or, in severe cases, pancreas transplantation. However, frequent episodes of hypoglycemia are common in patients on life-long insulin therapy, and whole pancreas transplantation is an invasive surgical procedure with significant risks. Islet cell transplantation is an attractive alternative to these traditional treatments. However, two of the major limiting factors in the widespread use of islet cell transplantation clinically are the availability of a sufficient number of islets and the inability of current immunosuppressive treatments to protect transplanted islets in the long term.β-cell regeneration is essential to the widespread use of islet transplantation in treating type 1 diabetes.As of 2010, an estimated 1.5 million people in the United States were diagnosed with insulin-dependent diabetes [Despite using 2-3 pancreata for each recipient, however, results reported by the Edmonton group have shown that the rate of success for functional islet grafts in the clinical setting is approximately 80% after 1 year, but only 10% after 5 years . Effectsβ-cell function and insulin sensitivity. Therefore, despite offering protection from host immune attack, these agents themselves can diminish graft function and contribute to failure of the transplanted islets. Additionally, recipients of islet grafts still suffer the autoimmune effects of diabetes development that led to beta cell destruction initially, thereby affecting function of transplanted islets in the long term.Many immunosuppressive protocols used in islet cell transplantation to date have relied on calcineurin inhibitors that have been shown to negatively affect pancreatic Incretin mimetics like exenatide are a potentially useful treatment in combination with islet cell transplantation. Exenatide is an analog to glucagon-like peptide 1 (GLP-1) and binds to the GLP-1 receptor on beta cells. GLP-1 and its analogs have been shown to increase insulin production by beta cells in response to glucose , 6 as weThese far-reaching effects of exenatide on islet cells lead us to believe that exenatide treatment in combination with islet cell transplantation may result in improved long-term islet graft function. Therefore, we propose that pre-treatment with exenatide will improve graft outcomes compared to treatment with more conventional immunosuppression.Macaca fascicularis), aged 2–4 years, were obtained from Charles River Laboratories  or Covance Research . Animals had a starting weight of 4.35 kg ±1.00 (range 2.5–6.1). Animals were housed in individual cages (28 × 30 × 24 in) and given a continuous water supply. Animals were fasted for 12 hours prior to surgery and intravenous glucose tolerance tests (IVGTTs) but were otherwise fed with a regular primate diet supplemented with fresh produce. The procedures described in this study were conducted according to the guidelines set forth in the “Guide for the Care and Use of Laboratory Animals” and by the Institutional Animal Care and Use Committee (IACUC) and University Laboratory Animal Resources (ULAR) [Cynomolgus monkeys (s (ULAR) . The OSUn = 3) was treated with 5 μg exenatide twice daily (bid)  subcutaneously on days −2 to the study endpoint, where day 0 was the day of transplant. Group 2 (n = 3) was also treated with exenatide (5 μg bid subq) to study endpoint, but beginning on the day of transplant. Group 3 (n = 5) was treated with the following immunosuppression regimen. Induction therapy consisted of rabbit antithymocyte globulin (ATG)  at a dose of 1.5 mg/kg intravenously on days 0–3 and prednisone  tapered from 25 to 5 mg over days 0–3. Immunosuppression maintenance consisted of 25 mg cyclosporine (CSA)  and 250 mg mycophenolate mofetil (MMF)  given orally from day 0 to the study endpoint. Group 4 (n = 4) was untreated and served as the control group.All animals underwent total pancreatectomies to induce diabetes and were transplanted with islet allografts. The average dose of islets transplanted was 12,110 IEq/kg ±7442.8. Group 1  without heparin were slowly injected over 10 minutes. The catheter was removed and the vein ligated. The incision was closed, and animals recovered under close observation, and Buprenex  was given as a postoperative analgesic.Islet isolation was performed according to the modified human islet isolation protocol previously described . Islet nμL drop of blood was drawn from the animal's tail for measurement on a standard glucometer . Fasting blood glucose (FBG) was then measured prior to morning feeding once daily through day 7 following transplant and weekly thereafter . Animals with a fasting blood glucose measuring 300 mg/dL or greater were monitored daily and given 2 units of NPH insulin . Intravenous glucose tolerance tests (IVGTTs) were performed prior to pancreatectomy on day 0 and after transplant on days 10 and 90. The study endpoint was considered 90 days after transplant for Groups 1–3 and 10 days after transplant for the untreated Group 4 . However, two animals in group 1 (exenatide pre-treatment) were monitored beyond 90 days and an additional IVGTT was performed on day 220. Body weights for animals were monitored throughout the study. At the study endpoint, all animals were euthanized following a final IVGTT. Postmortem analysis of the animals' anatomies was performed to visually confirm that no pancreatic tissue remained in the abdomen.For the first 48 hours subsequent to transplant, blood glucose was measured twice daily using the tail prick method. A 10 Animals were fasted 12 hours prior to each IVGTT. Baseline glucose and insulin were measured at times −5 and 0. A 50% solution of glucose was administered intravenously at time 0. Blood was drawn at times 1, 3, 5, 10, 15, 20, 25, and 30 minutes after glucose administration for measurement of blood glucose and serum insulin levels.μm) and stained for insulin using a 1 : 100 dilution of guinea-pig polyclonal insulin antibody  and for CD3 using a 1 : 400 dilution of rabbit polyclonal CD3 antibody . Images were taken at 20x magnification.Upon euthanasia, livers were removed from all animals and preserved in buffered formalin. Paraffin-embedded sections of liver were cut (4 n = 5 islets per replicate × 3 replicates) and incubated at 37°C in either “high” (16.7 mM) or “low” (1.67 mM) glucose for 1 hour. Insulin released in the supernatant was measured using an ELISA kit for human insulin . An insulin stimulation index (SI = high/low glucose insulin release) was determined for each sample.Freshly isolated islets were handpicked (G) were used as a measure of glucose tolerance and insulin sensitivity for IVGTTs. kG was calculated as the negative slope of the linear regression for the natural logarithm of glucose from 10 to 30 minutes. Acute insulin response to glucose (AIRg) was used as a measure of first phase insulin secretion during IVGTTs. AIRg was calculated as the mean of insulin at time 3–5 minutes minus the mean at time 0. Beta