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Phytophthora infestans is the causal agent of potato late blight, a devastating disease of tomato and potato and a threat to global food security. Early detection and intervention is essential for effective management of the pathogen. We developed a loop-mediated isothermal amplification (LAMP) assay for P. infestans and compared this assay to conventional PCR, real-time LAMP, and droplet digital PCR for detection of P. infestans. The LAMP assay was specific for P. infestans on potato and tomato and did not amplify other potato- or tomato-infecting Phytophthora species or other fungal and bacterial pathogens that infect potato and tomato. The detection threshold for SYBR Green LAMP and real-time LAMP read with hydroxynaphthol blue and EvaGreen was 1 pg/µl. In contrast, detection by conventional PCR was 10 pg/µl. Droplet digital PCR had the lowest detection threshold (100 fg/µl). We adapted the LAMP assay using SYBR Green and a mobile reader (mReader) for use in the field. Detection limits were 584 fg/µl for SYBR Green LAMP read on the mReader, which was more sensitive than visualization with the human eye. The mobile platform records geospatial coordinates and data from positive pathogen detections can be directly uploaded to a cloud database. Data can then be integrated into disease surveillance networks. This system will be useful for real-time detection of P. infestans and will improve the timeliness of reports into surveillance systems such as USABlight or EuroBlight.

Among the canonical transient receptor potential (TRPC) channels, the TRPC4 non-selective cation channel is one of the most abundantly expressed subtypes within mammalian corticolimbic brain regions, but its functional and behavioral role is unknown. To identify a function for TRPC4 channels we compared the performance of rats with a genetic knockout of the trpc4 gene (trpc4 KO) to wild-type (WT) controls on the acquisition of simple and complex learning for natural rewards, and on cocaine self-administration (SA). Despite the abundant distribution of TRPC4 channels through the corticolimbic brain regions, we found trpc4 KO rats exhibited normal learning in Y-maze and complex reversal shift paradigms. However, a deficit was observed in cocaine SA in the trpc4 KO group, which infused significantly less cocaine than WT controls despite displaying normal sucrose SA. Given the important role of ventral tegmental area (VTA) dopamine neurons in cocaine SA, we hypothesized that TRPC4 channels may regulate basal dopamine neuron excitability. Double-immunolabeling showed a selective expression of TRPC4 channels in a subpopulation of putative dopamine neurons in the VTA. Ex vivo recordings of spontaneous VTA dopamine neuronal activity from acute brain slices revealed fewer cells with high-frequency firing rates in trpc4 KO rats compared to WT controls. Since deletion of the trpc4 gene does not impair learning involving natural rewards, but reduces cocaine SA, these data demonstrate a potentially novel role for TRPC4 channels in dopamine systems and may offer a new pharmacological target for more effective treatment of a variety of dopamine disorders.

The initial reinforcing properties of drugs of abuse, such as cocaine, are largely attributed to their ability to activate the mesolimbic dopamine system. Resulting increases in extracellular dopamine in the nucleus accumbens (NAc) are traditionally thought to result from cocaine’s ability to block dopamine transporters (DATs). Here we demonstrate that cocaine also interacts with the immunosurveillance receptor complex, Toll-Like Receptor 4 (TLR4), on microglial cells to initiate central innate immune signaling. Disruption of cocaine signaling at TLR4 suppresses cocaine-induced extracellular dopamine in the NAc, as well as cocaine conditioned place preference and cocaine self-administration. These results provide a novel understanding of the neurobiological mechanisms underlying cocaine reward/reinforcement that includes a critical role for central immune signaling, and offer a new target for medication development for cocaine abuse treatment.

Extinction is a form of inhibitory learning viewed as an essential process in suppressing conditioned responses to drug cues, yet there is little information concerning experiential variables that modulate its formation. Coping factors play an instrumental role in determining how adverse life events impact the transition from casual drug use to addiction. Here we provide evidence in rat that prior exposure to controllable stress accelerates the extinction of cocaine-seeking behavior relative to uncontrollable or no stress exposure. Subsequent experimentation using high-speed optogenetic tools determined if the infralimbic region (IL) of the ventral medial prefrontal cortex mediates the impact of controllable stress on cocaine-seeking behavior. Photoinhibition of pyramidal neurons in the IL during coping behavior did not interfere with subject's ability to control the stressor, but prevented the later control-induced facilitation of extinction. These results provide strong evidence that the degree of behavioral control over adverse events, rather than adverse events per se, potently modulates the extinction of cocaine-seeking behavior, and that controllable stress engages prefrontal circuitry that primes future extinction learning.

Standard-of-practice search management requires that the probability of detection (POD) be determined for each search resource after a task. To calculate the POD, a detection index (W) is obtained by field experiments. Because of the complexities of the land environment, search planners need a way to estimate the value of W without conducting formal experiments. We demonstrate a robust empirical correlation between detection range (Rd) and W, and argue that Rd may reliably be used as a quick field estimate for W.We obtained the average maximum detection range (AMDR), Rd, and W values from 10 detection experiments conducted throughout North America. We measured the correlation between Rd and W, and tested whether the apparent relationship between W and Rd was statistically significant.On average we found W ≈ 1.645 × Rd with a strong correlation (R(2) = .827). The high-visibility class had W ≈ 1.773 × Rd (also R(2) = .867), the medium-visibility class had W ≈ 1.556 × Rd (R(2) = .560), and the low-visibility had a correction factor of 1.135 (R(2) = .319) for Rd to W. Using analysis of variance and post hoc testing, only the high- and low-visibility classes were significantly different from each other (P.01). We also found a high correlation between the AMDR and Rd (R(2) = .9974).Although additional experiments are required for the medium- and low-visibility search objects and in the dry-domain ecoregion, we suggest search planners use the following correction factors to convert field-measured Rd to an estimate of the effective sweep width (W): high-visibility W = 1.8 × Rd; medium-visibility W = 1.6 × Rd; and low-visibility W = 1.1 × Rd.

