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.
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.
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.
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.
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.
glutamate transporter; alcohol; reward; endocannabinoid
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.
dopamine receptor; dystonia; long-term depression; torsinA; trihexyphenidyl
α-synuclein(α-syn) plays a prominent role in the degeneration of midbrain dopaminergic (mDA) neurons in Parkinson disease (PD). However, only a few studies on α-syn have been carried out in the mDA neurons in vivo, which may be attributed to a lack of α-syn transgenic mice that develop PD-like severe degeneration of mDA neurons. To gain mechanistic insights into the α-syn-induced mDA neurodegeneration, we generated a new line of tetracycline-regulated inducible transgenic mice that overexpressed the PD-related α-syn A53T missense mutation in the mDA neurons. Here we show that the mutant mice developed profound motor disabilities and robust mDA neurodegeneration, resembling some key motor and pathological phenotypes of PD. We further systematically examined the subcellular abnormalities appeared in the mDA neurons of mutant mice, and observed a profound decrease of dopamine release, the fragmentation of Golgi apparatus, and impairments of autophagy/lysosome degradation pathways in these neurons. To further understand the specific molecular events leading to the α-syn-dependent degeneration of mDA neurons, we found that over-expression of α-syn promoted a proteasome-dependent degradation of nuclear receptor related 1 protein (Nurr1); while inhibition of Nurr1 degradation ameliorated the α-syn-induced loss of mDA neurons. Given that Nurr1 plays an essential role in maintaining the normal function and survival of mDA neurons, our studies suggest that the α-syn-mediated suppression of Nurr1 protein expression may contribute to the preferential vulnerability of mDA neurons in the pathogenesis of PD.
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).
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.
Using a combination of immunohistochemistry, electrophysiology, DiI labeling, and retrograde tracing, we investigated the phenotypes of GFP-expressing cells in the GAD1-GFP mice.
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.
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.
Basal Ganglia; Patch; GABA; Medium Spiny Neuron; BAC Transgenic
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.
The neurological effects of organophosphate pesticides, commonly used on foods and in households, are an important public health concern. Furthermore, subclinical exposure to combinations of organophosphates is implicated in Gulf War illness. Here we characterized the effects of the broadly-used insecticide chlorpyrifos on dopamine and glutamatergic neurotransmission effectors in corticostriatal motor/reward circuitry. Chlorpyrifos potentiated PKA-dependent phosphorylation of the striatal protein DARPP-32 and the GluR1 subunit of AMPA receptors in mouse brain slices. It also increased GluR1 phosphorylation by PKA when administered systemically. This correlated with enhanced glutamate release from cortical projections in rat striatum. Similar effects were induced by the sarin congener, diisopropyl fluorophosphate, alone or in combination with the putative neuroprotectant, pyridostigmine bromide and the pesticide DEET. This combination, meant to mimic the neurotoxicant exposure encountered by veterans of the 1991 Persian Gulf War, also induced hyperphosphorylation of the neurofibrillary tangle-associated protein tau. Diisopropyl fluorophosphate and pyrodostigmine bromide, alone or in combination, also increased the aberrant activity of the protein kinase, Cdk5, as indicated by conversion of its activating cofactor p35 to p25. Thus consistent with recent findings in humans and animals, organophosphate exposure causes dysregulation in the motor/reward circuitry and invokes mechanisms associated with neurological disorders and neurodegeneration.
insecticide; dopamine; neurotoxicity; organophosphate; chlorpyrifos; Gulf War Illness
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.
PET; fMRI; electrophysiology; self-administration; Fyn kinase; c-Fos
Ca2+/calmodulin-dependent protein kinase II (CaMKII) is abundant in striatal medium spiny neurons (MSNs). CaMKII is dynamically regulated by changes in dopamine signaling, as occurs in Parkinson's disease as well as addiction. Although CaMKII has been extensively studied in the hippocampus where it regulates excitatory synaptic transmission, relatively little is known about how it modulates neuronal function in the striatum. Therefore, we examined the impact of selectively overexpressing an EGFP-fused CaMKII inhibitory peptide (EAC3I) in striatal medium spiny neurons (MSNs) using a novel transgenic mouse model. EAC3I-expressing cells exhibited markedly decreased excitatory transmission, indicated by a decrease in the frequency of spontaneous excitatory postsynaptic currents (sEPSCs). This decrease was not accompanied by changes in the probability of release, levels of glutamate at the synapse, or changes in dendritic spine density. CaMKII regulation of the AMPA receptor subunit GluA1 is a major means by which the kinase regulates neuronal function in the hippocampus. We found that the decrease in striatal excitatory transmission seen in the EAC3I mice is mimicked by deletion of GluA1. Further, while CaMKII inhibition decreased excitatory transmission onto MSNs, it increased their intrinsic excitability. These data suggest that CaMKII plays a critical role in setting the excitability rheostat of striatal MSNs by coordinating excitatory synaptic drive and the resulting depolarization response.
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.
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.
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.
5-Hydroxytryptamine type 3 (5-HT3) receptors; Fluorophore assisted light inactivation; Constitutive internalization; Electrophysiology
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.
long-term depression; CB1; serotonin; 5-HT1b; adenosine; Parkinson’s disease; medium spiny neuron
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.
docosahexaenoic acid (DHA); N-docosahexaenoylethanolamide (DEA); hippocampus; neurite growth; neuron; synaptogenesis
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.
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.
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
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.
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.
Long-term plasticity; Dopamine; Glutamate; Endocannabinoid; Instrumental learning; Skill Learning
Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.