cell function was measured by the mathematical assessment of glucose regulation called the homeostasis model assessment (HOMA-%B). HOMA-%B was calculated as the average basal insulin (μIU/mL) multiplied by 20 and divided by the average basal glucose (mmol/mL) minus 3.5 [Area under the curve (AUC) was determined for all IVGTT glucose curves. Glucose disappearance rate constants  were used for parametric analysis. A probability value (P) of less than 0.05 is considered statistically significant.All results are expressed as mean ± SEM unless otherwise stated. Student's Fasting blood glucose levels were used as a determinant for islet graft function posttransplant. Animals in the untreated control group showed elevated blood glucose levels by day 1 posttransplant , with thResults from IVGTTs also indicated functioning islet allografts in animals pre-treated with exenatide or treated with ATG/CSA/MMF, whereas untreated animals had elevated glucose levels and reduced insulin production. Animals receiving exenatide treatment after transplant only also showed impaired insulin production and abnormal glucose curves Figures  and 3. BμU/mL ±19.2 versus 61.0 μU/mL ±40.0) (P = 0.01) . This did drop after transplant to 105 μU/mL ±19.0 at 90 days but continued to remain significantly higher than other treatment groups, including those treated with immunosuppression (First phase insulin secretion (AIRg) in untreated recipients was severely reduced at 10 days after transplant compared to baseline (9.53 L ±40.0) . Beta ce = 0.01) . Islet rmL ±18.1 . SimilarmL ±18.1 . On the ansplant . Beta ceansplant . Animalspression . These rpression .It is known that exenatide increases glucose-dependent insulin secretion. However, in addition to this effect, exenatide has been shown in other studies to promote beta cell regeneration and neogenesis as well. Xu et al. described that treatment of rats with exendin-4 after partial pancreatectomy prevented the development of diabetes with evidence of both replication of preexisting beta cells as well as neogenesis of beta cells from ductal progenitor cells . AnotherIn vitro analysis of INS-1 cells has shown complete abrogation of apoptosis following pre-treatment with exenatide. This same study also showed a decrease in the oxidative stress-inducing thioredoxin interacting protein (TXNIP) and the apoptotic factors caspase-3 and Bax after in vivo treatment with exenatide [Additionally, exenatide has been shown to decrease apoptosis of beta cells. xenatide .A combination of these effects of exenatide on beta cell function, regeneration, and survival likely play a role in the maintenance of islet graft function in the absence of immunosuppression treatment. Further analysis will need to be done, however, to evaluate the roles of these effects on allogeneic graft survival in the nonhuman primate.Liver sections from animals in each group were stained for insulin and CD3 at the conclusion of the study. In animals that were pre-treated with exenatide, islet grafts were evident in the liver as indicated by areas of positive insulin staining. CD3 staining yielded few positive cells in these grafts, suggesting a lack of T-cell infiltration Figures . This coIn vitro glucose stimulation assays performed on freshly isolated islets in this study indicated a significant improvement in islet function for islets pre-treated with exenatide compared to those that did not receive exenatide treatment. The average stimulation index for islets isolated from animals receiving in vivo exenatide pre-treatment was 2.98 ± 1.85. For islets isolated from untreated donor animals (not previously treated with exenatide), the average stimulation index was 0.52 ± 0.32. This was significantly lower than that of exenatide-treated animals (P = 0.04)  . This suin vitro that pre-treatment with exendin-4 improves protection of beta cells from apoptosis [in vitro and in vivo) that has the potential to protect islet cells for transplantation. In a clinical setting, however, cadaveric transplantation would not allow for in vivo pre-treatment of islets. This is a limitation to this current protocol and suggests that further study is necessary.Additionally, even at day 0 prior to transplantation, insulin secretion and glucose tolerance were better in exenatide pre-treated animals compared to other groups. Serum insulin levels produced in response to intravenous glucose administration were also significantly higher in animals pre-treated with exenatide . Becausepoptosis , 14. ThuThere was some initial concern regarding potential side effects of exenatide treatment at this dose in the nonhuman primate. It has been shown previously that exenatide treatment can result in decreased gastric emptying and weight loss. Therefore, animal diet and weight were monitored closely throughout the study. For approximately 2–4 weeks after the start of exenatide treatment, decreased appetite was noted compared to animals not treated with exenatide. This resulted in an average 16.9% weight loss in these animals at day 10 posttransplant. However, after 2–4 weeks of exenatide treatment, the animals' appetites returned to normal, and healthy weight gain was resumed with an average weight gain of 7.59% by day 90. This was compared to an average total weight gain of 10.9% at day 90 in animals not treated with exenatide.In vitro studies by other groups have shown that exenatide reduces expression of JAK-1 and STAT-1, which mediate transcriptional effects of interferon-γ (INF-γ) involved in beta cell apoptosis in the autoimmune development of type 1 diabetes. Microarray analysis and quantitative real-time PCR showed decreases in JAK-1 and STAT-1 expression in both INS-1 cells and isolated human islets after coculture with exenatide [xenatide . AdditioIn summary, we have shown in this study that exenatide can improve graft function relative to treatment with immunosuppression and that, in fact, exenatide treatment alone is sufficient in itself in protecting islet allografts from rejection. However, pre-treatment of donors and/or recipients appears to be necessary to achieve this level of graft function. When exenatide treatment of the recipient is not initiated until day 0 and transplanted islets are also untreated, graft function becomes impaired and more closely resembles that of grafts in untreated animals. In addition, this is a pilot study, and the mechanisms of action have not been elucidated. We believe, however, that the roles of exenatide in insulin secretion, beta cell regeneration, and protection from apoptosis are crucial to the success of graft survival. This, along with its potential to protect islets from autoimmune attack, makes exenatide a useful treatment for long-term success in islet transplantation in type 1 diabetic patients. Further studies are needed to test this intriguing hypothesis in type 1 diabetic patients who are candidates for islet cell transplantation.


