Cesar Labarca

Neuronal P2X transmitter-gated cation channels change their ion selectivity in seconds.

Fast synaptic transmission depends on the selective ionic permeability of transmitter-gated ion channels. Here we show changes in the ion selectivity of neuronal P2X transmitter-gated cation channels as a function of time (on the order of seconds) and previous ATP exposure. Heterologously expressed P2X2, P2X2/P2X3 and P2X4 channels as well as native neuronal P2X channels possess various combinations of mono- or biphasic responses and permeability changes, measured by NMDG+ and fluorescent dye. Furthermore, in P2X4 receptors, this ability to alter ion selectivity can be increased or decreased by altering an amino-acid residue thought to line the ion permeation pathway, identifying a region that governs this activity-dependent change.

Steady states, charge movements, and rates for a cloned GABA transporter expressed in Xenopus oocytes

Voltage-clamp analysis was applied to study the currents associated with the uptake of extracellular gamma-aminobutyric acid (GABA) by the cloned transporter GAT1 expressed at high efficiency in Xenopus oocytes. Steady-state GABA currents were increased at higher extracellular [GABA], [Na+], and [Cl-] and at more negative potentials. The Hill coefficient for Na+ exceeded unity, suggesting the involvement of two Na+ ions. In the absence of GABA, voltage jumps produced transient currents that behaved like capacitive charge movements; these were suppressed by the uptake inhibitor SKF-89976A, were shifted to more negative potentials at lower external [Na+] and [Cl-], and had an effective valence of 1.1 elementary charge. A turnover rate per transporter of 6-13/s at maximal [GABA] (-80 mV, 96 mM NaCl, 22 degrees C) is given both by the kinetics of voltage jump relaxations and by the ratio between the maximal GABA currents and the charge movements. These quantitative data are necessary for evaluating the roles of GAT1 in synaptic function.

Chronic Nicotine Cell Specifically Upregulates Functional α4* Nicotinic Receptors: Basis for Both Tolerance in Midbrain and Enhanced Long-Term Potentiation in Perforant Path

Understanding effects of chronic nicotine requires identifying the neurons and synapses whose responses to nicotine itself, and to endogenous acetylcholine, are altered by continued exposure to the drug. To address this problem, we developed mice whose α4 nicotinic receptor subunits are replaced by normally functioning fluorescently tagged subunits, providing quantitative studies of receptor regulation at micrometer resolution. Chronic nicotine increased α4 fluorescence in several regions; among these, midbrain and hippocampus were assessed functionally. Although the midbrain dopaminergic system dominates reward pathways, chronic nicotine does not change α4* receptor levels in dopaminergic neurons of ventral tegmental area (VTA) or substantia nigra pars compacta. Instead, upregulated, functional α4* receptors localize to the GABAergic neurons of the VTA and substantia nigra pars reticulata. In consequence, GABAergic neurons from chronically nicotine-treated mice have a higher basal firing rate and respond more strongly to nicotine; because of the resulting increased inhibition, dopaminergic neurons have lower basal firing and decreased response to nicotine. In hippocampus, chronic exposure to nicotine also increases α4* fluorescence on glutamatergic axons of the medial perforant path. In hippocampal slices from chronically treated animals, acute exposure to nicotine during tetanic stimuli enhances induction of long-term potentiation in the medial perforant path, showing that the upregulated α4* receptors in this pathway are also functional. The pattern of cell-specific upregulation of functional α4* receptors therefore provides a possible explanation for two effects of chronic nicotine: sensitization of synaptic transmission in forebrain and tolerance of dopaminergic neuron firing in midbrain.

Point mutant mice with hypersensitive α4 nicotinic receptors show dopaminergic deficits and increased anxiety

Knock-in mice were generated that harbored a leucine-to-serine mutation in the α4 nicotinic receptor near the gate in the channel pore. Mice with intact expression of this hypersensitive receptor display dominant neonatal lethality. These mice have a severe deficit of dopaminergic neurons in the substantia nigra, possibly because the hypersensitive receptors are continuously activated by normal extracellular choline concentrations. A strain that retains the neo selection cassette in an intron has reduced expression of the hypersensitive receptor and is viable and fertile. The viable mice display increased anxiety, poor motor learning, excessive ambulation that is eliminated by very low levels of nicotine, and a reduction of nigrostriatal dopaminergic function upon aging. These knock-in mice provide useful insights into the pathophysiology of sustained nicotinic receptor activation and may provide a model for Parkinsons disease.

