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1.  Voltage drives diverse endocannabinoid signals to mediate striatal microcircuit-specific plasticity 
Nature neuroscience  2013;16(9):1275-1283.
The dorsolateral striatum and cannabinoid type 1 receptor (CB1) signaling mediate habitual action learning, which is thought to require a balance of activity in the direct and indirect striatal output pathways. However, very little is known about how the high CB1-expressing striatal inhibitory microcircuitry might contribute to long-term plasticity capable of sculpting direct/indirect pathway output. Using optogenetic and molecular interrogation of striatal GABAergic microcircuits, we describe novel mechanisms of voltage-dependent long-term depression of inhibitory synapses (iLTD) onto mouse and rat medium spiny projection neurons (MSNs). This iLTD involves recruitment of different endocannabinoid types and shows both presynaptic and postsynaptic selectivity for MSN subtypes, ultimately resulting in a powerful disinhibition of direct pathway MSNs. These results indicate a new role for voltage states in gating circuit-specific forms of synaptic plasticity and illuminate possible circuit dynamics underlying action control.
doi:10.1038/nn.3478
PMCID: PMC3758434  PMID: 23892554
2.  GluN2B in corticostriatal circuits governs choice learning and choice shifting 
Nature neuroscience  2013;16(8):1101-1110.
A choice that reliably produces a preferred outcome can be automated to liberate cognitive resources for other tasks. Should an outcome become less desirable, behavior must adapt in parallel or become perseverative. Corticostriatal systems are known to mediate choice learning and flexibility, but the molecular mechanisms subserving the instantiation of these processes are not well understood. We integrated mouse behavioral, immunocytochemical, in vivo electrophysiological, genetic, and pharmacological approaches to study choice. We found that the dorsal striatum (DS) was increasingly activated with choice learning, whereas reversal of learned choice engaged prefrontal regions. In vivo, DS neurons showed activity associated with reward anticipation and receipt that emerged with learning and relearning. Corticostriatal or striatal GluN2B gene deletion, or DS-restricted GluN2B antagonism, impaired choice learning, whereas cortical GluN2B deletion or OFC GluN2B antagonism impaired shifting. Our convergent data demonstrate how corticostriatal GluN2B circuits govern the ability to learn and shift choice behavior.
doi:10.1038/nn.3457
PMCID: PMC3725191  PMID: 23831965
3.  Repeated Binge-Like Ethanol Drinking Alters Ethanol Drinking Patterns and Depresses Striatal GABAergic Transmission 
Neuropsychopharmacology  2013;39(3):579-594.
Repeated cycles of binge alcohol drinking and abstinence are key components in the development of dependence. However, the precise behavioral mechanisms underlying binge-like drinking and its consequences on striatal synaptic physiology remain unclear. In the present study, ethanol and water drinking patterns were recorded with high temporal resolution over 6 weeks of binge-like ethanol drinking using the ‘drinking in the dark' (DID) protocol. The bottle exchange occurring at the beginning of each session prompted a transient increase in the drinking rate that might facilitate the acquisition of ethanol binge-like drinking. Ethanol drinking mice also displayed a ‘front-loading' behavior, in which the highest rate of drinking was recorded during the first 15 min. This rate increased over weeks and paralleled the mild escalation of blood ethanol concentrations. GABAergic and glutamatergic transmission in the dorsal striatum were examined following DID. Spontaneous glutamatergic transmission and the density of dendritic spines were unchanged after ethanol drinking. However, the frequency of GABAA receptor-mediated inhibitory postsynaptic currents was depressed in medium spiny neurons of ethanol drinking mice. A history of ethanol drinking also increased ethanol preference and altered the acute ethanol effects on GABAergic transmission differentially in dorsolateral and dorsomedial striatum. Together, the study shows that the bottle exchange during DID promotes fast, voluntary ethanol drinking and that this intermittent pattern of ethanol drinking causes a depression of GABAergic transmission in the dorsal striatum.
doi:10.1038/npp.2013.230
PMCID: PMC3895236  PMID: 23995582
Repeated binge-like ethanol drinking alters ethanol drinking patterns and depresses striatal GABAergic transmission; alcohol; drinking in the dark; dendritic spines; dopamine; striatum; dorsolateral striatum
4.  Mutations M287L and Q266I in the Glycine Receptor α1 Subunit Change Sensitivity to Volatile Anesthetics in Oocytes and Neurons, but Not the Minimal Alveolar Concentration in Knockin Mice 
Anesthesiology  2012;117(4):765-771.
Background
Volatile anesthetics (VAs) alter the function of key central nervous system proteins but it is not clear which, if any, of these targets mediates the immobility produced by VAs in the face of noxious stimulation. A leading candidate is the glycine receptor, a ligand-gated ion channel important for spinal physiology. VAs variously enhance such function, and blockade of spinal GlyRs with strychnine affects the minimal alveolar concentration (an anesthetic EC50) in proportion to the degree of enhancement.