Matched molecular pairs (MMPs) have previously been used to extract chemical transformations and study their effect on molecular properties such as activity ,2. Chemi


Hypocretins (hcrt), also known as orexins, play a critical role in reward-seeking behavior for natural rewards and drugs of abuse. The mesolimbic dopamine pathway that projects from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) is critically involved in the neural mechanisms underlying reward-seeking and motivation. Hcrt immunopositive fibers densely project to the shell of the nucleus accumbens (NAcSh), suggesting that the NAcSh might be a site for the interaction between hcrt and dopaminergic modulation of reward-seeking behavior. While it is known that hcrt action in the VTA can increase dopamine in the NAc, it has not been determined if hcrt released locally at dopaminergic terminals in the NAcSh can modulate dopamine concentration. Here, we use fast scan cyclic voltammetry (FSCV) in forebrain slices containing the NAcSh to determine whether hcrt can alter evoked dopamine concentration. We found bath application of hcrt-1 increases phasically evoked dopamine release, without altering reuptake at dopamine terminals in the NAcSh. Hcrt-1-induced potentiation of dopamine concentration was inhibited by SB334867, a hcrt receptor 1 antagonist, as well as ionotropic glutamate receptor antagonists, AP-5, CNQX and DNQX. Taken together, these results suggest that local hcrt-1 can modulate dopamine in the NAcSh and may play a role in reward-seeking and appetitive behaviors. The hypocretins (hcrt), also known as orexins, are neuropeptides produced solely in the lateral hypothalamic and perifornical areas  project to the nucleus accumbens (NAc) via the medial forebrain bundle  at single pulse or phasic burst-like stimulation frequencies.Hcrt-1 in the VTA increases dopamine release in the NAc and prefrontal cortex  saturated with 95% O2/5% CO2 using a vibratome . Slices were incubated in warm (31.5°C) 95% O2/5% CO2 oxygenated aCSF  for at least 1 h and transferred to a recording chamber that was constantly perfused (gravity flow: 1.5 ml/min) with oxygenated aCSF (30–32°C). The slices were allowed to equilibrate in the recording chamber with the superfusion medium for an additional 20–30 min before experimentation.Male C57BL/2J mice (25–30 g) were anesthetized with halothane or isoflurane, decapitated, and brains were extracted and prepared for slicing. Coronal slices (250 μm) containing the NAcSh were cut in ice-cold sucrose-containing artificial cerebrospinal fluid (aCSF) solution  were pulled in glass electrodes and cut to a final exposed length of ~150 μm. Triangular waveforms (holding at −0.4 V) at 10 Hz (−0.4 to 1.0 V vs. Ag/AgCl at 400 V/s scan rate) were used. Catecholamine release was evoked using electrical stimulation applied with a bipolar stimulating electrode positioned flush with the tissue for local surface stimulation. To evoke [DA]o in the NAcSh, either a single pulse was delivered or 100 Hz, 5 pulses to mimic phasic dopamine release  reconstituted in DMSO and stored in aliquots, protected from light, at −20°C. Prior to the experiment, these compounds were diluted in aCSF and used at 1/1000 working concentrations of DMSO. SB334867 was purchased from Tocris (Ellisville) reconstituted in DMSO and stored in aliquots at −20°C. Prior to the experiment, SB334867 was diluted in aCSF and used at 1/1000 working concentrations of DMSO. Our previous work has demonstrated that 1/1000 DMSO does not influence evoked [DA]o , reconstituted in ddHt-test comparing a time-point on the baseline prior to drug application to a time-point after drug application. For multiple comparisons, One-Way ANOVA was used unless otherwise indicated. A difference of p < 0.05 was considered significant. Statistical tests were performed with GraphPad Prism v.5.Values listed are means ± SEM. Statistical significance was assessed using a paired student o at dopaminergic terminals in the NAcSh where there is significant density of hcrt-immunopositive axonal fibers . No significant change occurred in evoked [DA]o over the duration of the experiment in the absence of hcrt-1 . The decay of the current, represented by tau (τ), has been demonstrated to be positively correlated with Km, suggesting that τ is an appropriate measurement of dopamine uptake . Decay of evoked dopamine did not significantly change over the course of the experiment in the absence of hcrt-1 .To determine if hcrt-1 could modulate [DA]o under phasic conditions, we evoked dopamine release every 5 min with 100 Hz, 5 pulses, a parameter which increases dopamine concentration relative to a single pulse  compared to a single pulse . To test if peak [DA]o was altered during hcrt-1 application, we evoked [DA]o 2.5 min after initiation of the 5 min bath application. Surprisingly, there was no significant increase in phasically evoked [DA]o immediately after hcrt-1 application . To determine if the increase in [DA]o was due to a change in uptake, we measured the decay of evoked DA under phasic conditions. There was no significant difference in τ 25 min after application of hcrt-1 . Taken together, these results suggest that hcrt-1 increases dopamine release only under phasic release conditions.Phasic dopamine release accompanies reward-predicting stimuli  while evoking [DA]o under phasic conditions. In slices preincubated for 30 min with SB334867, hcrt-1 did not alter phasically evoked [DA]o , suggesting that striatal hcrt-R1s are required for hcrt-1-mediated potentiation of [DA]o.To determine if hcrt-1-induced potentiation of striatal [DA]o in the NAcSh, we repeated the experiment in the presence of CNQX (10 μM) and AP-5 (50 μM) to block α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/Kainate and NMDA receptors, respectively. Application of excitatory amino acid receptor antagonists significantly attenuated phasically evoked dopamine by 12 ± 4% . The effect of hcrt-1 on [DA]o was abolished in the presence of CNQX and AP-5 . Application of the AMPA/Kainate receptor antagonist, DNQX, inhibited phasically evoked dopamine by 14 ± 2% . The effect of hcrt-1 on [DA]o was also abolished in the presence of DNQX . Taken together, these data suggest that hcrt-1 activation of glutamatergic inputs is required for hcrt-1-mediated increase in dopamine release under phasic conditions.Interestingly, hcrt-1 modulation of dopamine release occurs only under phasic conditions and not single pulse, suggesting that hcrt-1 may not be directly modulating dopamine terminals to promote release. One possibility is that hcrt-1 could be acting at its receptors on glutamatergic terminals within the NAcSh to increase dopamine release indirectly. To test the contribution of glutamatergic signaling to hcrt-1 modulation of [DA]o under phasic conditions reached a maximum 15 after hcrt-1 application. This delayed effect is likely due to the necessity for the 33 amino acid peptide to penetrate the tissue to reach its