Sample collection and amino acids analysis of extracellular fluid of mouse brain slices with low flow push-pull perfusion.

Brain tissue slices are a common neuroscience model that allows relatively sophisticated analysis of neuronal networks in a simplified preparation. Most experimental methodology utilizes electrophysiological tools to probe these model systems. The work here demonstrates the adaptation of low-flow push-pull perfusion sampling (LFPS) to a brain slice system. LFPS is used to sample from the hippocampus of mouse brain slices. Perfusate amino acid levels are quantified following sampling with capillary electrophoresis. Glutamate was measured from the CA1 region of the hippocampus in slices taken from a cystine-glutamate transporter deletion mutant, xCT(-/-), and the background strain C57BL/6J. Sampling is performed over up to 6.5 h with standard tissue slice preparation and experimentation methods. Four amino acids were quantified to demonstrate the ability to perform LFPS and show good agreement with published literature. Perfusate glutamate levels are found to be significantly lower with xCT(-/-) slices (1.9(±0.5) µM) relative to controls (4.90(±1.1) µM). But, experiments with control slices show a significant decrease in glutamate over the 6 h sampling period that are not seen with xCT(-/-) slices. Increasing the LFPS sample collection rate during the first 90 min of sampling did not show a sampling artifact in perfusate glutamate content. Sampling immediately following slicing did not show an early increasing glutamate level that would be indicative of a significant contribution from blood or tissue damage. The data presented here show a complementarity to electrophysiological studies of tissue slices. The ability to characterize extracellular fluid chemical content with LFPS in these slices provides an alternative data stream for probing neurochemical signaling networks in brain tissue slices.

Drosophila alpha- and beta-spectrin mutations disrupt presynaptic neurotransmitter release.

Spectrins are plasma membrane-associated cytoskeletal proteins implicated in several aspects of synaptic development and function, including presynaptic vesicle tethering and postsynaptic receptor aggregation. To test these hypotheses, we characterized Drosophila mutants lacking either alpha- or beta-spectrin. The Drosophila genome contains only one alpha-spectrin and one conventional beta-spectrin gene, making it an ideal system to genetically manipulate spectrin levels and examine the resulting synaptic alterations. Both spectrin proteins are strongly expressed in the Drosophila neuromusculature and highly enriched at the glutamatergic neuromuscular junction. Protein null alpha- and beta-spectrin mutants are embryonic lethal and display severely disrupted neurotransmission without altered morphological synaptogenesis. Contrary to current models, the absence of spectrins does not alter postsynaptic glutamate receptor field function or the ultrastructural localization of presynaptic vesicles. However, the subcellular localization of numerous synaptic proteins is disrupted, suggesting that the defects in presynaptic neurotransmitter release may be attributable to inappropriate assembly, transport, or localization of proteins required for synaptic function.

Presynaptic glutamic acid decarboxylase is required for induction of the postsynaptic receptor field at a glutamatergic synapse.

We have systematically screened EMS-mutagenized Drosophila for embryonic lethal strains with defects in glutamatergic synaptic transmission. Surprisingly, this screen led to the identification of several alleles with missense mutations in highly conserved regions of Dgad1. Analysis of these gad mutants reveals that they are paralyzed owing to defects in glutamatergic transmission at the neuromuscular junction. Further electrophysiological and immunohistochemical examination reveals that these mutants have greatly reduced numbers of postsynaptic glutamate receptors in an otherwise morphologically normal synapse. By overexpressing wild-type Dgad1 in selected neurons, we show that GAD is specifically required in the presynaptic neuron to induce a postsynaptic glutamate receptor field, and that the level of postsynaptic receptors is closely dependent on presynaptic GAD function. These data demonstrate that GAD plays an unexpected role in glutamatergic synaptogenesis.

Surprises from Drosophila: genetic mechanisms of synaptic development and plasticity.

Drosophila are excellent models for the study of synaptic development and plasticity, thanks to the availability and applicability of a wide variety of powerful molecular, genetic, and cell-biology techniques. Three decades of study have led to an intimate understanding of the sequence of events leading to a functional and plastic synapse, yet many of the molecular mechanisms underlying these events are still poorly understood. Here, we provide a review of synaptogenesis at the Drosophila glutamatergic neuromuscular junction (NMJ). Next, we discuss the role of two proteins that forward genetic screens in Drosophila have revealed to play crucial-and completely unexpected-roles in NMJ development and plasticity: the origin of replication complex protein Latheo, and the enzyme glutamate decarboxylase. The requirement for these proteins at the NMJ highlights the fact that synaptic development and plasticity involves intense inter- and intracellular signaling about which we know almost nothing.

Slow inactivation in human cardiac sodium channels.

The available pool of sodium channels, and thus cell excitability, is regulated by both fast and slow inactivation. In cardiac tissue, the requirement for sustained firing of long-duration action potentials suggests that slow inactivation in cardiac sodium channels may differ from slow inactivation in skeletal muscle sodium channels. To test this hypothesis, we used the macropatch technique to characterize slow inactivation in human cardiac sodium channels heterologously expressed in Xenopus oocytes. Slow inactivation was isolated from fast inactivation kinetically (by selectively recovering channels from fast inactivation before measurement of slow inactivation) and structurally (by modification of fast inactivation by mutation of IFM1488QQQ). Time constants of slow inactivation in cardiac sodium channels were larger than previously reported for skeletal muscle sodium channels. In addition, steady-state slow inactivation was only 40% complete in cardiac sodium channels, compared to 80% in skeletal muscle channels. These results suggest that cardiac sodium channel slow inactivation is adapted for the sustained depolarizations found in normally functioning cardiac tissue. Complete slow inactivation in the fast inactivation modified IFM1488QQQ cardiac channel mutant suggests that this impairment of slow inactivation may result from an interaction between fast and slow inactivation.

