Luis G. Aguayo

Ethanol potentiates the GABAA-activated Cl− current in mouse hippocampal and cortical neurons

The effects of ethanol (1-80 mM) on GABA-activated Cl- current (IGABA) were studied in cultured mammalian hippocampal and cortical neurons. Patch-clamp recordings revealed that ethanol potentiated the Cl- current in a concentration-dependent manner (1-40 mM) in the majority of the cells studied. No higher degree of potentiation was found by increasing the concentration of ethanol to 80 mM. This study demonstrates that ethanol can potentiate IGABA in mammalian central neurons.

β-Amyloid Causes Depletion of Synaptic Vesicles Leading to Neurotransmission Failure

Alzheimer disease is a progressive neurodegenerative brain disorder that leads to major debilitating cognitive deficits. It is believed that the alterations capable of causing brain circuitry dysfunctions have a slow onset and that the full blown disease may take several years to develop. Therefore, it is important to understand the early, asymptomatic, and possible reversible states of the disease with the aim of proposing preventive and disease-modifying therapeutic strategies. It is largely unknown how amyloid β-peptide (Aβ), a principal agent in Alzheimer disease, affects synapses in brain neurons. In this study, we found that similar to other pore-forming neurotoxins, Aβ induced a rapid increase in intracellular calcium and miniature currents, indicating an enhancement in vesicular transmitter release. Significantly, blockade of these effects by low extracellular calcium and a peptide known to act as an inhibitor of the Aβ-induced pore prevented the delayed failure, indicating that Aβ blocks neurotransmission by causing vesicular depletion. This new mechanism for Aβ synaptic toxicity should provide an alternative pathway to search for small molecules that can antagonize these effects of Aβ.

High-affinity sodium-vitamin C co-transporters (SVCT) expression in embryonic mouse neurons.

The sodium–vitamin C co‐transporters SVCT1 and SVCT2 transport the reduced form of vitamin C, ascorbic acid. High expression of the SVCT2 has been demonstrated in adult neurons and choroid plexus cells by in situ hybridization. Additionally, embryonic mesencephalic dopaminergic neurons express the SVCT2 transporter. However, there have not been molecular and kinetic analyses addressing the expression of SVCTs in cortical embryonic neurons. In this work, we confirmed the expression of a SVCT2‐like transporter in different regions of the fetal mouse brain and in primary cultures of neurons by RT‐PCR. Kinetic analysis of the ascorbic acid uptake demonstrated the presence of two affinity constants, 103 µm and 8 µm. A Km of 103 µm corresponds to a similar affinity constant reported for SVCT2, while the Km of 8 µm might suggest the expression of a very high affinity transporter for ascorbic acid. Our uptake analyses also suggest that neurons take up dehydroascorbic acid, the oxidized form of vitamin C, through the glucose transporters. We consider that the early expression of SVCTs transporters in neurons is important in the uptake of vitamin C, an essential molecule for the fetal brain physiology. Vitamin C that is found at high concentration in fetal brain may function in preventing oxidative free radical damage, because antioxidant radical enzymes mature only late in the developing brain.

Sodium vitamin C cotransporter SVCT2 is expressed in hypothalamic glial cells.

Kinetic analysis of vitamin C uptake demonstrated that different specialized cells take up ascorbic acid through sodium–vitamin C cotransporters. Recently, two different isoforms of sodium–vitamin C cotransporters (SVCT1/SLC23A1 and SVCT2/SLC23A2) have been cloned. SVCT2 was detected mainly in choroidal plexus cells and neurons; however, there is no evidence of SVCT2 expression in glial and endothelial cells of the brain. Certain brain locations, including the hippocampus and hypothalamus, consistently show higher ascorbic acid values compared with other structures within the central nervous system. However, molecular and kinetic analysis addressing the expression of SVCT transporters in cells isolated from these specific areas of the brain had not been done. The hypothalamic glial cells, or tanycytes, are specialized ependymal cells that bridge the cerebrospinal fluid with different neurons of the region. Our hypothesis postulates that SVCT2 is expressed selectively in tanycytes, where it is involved in the uptake of the reduced form of vitamin C (ascorbic acid), thereby concentrating this vitamin in the hypothalamic area. In situ hybridization and optic and ultrastructural immunocytochemistry showed that the transporter SVCT2 is highly expressed in the apical membranes of mouse hypothalamic tanycytes. A newly developed primary culture of mouse hypothalamic tanycytes was used to confirm the expression and function of the SVCT2 isoform in these cells. The results demonstrate that tanycytes express a high‐affinity transporter for vitamin C. Thus, the vitamin C uptake mechanisms present in the hypothalamic glial cells may perform a neuroprotective role concentrating vitamin C in this specific area of the brain.