Lithium has been used extensively for mood stabilization, and it is particularly efficacious in the treatment of bipolar mania. Like other drugs used in the treatment of psychiatric diseases, it has little effect on the mood of healthy individuals. Our previous studies found that mice with a mutation in the Clock gene (ClockΔ19) have a complete behavioral profile that is very similar to human mania, which can be reversed with chronic lithium treatment. However, the cellular and physiological effects that underlie its targeted therapeutic efficacy remain unknown. Here we find that ClockΔ19 mice have an increase in dopaminergic activity in the ventral tegmental area (VTA), and that lithium treatment selectively reduces the firing rate in the mutant mice with no effect on activity in wild-type mice. Furthermore, lithium treatment reduces nucleus accumbens (NAc) dopamine levels selectively in the mutant mice. The increased dopaminergic activity in the Clock mutants is associated with cell volume changes in dopamine neurons, which are also rescued by lithium treatment. To determine the role of dopaminergic activity and morphological changes in dopamine neurons in manic-like behavior, we manipulated the excitability of these neurons by overexpressing an inwardly rectifying potassium channel subunit (Kir2.1) selectively in the VTA of ClockΔ19 mice and wild-type mice using viral-mediated gene transfer. Introduction of this channel mimics the effects of lithium treatment on the firing rate of dopamine neurons in ClockΔ19 mice and leads to a similar change in dopamine cell volume. Furthermore, reduction of dopaminergic firing rates in ClockΔ19 animals results in a normalization of locomotor- and anxiety-related behavior that is very similar to lithium treatment; however, it is not sufficient to reverse depression-related behavior. These results suggest that abnormalities in dopamine cell firing and associated morphology underlie alterations in anxiety-related behavior in bipolar mania, and that the therapeutic effects of lithium come from a reversal of these abnormal phenotypes.

We previously reported that the activity of mesolimbic dopamine neurons of the ventral tegmental area (VTA) is a key determinant of behavioral susceptibility vs resilience to chronic social defeat stress. However, this was based solely onex vivomeasurements, and thein vivofiring properties of VTA dopamine neurons in susceptible and resilient mice, as well as the effects of antidepressant treatments, remain completely unknown. Here, we show that chronic (10 d) social defeat stress significantly increased thein vivospontaneous firing rates and bursting events in susceptible mice but not in the resilient subgroup. Both the firing rates and bursting events were significantly negatively correlated with social avoidance behavior, a key behavioral abnormality induced by chronic social defeat stress. Moreover, the increased firing rates, bursting events, and avoidance behavior in susceptible mice were completely reversed by chronic (2 week), but not acute (single dose), treatments with the antidepressant medication fluoxetine (20 mg/kg). Chronic social defeat stress increased hyperpolarization-activated cation current (Ih) in VTA dopamine neurons, an effect that was also normalized by chronic treatment with fluoxetine. As well, local infusion of Ihinhibitors ZD7288 (0.1 μg) or DK-AH 269 (0.6 μg) into the VTA exerted antidepressant-like behavioral effects. Together, these data suggest that the firing patterns of mesolimbic dopamine neuronsin vivomediate an individual's responses to chronic stress and antidepressant action.

Excessive inhibition of brain neurons in primary or slice cultures can induce homeostatic intrinsic plasticity, but the functional role and underlying molecular mechanisms of such plasticity are poorly understood. Here, we developed an ex vivo locus coeruleus (LC) slice culture system and successfully recapitulated the opiate-induced homeostatic adaptation in electrical activity of LC neurons seen in vivo. We investigated the mechanisms underlying this adaptation in LC slice cultures by use of viral-mediated gene transfer and genetic mutant mice. We found that short-term morphine treatment of slice cultures almost completely abolished the firing of LC neurons, whereas chronic morphine treatment increased LC neuronal excitability as revealed during withdrawal. This increased excitability was mediated by direct activation of opioid receptors and up-regulation of the cAMP pathway and accompanied by increased cAMP response-element binding protein (CREB) activity. Overexpression of a dominant negative CREB mutant blocked the increase in LC excitability induced by morphine- or cAMP-pathway activation. Knockdown of CREB in slice cultures from floxed CREB mice similarly decreased LC excitability. Furthermore, the ability of morphine or CREB overexpression to up-regulate LC firing was blocked by knockout of the CREB target adenylyl cyclase 8. Together, these findings provide direct evidence that prolonged exposure to morphine induces homeostatic plasticity intrinsic to LC neurons, involving up-regulation of the cAMP-CREB signaling pathway, which then enhances LC neuronal excitability.