Methods
We produced single amino acid mutations into the glycine receptorα1 subunit that increased (M287L, third transmembrane region) or decreased (Q266I, second transmembrane region) sensitivity to isoflurane in recombinant receptors, and introduced such receptors into mice. The resulting knockin mice presented impaired glycinergic transmission, but heterozygous animals survived to adulthood, and we determined the effect of isoflurane on glycine-evoked responses of brain stem neurons from the knockin mice, and the minimal alveolar concentration for isoflurane and other VAs in the immature and mature knockin mice.
Results
Studies of glycine-evoked currents in brain stem neurons from knock-in mice confirmed the changes seen with recombinant receptors. No increases in the minimal alveolar concentration were found in knockin mice, but the minimal alveolar concentration for isoflurane and enflurane (but not halothane) decreased in 2-week-old Q266I mice. This change is opposite to the one expected for a mutation that decreases the sensitivity to volatile anesthetics.
Conclusion
Taken together, these results indicate that glycine receptors containing the α1 subunit are not likely to be crucial for the action of isoflurane and other VAs.
doi:10.1097/ALN.0b013e31826a0d93
PMCID: PMC3447119  PMID: 22885675
5.  Reduced alcohol intake and reward associated with impaired endocannabinoid signaling in mice with a deletion of the glutamate transporter GLAST 
Neuropharmacology  2012;63(2):181-189.
A hyperglutamatergic state has been hypothesized to drive escalation of alcohol intake. This hypothesis predicts that an impairment of glutamate clearance through inactivation of the astrocytic glutamate transporter, GLAST (EAAT1), will result in escalation of alcohol consumption. Here, we used mice with a deletion of GLAST to test this prediction. WT and GLAST KO mice were tested for alcohol consumption using two-bottle free-choice drinking. Alcohol reward was evaluated using conditioned place preference (CPP). Sensitivity to depressant alcohol effects was tested using the accelerating rotarod, alcohol-induced hypothermia, and loss of righting reflex. Extracellular glutamate was measured using microdialysis, and striatal slice electrophysiology was carried out to examine plasticity of the cortico-striatal pathway as a model system in which adaptations to the constitutive GLAST deletion can be studied. Contrary to our hypothesis, GLAST KO mice showed markedly decreased alcohol consumption, and lacked CPP for alcohol, despite a higher locomotor response to this drug. Alcohol-induced ataxia, hypothermia, and sedation were unaffected. In striatal slices from GLAST KO mice, long-term depression (LTD) induced by high frequency stimulation, or by post-synaptic depolarization combined with the L-type calcium channel activator FPL 64176 was absent. In contrast, normal synaptic depression was observed after application of the cannabinoid 1 (CB1) receptor agonist WIN55,212-2. Constitutive deletion of GLAST unexpectedly results in markedly reduced alcohol consumption and preference, associated with markedly reduced alcohol reward. Endocannabinoid signaling appears to be down-regulated upstream of the CB1 receptor as a result of the GLAST deletion, and is a candidate mechanism behind the reduction of alcohol reward observed.
doi:10.1016/j.neuropharm.2012.01.027
PMCID: PMC3372600  PMID: 22342743
glutamate transporter; alcohol; reward; endocannabinoid
6.  Subsets of Spiny Striosomal Striatal Neurons Revealed in the Gad1-GFP BAC Transgenic Mouse 
Basal Ganglia  2011;1(4):201-211.
Objective
To characterize GFP-expressing cells in the striatum of Cb6-Tg(Gad1-EGFP)G42Zjh/J mice, in which the Gad1 (also referred to as GAD67) promoter drives GFP expression (Gad1-GFP mouse).
Background
GFP-expressing cells of the GAD1-GFP mouse have been described to be a population of parvalbumin-positive basket interneurons residing in the cerebral cortex and the cerebellum. However, the cells in the dorsal striatum of these mice have not been characterized.
Methods
Using a combination of immunohistochemistry, electrophysiology, DiI labeling, and retrograde tracing, we investigated the phenotypes of GFP-expressing cells in the GAD1-GFP mice.
Results
A small number of striatal neurons express GFP in these mice. In the mature striatum, these cells are preferentially located in the lateral striatum with a strong expression in the lateral striatal streak. The GAD1-GFP positive neurons are distinct from the standard fast-spiking and low-threshold-spiking GAD-67 expressing striatal interneurons and appear to be a subset of medium spiny neurons. These neurons are generally colocalized with striosomal markers such as dynorphin, mu-opioid receptors, as well as CB1 and calretinin-immunopositive fibers. Striatal Gad1-GFP neurons can be separated into two groups based on the shape of the somata and patterns of action potential firing. Retrograde labeling indicated that a proportion of these cells are projection neurons.