Modulation of glycine-activated ion channel function by G-protein |[beta]||[gamma]| subunits

Glycine receptors (GlyRs), together with GABAA and nicotinic acetylcholine (ACh) receptors, form part of the ligand-activated ion channel superfamily and regulate the excitability of the mammalian brain stem and spinal cord. Here we report that the ability of the neurotransmitter glycine to gate recombinant and native ionotropic GlyRs is modulated by the G protein βγ dimer (Gβγ). We found that the amplitude of the glycine-activated Cl− current was enhanced after application of purified Gβγ or after activation of a G protein–coupled receptor. Overexpression of three distinct G protein α subunits (Gα), as well as the Gβγ scavenger peptide ct-GRK2, significantly blunted the effect of G protein activation. Single-channel recordings from isolated membrane patches showed that Gβγ increased the GlyR open probability (nPo). Our results indicate that this interaction of Gβγ with GlyRs regulates both motor and sensory functions in the central nervous system.

Effects of nerve growth factor on TTX- and capsaicin-sensitivity in adult rat sensory neurons

We have investigated the effects of nerve growth factor (NGF, 2.5 ng/ml for 1-2 weeks) on enriched adult rat dorsal root ganglion (DRG) neurons maintained in cell culture in defined media. Whole-cell recordings in cells cultured in the absence and presence of NGF revealed no significant difference in resting membrane potential and input resistance. However, the threshold for spike generation was significantly lower in untreated cells than in treated cells; -25 +/- 1.1 mV vs -19 +/- 2.2 mV, respectively. The sensitivity of the Na+ spike to tetrodotoxin (TTX, 1 microM) was different in cells cultured in the absence or presence of NGF. For example, spikes were abolished by TTX in 100% of untreated cells, while in NGF-treated cells the spike was abolished in only 41% of the neurons. Chemosensitivity of DRG neurons was also different in the absence and presence of NGF. For example, the percent of neurons in which a current activated by 8-methyl-N-vanillyl-6-nonenamide (capsaicin, 500 nM) was detected, increased from 18% in untreated cells to 55% in NGF-treated cells. NGF did not influence the number of cells surviving. The results indicate that NGF can regulate TTX and capsaicin sensitivity in these adult rat sensory neurons. Our experimental protocol indicates that this effect is not mediated by a factor in the serum or released from non-neuronal cells.

Mechanistic Insights and Functional Determinants of the Transport Cycle of the Ascorbic Acid Transporter SVCT2 ACTIVATION BY SODIUM AND ABSOLUTE DEPENDENCE ON BIVALENT CATIONS

We characterized the human Na+-ascorbic acid transporter SVCT2 and developed a basic model for the transport cycle that challenges the current view that it functions as a Na+-dependent transporter. The properties of SVCT2 are modulated by Ca2+/Mg2+ and a reciprocal functional interaction between Na+ and ascorbic acid that defines the substrate binding order and the transport stoichiometry. Na+ increased the ascorbic acid transport rate in a cooperative manner, decreasing the transport Km without affecting the Vmax, thus converting a low affinity form of the transporter into a high affinity transporter. Inversely, ascorbic acid affected in a bimodal and concentration-dependent manner the Na+ cooperativity, with absence of cooperativity at low and high ascorbic acid concentrations. Our data are consistent with a transport cycle characterized by a Na+:ascorbic acid stoichiometry of 2:1 and a substrate binding order of the type Na+:ascorbic acid:Na+. However, SVCT2 is not electrogenic. SVCT2 showed an absolute requirement for Ca2+/Mg2+ for function, with both cations switching the transporter from an inactive into an active conformation by increasing the transport Vmax without affecting the transport Km or the Na+ cooperativity. Our data indicate that SVCT2 may switch between a number of states with characteristic properties, including an inactive conformation in the absence of Ca2+/Mg2+. At least three active states can be envisioned, including a low affinity conformation at Na+ concentrations below 20 mm and two high affinity conformations at elevated Na+ concentrations whose Na+ cooperativity is modulated by ascorbic acid. Thus, SVCT2 is a Ca2+/Mg2+-dependent transporter.