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.
Docosahexaenoic acid (DHA, 22:6n-3), the major polyunsaturated fatty acid accumulated in the brain during development, has been implicated in learning and memory, but underlying cellular mechanisms are not clearly understood. Here, we demonstrate that DHA significantly affects hippocampal neuronal development and synaptic function in developing hippocampi. In embryonic neuronal cultures, DHA supplementation uniquely promoted neurite growth, synapsin puncta formation and synaptic protein expression, particularly synapsins and glutamate receptors. In DHA-supplemented neurons, spontaneous synaptic activity was significantly increased, mostly because of enhanced glutamatergic synaptic activity. Conversely, hippocampal neurons from DHA-depleted fetuses showed inhibited neurite growth and synaptogenesis. Furthermore, n-3 fatty acid deprivation during development resulted in marked decreases of synapsins and glutamate receptor subunits in the hippocampi of 18-day-old pups with concomitant impairment of long-term potentiation, a cellular mechanism underlying learning and memory. While levels of synapsins and NMDA receptor subunit NR2A were decreased in most hippocampal regions, NR2A expression was particularly reduced in CA3, suggesting possible role of DHA in CA3-NMDA receptor-dependent learning and memory processes. The DHA-induced neurite growth, synaptogenesis, synapsin, and glutamate receptor expression, and glutamatergic synaptic function may represent important cellular aspects supporting the hippocampus-related cognitive function improved by DHA.
docosahexaenoic acid; hippocampal development; long-term potentiation; neurite growth; synaptic function; synaptogenesis
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.
LTP; LTD; fear; hippocampus; cortex; mice
Glutamate receptors are important target molecules of the acute effect of ethanol. We studied ethanol sensitivity of homomeric GluR-D receptors expressed in HEK293 cells, and examined whether recently discovered transmembrane AMPA receptor regulatory proteins (TARPs) affect ethanol sensitivity. Co-expression of the TARPs, stargazin and γ4, increased the time constant (τ-value) of current decay in the presence of agonist, thus slowing the onset of desensitization and increasing the steady-state current. Ethanol produced less inhibition of the peak current than the steady-state current for all types of the GluR-D receptors. In addition, ethanol concentration-dependently accelerated the rate of desensitization, measured as the τ-value of fast decay of peak current. This effect was enhanced with co-expression of TARPs. The recovery from desensitization was slowed down by co-expression of γ4, but ethanol did not affect this process in any GluR-D combination. The results support the idea that increased desensitization is an important mechanism in the ethanol inhibition of AMPA receptors, and indicate that co-expression of TARPs can alter this effect of ethanol.
GluA4 receptors; Alcohol; Protein interactions; Recombinant receptors; Patch-clamp
The learning of new skills is characterized by an initial phase of rapid improvement in performance and a phase of more gradual improvements as skills are automatized and performance asymptotes. Using in vivo striatal recordings, we observed region-specific changes in neural activity during the different phases of skill learning, with the associative or dorsomedial striatum being preferentially engaged early in training and the sensorimotor or dorsolateral striatum being engaged later in training. Ex vivo recordings from medium spiny striatal neurons in brain slices of trained mice revealed that the changes observed in vivo corresponded to regional- and training-specific changes in excitatory synaptic transmission in the striatum. Furthermore, the potentiation of glutamatergic transmission observed in dorsolateral striatum after extensive training was preferentially expressed in striatopallidal neurons, rather than striatonigral neurons. These findings demonstrate that region- and pathway-specific plasticity sculpts the circuits involved in the performance of the skill as it becomes automatized.
The novel endocannabinoid-like lipid N-arachidonoyl L-serine (ARA-S) causes vasodilation through both endothelium-dependent and -independent mechanisms. We have analyzed the vasorelaxant effect of ARA-S in isolated vascular preparations and its effects on Ca2+-activated K+ currents in human embryonic kidney cells stably transfected with the α-subunit of the human, large conductance Ca+-activated K+ (BKCa) channel (HEK293hSlo cells). ARA-S caused relaxation of rat isolated, intact and denuded, small mesenteric arteries pre-constricted with phenylephrine (pEC50: 5.49 and 5.14, respectively), whereas it caused further contraction of vessels pre-constricted with KCl (pEC50 5.48 and 4.82, respectively). Vasorelaxation by ARA-S was inhibited by 100 nM iberiotoxin. In HEK293hSlo cells, ARA-S and its enantiomer N-arachidonoyl-D-serine enhanced the whole cell outward K+ current with similar potency (pEC50: 5.63 and 5.32, respectively). The potentiation was not altered by β1 subunit or mediated by ARA-S metabolites, stimulation of known cannabinoid receptors, G proteins, protein kinases or Ca2+-dependent processes; it was lost after patch excision or following membrane cholesterol depletion, but was restored after cholesterol reconstitution. BKCa currents were also enhanced by anandamide (pEC50: 5.27) but inhibited by another endocannabinoid, virodhamine (pIC50: 6.35), or by the synthetic cannabinoid O-1918 (pIC50: 6.59), which blocks ARA-S-induced vasodilation. We conclude that (i) ARA-S directly activates BKCa channels. (ii) This interaction does not involve cannabinoid receptors or cytosolic factors but is dependent on the presence of membrane cholesterol. (iii) Direct BKCa channel activation likely contributes to the endothelium-independent component of ARA-S-induced mesenteric vasorelaxation. (iv) O-1918 is a BKCa channel inhibitor.