Conclusions
The examination of GAD1-GFP cells in these mice revealed 2 subpopulations of ventral striosomal striatal medium spiny neurons, based on morphology, patch-matrix segregation and membrane properties.
doi:10.1016/j.baga.2011.11.002
PMCID: PMC3225898  PMID: 22140656
Basal Ganglia; Patch; GABA; Medium Spiny Neuron; BAC Transgenic
7.  Endocannabinoid–Dopamine Interactions in Striatal Synaptic Plasticity 
The nigrostriatal dopaminergic system is implicated in action control and learning. A large body of work has focused on the contribution of this system to modulation of the corticostriatal synapse, the predominant synapse type in the striatum. Signaling through the D2 dopamine receptor is necessary for endocannabinoid-mediated depression of corticostriatal glutamate release. Here we review the known details of this mechanism and discuss newly discovered signaling pathways interacting with this system that ultimately exert dynamic control of cortical input to the striatum and striatal output. This topic is timely with respect to Parkinson’s disease given recent data indicating changes in the striatal endocannabinoid system in patients with this disorder.
doi:10.3389/fphar.2012.00066
PMCID: PMC3329863  PMID: 22529814
long-term depression; CB1; serotonin; 5-HT1b; adenosine; Parkinson’s disease; medium spiny neuron
8.  An anticholinergic reverses motor control and corticostriatal LTD deficits in Dyt1 ΔGAG knock-in mice 
Behavioural brain research  2011;226(2):465-472.
DYT1 early-onset generalized torsion dystonia is an inherited movement disorder associated with mutations in DYT1 that codes for torsinA protein. The most common mutation seen in this gene is a trinucleotide deletion of GAG. We previously reported a motor control deficit on a beam-walking task in our Dyt1 ΔGAG knock-in heterozygous mice. In this report we show the reversal of this motor deficit with the anticholinergic trihexyphenidyl (THP), a drug commonly used to treat movement problems in dystonia patients. THP also restored the reduced corticostriatal long-term depression (LTD) observed in these mice. Corticostriatal LTD has long been known to be dependent on D2 receptor activation. In this mouse model, striatal D2 receptors were expressed at lower quantities in comparison to wild-type mice. Furthermore, the mice were also partially resistant to FPL64176, an agonist of L-type calcium channels that have been previously reported to cause severe dystonic-like symptoms in wild-type mice. Our findings collectively suggest that altered communication between cholinergic interneurons and medium spiny neurons is responsible for the LTD deficit and that this synaptic plasticity modification may be involved in the striatal motor control abnormalities in our mouse model of DYT1 dystonia.
doi:10.1016/j.bbr.2011.10.002
PMCID: PMC3225290  PMID: 21995941
dopamine receptor; dystonia; long-term depression; torsinA; trihexyphenidyl
9.  A Common Molecular Basis for Exogenous and Endogenous Cannabinoid Potentiation of Glycine Receptors* 
The Journal of Neuroscience  2012;32(15):5200-5208.
Both exogenous and endogenous cannabinoids can allosterically modulate glycine receptors (GlyRs). However, little is known about the molecular basis of cannabinoid-GlyR interactions. Here we report that sustained incubation with endocannabinoid anandamide (AEA) substantially increased the amplitude of Gly-activated current in both rat cultured spinal neurons and in HEK-293 cells expressing human α1, rat α2 and α3 GlyRs. While the α1 and α3 subunits were highly sensitive to AEA-induced potentiation, the α2 subunit was relatively insensitive to AEA. Switching a serine at 296 and 307 in the TM3 of the α1 and α3 subunits with an alanine (A) at the equivalent position in the α2 subunit converted the α1/α3 AEA sensitive receptors to sensitivity resembling that of α2. The S296 residue is also critical for exogenous cannabinoid-induced potentiation of IGly. The magnitude of AEA potentiation decreased with removal of either the hydroxyl or oxygen groups on AEA. While desoxy-AEA was significantly less potent in potentiating IGly, desoxy-AEA inhibited potentiation produced by both Δ9-tetrahydrocannabinol (THC), a major psychoactive component of marijuana, and AEA. Similarly, didesoxy-THC, a modified THC with removal of both hydroxyl/oxygen groups, did not affect IGly when applied alone but inhibited the potentiation of IGly induced by AEA and THC. These findings suggest that exogenous and endogenous cannabinoids potentiate GlyRs via a hydrogen bonding-like interaction. Such a specific interaction likely stems from a common molecular basis involving the S296 residue in the TM3 of the α1 and α3 subunits.