receptors. Interestingly, hcrt-1 modulates NMDA receptors on VTA dopamine neurons with a similar time course (Borgland et al., o was due to an increase in dopamine release as opposed to modulation of reuptake mechanisms as there was no effect of hcrt-1 on the decay of evoked dopamine. Taken together, in addition to hcrt-1 acting at the VTA to increase dopamine concentration in the NAc, hcrt-1 can also act at terminals to modulate release.While several studies have demonstrated that hcrt-1 administered into the VTA can increase dopamine concentration in the NAcSh (Vittoz et al., o under phasic conditions, but not after a single pulse repeated every 2 min. Therefore, it is likely that hcrt-1 is acting indirectly in the NAcSh to increase terminal dopamine release. One possibility is that hcrt-1 modulates GABAergic activity onto dopamine terminals in the NAcSh. While it has not been demonstrated how dopamine concentration is modulated by GABA in the NAcSh, GABA is reported to increase [DA]o in the dorsal striatum (Avshalumov et al., o. Consistent with this idea, we demonstrated that application of AMPA or NMDA receptor antagonists inhibited a hcrt-1-induced increase of [DA]o.We observed that hcrt-1 modulated evoked [DA]o. In contrast to the present study, phasically evoked dopamine from dorsal striatum slices was increased in the presence of the AMPA receptor antagonist, GYKI-52466 (Avshalumov et al., in vivo voltammetry in the NAc (Svensson et al., Dopamine spillover from dopaminergic synapses onto MSNs not only activates receptors on MSNs several micrometers away (Garris et al., Hcrt-1 acts with similar efficacy at both hcrt-R1 and hcrt-R2 (Sakurai et al., o in the NAcSh. This effect required activation of AMPA receptors and hcrt-R1. This study demonstrates an additional mechanism by which hcrt-1 can modulate dopamine release and potentially influence appetitive behaviors.Dense staining of hcrt terminals in the NAcSh has been observed (Peyron et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


There is compelling evidence that acute ethanol exposure stimulates ventral tegmental area (VTA) dopamine cell activity and that VTA-dependent dopamine release in terminal fields within the nucleus accumbens plays an integral role in the regulation of ethanol drinking behaviors. Unfortunately, due to technical limitations, the specific temporal dynamics linking VTA dopamine cell activation and ethanol self-administration are not known. In fact, establishing a causal link between specific patterns of dopamine transmission and ethanol drinking behaviors has proven elusive. Here, we sought to address these gaps in our knowledge using a newly developed viral-mediated gene delivery strategy to selectively express Channelrhodopsin-2 (ChR2) on dopamine cells in the VTA of wild-type rats. We then used this approach to precisely control VTA dopamine transmission during voluntary ethanol drinking sessions. The results confirmed that ChR2 was selectively expressed on VTA dopamine cells and delivery of blue light pulses to the VTA induced dopamine release in accumbal terminal fields with very high temporal and spatial precision. Brief high frequency VTA stimulation induced phasic patterns of dopamine release in the nucleus accumbens. Lower frequency stimulation, applied for longer periods mimicked tonic increases in accumbal dopamine. Notably, using this optogenetic approach in rats engaged in an intermittent ethanol drinking procedure, we found that tonic, but not phasic, stimulation of VTA dopamine cells selectively attenuated ethanol drinking behaviors. Collectively, these data demonstrate the effectiveness of a novel viral targeting strategy that can be used to restrict opsin expression to dopamine cells in standard outbred animals and provide the first causal evidence demonstrating that tonic activation of VTA dopamine neurons selectively decreases ethanol self-administration behaviors. Alcoholism is a devastating socio-economic problem estimated to account for 4% of the global burden of disease. Despite the widespread use and abuse of alcohol, little is known about the neurocircuitry responsible for the development and progression of alcoholism. Ethanol affects brain function by modulating numerous neurotransmitter and neuromodulator systems, including, but not limited to, GABA Koob, , glutamaUntil recently it has been impossible to parse the causal role of specific neurotransmitter systems in the regulation of ethanol drinking behaviors. Optogenetics provides a means to experimentally control the activation of specific neuronal sub-populations in heterogeneous brain regions where multiple neuronal subtypes exist and to do so with exquisite temporal precision. While several transgenic strategies have been developed to target opsins to dopaminergic neurons in rats and mice, we have developed a novel viral vector approach that can theoretically be used in any rodent species or strain. In this approach, the expression of channelrhodopsin-2 (ChR2) is driven by a tyrosine hydroxylase (TH) promoter, which has previously been shown to restrict expression primarily to dopaminergic neurons  construct, in which ChR2 expression is restricted to TH-positive neurons into the rat VTA and characterized the effect of tonic and phasic activation of VTA dopaminergic neurons on dopamine release in the nucleus accumbens and ethanol drinking behaviors. Our data confirm that the viral strategy selectively targets ChR2 expression to dopamine neurons within the VTA and that low (5 Hz) and high (50 Hz) frequency stimulation of VTA dopamine neurons results in distinct patterns of accumbal dopamine release. Moreover, ethanol drinking behaviors were selectively reduced by tonic, but not phasic, VTA dopamine release. Collectively, these data demonstrate a novel strategy that successfully targets opsins to VTA dopamine cells in standard outbred rats and provide new insight in the specific temporal dynamics of dopamine signaling that regulate ethanol drinking behaviors.Viruses were packaged using a standard triple transfection packaging protocol to generate pseudotyped AAV2/10  and xylazine hydrochloride  and placed in a stereotaxic frame. The scalp was shaved, swabbed with iodine and a central incision made to expose the skull. Two small holes were drilled and 2 skull screws were placed in to secure a cement cap. A third hole was drilled above the right VTA  and an optic-fluid cannula (OFC)  was implanted . Finally, 1.3 μl of virus that expresses ChR2-EYFP with expression driven by a TH promoter was slowly injected into the VTA  over 3 min via a Hamilton syringe connected to the OFC. The exposed skull was coated with dental cement secured by skull screws and upon drying the animals were returned to their home cages for recovery.Ethanol drinking was assessed using an intermittent home-cage drinking procedure which we and others have previously shown to engender relatively high levels of ethanol intake in male Long-Evans rats Wise, . Brieflyin