A defect in skeletal muscle sodium channel deactivation exacerbates hyperexcitability in human paramyotonia congenita.

1. Paramyotonia congenita (PC) is a human hereditary disorder wherein missense mutations in the skeletal muscle sodium channel lead to cold-exacerbated muscle hyperexcitability. The most common site for PC mutations is the outermost arginine of domain i.v. segment 4 (human R1448, rat R1441). 2. We examined the rat homologues of two PC mutants with changes at this site: R1441P and R1441C. The R-->P mutation leads to the most clinically severe form of the disease. Since PC has so far been attributed to defects in fast inactivation, we expected the R-->P substitution to have a more dramatic effect on fast inactivation than R-->C. Both mutants (R1441P and R1441C), however, had identical rates and voltage dependence of fast inactivation and activation. 3. R1441P and R1441C also had slowed deactivation, compared with wild-type, raising the possibility that slowed deactivation, in combination with defective fast inactivation, might be a contributing cause of paramyotonia congenita. Furthermore, deactivation was slower in R1441P than in R1441C, suggesting that the worse phenotype of the human R-->P mutation is due to a greater effect on deactivation, and supporting our hypothesis that slowed sodium channel deactivation contributes to paramyotonia congenita. 4. We show that the downstroke of the muscle action potential produced a sodium tail current, and thus slowed deactivation opposes repolarization and therefore leads to hyperexcitability. Hyperexcitability due to slowed deactivation, which has previously been overlooked, also predicts the temperature sensitivity of PC, which has otherwise not been adequately explained.

Defective fast inactivation recovery and deactivation account for sodium channel myotonia in the I1160V mutant.

The skeletal muscle sodium channel mutant I1160V cosegregates with a disease phenotype producing myotonic discharges (observed as muscle stiffness) that are worsened by elevated K+ levels but unaffected by cooling. The I1160V alpha-subunit was co-expressed with the beta1-subunit in Xenopus oocytes. An electrophysiological characterization was undertaken to examine the underlying biophysical characteristics imposed by this mutation. Two abnormalities were found. 1) The voltage dependence of steady-state fast inactivation was reduced in I1160V, which resulted in faster rates of closed-state fast inactivation onset and recovery in I1160V compared with wild-type channels. 2) The rates of deactivation were slower in I1160V than in wild-type channels. Using a computer-simulated model, the combination of both defects elicited myotonic runs under conditions of elevated K+, consistent with the observed phenotype of the mutant.

Human Na+ channel fast and slow inactivation in paramyotonia congenita mutants expressed in Xenopus laevis oocytes.

1. Paramyotonia congenita (PC) is a human hereditary disease caused by one or more amino acid substitutions in skeletal muscle sodium channels. Using macropatches, the effect of PC mutations R1448C and T1313M were compared with wild-type (WT) in Xenopus oocytes coinjected with both alpha- and beta-subunits of human skeletal muscle (SkM1) sodium channels. 2. Slow inactivation in either T1313M or R1448C was not different from WT. Fast inactivation in both PC mutants, however, was significantly altered. 3. Commonly used biophysical protocols (such as I-V curves, steady-state inactivation curves, and measurements of inactivation rates) did not uniformly indicate that hyperexcitability should result from T1313M or R1448C. In fact, the only alteration of fast inactivation common to T1313M and R1448C that predicted cellular hyperexcitability was slowed open-state inactivation, compared with WT. 4. To test whether this alteration was sufficient to cause the phenotypic hyperexcitability, we used a novel voltage command that simulated muscle membrane activity. With this protocol, we found that R1448C and T1313M were similar in that they maintained a significantly higher channel availability during high frequency activity, compared with WT.

Interaction between fast and slow inactivation in Skm1 sodium channels.

Rat skeletal muscle (Skm1) sodium channel alpha and beta 1 subunits were coexpressed in Xenopus oocytes, and resulting sodium currents were recorded from on-cell macropatches. First, the kinetics and steady-state probability of both fast and slow inactivation in Skm1 wild type (WT) sodium channels were characterized. Next, we confirmed that mutation of IFM to QQQ (IFM1303QQQ) in the DIII-IV 'inactivation loop' completely removed fast inactivation at all voltages. This mutation was then used to characterize Skm1 slow inactivation without the presence of fast inactivation. The major findings of this paper are as follows: 1) Even with complete removal of fast inactivation by the IFM1303QQQ mutation, slow inactivation remains intact. 2) In WT channels, approximately 20% of channels fail to slow-inactivate after fast-inactivating, even at very positive potentials. 3) Selective removal of fast inactivation by IFM1303QQQ allows slow inactivation to occur more quickly and completely than in WT. We conclude that fast inactivation reduces the probability of subsequent slow inactivation.