doi:10.1523/JNEUROSCI.6347-11.2012
PMCID: PMC3334839  PMID: 22496565
10.  STRIATAL INVOLVEMENT IN HUMAN ALCOHOLISM AND ALCOHOL CONSUMPTION, AND WITHDRAWAL IN ANIMAL MODELS 
This report addresses recent advances in our understanding of the role of the dorsolateral (DLS), dorsomedial (DMS), and ventral striatum in behavioral responses to alcohol, including alcohol craving in abstinent alcoholics, and alcohol consumption and withdrawal in rat, mouse and nonhuman primate models. Recent data are discussed showing that reduced neuronal activity as well as dysfunctional connectivity between the ventral striatum and the dorsolateral prefrontal cortex is associated with alcohol craving and impairment of new learning processes in abstinent alcoholics. Emerging results show that, within the DLS of mice and nonhuman primates withdrawn from alcohol after chronic exposure, glutamatergic transmission in striatal projection neurons (medium spiny neurons, MSNs) is increased, while GABAergic transmission is decreased. Glutamatergic transmission in DMS MSNs is also increased in ethanol withdrawn rats. Ex vivo or in vivo ethanol exposure and withdrawal causes a long-lasting increase in NR2B subunit-containing NMDA receptor activity in the DMS, contributing to ethanol-drinking. Analyses of neuronal activation associated with alcohol withdrawal and site-directed lesions implicate the rostroventral caudate putamen (rvCP), a ventrolateral segment of the DMS, in genetically determined differences in risk for alcohol withdrawal in mice. The influence of the identified risk factor on alcohol withdrawal may be involved in physical association of the multi-PDZ domain protein, MPDZ, with 5-HT2C receptors and/or NR2B within the rvCP. Taken together, these studies increase our understanding of the role of dopaminergic, glutamatergic and GABAergic signaling within different regions of the striatum in alcohol craving, consumption, dependence and withdrawal in humans and animal models.
doi:10.1111/j.1530-0277.2011.01520.x
PMCID: PMC3276303  PMID: 21615425
PET; fMRI; electrophysiology; self-administration; Fyn kinase; c-Fos
11.  Cocaine supersensitivity and enhanced motivation for reward in mice lacking dopamine D2 autoreceptors 
Nature neuroscience  2011;14(8):1033-1038.
Dopamine (DA) D2 receptors expressed in DA neurons (D2 autoreceptors) exert a negative feedback regulation that reduces DA neuron firing, DA synthesis and DA release. As D2 receptors are mostly expressed in postsynaptic neurons, pharmacological and genetic approaches have been unable to definitively address the in vivo contribution of D2 autoreceptors to DA-mediated behaviors. We found that midbrain DA neurons from mice deficient in D2 autoreceptors (Drd2loxP/loxP; Dat+/IRES-cre, referred to as autoDrd2KO mice) lacked DA-mediated somatodendritic synaptic responses and inhibition of DA release. AutoDrd2KO mice displayed elevated DA synthesis and release, hyperlocomotion and supersensitivity to the psychomotor effects of cocaine. The mice also exhibited increased place preference for cocaine and enhanced motivation for food reward. Our results highlight the importance of D2 autoreceptors in the regulation of DA neurotransmission and demonstrate that D2 autoreceptors are important for normal motor function, food-seeking behavior, and sensitivity to the locomotor and rewarding properties of cocaine.
doi:10.1038/nn.2862
PMCID: PMC3175737  PMID: 21743470
12.  Selective activation of cholinergic interneurons enhances accumbal phasic dopamine release: setting the tone for reward processing 
Cell reports  2012;2(1):33-41.
Dopamine (DA) plays a critical role in motor control, addiction and reward-seeking behaviors, and its release dynamics have traditionally been linked to changes in midbrain dopamine neuron activity. Here, we report that selective endogenous cholinergic activation achieved via in vitro optogenetic stimulation of nucleus accumbens (NAc), a terminal field of dopaminergic neurons, elicits real-time DA release. This mechanism occurs via direct actions on DA terminals, does not require changes in neuron firing within the midbrain and is dependent on glutamatergic receptor activity. More importantly, we demonstrate that in vivo selective activation of cholinergic interneurons (CINs) is sufficient to elicit DA release in the NAc. Therefore, the control of accumbal extracellular DA levels by endogenous cholinergic activity results from a complex convergence of neurotransmitter/neuromodulator systems that may ultimately synergize to drive motivated behavior.
doi:10.1016/j.celrep.2012.05.011
PMCID: PMC3408582  PMID: 22840394
13.  Fluorophore assisted light inactivation (FALI) of recombinant 5-HT3A receptor constitutive internalization and function 
Fluorescent proteins and molecules are now widely used to tag and visualize proteins resulting in an improved understanding of protein trafficking, localization, and function. In addition, fluorescent tags have also been used to inactivate protein function in a spatially and temporally-defined manner, using a technique known as fluorophore-assisted light inactivation (FALI) or chromophore-assisted light inactivation (CALI). In this study we tagged the serotonin3 A subunit with the α-bungarotoxin binding sequence (BBS) and subsequently labeled 5-HT3A/BBS receptors with fluorescently conjugated α-bungarotoxin in live cells. We show that 5-HT3A/BBS receptors are constitutively internalized in the absence of an agonist and internalization as well as receptor function are inhibited by fluorescence. The fluorescence-induced disruption of function and internalization was reduced with oxygen radical scavengers suggesting the involvement of reactive oxygen species, implicating the FALI process. Furthermore, these data suggest that intense illumination during live-cell microscopy may result in inadvertent FALI and inhibition of protein trafficking.