vitro with known concentrations of dopamine (2–5 μM). Dopamine uptake was determined from the clearance rate of dopamine and was assumed to follow Michaelis-Menten kinetics. The changes in dopamine during and after optical or electrical stimulation were fit using the equation:f is the stimulation frequency (Hz), [DA]p is the concentration of dopamine released per stimulus pulse, and Vmax is the maximal rate of dopamine uptake, which is proportional to the number of available DAT proteins. The baseline value of Km was calculated to be between 0.16–0.2 μM, a value determined in rat brain synaptosomes and commonly used in the analysis of voltammetric data  and placed in a stereotaxic frame. The cement above the striatum was removed and a hole for a carbon fiber electrode  insertion was drilled . An Ag/AgCl reference electrode was implanted in the contralateral hemisphere and a carbon fiber electrode was positioned in the striatum . An optic-fluid cannula  with fiber 200 μm in diameter  was connected to the laser . The reference and carbon fiber electrodes were connected to a voltammetric amplifier  and voltammetric recordings were made at the carbon fiber electrode every 100 ms by applying a triangular waveform . Data were digitized  and stored on a computer. Light evoked dopamine release was identified by the background-subtracted cyclic voltammogram. Carbon fiber microelectrodes were calibrated The optical setup consisted of a laser at wavelength 473 nm  with a maximum power output of 100 mW. The laser was modulated using the TTL input control port on the laser power supply with the control signals provided by a programmable function generator (Hewlett-Packard model 8116A). The start of the whole optical pulse procedure was initiated by manually firing a pulse generator (Systron Donner Model 100C) that then triggered a digital delay generator (SRS Model DG535) that was used to gate the function generator. The main purpose of the digital delay generator was to make sure that the function generator was properly gated to only select out a finite number of pulses from the continuous waveforms normally produced by the function generator. The function generator would produce a series of either 5 Hz or 50 Hz square pulses, and the total number of pulses in one data stream (50 and 250 respectively) would be controlled by the digital delay generator, since the temporal length of a gate pulse sent to the function generator would determine the number of square pulses the function generator would produce for each trigger. Each pulse within a series of pulses had a temporal width of 4 ms. The laser power output was measured using a commercial power meter (Newport Model 1815C). Synchronization of the laser to the voltammetric recording was achieved by manually triggering the laser 5 s after initiating the voltammetric recording that was controlled through an electronic controller instrument that was built by the Electronics Design Facility at the University of North Carolina at Chapel Hill. The light paradigm was 50 light pulses at 50 Hz with 30 s intervals for phasic stimulation and 250 light pulses at 5 Hz with 1 s intervals for tonic stimulation.Invitrogen #A6455) at 1:2000. All antibodies were diluted in PBS + 0.3% triton X-100. The next day the sections were rinsed 3 times for 10 min each in PBS and then incubated with secondary antibodies consisting of Alexa 555 donkey anti-mouse  and Alexa 488 goat anti-rabbit  for 2 h at room temperature while shaking. Sections were rinsed again in PBS three times for 10 min, then mounted on slides and coverslipped using Prolong Gold media. Slides were visualized on a Zeiss Axio Observer Confocal microscope.Rats were deeply anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (100 mg/kg) and transcardially perfused with 10% normal buffered formalin. Brains were removed and incubated overnight with fix at 4°C and then transferred and incubated again overnight in 25% sucrose until the brains sank. They were sectioned at 50 microns on a dry ice chilled stage using an American Optical 860 sliding microtome. Floating coronal sections containing the midbrain were processed for immunohistochemistry. Briefly, sections were rinsed in PBS for 5 min, and again in PBS + 0.5% triton X-100 three times for 10 min. Sections were incubated with primary antibody overnight at 4°C while shaking. Primary antibodies consisted of a mouse anti-tyrosine hydroxylase (ImmunoStar #22941) at a 1:4000 dilution and a rabbit anti-GFP . The data are presented as mean ± SEM and the criterion for significance was set at P < 0.05.Data were analyzed in GraphPad Prism . A two-way ANOVA was performed for the neurochemical studies characterizing differences between the effect of tonic and phasic light activation on accumbal dopamine efflux and for the effect of these stimulation patterns on the number of ethanol and water licks. The effects of optical stimulations on other ethanol drinking parameters  were analyzed by one-way analysis of variance. Bonferroni's tests were used for all To determine the contribution of VTA dopaminergic neurotransmission to alcohol seeking behavior, we generated AAVs that target dopaminergic neurons in the VTA. A TH promoter was used to restrict expression of ChR2 to TH-positive neurons. When injected directly into the VTA, expression was restricted to TH-positive neurons as revealed by immunohistochemical staining Figure . Robust in vivo fast-scan cyclic voltammetry (FSCV) to confirm that the level of ChR2 expression achieved in the VTA is sufficient for optical stimulation of dopamine release in the rat nucleus accumbens. Figure Vmax and apparent Km (Michaelis-Menten constant) were indistinguishable from those previously reported for the rat nucleus accumbens using FSCV in vitro and in vivo  was 24 ± 6 nM, which is less than the amount dopamine released by an electrical pulse of the same length (4 ms).We first employed F = 71.21; p < 0.0001]. Changes in dopamine concentration over time were also significantly different .Finally, we examined various parameters of optical stimulation of the VTA, which allows us to induce phasic (a relatively large but transient rise of extracellular dopamine concentration) and tonic (relatively low but sustained dopamine increase) release patterns in the nucleus accumbens. We found that high frequency (50 Hz), brief (1s) stimulation could mimic phasic dopamine responses, while low frequency (5 Hz), long lasting (50 s) stimulation resulted in a tonic elevation in dopamine concentration Figure . As expen = 5). High drinking rats were selected for these initial studies to explore the effects of optogenetic manipulation of the VTA on the voluntary consumption of pharmacologically meaningful doses of ethanol  = 55.29; p < 0.0001]. Taking advantage of the stable drinking patterns, we explored the effects of tonic and phasic optical stimulation of dopaminergic neurons in the VTA on drinking behaviors . No significant change (p > 0.05) in