In vivo incorporation of unnatural amino acids into ion channels in Xenopus oocyte expression system

A general method for the incorporation of unnatural amino acids into ion channels and membrane receptors using a Xenopus oocyte expression system has been described. A large number of unnatural amino acids have been incorporated into the nAChR, GIRK, and Shaker K+ channels. Continuing efforts focus on incorporating unnatural amino acids that differ substantially from the natural amino acids, for example, residues that include fluorophores. In addition, we are addressing the feasibility of incorporating unnatural amino acids into ion channels and membrane receptors in mammalian cells.

Seizures and enhanced cortical GABAergic inhibition in two mouse models of human autosomal dominant nocturnal frontal lobe epilepsy.

Selected mutations in the human α4 or β2 neuronal nicotinic acetylcholine receptor subunit genes cosegregate with a partial epilepsy syndrome known as autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). To examine possible mechanisms underlying this inherited epilepsy, we engineered two ADNFLE mutations (Chrna4S252F and Chrna4+L264) in mice. Heterozygous ADNFLE mutant mice show persistent, abnormal cortical electroencephalograms with prominent delta and theta frequencies, exhibit frequent spontaneous seizures, and show an increased sensitivity to the proconvulsant action of nicotine. Relative to WT, electrophysiological recordings from ADNFLE mouse layer II/III cortical pyramidal cells reveal a >20-fold increase in nicotine-evoked inhibitory postsynaptic currents with no effect on excitatory postsynaptic currents. i.p. injection of a subthreshold dose of picrotoxin, a use-dependent γ-aminobutyric acid receptor antagonist, reduces cortical electroencephalogram delta power and transiently inhibits spontaneous seizure activity in ADNFLE mutant mice. Our studies suggest that the mechanism underlying ADNFLE seizures may involve inhibitory synchronization of cortical networks via activation of mutant α4-containing nicotinic acetylcholine receptors located on the presynaptic terminals and somatodendritic compartments of cortical GABAergic interneurons.

Novel Seizure Phenotype and Sleep Disruptions in Knock-In Mice with Hypersensitive α4* Nicotinic Receptors

A leucine to alanine substitution (L9′A) was introduced in the M2 region of the mouse α4 neuronal nicotinic acetylcholine receptor (nAChR) subunit. Expressed in Xenopus oocytes, α4(L9′A)β2 nAChRs were ≥30-fold more sensitive than wild type (WT) to both ACh and nicotine. We generated knock-in mice with the L9′A mutation and studied their cellular responses, seizure phenotype, and sleep-wake cycle. Seizure studies on α4-mutated animals are relevant to epilepsy research because all known mutations linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) occur in the M2 region of α4or β2 subunits. Thalamic cultures and synaptosomes from L9′A mice were hypersensitive to nicotine-induced ion flux. L9′A mice were ∼15-fold more sensitive to seizures elicited by nicotine injection than their WT littermates. Seizures in L9′A mice differed qualitatively from those in WT: L9′A seizures started earlier, were prevented by nicotine pretreatment, lacked EEG spike-wave discharges, and consisted of fast repetitive movements. Nicotine-induced seizures in L9′A mice were partial, whereas WT seizures were generalized. When L9′A homozygous mice received a 10 mg/kg nicotine injection, there was temporal and phenomenological separation of mutant and WT-like seizures: an initial seizure ∼20 s after injection was clonic and showed no EEG changes. A second seizure began 3-4 min after injection, was tonic-clonic, and had EEG spike-wave activity. No spontaneous seizures were detected in L9′A mice during chronic video/EEG recordings, but their sleep-wake cycle was altered. Our findings show that hypersensitive α4* nicotinic receptors in mice mediate changes in the sleep-wake cycle and nicotine-induced seizures resembling ADNFLE.

Nicotine-induced dystonic arousal complex in a mouse line harboring a human autosomal-dominant nocturnal frontal lobe epilepsy mutation.