Background Circadian rhythm abnormalities are strongly associated with bipolar disorder; however the role of circadian genes in mood regulation is unclear. Previously, we reported that mice with a mutation in the Clock gene ( Clock Δ19) display a behavioral profile that is strikingly similar to bipolar patients in the manic state. Methods Here, we used RNA interference and viral-mediated gene transfer to knock down Clock expression specifically in the ventral tegmental area (VTA) of mice. We then performed a variety of behavioral, molecular, and physiological measures. Results We found that knockdown of Clock , specifically in the VTA, results in hyperactivity and a reduction in anxiety-related behavior, which is similar to the phenotype of the Clock Δ19 mice. However, VTA-specific knockdown also results in a substantial increase in depression-like behavior, creating an overall mixed manic state. Surprisingly, VTA knockdown of Clock also altered circadian period and amplitude, suggesting a role for Clock in the VTA in the regulation of circadian rhythms. Furthermore, VTA dopaminergic neurons expressing the Clock short hairpin RNA have increased activity compared with control neurons, and this knockdown alters the expression of multiple ion channels and dopamine-related genes in the VTA that could be responsible for the physiological and behavioral changes in these mice. Conclusions Taken together, these results suggest an important role for Clock in the VTA in the regulation of dopaminergic activity, manic and depressive-like behavior, and circadian rhythms.

SummarySubiculum, the primary efferent pathway of hippocampus, participates in memory for spatial tasks, relapse to drug abuse, and temporal lobe seizures. Subicular pyramidal neurons exhibit low-threshold burst firing driven by a spike afterdepolarization. Here we report that burst firing can be regulated by stimulation of afferent projections to subiculum. Unlike synaptic plasticity, burst plasticity did not require synaptic depolarization, activation of AMPA or NMDA receptors, or action potential firing. Rather, enhancement of burst firing required synergistic activation of group I, subtype 1 metabotropic glutamate receptors (mGluRs) and muscarinic acetylcholine receptors (mAChR). When either of these receptors was blocked, a suppression of bursting was revealed, which in turn was blocked by antagonists of group I, subtype 5 mGluRs. These results indicate that the output of subiculum can be strongly and bidirectionally regulated by activation of glutamatergic inputs within the hippocampus and cholinergic afferents from the medial septum.

The ventral subiculum (vSUB) is an interface between the hippocampal formation and structures in the brain reward circuitry, such as the nucleus accumbens (NAc) and prefrontal cortex (PFC). The vSUB powerfully activates the dopamine system, particularly in response to novelty. This activity is both necessary and sufficient to elevate nucleus accumbens dopamine levels triggered by a novel stimulus. Direct stimulation of the vSUB increases the population of active dopamine neurons in the ventral tegmental area and dopamine levels in the accumbens via a multisynaptic route relayed through the nucleus accumbens. Furthermore, activity in the vSUB is correlated with drug-seeking behaviour such that vSUB inhibition attenuates cocaine-primed reinstatement of drug-seeking, while brief vSUB activation triggers reinstatement cocaine-seeking. We report that acute cocaine alters vSUB pyramidal neuron activity by inducing a frequency-dependent output mode transition from bursting to single-spiking. We suggest that under normal conditions bursting and output mode switching (bursting to single-spiking) may be needed for proper routing of information in and out of the vSUB. We propose that psychostimulants disrupt bursting and output mode switching leading to inappropriate dopamine/novelty signaling that is necessary to set motivational states and direct attention and ultimately, behaviour.

Although there are clear interactions between circadian rhythms and drug addiction, mechanisms for such interactions remain unknown. Here we establish a role for the Clock gene in regulating the brain's reward circuit. Mice lacking a functional Clock gene display an increase in cocaine reward and in the excitability of dopamine neurons in the midbrain ventral tegmental area, a key brain reward region. These phenotypes are associated with increased expression and phosphorylation of tyrosine hydroxylase (the rate-limiting enzyme in dopamine synthesis), as well as changes in several genes known to regulate dopamine activity in the ventral tegmental area. These findings demonstrate the involvement of a circadian-associated gene, Clock , in regulating dopamine function and cocaine reward.

Dopamine (DA) modulation of excitability in medial prefrontal cortex (mPFC) pyramidal neurons has attracted considerable attention because of the involvement of mPFC DA in several neuronal disorders. Here, we focused on DA modulation of inwardly rectifying K+ current (IRKC) in pyramidal neurons acutely dissociated from rat mPFC. A Cs+-sensitive whole-cell IRKC was elicited by hyperpolarizing voltage steps from a holding potential of –50 mV. DA (20 μm) reduced IRKC amplitude, as did selective stimulation of DA D1 or D2 class receptors (D1Rs and D2Rs). D1Rs activate, whereas D2Rs inhibit, the adenylyl cyclase–cAMP–protein kinase A (PKA) signaling pathway. Suppression of IRKC by D2R stimulation was attributable to decreased PKA activity because similar inhibition was observed with PKA inhibitors, whereas enhancing PKA activity increased IRKC. This suggests that the DA D1R suppression of IRKC occurred through a PKA phosphorylation-independent process. Using outside-out patches of mPFC pyramidal neurons, which preclude involvement of cytosolic signaling molecules, we observed a Cs+-sensitive macroscopic IRKC that was suppressed by the membrane-permeable cyclic nucleotide Sp-cAMP but was unaffected by non-nucleotide modulators of PKA, suggesting direct interactions of the cyclic nucleotides with IRK channels. Our results indicate that DA suppresses IRKC through two mechanisms: D1R activation of cAMP and direct interactions of the nucleotide with IRK channels and D2R-mediated dephosphorylation of IRK channels. The DA modulation of IRKC indicates that ambient DA would tend to increase responsiveness to excitatory inputs when PFC neurons are near the resting membrane potential and may provide a mechanism by which DA impacts higher cognitive function. ![Figure 8.][1] Figure 8. Dopamine modulates two types of K+ currents in PFC neurons. DA modulates the VGKC and the IRKC via activation of DA D1- and D2-class receptors. Activation of D1R increases intracellular cAMP level by activation of adenylyl cyclase (AC). The intracellular cAMP may diverge into two pathways: (1) direct interaction with IRK channels and reduction of IRKC; (2) PKA-dependent phosphorylation of IRK channels and enhancement of the IRKC. Although D1R-mediated modulation of IRKC is bidirectional, the inhibitory effect predominates under normal conditions. Activation of D2R inhibits the adenylyl cyclase activity, reduces basal activity of the cAMP–PKA signal transduction pathway, and dephosphorylates IRK channels, resulting in suppression of IRKC. DA modulates the VGKC primarily through D1R, the activation of which stimulates the adenylyl cyclase–cAMP–PKA pathway and increases the phosphorylation of VGK channels, resulting in suppression of the D-type VGKC. P, Phosphorylation. [1]: pending:yes