doi:10.1016/j.mcn.2011.02.007
PMCID: PMC3172681  PMID: 21338684
5-Hydroxytryptamine type 3 (5-HT3) receptors; Fluorophore assisted light inactivation; Constitutive internalization; Electrophysiology
14.  Frequency-dependent inversion of net striatal output by endocannabinoid-dependent plasticity at different synaptic inputs 
Understanding how striatal neurons integrate glutamatergic and GABAergic inputs is essential for understanding the control of movement and the formation of striatal-based memories. Here we show that GABAergic synapses on striatal medium spiny neurons (MSNs) are more sensitive than glutamatergic synapses on the same cells to endocannabinoid (eCB) signaling, and that protocols that induce short-lasting cannabinoid 1 receptor (CB1R)-dependent depression at glutamatergic synapses are sufficient to induce LTD at GABAergic synapses. We also show that the frequency and duration of glutamatergic input are strong determinants of the net effect of eCB signaling, and key factors in determining if LTD has a net disinhibitory or inhibitory action in striatum. Plastic changes in net output from striatal MSNs are thus a complex function of disinhibitory and inhibitory LTD combined with other forms of synaptic plasticity such as long-term potentiation (LTP) at excitatory synapses.
doi:10.1523/JNEUROSCI.3842-08.2009
PMCID: PMC2744205  PMID: 19193884
anandamide; basal ganglia; LTD; synaptic plasticity; Gabaergic; glutamate
15.  The L293 Residue in Transmembrane Domain 2 of the 5-HT3A Receptor is a Molecular Determinant of Allosteric Modulation by 5-Hydroxyindole 
Neuropharmacology  2008;54(8):1153-1165.
Allosteric modulation of ligand-gated ion channels can play important roles in shaping synaptic transmission. The function of the 5-hydroxytryptamine (serotonin) type 3 (5-HT3) receptor, a member of the Cys-loop ligand-gated ion channel superfamily, is modulated by a variety of compounds such as alcohols, anesthetics and 5-hydroxyindole (5-HI). In the present study, the molecular determinants of allosteric modulation by 5-HI were explored in N1E-115 neuroblastoma cells expressing the native 5-HT3 receptor and HEK 293 cells transfected with the recombinant 5-HT3A receptor using molecular biology and whole-cell patch-clamp techniques. 5-HI potentiated 5-HT-activated currents in both N1E-115 cells and HEK 293 cells, and significantly decreased current desensitization and deactivation. Substitution of Leu293 (L293, L15′) in the second transmembrane domain (TM2) with cysteine (L293C) or serine (L293S) abolished 5-HI modulation. Other mutations in the TM2 domain, such as D298A and T284F, failed to alter 5-HI modulation. The L293S mutation enhanced dopamine efficacy and converted 5-HI into a partial agonist at the mutant receptor. These data suggest that 5-HI stabilizes the 5-HT3A receptor in the open state by decreasing both desensitization and 5-HT unbinding/channel closing; and L293 is a common site for both channel gating and allosteric modulation by 5-HI. Our observations also indicate existence of a second 5-HI recognition site on the 5-HT3A receptor which may overlap with the 5-HT binding site and is not involved in the positive modulation by 5-HI. These findings support the idea that there are two discrete sites for 5-HI allosteric modulation and direct activation in the 5-HT3A receptor.
doi:10.1016/j.neuropharm.2008.03.009
PMCID: PMC2515613  PMID: 18436267
5-HT3A receptor; allosteric modulation; gating; desensitization; structure-function; electrophysiology
16.  Electrophysiological properties and gap junction coupling of striatal astrocytes 
Neurochemistry international  2008;52(7):1365-1372.
The striatum is the biggest nucleus of the basal ganglia and receives input from almost all cortical regions, substantia nigra and the thalamus. Striatal neuronal circuitry is well characterized, but less is known about glial physiology. To this end, we evaluated astrocyte electrophysiological properties using whole-cell patch-clamp recording in dorsal striatal brain slices from P15 – P21 rat. The majority of cells (95%) were passive astrocytes that do not express any detectable voltage-gated channels. Passive astrocytes were subcategorized into three groups based on time dependent current properties. The observed proportion of the different astrocyte subtypes did not change within the age range evaluated here, but was modulated during reduction of specific conductances and gap junction coupling. Striatal astrocytes were extensively interconnected and closure of gap junctions with octanol (1 mM), carbenoxolone (100 µM) or increased intracellular calcium (2 mM), significantly altered intrinsic properties. When simultaneously blocking potassium channels and gap junction coupling almost no passive conductance was detected, implying that the major currents in striatal astrocytes derive from potassium and gap junction conductance. Uncoupling of the syncytium reduced currents activated in response to a hyperpolarizing pulse, suggesting that changes in gap junction coupling alters astrocyte electrophysiological responses. Our findings indicate that the prevalent gap junction coupling is vital for astrocyte function in the striatum, and that whole cell recordings will be distorted by currents activated in neighboring cells.
doi:10.1016/j.neuint.2008.02.006
PMCID: PMC2442918  PMID: 18396351
Astroglia; basal ganglia; carbenoxolone; glia; patch clamp
17.  N-Docosahexaenoylethanolamide promotes development of hippocampal neurons 
The Biochemical journal  2011;435(2):327-336.
