Research Projects

1. Site-Directed Mutagenesis to Define Sites of Action of Alcohol/Abused Solvents on NMDA Receptors

Our ongoing work (Ronald et al., 2001; Smothers and Woodward, 2006) described the effects of selected mutants on the sensitivity of NMDA receptors to alcohol. One of the mutants tested (F639A in the third transmembrane domain of the NR1 subunit) significantly reduced the inhibitory effects of alcohol when co-expressed with any of the NR2 subunits in oocytes or HEK cells. Altering selective amino acids in the TM4 domain of the NR1 subunit reversed this effect suggesting that residues in the TM3 and TM4 domains may define an alcohol site of action. We are currently using the cysteine trapping method to test whether these residues are physically involved in defining an alcohol binding site. This technique involves expressing a receptor carrying a cysteine at the site of interest and exposing the cells to a reagent that will covalently bind to the cysteine thus occluding the site.

In related studies with Dr. Adron Harris's group at the Univ. of Texas (Ogata et al. 2006), we have also shown that the NR1(F639A) and NR2A(A825W) mutants also affect the sensitivity of NMDA receptors to a variety of volatile anesthetics including isoflurane, halothane, chlorform, cyclopropane and xenon. The inhibition by ketamine and benzene, a volatile organic solvent, were not altered by these mutations.

Although the F639A NR1 mutant reduces the effects of inhibitory effect of ethanol on receptors when expressed alone in oocytes or HEK cells, it is unknown whether it confers reduced sensitivity in the presence of the wild-type NR1 subunit. To address this question, we have transfected HEK cells or hippocampal neurons with different NR1 subunits and measured the ethanol sensitivity of these receptors.

To identify cells expressing recombinant NMDA subunits, we co-transfect with cDNA plasmids encoding fluorescent proteins (GFP or DsRed) and visualize the transfected cells by epifluorescent microscopy. In the left panel above, HEK293 cells were transfected with two cDNA plasmids encoding NR1-DsRed and Src-GFP fusion proteins. The DsRed or GFP protein is in frame with the C-terminus of the NR1 or Src protein. Fluorescent images (40X, non-confocal) were taken at each excitation/emission maximum and then merged. The middle panel shows primary hippocampal neurons transfected with an eGFP expressing vector under control of the neuron-specific promoter synapsin. This image was taken approximately 1 month after transfection showing long-term expression of the GFP signal. The right panel shows expression of eGFP in CA1 neurons in a hippocampal slice following infection with a lentivirus under control of the synapsin promoter. Using similar approaches in combination with patch-clamp electrophysiology, we have shown that NMDA responses measured in neurons co-transfected with the F639A NR1 mutant also show a reduced sensitivity to alcohol.

We have a variety of GFP and DsRed tagged proteins that we are using to study receptor expression and localization. Click here for a list of constructs.

We are collaborating with Dr. Gregg Homanics at the University of Pittsburgh to produce mice in which the genes that code for wild-type NMDA receptor subunits are replaced with those containing alcohol/anesthetic insensitive sites. These knock-in animals will be invaluable in determining the role of NMDA receptors in mediating the varioius effects of alcohols and anesthetics. Characterization of the first generation of these animals is currently underway.

4. Determination of the Sensitivity of Brain Ion Channels to Abused Solvents

Previous studies from our lab have shown that ion channels that are sensitive to alcohol are also sensitive to solvents that are commonly used as drugs of abuse. A major project in the lab is the determination of the sensitivity of brain ion channels to toluene and other widely abused solvents,. In initial studies, we showed that toluene inhibits NMDA but not non-NMDA receptors at concentrations that are obtained after voluntary inhalation of abused solvents (Cruz et al., 1997; 2000). As shown in the figures below, the most solvent-sensitive NMDA subtype is composed of NR2B subunits with NR2A and NR2C containing receptors being approximately 10X less sensitive.

In more recent studies, we have also investigated the effects of abused solvents on a variety of other ion channels including the neuronal nicotinic receptors (Bale et al., 2002), voltage-gated calcium channels (Tillar et al., 2002; Shafer et al., 2005) and two classes of potassium channels (Del Re et al., 2006).

The toluene sensitivity of nAchRs is also subunit-dependent and the most sensitive subunit combination that we have tested to date is the receptor with homomeric a7 receptors being nearly 10-times less affected by toluene. In addition, substitution of b4 for b2 subunits in heteromeric combinations dramatically lowers the toluene sensitivity of these receptors.

VSCC's expressed in PC-12 cells were also inhibited by toluene and this inhibition was enhanced after treatment with NGF. Both BK and GirK channels were inhibited by toluene; a finding that is opposite to that found for ethanol.

The solvent sensitivity profile of recombinant ion channels has been validated using primary cultures of rat hippocampal neurons (Bale et al., 2005). In these neurons, toluene dose-dependently inhibits NMDA induced currents at concentrations that do not affect currents elicited by application of AMPA or kainate. Prolonged (4 days) exposure of neurons to toluene induces a compensatory increase in glutamatergic signaling with a comcomitant decrease in inhibitory GABA signaling.

5. Effects of Chronic Alcohol/Solvent Exposure on NMDA-Receptor Function and Distribution in Neurons

In a collaborative effort with Dr. Judson Chandler here at MUSC, we have examined the effects of exposing cultured hippocampal neurons to alcohol or toluene for prolonged periods of time (days-weeks). In these studies, Dr. Chandler's lab receptor uses confocal microscopy to map the distribution and localization of antibody-labelled NMDA receptor subunits. Synaptic receptors are identified as those co-localized with synapsin or other markers of synapse formation.

Our lab examines the effects of chronic alcohol/solvent exposure on the functional properties of these neurons. As shown in the example above, we use patch-clamp electrophysiology to measure the frequency and amplitude of post-synaptic currents (EPSCs and IPSCs) that arise from the spontaneous release of glutamate or GABA at synapses in these cultures. These studies have shown that chronic treatment of neurons with ethanol (Carpenter-Hyland et al., 2004) or toluene (Bale et al., 2005) enhances the expression and synaptic localization of NMDA receptors.

We have recently developed the visualized slice-patch clamp recording technique in order to investigate the effects of acute and chronic ethanol on spontaneous glutamatergic synaptic transmission in acute and cultured brain slices. Neurons in brain slices retain many of the intrinsic network connections that exist in the intact brain and are amenable to patch-clamp recording. For example, shown below are traces from deep-layer pyramidal neurons from the prefrontal cortex maintained in a novel triple slice co-culture containing slices of cortex, hippocampus and midbrain (left panel). These neurons display spontaneous and evoked periods of persistent activity characterized by sudden plateau depolarizations (up-states) accompanied by spike firing. As reported in our recent Journal of Neuroscience paper (Tu et al., 2007), ethanol inhibits these up-states and following washout, neurons often display an enhanced period of persistent activity . The mechanisms underlying these effects are currently under study in a project funded by the NIAAA P50 Alcohol Research Center.