Psychostimulant drugs such as amphetamine are prescribed to increase vigilance, suppress appetite, and treat attention disorders, but they powerfully activate the dopamine system and have serious abuse potential. Repeated psychostimulant exposure induces neuronal plasticity within the mesolimbic dopamine system. Here we present evidence that repeated amphetamine exposure results in a suppression of intrinsic neuronal excitability in the ventral subiculum, a hippocampal region that activates dopamine neurotransmission. We used patch-clamp recordings from brain slices obtained at different times after withdrawal from repeated amphetamine exposure to determine the long-term effects of amphetamine on subicular excitability. Using several postsynaptic indices of sodium channel function, our results show that excitability is decreased for days, but not weeks, after repeated amphetamine exposure. The resulting increase in action potential threshold and decrease in postsynaptic amplification of excitatory synaptic input provide the first direct evidence that psychostimulants induce plasticity of hippocampal output and suggest one mechanism by which drug withdrawal may influence limbic dopamine-dependent learning and memory.

In order to parse the causal elements underlying complex behaviors and decision-making processes, appropriate behavioral methods must be developed and used in concurrence with molecular, pharmacological, and electrophysiological approaches. Presented is a protocol for a novel Go/No-Go behavioral paradigm to study the brain attention and motivation/reward circuitry in awake, head-restrained rodents. This experimental setup allows: (1) Pharmacological and viral manipulation of various brain regions via targeted guide cannula; (2) Optogenetic cell-type specific activation and silencing with simultaneous electrophysiological recording and; (3) Repeated electrophysiological single and multiple unit recordings during ongoing behavior. The task consists of three components. The subject first makes an observing response by initiating a trial by lever pressing in response to distinctive Go or No-Go tones. Then, after a variable delay period, the subject is presented with a challenge period cued by white noise during which they must respond with a lever press for the Go condition or withhold from lever pressing for the duration of the cue in the No-Go condition. After correctly responding during the challenge period (Challenge) and a brief delay, a final reward tone of the same frequency as the initiation tone is presented and sucrose reward delivery is available and contingent upon lever pressing. Here, we provide a novel procedure and validating data set that allows researchers to study and manipulate components of behavior such as attention, motivation, impulsivity, and reward-related working memory during an ongoing operant behavioral task while limiting interference from non task-related behaviors.

Previous studies using a variety of drugs with different affinities for the dopamine (DA) D3 receptor suggested that this receptor is involved in regulating motor activity and hypothermia. However, the in vivo selectivity of many of these compounds has been repeatedly questioned. To examine the precise roles of the DA D3 receptor in motor activity and hypothermic responses, we used mutant mice lacking the DA D3 receptor to evaluate the in vivo effects of several putative D3 receptor-selective agonists and antagonists. Using automated photocell activity chambers, we observed that the decreases in locomotor activity produced by putative D3 receptor-selective agonists as well as increases in locomotor activity produced by putative D3 receptor antagonists are identical in D3 receptor mutant and wild-type mice. In addition, the hypothermia produced by the putative D3 receptor-selective agonist PD 128907 is identical in both groups of mice. Based on these findings, we propose that D3 receptors are unlikely to be involved in these effects and we caution that the putative D3 ligands that have been used to reach conclusions regarding the functional roles of D3 receptors lack the necessary in vivo selectivity to support such conclusions.

Dopaminergic neurons exert a major modulatory effect on the forebrain. Dopamine and adenosine 3′,5′-monophosphate–regulated phosphoprotein (32 kilodaltons) (DARPP-32), which is enriched in all neurons that receive a dopaminergic input, is converted in response to dopamine into a potent protein phosphatase inhibitor. Mice generated to contain a targeted disruption of the DARPP-32 gene showed profound deficits in their molecular, electrophysiological, and behavioral responses to dopamine, drugs of abuse, and antipsychotic medication. The results show that DARPP-32 plays a central role in regulating the efficacy of dopaminergic neurotransmission.