DHA (docosahexaenoic acid, C22:6,n−3) has been shown to promote neurite growth and synaptogenesis in embryonic hippocampal neurons, supporting the importance of DHA known for hippocampus-related learning and memory function. In the present study, we demonstrate that DHA metabolism to DEA (N-docosahexaenoylethanolamide) is a significant mechanism for hippocampal neuronal development, contributing to synaptic function. We found that a fatty acid amide hydrolase inhibitor URB597 potentiates DHA-induced neurite growth, synaptogenesis and synaptic protein expression. Active metabolism of DHA to DEA was observed in embryonic day 18 hippocampal neuronal cultures, which was increased further by URB597. Synthetic DEA promoted hippocampal neurite growth and synaptogenesis at substantially lower concentrations in comparison with DHA. DEA-treated neurons increased the expression of synapsins and glutamate receptor subunits and exhibited enhanced glutamatergic synaptic activity, as was the case for DHA. The DEA level in mouse fetal hippocampi was altered according to the maternal dietary supply of n−3 fatty acids, suggesting that DEA formation is a relevant in vivo process responding to the DHA status. In conclusion, DHA metabolism to DEA is a significant biochemical mechanism for neurite growth, synaptogenesis and synaptic protein expression, leading to enhanced glutamatergic synaptic function. The novel DEA-dependent mechanism offers a new molecular insight into hippocampal neurodevelopment and function.
doi:10.1042/BJ20102118
PMCID: PMC3169088  PMID: 21281269
docosahexaenoic acid (DHA); N-docosahexaenoylethanolamide (DEA); hippocampus; neurite growth; neuron; synaptogenesis
18.  Editorial: Tonic for What Ails Us? High Affinity GABAA Receptors and Alcohol 
Alcohol (Fayetteville, N.Y.)  2007;41(3):139-143.
Ethanol interactions with gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the brain, play key roles in acute intoxication. However, the exact mechanisms of these ethanol interactions have been the subject of considerable confusion and controversy. Many studies suggest that ethanol potentiates the function of the type A GABA receptor (GABAA-R). However, these findings have not been consistently replicated in experiments that directly examined ethanol effects on GABAA-R-mediated ion current. Differences in ethanol sensitivity of different GABAA-R subtypes have been invoked as a potential explanation for the inconsistent findings, and recent work suggests that GABAA-Rs that contain the delta subunit and/or mediate tonic extrasynaptic GABA responses may be especially ethanol-sensitive. However, considerable disagreement has arisen over these findings. This special issue of Alcohol contains articles from eight research groups who are examining this issue. The authors present their work, their views on the present state of this area of alcohol research, and their ideas about how to proceed with future studies that may help to address the present confusion and controversy. This editorial provides an introduction to this line of research and the current findings and controversies.
doi:10.1016/j.alcohol.2007.03.008
PMCID: PMC2043151  PMID: 17521844
Ethanol; Synaptic Inhibition; Intoxication; RO15-4513
19.  Retrograde Endocannabinoid Signaling in a Postsynaptic Neuron/Synaptic Bouton Preparation from Basolateral Amygdala 
Retrograde synaptic signaling by endogenous cannabinoids (endocannabinoids) is a recently discovered form of neuromodulation in the brain. In the basolateral amygdala (BLA), endocannabinoid signaling has been implicated in learning and memory, specifically in extinction of aversive memories. To examine retrograde endocannabinoid signaling in this brain region, BLA neurons were freshly isolated using an enzyme-free procedure. These isolated neurons retain attached functional excitatory and inhibitory synaptic boutons. Spontaneous GABAergic IPSCs (sIPSCs) were isolated from these freshly isolated neurons and a 4 s step of depolarization from –60 to 0 mV produced suppression of sIPSC frequency and amplitude. A similar depolarization-induced suppression of inhibition (DSI) was observed in neurons in BLA slices. DSI in the single-cell preparation was abolished by the CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide, and DSI duration was shortened in the presence of 2-methyl-6-(phenylethynyl) pyridine, an mGluR5 (metabotropic glutamate receptor 5) antagonist. The initial decrease in sIPSCs induced by the DSI procedure was greatly attenuated in recordings with 20 mM BAPTA containing postsynaptic internal solution, but a delayed-onset decrease was observed under this recording condition. A CB1 agonist decreased sIPSC frequency and amplitude, whereas CB1 antagonists increased these responses. The antagonist-induced increase was abolished in 20 mM BAPTA-filled cells. These data provide solid evidence for retrograde endocannabinoid signaling in the BLA and also indicate that this retrograde signaling requires only a postsynaptic neuron and attached synaptic boutons.