Changes in the properties of developing glycine receptors in cultured mouse spinal neurons

We studied several neurophysiological properties of in vitro maturing glycine receptors in mouse spinal cord neurons cultured for various times: 3–7 days (early), 10–12 days (intermediate), and 17–24 days (mature), using whole‐cell and gramicidin‐perforated techniques. The glycine‐activated Cl− conductance increased about 6‐fold during in vitro development, and the current density increased from 177 ± 42 pA/pF in early to 504 ± 74 pA/pF in mature neurons. The sensitivity to glycine increased transiently from 39 ± 2.8 μM in early neurons to 29 ± 1 μM in intermediate neurons. Using whole‐cell recordings, we found that ECl did not change during development. With the gramicidin‐perforated technique, on the other hand, ECl shifted from −27 to −52 mV with development. Thus, immature neurons were depolarized by the activation of glycine receptors, whereas mature neurons were hyperpolarized. The current decayed (desensitized) during the application of 500 μM glycine. The decay was single exponential and the time constant increased from 2,212 ± 139 msec in early neurons to 4,580 ± 1,071 msec in mature neurons. Picrotoxin (10 μM) inhibited the current to a larger extent in early neurons (46 ± 6% of control), and the sensitivity of these receptors to strychnine (IC50) increased from 23 ± 3 nM to 9 ± 1 nM in mature neurons. In conclusion, several properties of spinal glycine receptors changed during in vitro neuronal maturation. This indicates that, similar to GABAA receptors, the functions of these receptors are developmentally regulated. These changes should affect the excitability of spinal neurons as well as other maturation processes. Synapse 28:185–194, 1998.

Molecular Determinants for G Protein βγ Modulation of Ionotropic Glycine Receptors

The ligand-gated ion channel superfamily plays a critical role in neuronal excitability. The functions of glycine receptor (GlyR) and nicotinic acetylcholine receptor are modulated by G protein βγ subunits. The molecular determinants for this functional modulation, however, are still unknown. Studying mutant receptors, we identified two basic amino acid motifs within the large intracellular loop of the GlyR α1 subunit that are critical for binding and functional modulation by Gβγ. Mutations within these sequences demonstrated that all of the residues detected are important for Gβγ modulation, although both motifs are necessary for full binding. Molecular modeling predicts that these sites are α-helixes near transmembrane domains 3 and 4, near to the lipid bilayer and highly electropositive. Our results demonstrate for the first time the sites for G protein βγ subunit modulation on GlyRs and provide a new framework regarding the ligand-gated ion channel superfamily regulation by intracellular signaling.

Canonical Wnt3a modulates intracellular calcium and enhances excitatory neurotransmission in hippocampal neurons

A role for Wnt signal transduction in the development and maintenance of brain structures is widely acknowledged. Recent studies have suggested that Wnt signaling may be essential for synaptic plasticity and neurotransmission. However, the direct effect of a Wnt protein on synaptic transmission had not been demonstrated. Here we show that nanomolar concentrations of purified Wnt3a protein rapidly increase the frequency of miniature excitatory synaptic currents in embryonic rat hippocampal neurons through a mechanism involving a fast influx of calcium from the extracellular space, induction of post-translational modifications on the machinery involved in vesicle exocytosis in the presynaptic terminal leading to spontaneous Ca2+ transients. Our results identify the Wnt3a protein and a member of its complex receptor at the membrane, the low density lipoprotein receptor-related protein 6 (LRP6) coreceptor, as key molecules in neurotransmission modulation and suggest cross-talk between canonical and Wnt/Ca2+ signaling in central neurons.