Optogenetic methods have emerged as a powerful tool for elucidating neural circuit activity underlying a diverse set of behaviors across a broad range of species. Optogenetic tools of microbial origin consist of light-sensitive membrane proteins that are able to activate (e.g., channelrhodopsin-2, ChR2) or silence (e.g., halorhodopsin, NpHR) neural activity ingenetically-defined cell types over behaviorally-relevant timescales. We first demonstrate a simple approach for adeno-associated virus-mediated delivery of ChR2 and NpHR transgenes to the dorsal subiculum and prelimbic region of the prefrontal cortex in rat. Because ChR2 and NpHR are genetically targetable, we describe the use of this technology to control the electrical activity of specific populations of neurons (i.e., pyramidal neurons) embedded in heterogeneous tissue with high temporal precision. We describe herein the hardware, custom software user interface, and procedures that allow for simultaneous light delivery and electrical recording from transduced pyramidal neurons in an anesthetized in vivo preparation. These light-responsive tools provide the opportunity for identifying the causal contributions of different cell types to information processing and behavior.

The canonical transient receptor potential (TRPC) family of Ca2+ permeable, non-selective cation channels is abundantly expressed throughout the brain, and plays a pivotal role in modulating cellular excitability. Unlike other TRPC channels, TRPC4 subtype expression in the adult rodent brain is restricted to a network of structures that receive dopaminergic innervation, suggesting an association with motivation- and reward-related behaviors. We hypothesized that these channels may play a critical role in dopamine-dependent drug-seeking behaviors. Here, we gathered data testing trpc4 knockout (KO) rats and wild-type (WT) littermates in the acquisition of a natural sucrose reward (10 days), and cocaine self-administration (13 days) at 0.5 mg/kg/infusion. Rats lacking the trpc4 gene (trpc4-KO) learned to lever press for sucrose to a similar degree as their WT controls. However, when they were switched to cocaine, the trpc4-KO rats had substantially reduced cocaine-paired lever pressing compared to WT controls. No obvious group differences in inactive lever pressing were observed, for any time, during cocaine self-administration.

Canonical transient receptor potential (TRPC) channels are a family of non-selective cation channels that play a crucial role in modulating neuronal excitability due to their involvement in intracellular Ca2+ regulation and dendritic growth. TRPC5 channels a) are one of the two most prevalent TRPC channels in the adult rodent brain; b) are densely expressed in deep layer pyramidal neurons of the prefrontal cortex (PFC); and c) modulate neuronal persistent activity necessary for working memory and attention. In order to evaluate the causal role of TRPC5 in motivation/reward-related behaviors, conditional forebrain TRPC5 knock-down (trpc5-KD) mice were generated and trained to nose-poke for intravenous cocaine. Here we present a data set containing the first 6 days of saline or cocaine self-administration in wild type (WT) and trpc5-KD mice. In addition, we also present a data set showing the dose-response to cocaine after both groups had achieved similar levels of cocaine self-administration. Compared to WT mice, trpc5-KD mice exhibited an apparent increase in self-administration on the first day of cocaine testing without prior operant training. There were no apparent differences between WT and trpc5-KD mice for saline responding on the first day of training. Both groups showed similar dose-response sensitivity to cocaine after several days of achieving similar levels of cocaine intake.

Canonical transient receptor potential (TRPC) channels are a family of non-selective cation channels that play a crucial role in modulating neuronal excitability due to their involvement in intracellular Ca2+ regulation and dendritic growth. TRPC5 channels a) are one of the two most prevalent TRPC channels in the adult rodent brain; b) are densely expressed in deep layer pyramidal neurons of the prefrontal cortex (PFC); and c) modulate neuronal persistent activity necessary for working memory and attention. In order to evaluate the causal role of TRPC5 in motivation/reward-related behaviors, conditional forebrain TRPC5 knock-down (trpc5-KD) mice were generated and trained to nose-poke for intravenous cocaine. Here we present a data set containing the first 6 days of saline or cocaine self-administration in wild type (WT) and trpc5-KD mice. In addition, we also present a data set showing the dose-response to cocaine after both groups had achieved similar levels of cocaine self-administration. Compared to WT mice, trpc5-KD mice exhibited an apparent increase in self-administration on the first day of cocaine testing without prior operant training. There were no apparent differences between WT and trpc5-KD mice for saline responding on the first day of training. Both groups showed similar dose-response sensitivity to cocaine after several days of achieving similar levels of cocaine intake.

The brain dopamine system is thought to be the major target for the neuropharmacological actions of psychomotor stimulants such as cocaine. To investigate the mechanisms of cocaine action, we used a genetic approach, the gene-targeting technique, and generated D1 dopamine receptor mutant mice. Locomotor activity analysis in response to cocaine indicates that, in contrast to control mice which showed a dose-dependent increase in locomotion, D1 receptor mutant mice exhibited a dose-dependent decrease, suggesting that D1 receptors play an essential role in mediating such effects. Extracellular single unit recording of dopamine sensitive nucleus accumbens neurons in the D1 receptor mutant mice and control mice revealed a marked reduction in the inhibitory effects of cocaine and dopamine on the generation of action potentials, suggesting that D1 receptors play a fundamental role in cocaine- and dopamine-mediated neurophysiological effects within the nucleus accumbens. From these analyses, we conclude that the D1 dopamine receptor plays essential roles in mediating these effects of cocaine. In the future, the use of this powerful genetic approach will be essential for elucidating the molecular components of the signal transduction pathway leading to anatomical, cellular and behavioral changes upon cocaine administration and dopamine neurotransmission.

