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.circadian rhythms ͉ dopamine ͉ drug addiction ͉ tyrosine hydroxylase D rug addiction is associated with disruptions in sleep and circadian rhythmicity (1-3). Moreover, in animal models of addiction, several reward-related behaviors exhibit clear circadian regulation. For example, levels of drug self administration and drug-induced locomotor sensitization vary according to the day͞night cycle (4-6). These observations suggest interactions between the brain's circadian and reward systems.Although many of the genes involved in circadian rhythms are expressed outside the suprachiasmatic nucleus (SCN), the brain's master circadian pacemaker, and are found in limbic regions of the brain, little is known about their function in these other brain regions. The first indication that circadian-associated genes may be involved in drug-related behaviors came from studies in Drosophila, which showed that behavioral sensitization to cocaine depended on expression of Period, Clock, Cycle, and Doubletime (7). More recently, it was reported that locomotor sensitization and conditioned preference for cocaine are abnormal in mice lacking the Period-1 (mPer1) or Period-2 (mPer2) gene (6). These genes are induced as well by cocaine in the dorsal striatum and nucleus accumbens, brain regions important for cocaine's behavioral effects (8, 9). Although these findings support a role for circadian-associated genes in behavioral responses to drugs of abuse, little is known about the mechanisms by which these genes function, or are regulated, within the brain's reward and motor circuits.Cocaine and other drugs of abuse produce their behavioral effects in part by modulating dopamine neurotransmission in the midbrain ventral tegmental area (VTA), a key component of the brain's reward circuit (10). Several interactions between dopamine and circadian function have been reported. For example, dopamine neurons in the retina regulate adaptations to light (11). Moreover, dopamine D1 receptors in the prenatal SCN are necessary for synchronizing the master circadian clock during development (12). However, a direct link between circadian genes and the VTA dopamine reward system has not been described. CLOCK is a member of the basic helix-loop-helix-PAS (PER-ARNT-SIM) transc...

Autophagy is crucial for neuronal integrity. Loss of key autophagic components leads to progressive neurodegeneration and structural defects in pre- and postsynaptic morphologies. However, the molecular mechanisms regulating autophagy in the brain remain elusive. Similarly, while it is widely accepted that protein turnover is required for synaptic plasticity, the contribution of autophagy to the degradation of synaptic proteins is unknown. Here, we report that BDNF signaling via the tropomyosin receptor kinase B (TrkB) and the phosphatidylinositol-3' kinase (PI3K)/Akt pathway suppresses autophagy in vivo. In addition, we demonstrate that suppression of autophagy is required for BDNF-induced synaptic plasticity and for memory enhancement under conditions of nutritional stress. Finally, we identify three key remodelers of postsynaptic densities as cargo of autophagy. Our results establish autophagy as a pivotal component of BDNF signaling, which is essential for BDNF-induced synaptic plasticity. This molecular mechanism underlies behavioral adaptations that increase fitness in times of scarcity.