the number of water licks was found, while a noticeable trend (t = 2.4) toward a decrease in ethanol licking following 5 Hz stimulation was evident  in which optical stimulation was performed in a randomized order. This design allowed us to avoid possible confounds such as a habituation. In this larger cohort of animals, tonic activation of VTA dopamine cell bodies significantly affects ethanol drinking behaviors. Figure t = 3.45; p < 0.01) and the amount of ethanol consumed , when rats were stimulated at 5 Hz in the drinking cage. In marked contrast, phasic stimulation of VTA dopamine cells during the first 10 min of the drinking session had no effect on any measures of ethanol intake. Moreover, no significant changes in ethanol intake measures were observed when subjects received tonic stimulation in their home cages just prior to the drinking sessions (p > 0.05). Finally, we found a significant two-fold delay for the first ethanol lick when rats were stimulated with the frequency that induces tonic dopamine release . No significant changes in this parameter were evident during other stimulating protocols.Ten days following the injection, ethanol intake returned to baseline levels. Figure t Figure . As expeIt is increasingly clear that revealing the precise contribution of dopamine neurons to specific behaviors requires more sophisticated control over their activity than traditional techniques can provide   optogenetic activation of VTA dopamine cells can induce measurable phasic dopamine response in the mouse nucleus accumbens and is sufficient to induce conditional place preference  and consummatory measures of ethanol drinking are not correlated (Samson and Czachowski, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In asthmatic patients, inhaled dopamine induces bronchodilation. Although the Gi-coupled dopamine D2 receptor is expressed and sensitizes adenylyl cyclase activity in airway smooth muscle (ASM) cells, the Gs-coupled dopamine D1-like receptor subtypes have never been identified on these cells. Activation of Gs-coupled receptors stimulates cyclic AMP (cAMP) production through the stimulation of adenylyl cyclase, which promotes ASM relaxation. We questioned whether the dopamine D1-like receptor is expressed on ASM, and modulates its function through Gs-coupling.Dopamine signaling is mediated by G1-like receptor subtypes in both native human and guinea pig ASM tissue and cultured human ASM (HASM) cells was measured. To characterize the stimulation of cAMP through the dopamine D1 receptor, HASM cells were treated with dopamine or the dopamine D1-like receptor agonists (A68930 or SKF38393) before cAMP measurements. To evaluate whether the activation of dopamine D1 receptor induces ASM relaxation, guinea pig tracheal rings suspended under isometric tension in organ baths were treated with cumulatively increasing concentrations of dopamine or A68930, following an acetylcholine-induced contraction with or without the cAMP-dependent protein kinase (PKA) inhibitor Rp-cAMPS, the large-conductance calcium-activated potassium (BKCa) channel blocker iberiotoxin, or the exchange proteins directly activated by cAMP (Epac) antagonist NSC45576.The mRNA and protein expression of dopamine D1 and D5 receptors were detected in native human ASM tissue and cultured HASM cells. Immunoblots confirmed the protein expression of the dopamine D1 receptor in both native human and guinea pig ASM tissue and cultured HASM cells. The dopamine D1 receptor was also immunohistochemically localized to both human and guinea pig ASM. The dopamine D1-like receptor agonists stimulated cAMP production in HASM cells, which was reversed by the selective dopamine D1-like receptor antagonists SCH23390 or SCH39166. A68930 relaxed acetylcholine-contracted guinea pig tracheal rings, which was attenuated by Rp-cAMPS but not by iberiotoxin or NSC45576.Messenger RNA encoding the dopamine D1 receptors are expressed on ASM and regulate smooth muscle force via cAMP activation of PKA, and offer a novel target for therapeutic relaxation of ASM.These results demonstrate that the dopamine D Lysed cells were centrifuged  and an aliquot of the supernatant was subjected to protein analysis and storing at -80°C. Freshly dissected native human and guinea pig airway smooth muscle, and guinea pig brain cerebral cortex was homogenized (Tekmar Ultra Turrax T25 high-speed homogenizer set at top speed for 30 s) in cold (4°C) buffer . The homogenate was filtered through 125-μm Nitex mesh and centrifuged twice at 500 g for 15 min. The supernatant was transferred into new tubes and centrifuged at 50,000 g for 30 min at 4°C. The final membrane pellet was resuspended in the same buffer for protein concentration determinations and stored at -80°C. Each sample was solubilized by heating at 95°C for 10 min in sample buffer before use.Confluent cultures of HASM cells were rinsed with cold phosphate-buffered saline (PBS), and mechanically scraped from the surface of the T75 culture flask in the presence of protease inhibitor cocktail III. Cells were pelleted  or the dopamine D5 receptor protein  overnight at 4°C. After washing three times with TBST, membranes were incubated for 1 hr at room temperature with HRP-labeled secondary anti-rabbit antibodies . The signals from the immunoreactive bands were detected by ECL Plus  and were captured using chemiluminescent image analyzer .All the samples and positive controls were electrophoresed (8% SDS-PAGE) and transferred to PVDF membranes. 5% membrane blocking agent  in Tris-buffered saline with 0.1% Tween 20 (TBST) was used to block non-specific binding for 1 hr at room temperature. Membranes were then probed with antibodies directed against the dopamine D1 receptor protein  or dopamine D5 receptor protein , in 2% normal goat serum in PBS with 0.1% Triton X-100 (PBST). A parallel tracheal ring section was incubated with an isotype-specific rabbit IgG antibody as a negative control. After overnight incubation at 4°C, slides were washed three times with PBST and primary antibodies were detected using biotinylated anti-rabbit antibodies  at a concentration of 1:100. After incubation with ABC-HRP complex  for 30 min, the antigen antibody complex was then visualized with the peroxidase substrate kit (DAB) . Sections were counterstained with hematoxylin , dried, dehydrated in a graded alcohol series and xylene, and cover slipped using Poly-mount .Human and guinea pig tracheal rings were fixed, paraffin embedded, sectioned and rehydrated as previously described . Heat-me1 receptor-mediated cAMP production in primary cultured HASM cells  was measured using a HitHunter™ cAMP XS+ Assay kit according to the manufacturer’s instructions. Briefly, HASM cells in white 96-well plates were washed twice with warm PBS (37°C). The cells were incubated with dopamine (1 μM), or the dopamine D1-like receptor agonists (A68930 or SKF38393) (1 μM) for 20 min at 37°C. To examine the effect of dopamine on cAMP production in the presence