doi:10.1523/JNEUROSCI.1148-05.2005
PMCID: PMC1352167  PMID: 15987949
mGluR5; GABAergic transmission; CB1 receptors; depolarization-induced suppression of inhibition; IPSC; basolateral amygdala
20.  Serotonin Induces Long-Term Depression at Corticostriatal Synapses 
The striatum has important roles in motor control and action learning and, like many brain regions, receives multiple monoaminergic inputs. We have examined serotonergic modulation of rat and mouse corticostriatal neurotransmission and find that serotonin (5-HT) activates 5-HT1b receptors resulting in a long-term depression (LTD) of glutamate release and striatal output that we have termed 5-HT-LTD. 5-HT-LTD is presynaptically mediated, cAMP pathway-dependent, and inducible by endogenous striatal 5-HT, as revealed by application of a selective 5-HT reuptake inhibitor (SSRI). 5-HT-LTD is mutually occlusive with dopamine/endocannabinoid-dependent LTD, suggesting that these two forms of LTD act on the same corticostriatal terminals. Thus, serotonergic and dopaminergic mechanisms exist that may interact to persistently sculpt corticostriatal circuits, potentially influencing action learning and striatal-based disorders.
doi:10.1523/JNEUROSCI.6250-10.2011
PMCID: PMC3113491  PMID: 21593324
21.  Synaptic and Morphological Neuroadaptations in the Putamen Associated with Long-Term, Relapsing Alcohol Drinking in Primates 
Neuropsychopharmacology  2011;36(12):2513-2528.
Alcoholism and alcohol use disorders are characterized by several months to decades of heavy and problematic drinking, interspersed with periods of abstinence and relapse to heavy drinking. This alcohol-drinking phenotype was modeled using macaque monkeys to explore neuronal adaptations in the striatum, a brain region controlling habitual behaviors. Prolonged drinking with repeated abstinence narrowed the variability in daily intake, increased the amount of ethanol consumed in bouts, and led to higher blood ethanol concentrations more than twice the legal intoxication limit. After the final abstinence period of this extensive drinking protocol, we found a selective increase in dendritic spine density and enhanced glutamatergic transmission in the putamen, but not in the caudate nucleus. Intrinsic excitability of medium-sized spiny neurons was also enhanced in the putamen of alcohol-drinking monkeys in comparison with non-drinkers, and GABAeric transmission was selectively suppressed in the putamen of heavy drinkers. These morphological and physiological changes indicate a shift in the balance of inhibitory/excitatory transmission that biases the circuit toward an enduring increase in synaptic activation of putamen output as a consequence of prolonged heavy drinking/relapse. The resultant potential for increased putamen activation may underlie an alcohol-drinking phenotype of regulated drinking and sustained intoxication.
doi:10.1038/npp.2011.140
PMCID: PMC3194078  PMID: 21796110
self-administration monkeys; alcohol; caudate/putamen; synaptic morphology; glutamatergic transmission; GABAergic transmission; behavioral science; alcohol & alcoholism; animal models; neurophysiology; self-administration monkeys; alcohol; caudate/putamen; synaptic morphology; glutamatergic transmission
22.  Endocannabinoids rein in pain outside the brain 
Nature neuroscience  2010;13(10):1155-1156.
Enhancers of endocannabinoid signaling are potential analgesics, but they cause unacceptable psychiatric side effects. A new study reports an inhibitor of endocannabinoid breakdown that has analgesic activity and cannot enter the CNS.
doi:10.1038/nn1010-1155
PMCID: PMC3172675  PMID: 20877276
23.  Neurotransmitter Roles in Synaptic Modulation, Plasticity and Learning in the Dorsal Striatum 
Neuropharmacology  2010;58(7):951-961.
The dorsal striatum is a large forebrain region involved in action initiation, timing, control, learning and memory. Learning and remembering skilled movement sequences requires the dorsal striatum, and striatal subregions participate in both goal-directed (action-outcome) and habitual (stimulus-response) learning. Modulation of synaptic transmission plays a large part in controlling input to as well as the output from striatal medium spiny projection neurons (MSNs). Synapses in this brain region are subject to short-term modulation, including allosteric alterations in ion channel function and prominent presynaptic inhibition. Two forms of long-term synaptic plasticity have also been observed in striatum, long-term potentiation (LTP) and long-term depression (LTD). LTP at glutamatergic synapses onto MSNs involves activation of NMDA-type glutamate receptors and D1 dopamine or A2A adenosine receptors. Expression of LTP appears to involve postsynaptic mechanisms. LTD at glutamatergic synapses involves retrograde endocannabinoid signaling stimulated by activation of metabotropic glutamate receptors (mGluRs) and D2 dopamine receptors. While postsynaptic mechanisms participate in LTD induction, maintained expression involves presynaptic mechanisms. A similar form of LTD has also been observed at GABAergic synapses onto MSNs. Studies have just begun to examine the roles of synaptic plasticity in striatal-based learning. Findings to date indicate that molecules implicated in induction of plasticity participate in these forms of learning. Neurotransmitter receptors involved in LTP induction are necessary for proper skill and goal-directed instrumental learning. Interestingly, receptors involved in LTP and LTD at glutamatergic synapses onto MSNs of the “indirect pathway” appear to have important roles in habit learning. More work is needed to reveal if and when synaptic plasticity occurs during learning and if so what molecules and cellular processes, both short- and long-term, contribute to this plasticity.