Neurofibromatosis type I (NF1) is one of the most common singlegene causes of learning disabilities. Here, we use behavioral working memory probes and electrophysiological studies in a mouse model of NF1 (Nf1 heterozygous null mutants; Nf1 +/− ) to demonstrate that (i) Neurofibromin regulates prefrontal and striatal inhibitory networks, specifically activity-dependent GABA release and (ii) is required for working memory performance, with inhibitiondependent working memory deficits seen in Nf1 +/− mice. We find that increased inhibition in medial prefrontal cortex (mPFC) is sufficient to alter persistent activity in a biophysical model of an mPFC microcircuit, suggesting a possible mechanism for Nf1 +/− working memory deficits. Accordingly, working memory assays applied during functional MRI (fMRI) studies in human subjects with NF1 reveal hypoactivation of corticostriatal networks, which is associated with impaired working memory performance. Collectively, these integrative mouse and human studies reveal molecular and cellular mechanisms contributing to working memory deficits in NF1.GABA | Ras | prefrontal cortex | learning disability | neurofibromatosis type I N eurofibromatosis type 1 (NF1) is a valuable model for understanding mechanisms of learning disabilities (1). NF1 is a common genetic disorder (incidence 1:3,000) that results from mutations in a single gene (Nf1) that encodes the neurofibromin protein (2, 3). Specific deficits in the domains of visuospatial and executive functions are among the most common cognitive deficits associated with this syndrome (1,4,5). Previous mechanistic studies in a mouse model of NF1 (Nf1 heterozygous null mutants or Nf1 +/− ) demonstrated that neurofibromin modulates Rasdependent GABA release in the hippocampus, which in turn modulates long-term potentiation (LTP) and hippocampaldependent learning (6, 7). However, the mechanisms underlying frontal executive dysfunction in NF1, including prominent working memory deficits (5), are unknown. Therefore, to investigate mechanisms underlying working memory deficits associated with the NF1 mutation we carried out parallel experiments in mice and humans.Working memory is a cognitive construct involving the ability to hold and update information transiently in mind in the service of higher-order cognitive activities. Executive functions, including working memory, are thought to depend on common corticostriatal networks (8-11). Therefore, our experiments focused on frontal corticostriatal circuitry, with an emphasis on the dorsolateral prefrontal cortex (DLPFC) in humans, thought to be critical for working memory (12), and its functionally analogous structure in rodents, the medial prefrontal cortex (mPFC) (13,14).Here, we report Ras-dependent increases in GABA release in the mPFC and striatum of the Nf1 +/− mouse model. Increased GABAergic inhibition is likely to be responsible for the working memory deficits that we found in the Nf1 +/− mice because these deficits could be reversed with a drug that decreased inhibition. Further,...

The canonical transient receptor potential (TRPC) channels are a family of non-selective cation channels that are activated by increases in intracellular Ca2+ and Gq/phospholipase C-coupled receptors. We used quantitative real-time PCR, in situ hybridization, immunoblots and patch-clamp recording from several brain regions to examine the expression of the predominant TRPC channels in the rodent brain. Quantitative real-time PCR of the seven TRPC channels in the rodent brain revealed that TRPC4 and TRPC5 channels were the predominant TRPC subtypes in the adult rat brain. In situ hybridization histochemistry and immunoblotting further resolved a dense corticolimbic expression of the TRPC4 and TRPC5 channels. Total protein expression of HIP TRPC4 and 5 proteins increased throughout development and peaked late in adulthood (6–9 weeks). In adults, TRPC4 expression was high throughout the frontal cortex, lateral septum (LS), pyramidal cell layer of the hippocampus (HIP), dentate gyrus (DG), and ventral subiculum (vSUB). TRPC5 was highly expressed in the frontal cortex, pyramidal cell layer of the HIP, DG, and hypothalamus. Detailed examination of frontal cortical layer mRNA expression indicated TRPC4 mRNA is distributed throughout layers 2–6 of the prefrontal cortex (PFC), motor cortex (MCx), and somatosensory cortex (SCx). TRPC5 mRNA expression was concentrated specifically in the deep layers 5/6 and superficial layers 2/3 of the PFC and anterior cingulate. Patch-clamp recording indicated a strong metabotropic glutamate-activated cation current-mediated depolarization that was dependent on intracellular Ca2+and inhibited by protein kinase C in brain regions associated with dense TRPC4 or 5 expression and absent in regions lacking TRPC4 and 5 expression. Overall, the dense corticolimbic expression pattern suggests that these Gq/PLC coupled nonselective cation channels may be involved in learning, memory, and goal-directed behaviors.

Intrinsic properties of neurons that enable them to maintain depolarized, persistently activated states in the absence of sustained input are poorly understood. In short-term memory tasks, individual prefrontal cortical (PFC) neurons are capable of maintaining persistent action potential output during delay periods between informative cues and behavioral responses. Dopamine and drugs of abuse alter PFC function and working memory possibly by modulating intrinsic neuronal properties. Here we use patch-clamp recording of layer 5 PFC pyramidal neurons to identify an action potential burst-evoked intrinsic mGluR5-mediated postsynaptic depolarization that initiates an activated state. Depolarization occurs in the absence of recurrent synaptic activity and is reduced by a postsynaptic dopamine D1/5 receptor pathway. The depolarization is substantially diminished following behavioral sensitization to cocaine; moreover the D1/5 receptor modulation is lost. We propose the burst-evoked intrinsic depolarization to be a novel form of short-term cellular memory that is modulated by dopamine and cocaine experience.