Behavioral control over a stressful event reduces the negative consequences of not only that event, but also future stressful events. Plasticity in the prelimbic (PL) medial prefrontal cortex is critical to this process, but the nature of the changes induced is unknown. We used patch-clamp recording to measure the intrinsic excitability of PL pyramidal neurons in acute slices from rats exposed to either escapable stress (ES), for which rats had behavioral control over tail-shock termination, or inescapable stress (IS) without control. Shortly after exposure (2 h) to tail-shock stress, neurons in the ES group had larger action potential (AP) amplitude and faster AP rise rate, larger postspike afterdepolarization, and reduced membrane time constant. No significant effects of IS were observed. We developed a conductance-based computer model using the simulation tool NEURON. The computer model simulated the observed changes in the ES group with increases in Na+conductance (gNa) and T-type Ca2+conductance (gCaT). The empirical and computational results indicate that behavioral control over stress, but not stress itself, increases PL pyramidal neuron excitability by increasing intrinsic membrane excitability. It is proposed that plasticity of excitability is important to the behavioral effects of controllable stressor exposure.

Precise spatial and temporal manipulation of neural activity in specific genetically defined cell populations is now possible with the advent of optogenetics. The emerging field of optogenetics consists of a set of naturally-occurring and engineered light-sensitive membrane proteins that are able to activate (e.g., channelrhodopsin-2, ChR2) or silence (e.g., halorhodopsin, NpHR) neural activity. Here we demonstrate the technique and the feasibility of using novel adeno-associated viral (AAV) tools to activate (AAV-CaMKll{\alpha}-ChR2-eYFP) or silence (AAV-CaMKll{\alpha}-eNpHR3.0-eYFP) neural activity of rat prefrontal cortical prelimbic (PL) pyramidal neurons in vivo. In vivo single unit extracellular recording of ChR2-transduced pyramidal neurons showed that delivery of brief (10 ms) blue (473 nm) light-pulse trains up to 20 Hz via a custom fiber optic-coupled recording electrode (optrode) induced spiking with high fidelity at 20 Hz for the duration of recording (up to two hours in some cases). To silence spontaneously active neurons we transduced them with the NpHR construct and administered continuous green (532 nm) light to completely inhibit action potential activity for up to 10 seconds with 100% fidelity in most cases. These versatile photosensitive tools combined with optrode recording methods provide experimental control over activity of genetically defined neurons and can be used to investigate the functional relationship between neural activity and complex cognitive behavior., Comment: 4 pages, 4 figures F1000Research article

Here we describe the first report of using low-cost cellular or web-based digital cameras to image and quantify standardized rapid immunoassay strips as a new point-of-care diagnostic and forensics tool with health applications. Quantitative ratiometric pixel density analysis (QRPDA) is an automated method requiring end-users to utilize inexpensive (~ $1 USD/each) immunotest strips, a commonly available web or mobile phone camera or scanner, and internet or cellular service. A model is described whereby a central computer server and freely available IMAGEJ image analysis software records and analyzes the incoming image data with time-stamp and geo-tag information and performs the QRPDA using custom JAVA based macros ( http://www.neurocloud.org ). To demonstrate QRPDA we developed a standardized method using rapid immunotest strips directed against cocaine and its major metabolite, benzoylecgonine. Images from standardized samples were acquired using several devices, including a mobile phone camera, web cam, and scanner. We performed image analysis of three brands of commercially available dye-conjugated anti-cocaine/benzoylecgonine (COC/BE) antibody test strips in response to three different series of cocaine concentrations ranging from 0.1 to 300 ng/ml and BE concentrations ranging from 0.003 to 0.1 ng/ml. This data was then used to create standard curves to allow quantification of COC/BE in biological samples. Across all devices, QRPDA quantification of COC and BE proved to be a sensitive, economical, and faster alternative to more costly methods, such as gas chromatography-mass spectrometry, tandem mass spectrometry, or high pressure liquid chromatography. The limit of detection was determined to be between 0.1 and 5 ng/ml. To simulate conditions in the field, QRPDA was found to be robust under a variety of image acquisition and testing conditions that varied temperature, lighting, resolution, magnification and concentrations of biological fluid in a sample. To determine the effectiveness of the QRPDA method for quantifying cocaine in biological samples, mice were injected with a sub-locomotor activating dose of cocaine (5 mg/kg; i.p.) and were found to have detectable levels of COC/BE in their urine (160.6 ng/ml) and blood plasma (8.1 ng/ml) after 15–30 minutes. By comparison rats self-administering cocaine in a 4 hour session obtained a final BE blood plasma level of 910 ng/ml with an average of 62.5 infusions. It is concluded that automated QRPDA is a low-cost, rapid and highly sensitive method for the detection of COC/BE with health, forensics, and bioinformatics application and the potential to be used with other rapid immunotest strips directed at several other targets. Thus, this report serves as a general reference and method describing the use of image analysis of lateral flow rapid test strips.