doi:10.1016/j.neuropharm.2010.01.008
PMCID: PMC2849868  PMID: 20096294
Long-term plasticity; Dopamine; Glutamate; Endocannabinoid; Instrumental learning; Skill Learning
24.  Hydrophobic Photolabeling Studies Identify the Lipid-Protein Interface of the 5-HT3A Receptor† 
Biochemistry  2009;48(39):9278-9286.
A HEK-293 cell line that stably expresses mouse 5-HT3ARs containing a C-terminal extension that confers high affinity binding of alpha-bungarotoxin (αBgTx) was established (αBgTx-5-HT3ARs) and used to purify αBgTx-5-HT3ARs in a lipid environment for use in structural studies using photoaffinity labeling. αBgTx-5-HT3ARs were expressed robustly (60 pmol [3H]BRL-43694 binding sites (~3 µg receptor) per mg protein and displayed the same functional properties as wild-type receptors (serotonin EC50 = 5.3) +/− 0.04 µM). While [125I]αBgTx bound to the αBgTx-5-HT3ARs with high affinity (Kd = 11 nM), application of nonradioactive αBgTx (up to 300 µM) had no effect on serotonin-induced current responses. αBgTx-5-HT3ARs were purified on an αBgTx-derivatized affinity column from detergent extracts in mg quantities and at ~25% purity. The hydrophobic photolabel 3-trifluoromethyl-3-(m-[125I]iodophenyl) diazirine ([125I]TID) was used to identify the amino acids at the lipid-protein interface of purified and lipid-reconstituted αBgTx-5-HT3ARs. [125I]TID photoincorporation into the αBgTx-5-HT3AR subunit was initially mapped to subunit proteolytic fragments of 8 kDa, containing the M4 transmembrane segment and ~60% of incorporated 125I, and 17 kDa, containing the M1–M3 transmembrane segments. Within the M4 segment, [125I]TID labeled Ser451, equivalent to the [125I]TID-labeled residue Thr422 at the lipid-exposed face of the Torpedo nicotinic acetylcholine receptor (nAChR) α1M4 α-helix. These results provide a first definition of the surface of the 5-HT3AR M4 helix that is exposed to lipid and establish that this surface is equivalent to the surface exposed to lipid in the Torpedo nAChR.
doi:10.1021/bi901208j
PMCID: PMC2778300  PMID: 19715355
25.  Loss of GluN2B-containing NMDA receptors in CA1 hippocampus and cortex impairs long-term depression, reduces dendritic spine density and disrupts learning 
N-methyl-D-aspartate receptors (NMDARs) are key mediators of certain forms of synaptic plasticity and learning. NMDAR complexes are heteromers composed of an obligatory GluN1 subunit and one or more GluN2 (GluN2A- GluN2D) subunits. Different subunits confer distinct physiological and molecular properties to NMDARs, but their contribution to synaptic plasticity and learning in the adult brain remains uncertain. Here, we generated mice lacking GluN2B in pyramidal neurons of cortex and CA1 subregion of hippocampus. We found that hippocampal principal neurons of adult GluN2B mutants had faster decaying NMDAR-mediated excitatory postsynaptic currents (EPSCs) than non-mutant controls, and were insensitive to GluN2B but not NMDAR antagonism. A sub-saturating form of hippocampal long-term potentiation (LTP) was impaired in the mutants, whereas a saturating form of LTP was intact. A NMDAR-dependent form of long-term depression (LTD) produced by low-frequency stimulation combined with glutamate transporter inhibition was abolished in the mutants. Additionally, mutants exhibited decreased dendritic spine density in CA1 hippocampal neurons as compared to controls. On multiple assays for corticohippocampal-mediated learning and memory (hidden platform Morris water maze, T-maze spontaneous alternation, Pavlovian trace fear conditioning), mutants were impaired. These data further demonstrate the importance of GluN2B for synaptic plasticity in the adult hippocampus and suggest a particularly critical role in LTD, at least the form studied here. The finding that loss of GluN2B was sufficient to cause learning deficits illustrates the contribution of GluN2B-mediated forms of plasticity to memory formation, with implications for elucidating NMDAR-related dysfunction in disease-related cognitive impairment.
doi:10.1523/JNEUROSCI.0640-10.2010
PMCID: PMC2869199  PMID: 20357110
LTP; LTD; fear; hippocampus; cortex; mice

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