Although the medial prefrontal cortex (mPFC) plays a critical role in cocaine addiction, the effects of chronic cocaine on mPFC neurons remain poorly understood. Here, we performed visualized current-clamp recordings to determine the effects of repeated cocaine administration on the membrane excitability of mPFC pyramidal neurons in rat brain slices. Following repeated cocaine administration (15 mg/kg/day i.p. for 5 days) with a 3-day withdrawal, alterations in membrane properties, including increased input resistance, reduced intensity of intracellular injected currents required for generation of Na ϩ -dependent spikes (rheobase), and an increased number of spikes evoked by depolarizing current pulses were observed in mPFC neurons. The current-voltage relationship was also altered in cocaine-pretreated neurons showing reduced outward rectification during membrane depolarization and decreased inward rectification during membrane hyperpolarization. Application of the K ϩ channel blocker Ba 2ϩ depolarized the resting membrane potential (RMP) and enhanced membrane potential response to injection of hyperpolarizing current pulses. However, the effects of Ba 2ϩ on RMP and hyperpolarized membrane potentials were significantly attenuated in cocaine-withdrawn neurons compared with saline-pretreated cells. These findings indicate that repeated cocaine administration increased the excitability of mPFC neurons after a short-term withdrawal, possibly via reducing the activity of the potassium inward rectifiers (K ir ) and voltage-gated K ϩ currents. Similar changes were also observed in cocaine-pretreated mPFC neurons after a long-term (2-3 weeks) withdrawal, revealing a persistent increase in excitability. These alterations in mPFC neuronal excitability may contribute to the development of behavioral sensitization and withdrawal effects following chronic cocaine exposure.

Working memory refers to the temporary storage of information and is strongly associated with the prefrontal cortex (PFC). Persistent activity of cortical neurons, namely the activity that persists beyond the stimulus presentation, is considered the cellular correlate of working memory. Although past studies suggested that this type of activity is characteristic of large scale networks, recent experimental evidence imply that small, tightly interconnected clusters of neurons in the cortex may support similar functionalities. However, very little is known about the biophysical mechanisms giving rise to persistent activity in small-sized microcircuits in the PFC. Here, we present a detailed biophysically—yet morphologically simplified—microcircuit model of layer V PFC neurons that incorporates connectivity constraints and is validated against a multitude of experimental data. We show that (a) a small-sized network can exhibit persistent activity under realistic stimulus conditions. (b) Its emergence depends strongly on the interplay of dADP, NMDA, and GABAB currents. (c) Although increases in stimulus duration increase the probability of persistent activity induction, variability in the stimulus firing frequency does not consistently influence it. (d) Modulation of ionic conductances (Ih, ID, IsAHP, IcaL, IcaN, IcaR) differentially controls persistent activity properties in a location dependent manner. These findings suggest that modulation of the microcircuit's firing characteristics is achieved primarily through changes in its intrinsic mechanism makeup, supporting the hypothesis of multiple bi-stable units in the PFC. Overall, the model generates a number of experimentally testable predictions that may lead to a better understanding of the biophysical mechanisms of persistent activity induction and modulation in the PFC.

Dietary intake of methyl donors, such as folic acid and methionine, shows considerable intra-individual variation in human populations. While it is recognized that maternal departures from the optimum of dietary methyl donor intake can increase the risk for mental health issues and neurological disorders in offspring, it has not been explored whether paternal dietary methyl donor intake influences behavioral and cognitive functions in the next generation. Here, we report that elevated paternal dietary methyl donor intake in a mouse model, transiently applied prior to mating, resulted in offspring animals (methyl donor-rich diet (MD) F1 mice) with deficits in hippocampus-dependent learning and memory, impaired hippocampal synaptic plasticity and reduced hippocampal theta oscillations. Gene expression analyses revealed altered expression of the methionine adenosyltransferase Mat2a and BK channel subunit Kcnmb2, which was associated with changes in Kcnmb2 promoter methylation in MD F1 mice. Hippocampal overexpression of Kcnmb2 in MD F1 mice ameliorated altered spatial learning and memory, supporting a role of this BK channel subunit in the MD F1 behavioral phenotype. Behavioral and gene expression changes did not extend into the F2 offspring generation. Together, our data indicate that paternal dietary factors influence cognitive and neural functions in the offspring generation.