of dopamine D2 receptor blockade, the cells were pretreated with the dopamine D2 receptor antagonist L-741626 (1 μM) for 30 min followed by incubation with dopamine (1 μM) for 20 min at 37°C. Incubation of the cells with L-741626 (1 μM) alone was also performed to determine whether L-741626 by itself exerts any effect on cAMP production. To examine the dose-dependent and time-course effects of the dopamine D1-like receptor agonist A68930 on cAMP production, the cells were incubated with A68930 (1 nM - 100 μM) for 10 - 60 min at 37°C. In separate experiments, the cells were pretreated with the dopamine D1-like receptor antagonists [SCH23390 (1 μM) or SCH39166 (1 μM)] or vehicle (PBS) for 30 min followed by incubation with A68930 (1 μM) for 20 min at 37°C. Then cAMP XS antibody reagent followed by the mixture of enzyme donor (ED)/Lysis/chemiluminescence (CL) working solution was added to each well. After incubation for 60 min at room temperature, cells were further incubated with the enzyme acceptor (EA) reagent for 3 hrs at room temperature. Luminescence signals were detected using a multimode microplate reader .The dopamine D50 or EC75 calculated for each ring). Following the achievement of a stable contraction , cumulatively increasing concentrations of dopamine (1 μM – 100 μM), the dopamine D1-like receptor agonist A68930 (1 μM - 100 μM) or the β-adrenoceptor agonist isoproterenol (0.5 nM – 1 μM) was incrementally added to the buffer in the baths. Control-contracted rings received vehicle (KH buffer) to serve as time controls for the A68930-induced relaxation. In separate experiments, rings were pretreated for 15 min with the dopamine D2 receptor selective antagonist L-741626 (10 μM), PKA inhibitor Rp-cAMPS (100 μM), large-conductance calcium-activated potassium (BKCa) channel blocker iberiotoxin (100 nM) or the Epac antagonist NSC45576 (250 μM) before the addition of cumulatively increasing concentrations of A68930 (1 μM – 100 μM) or isoproterenol (0.5 nM – 1 μM) to assess their effects on airway smooth muscle force.Guinea pig tracheas were dissected and equilibrated in organ baths as described . All rinP < 0.05 was considered significant.Statistical analysis was performed using repeated measures of ANOVA, followed by Bonferroni posttest comparison using GraphPad Instat 3.0.6 software . Data are presented as mean ± SEM; 1-like receptors (D1 and D5) in both freshly dissected native human airway smooth muscle tissue from the upper airways and in primary cultures of HASM cells from Lonza   of dopamine receptors are shown in Table 5 receptor PCR products were obtained confirming that PCR products were arising from cDNA and not from contaminating genomic DNA ], and in human whole kidney (positive control)   Figure A and 3E.1 receptor-mediated production of cAMP is well known in neurons. To examine whether dopamine D1 receptor agonists stimulate cAMP production in cultured HASM cells, we measured cAMP activity in the presence or absence of dopamine (1 μM) or the dopamine D1 receptor agonists (A68930 or SKF38393) (1 μM). Both dopamine D1-like receptor agonists significantly stimulated cAMP synthesis , while dopamine did not exert any effect   for 30 min, dopamine (1 μM) significantly stimulated cAMP synthesis , while L-741626 by itself did not exert any effect    or SCH39166 (1 μM)    , A68930 (100 μM) still relaxed tracheal rings to the similar degree (n = 7) compared with its relaxation after contraction with an EC50 concentration of acetylcholine (n = 9)  (data not shown in figures). Although A68930 is a potent highly selective dopamine D1 receptor agonist (EC50 = 2.1 nM), it also acts as an weaker agonist on the dopamine D2 receptor at higher concentration (EC50 = 3.92 μM) [2 receptor, we used the dopamine D2 receptor selective antagonist L-741626. Interestingly, under identical conditions, the pretreatment of rings with L-741626 (10 μM) significantly augmented the airway relaxation induced by A68930   . To conf1 receptor-mediated airway smooth muscle relaxation, we used Rp-cAMPS as a PKA inhibitor. The pretreatment of guinea pig airway rings with Rp-cAMPS (100 μM) significantly attenuated the relaxation induced by A68930    Figure . In cont) Figure .1 receptor-meidated airway relaxation, Epac antagonist NSC45576 was used. The pretreatment of guinea pig airway rings with NSC45576 (250 μM) did not block A68930-induced airway relaxation   is being examined. However, a limitation of this approach is that even with careful dissection, freshly isolated tissue could be contaminated by other cells types that could give rise to cDNA products during sensitive RT-PCR amplification. To confirm the expression of dopamine D1–like receptor in airway smooth muscle itself, we also evaluated its expression in primary cultured HASM cells which are a homogenous population of airway smooth muscle cells. In the present study, there was good agreement between the expression of dopamine D1-like receptor in freshly isolated native airway smooth muscle and from cultured HASM cells. These findings were in accordance with the previous findings using expression microarrays in that mRNA of both dopamine D1-like receptor subtypes was expressed in HASM cells [1 receptor protein was also identified in airway smooth muscle from native human airway smooth muscle, and cultured HASM cells from three independent sources. Immunohistochemical analysis also confirmed the expression of dopamine D1 receptor on airway smooth muscle in both human and guinea pig airway smooth muscle. On the other hand, the dopamine D5 receptor protein was not identified in both human and guinea pig native airway smooth muscle tissue, and cultured HASM cells by immunoblotting and immunohistochemistry. The discrepancy of the results obtained from RT-PCR and protein analyses on the expression of dopamine D5 receptor on airway smooth muscle is likely due to posttranscriptional regulation, differences in mRNA and protein turnover rates [5 mRNA by sensitive PCR techniques that does not translate into detectable levels of dopamine D5 protein. Several studies also described the correlation between mRNA and protein expression as moderately or weakly positive with correlation coefficients ranging from 0.2 to 0.5 [In addition to the expression in the central nervous system, numerous studies have also demonstrated the expression of functional dopamine Dl cortex -7,24-27.SM cells . Moreoveer rates ,30, or d2 to 0.5 .1 receptor on airway smooth muscle itself led us to question whether functional dopamine D1 receptors could modulate airway smooth muscle tone. First, we sought to assess its ability to increase cyclic AMP and relax airway smooth muscle. Many studies have demonstrated that the activation of Gs-coupled receptors  stimulates adenylyl cyclase activity, which leads to cAMP accumulation in airway smooth muscle [s-coupled receptors, our findings demonstrate that activation of the dopamine D1 receptor expressed on HASM cells with the endogenous ligand dopamine