Precise spatial and temporal manipulation of neural activity in specific genetically defined cell populations is now possible with the advent of optogenetics. The emerging field of optogenetics consists of a set of naturally-occurring and engineered light-sensitive membrane proteins that are able to activate (e.g. channelrhodopsin-2, ChR2) or silence (e.g. halorhodopsin, NpHR) neural activity. Here we demonstrate the technique and the feasibility of using novel adeno-associated viral (AAV) tools to activate (AAV-CaMKllα-ChR2-eYFP) or silence (AAV-CaMKllα-eNpHR3.0-eYFP) neural activity of rat prefrontal cortical prelimbic (PL) pyramidal neurons in vivo. In vivo single unit extracellular recording of ChR2-transduced pyramidal neurons showed that delivery of brief (10 ms) blue (473 nm) light-pulse trains up to 20 Hz via a custom fiber optic-coupled recording electrode (optrode) induced spiking with high fidelity at 20 Hz for the duration of recording (up to two hours in some cases). To silence spontaneously active neurons, we transduced them with the NpHR construct and administered continuous green (532 nm) light to completely inhibit action potential activity for up to 10 seconds with 100% fidelity in most cases. These versatile photosensitive tools, combined with optrode recording methods, provide experimental control over activity of genetically defined neurons and can be used to investigate the functional relationship between neural activity and complex cognitive behavior.

Kynurenic acid (KYNA), a classical ionotropic glutamate receptor antagonist is also purported to block the α7-subtype nicotinic acetylcholine receptor (α7* nAChR). Although many published studies cite this potential effect, few have studied it directly. In this study, the α7*-selective agonist, choline, was pressure-applied to interneurons in hippocampal subregions, CA1 stratum radiatum and hilus of acute brain hippocampal slices from adolescent to adult mice and adolescent rats. Stable α7* mediated whole-cell currents were measured using voltage-clamp at physiological temperatures. The effects of bath applied KYNA on spontaneous glutamatergic excitatory postsynaptic potentials (sEPSC) as well as choline-evoked α7* currents were determined. In mouse hilar interneurons, KYNA totally blocked sEPSC whole-cell currents in a rapid and reversible manner, but had no effect on choline-evoked α7* whole-cell currents. To determine if this lack of KYNA effect on α7* function was due to regional and/or species differences in α7* nAChRs, the effects of KYNA on choline-evoked α7* whole-cell currents in mouse and rat stratum radiatum interneurons were tested. KYNA had no effect on either mouse or rat stratum radiatum interneuron choline-evoked α7* whole-cell currents. Finally, to test whether the lack of effect of KYNA was due to unlikely slow kinetics of KYNA interactions with α7* nAChRs, recordings of a7*-mediated currents were made from slices that were prepared and stored in the presence of 1 mM KYNA (>90 minutes exposure). Under these conditions, KYNA had no measurable effect on α7* nAChR function. The results show that despite KYNA-mediated blockade of glutamatergic sEPSCs, two types of hippocampal interneurons that express choline-evoked α7* nAChR currents fail to show any degree of modulation by KYNA. Our results indicate that under our experimental conditions, which produced complete KYNA-mediated blockade of sEPSCs, claims of KYNA effects on choline-evoked α7* nAChR function should be made with caution.

TRPC4 ion channels are expressed extensively in corticolimbic brain regions and a subpopulation of midbrain dopamine neurons. TRPC4 knockout (KO) rats show reduced sociability and social exploration, but show no differences in simple and complex strategic learning compared to normal wild type (WT) rats. Using water reward, we found no differences between TRPC4-KO and WT rats in the break point on a progressive ratio schedule of reinforcement. Although deletion of the trpc4 gene alters social interaction/anxiety it does not appear to affect motivation for natural rewards. Current experiments are underway testing the role of trpc4 gene deletion on cocaine reward (see www.neuro-cloud.net/nature-precedings/klipec3 for updates and collaborations).

The number of emergency department visits concerning sport related TBI in youth increased 57% from 2001 to 2009 and while a fraction of that increase may be attributed to injuries that were once missed now being identified due to greater general awareness, the fact remains that there are nearly 200,000 children every year who suffer sport related concussion or other TBI serious enough to prompt a visit to the ED. 40% of sports related concussions involve children between the ages of 8 and 13, and in this group the rate of concussion doubled between 1997 and 2007. The risk of concussion is highest in football and there are nearly 67,000 diagnosed concussions in high school football every year. In other sports that males and females play, such as basketball, girls seem to be at a higher risk. In high schools, concussions account for 15% of all sports related injuries resulting in at least one day of play lost. The risk of a suffering a concussion when participating in a contact sport may be as high as 20% per season. In both girls’ and boys’ soccer and basketball 20% of the concussions were repeat concussions. These statistics just further illustrate the gravity of the problem of sport related concussions. Here we describe the need for more work on education, legislation and research in order to change in the concussion/mTBI culture.

Mobile Platform Informatics (MPI) and Smartphone Informatics (SPI) methods like Mobile Image Ratiometry (MIR) are potentially transformative point-of-use instantaneous analysis tools that are useful across a variety of industries. In agriculture, MIR-compatible immuno test strips allow early detection of a number of biotic stressors before devastating crop losses occur. Here we describe a low-cost and easy-to-use Smartphone and/or tablet-based protocol (Mobile Assay Inc., www.mobileassay.com) for the detection and on-sight instantaneous analysis of B. cinerea, a fungus that causes significant damage to a variety of plants and flowers. Early detection and tracking of the B. cinerea fungus before the visible gray mold appears has the potential to increase agricultural productivity especially in the developing world.