We generated a mouse line harboring an autosomal-dominant nocturnal frontal lobe epilepsy (ADNFLE) mutation: the α4 nicotinic receptor S248F knock-in strain. In this mouse, modest nicotine doses (1–2 mg/kg) elicit a novel behavior termed the dystonic arousal complex (DAC). The DAC includes stereotypical head movements, body jerking, and forelimb dystonia; these behaviors resemble some core features of ADNFLE. A marked Straub tail is an additional component of the DAC. Similar to attacks in ADNFLE, the DAC can be partially suppressed by the sodium channel blocker carbamazepine or by pre-exposure to a very low dose of nicotine (0.1 mg/kg). The DAC is centrally mediated, genetically highly penetrant, and, surprisingly, not associated with overt ictal electrical activity as assessed by (1) epidural or frontal lobe depth-electrode electroencephalography or (2) hippocampal c-fos-regulated gene expression. Heterozygous knock-in mice are partially protected from nicotine-induced seizures. The noncompetitive antagonist mecamylamine does not suppress the DAC, although it suppresses high-dose nicotine-induced wild-type-like seizures. Experiments on agonist-induced 86Rb+ and neurotransmitter efflux from synaptosomes and on α4S248Fβ2 receptors expressed in oocytes confirm that the S248F mutation confers resistance to mecamylamine blockade. Genetic background, gender, and mutant gene expression levels modulate expression of the DAC phenotype in mice. The S248F mouse thus appears to provide a model for the paroxysmal dystonic element of ADNFLE semiology. Our model complements what is seen in other ADNFLE animal models. Together, these mice cover the spectrum of behavioral and electrographic events seen in the human condition.

Increased Sensitivity to Agonist-Induced Seizures, Straub Tail, and Hippocampal Theta Rhythm in Knock-In Mice Carrying Hypersensitive α4 Nicotinic Receptors

We studied a strain of exon replacement mice (“L9′S knock-in”) whose α4 nicotinic receptor subunits have a leucine to serine mutation in the M2 region, 9′ position (Labarca et al., 2001); this mutation renders α4-containing receptors hypersensitive to agonists. Nicotine induced seizures at concentrations (1 mg/kg) approximately eight times lower in L9′S than in wild-type (WT) littermates. At these concentrations, L9′S but not WT showed increases in EEG amplitude and theta rhythm. L9′S mice also showed higher seizure sensitivity to the nicotinic agonist epibatidine, but not to the GABAAreceptor blocker and proconvulsant bicuculline. Dorsiflexion of the tail (Straub tail) was the most sensitive nicotine effect found in L9′S mice (0.1 mg/kg). The L9′S mice were hypersensitive to galanthamine- and tacrine-induced seizures and Straub tails. There were no apparent neuroanatomical differences between L9′S and WT mice in several brain regions. [125I]Epibatidine binding to brain membranes showed that the mutant allele was expressed at ∼25% of WT levels, presumably because of the presence of a neomycin selection cassette in a nearby intron. 86Rb efflux experiments on brain synaptosomes showed an increased fraction of function at low agonist concentrations in L9′S mice. These data support the possible involvement of gain-of-function α4 receptors in autosomal dominant nocturnal frontal-lobe epilepsy.

α4β2* Nicotinic Acetylcholine Receptors Modulate the Effects of Ethanol and Nicotine on the Acoustic Startle Response

BACKGROUND Ethanol modulates the functional activity of alpha4beta2 neuronal nicotinic cholinergic receptors (nAChR) when measured in vitro, but the potential role of alpha4beta2 nAChRs in regulating behavioral effects of ethanol is unknown. Recently, Tritto et al. (Tritto T, Stitzel JA, Marks MJ, Romm E, Collins AC (2002) Variability in response to nicotine in the LSxSS RI strains: potential role of polymorphisms in alpha4 and alpha6 nicotinic receptor genes. Pharmacogenetics 12:197-208) reported that a polymorphism (A529T) in the alpha4 nAChR subunit gene is associated with variability in nicotines effects on startle in the LSxSS recombinant inbred (RI) strains. Ethanol also alters the acoustic startle response. Thus, we evaluated the potential role of alpha4beta2 nAChRs in modulating ethanols effects on acoustic startle. METHODS The effects of ethanol on acoustic startle were determined in the LSxSS RI strains. In addition, the effects of ethanol and nicotine were also measured in alpha4 gain of function and beta2 null mutant mice. The beta2 mutants do not express the major variant of alpha4 nAChRs, alpha4beta2. RESULTS An association between the alpha4 A529T polymorphism and ethanols effects on startle was found in the LSxSS RI strains; those strains that express the A529 variant of alpha4 were more sensitive to ethanol-induced depression of startle. The alpha4 gain of function mutants were more sensitive to the effects of both nicotine and ethanol and the beta2 null mutants were less sensitive to both drugs. CONCLUSIONS alpha4beta2-containing nAChRs may play important roles in modulating the effects of both ethanol and nicotine on the acoustic startle response. We suggest that nAChR subunit genes should be evaluated as potential contributors to both alcoholism and tobacco abuse.