stimulated cAMP production in HASM cells but only when the dopamine D2-Gi coupled receptor was inhibited with L-741626. These findings suggest that dopamine exerts its effect equally on both dopamine D1-Gs coupled receptor and dopamine D2-Gi coupled receptor in HASM cells, and selective stimulation of the dopamine D1 receptor is required to stimulate cAMP production. Dopamine also relaxed acetylcholine-contracted guinea pig airway rings, however, contrary to the dopamine-stimulated cAMP production in HASM cells, the relaxation was produced without the blockade of dopamine D2 receptor (data not shown). These findings would suggest that the dopamine D2 receptor is more effectively coupled to Gi/adenylyl cyclase inhibition in the cultured HASM cells compared to intact tracheal rings. Another possibility is that in tracheal rings the dopamine-mediated relaxation is partially mediated through dopamine effects on β2-adrenoceptors. It was previously reported that dopamine-mediated relaxation of acetylcholine-contracted guinea pig trachea was partially blocked by the β-adrenoceptor antagonist propranolol [1 receptors and β2-adrenoceptors.Molecular identification of dopamine Dh muscle ,34,35. Ah muscle ,34,35. Ipranolol . These f1 receptor-specific effects on airway smooth muscle, the selective dopamine D1 receptor agonists A68930 and SKF38393 were used in the present study. Both agonists stimulated cAMP production in HASM cells, and A68930 induced airway smooth muscle relaxation in guinea pig tracheal rings. The specificities of A68930 (1 μM) at the dopamine D1 receptor in the present study were further supported by the finding that two different dopamine D1-like receptor antagonists SCH23390 or SCH39166 significantly blocked the effects of A68930 on cAMP production. However, treatment the HASM cells with A68930 at a higher concentration (100 μM) resulted in decrease in cAMP production compared with 1 μM of A68930. A68930 is a potent dopamine D1 receptor agonist (EC50 = 2.1 nM), but it also stimulates the Gi-coupled dopamine D2 receptor at higher concentrations (EC50 = 3.92 μM) [2 receptor expressed in HASM cells inhibited forskolin-stimulated adenylyl cyclase activity [1 receptors. This idea was further supported in the present study by demonstrating that the dopamine D2 receptor selective antagonist L-741626 significantly augmented A68930-induced guinea pig airway ring relaxation. In the present study, the concentration of A68930 which was required to produce significant relaxation in guinea pig airway ring was higher (≧ 20 μM) than that required to produce significant cAMP production in HASM cells (≧ 100 nM). Previous reports in tissue organ bath also showed that phenylephrine-contracted human corpus cavernosum smooth muscle strip without endothelium suspended in organ bath was dose-dependently relaxed by A68930 (EC50 = 3.5 ± 0.9 μM) [50 value of A68930-stimulated cAMP activity in LLC-PK1 cells was much lower (EC50 = 12.7 ± 2.7 nM) [To characterize the dopamine D3.92 μM) . We haveactivity , which c 0.9 μM) . In cont 2.7 nM) . These r1 receptor-mediated airway relaxation. However, several cAMP-mediated cellular events in airway smooth muscle are insensitive to PKA inhibition. Spicuzza et al. suggested that β2-adrenoceptor agonists suppressed acetylcholine-induced guinea pig airway contraction by mechanisms that are independent of PKA [et al. demonstrated the protein expression of two isoforms of Epac (Epac 1 and Epac2) in human and guinea pig trachea, and activation of Epac relaxes airway smooth muscle by decreasing MLC phosphorylation [2-adrenoceptor agonist relaxes airway smooth muscle through Epac rather than PKA [1 receptor agonist-induced airway relaxation. These results suggest that although both the dopamine D1 receptor and β2-adrenoceptor are Gs-coupled receptor, induce airway smooth muscle relaxation and increase cAMP, that the subsequent signaling targets of this cAMP elevation may not be identical following these receptors’ activations.Classically, most cAMP effects have been attributed to the activation of PKA. PKA has multiple intracellular targets that favor relaxation including phosphorylation of MLCK which leads to a decrease in its affinity for the calcium-calmodulin complex , resultit of PKA . Recentlt of PKA , and beerylation . They althan PKA . In cont2-adrenoceptor-mediated relaxation of airway smooth muscle [Ca channel causing plasma membrane hyperpolarization facilitating relaxation [Ca channel with iberiotoxin did not inhibit relaxation induced by a dopamine D1 receptor agonist.In addition to these cAMP-dependent mechanisms, a mechanism independent of cAMP is also relevant in βh muscle ,41, whiclaxation . However1 receptor agonist A68930. The first difference is that the relaxation evoked by isoproterenol and dopamine was rapid, while that evoked by A68930 was slow. These results suggest that the agonists’ interaction with the receptor can affect the speed of onset and duration of the induced relaxation. Variations in the molecular structure of agonists can affect the onset and duration of bronchodilation. For example, isoproterenol is a short-acting β agonist and has rapid onset, while the long-acting β2-adrenoceptor agonist salmeterol causes sustained relaxation in airway smooth muscle of at least 12 hrs after a single administration [1 agonists A68930 and A77636 contain an isochroman structure and are potent dopamine D1 agonists which exert long-lasting effects compared with dopamine in vivo. For example, A68930 elicited prolonged contralateral turning behavior in 6-hydroxydopamine lesioned rats (>20 hrs) [In the present study, there are several differences in the results from the cell-based cAMP assays and intact tissue organ bath studies with the dopamine Dstration , but thestration . There astration ,46. Thes>20 hrs) . In cont>20 hrs) ,48. Howes-coupled dopamine D1 receptors in human and guinea pig airway smooth muscle that can stimulate cAMP production in HASM cells and relax intact airway smooth muscle contracted with acetylcholine in the PKA-dependent manner. Although the Gs-coupled β2-adrenoceptor is a critical mediator of therapeutic relaxation of airway smooth muscle in diseases such as asthma and chronic obstructive pulmonary disease, less attention has focused on other Gs-coupled receptors on airway smooth muscle. Our findings suggest that the Gs-coupled dopamine D1 receptor may be a potential therapeutic target for relaxation of airway smooth muscle.In summary, our findings demonstrated for the first time the molecular expression of GThe authors declare that they have no competing interests.KM and CWE conceived and designed these studies; KM, YZ, DX, FM, FD and CWE performed experiments; KM, YZ, DX, FM, and CWE analyzed data; KM, YZ, DX, FM, and CWE interpreted results of experiments; KM and CWE prepared figures; KM, EM, and CWE drafted manuscript; KM, EM, and CWE edited and revised manuscript; All authors read and approved the final version of manuscript.