The TRPC4 ion channel is expressed extensively in corticolimbic and a subpopulation of midbrain dopamine neurons. While TRPC4 knockout (KO) rats exhibit reduced sociability and social exploration, little is known about the role of TRPC4 in motivation and learning. To identify a function for TRPC4 channels in learning processes we tested TRPC4 KO and normal wild type (WT) rats. TRPC4 KO and WT rats exhibited no differences in Y--maze learning or simple discrimination learning. Furthermore, on a more complex serial reversal shift task designed to assess strategic learning where the reward and non--reward cues were repeatedly reversed between training sessions both TRPC4 KO and WT rats performed equally well. Finally, we found no performance differences when using a conditional reversal shift task where a tone signals the reversal of reward and non-reward cues within sessions. These data suggest that although TRPC4 channels may play a role in social interaction/anxiety they exert a minimal role in simple and complex strategic learning.

Here we describe Mobile Image Ratiometry (MIR), a new method for the automated quantification of standardized rapid immunoassay strips using consumer-based mobile smartphone and tablet cameras. To demonstrate MIR we developed a standardized method using rapid immunotest strips directed against cocaine (COC) and its major metabolite, benzoylecgonine (BE). We performed image analysis of three brands of commercially available dye-conjugated anti-COC/BE antibody test strips in response to three different series of cocaine concentrations ranging from 0.1 to 300 ng/ml and BE concentrations ranging from 0.003 to 0.1 ng/ml. These data were then used to create standard curves to allow quantification of COC/BE in biological samples. MIR quantification of COC and BE proved to be a sensitive, economical, and faster alternative to more costly methods, such as gas chromatography-mass spectrometry, tandem mass spectrometry, or high pressure liquid chromatography. MIR is a valuable tool that provides instant data acquisition, tracking and analysis for the emerging field of mobile platform informatics (MPI).

In this paper, Arora and colleagues expand on their previous work on GIRK channels in the ventral tegmental area (VTA) presenting evidence that a single exposure to cocaine reduces inhibitory GABAergic transmission to dopamine (DA) neurons in the ventral tegmental area. Mice receiving i.p. injections of cocaine saw a short lived (1-5 days) decrease in GABAb mediated G-protein coupled inwardly-rectifying potassium (GIRK) currents in DA neurons in the VTA. This decrease parallels an NMDA-mediated increase in the frequency of glutamatergic neurotransmission. Chronic cocaine injections had no additional effects beyond those seen with single injections. Though they found no change in mRNA levels for GABAb receptors, GIRK channels, or RGS-2 (a G-protein regulator), immunoelectron microscopy indicated a decrease in levels of GIRK channels in the plasma membrane of the dendrites of VTA DA neurons. The cocaine-mediated decrease in GIRK currents was abolished in the presence of D2/3R antagonist sulpiride, but not in the presence of D1/5 antagonist SCH23390, indicating a link between D2/3 receptor activation and GIRK activity. Interestingly, the addition of quinpirole, a D2/3 agonist, elicited similar GIRK currents, though they were smaller than those mediated by GABAb receptors. Similarly, acute injections of cocaine significantly diminished quinpirole-evoked currents.

The nonselective cation channel TRPC4 has been shown to be present in high abundance in the corticolimbic regions of the brain and play a pivotal role in modulating cellular excitability due to their involvement in intracellular Ca2+ regulation. Recently we reported their involvement in socialization and regulating anxiety-like behaviors in rats. Given the important role for dopamine in modulating emotions involved in social anxiety we investigated whether TRPC4 protein and mRNA was found on dopaminergic neurons of the ventral tegmental area (VTA). Using emulsion autoradiography we found that TRPC4 mRNA is indeed present in the VTA and the substantia nigra. Additionally, immunohistochemistry verified it's presence on a subpopulation of dopamine neurons in the VTA. We confirmed these findings by testing Trpc4 knock-out rats in addition to wild-type animals. This novel finding suggests that TRPC4 plays a pivotal role in regulating dopamine release in a sub-population of neurons that may modulate emotional and cognitive responses in social situations., 2 pages 2 figures; Nature Precedings http://dx.doi.org/10.1038/npre.2011.6577.1 (2011)

Shyness and social anxiety are predominant features of some psychiatric disorders including autism, schizophrenia, anxiety and depression. Understanding the cellular and molecular determinants of sociability may reveal therapeutic approaches to treat individuals with these disorders and improve their quality of life. Previous experiments from our laboratory have identified selective mRNA and protein expression of a nonselective cation channel known as the canonical transient receptor potential channel 4 (TRPC4s) in brain regions implicated in emotional regulation and anxiety. TRPC4 is highly expressed in the corticolimbic regions of the mammalian brain. We hypothesized that robust corticolimbic expression of TRPC4 may regulate the brain’s response to emotion and anxiety resulting in changes in social interaction. Here we test trpc4 gene knockout rats in a model of social anxiety/interaction. We found that the Trpc4 knockout animals spent significantly less time exploring a juvenile intruder rat compared to their wild-type counterparts and Sprague-Dawley (SD) rats. Furthermore, Trpc4 wild-type (Fisher 344) rats explored the juvenile significantly less than the SD rats. These findings indicate that the trpc4 gene plays a role in modulating cellular excitability in specific regions of the brain associated sociality and/or anxiety.