Adrian M. Corbett

Immunolocalization of NaV1.2 channel subtypes in rat and cat brain and spinal cord with high affinity antibodies

High titer polyclonal antibodies were produced in rabbit against a peptide unique to NaV1.2 sodium channels. NaV1.2 antibodies displayed 500,000-fold greater affinity for the NaV1.2 peptide compared with NaV1.1 or NaV1.3 peptides from the same region. These antibodies, when coupled to Sepharose beads, retained saxitoxin binding sites from solubilized rat brain membranes. Eluted protein from this antibody-affinity column was recognized by antibodies directed against neuronal voltage-gated sodium channels. Rabbit antibodies, which had been partially purified, were used in immunocytochemical localization of the NaV1.2 channel in 50 microm rat brain slices at dilutions of 1:1000 or 1:2000. NaV1.2 channels were predominately localized in unmyelinated fibers in the cortex, hippocampus, spinal cord and hypothalamus. Varicosities were seen in fiber staining which may reflect true varicosities in the fiber or simply varying densities of sodium channels along the fiber. Cell body staining with the NaV1.2 antibody was primarily observed in the hypothalamus. Antibody staining in the cerebellum was complex, with staining observed primarily in posterior lobes and considerably lower amounts of staining observed in anterior lobes. Specific staining was limited to fibers located in the granule and molecular layer, in an orientation consistent with granule cell unmyelinated axon labeling.

Physiological characterization of the Na+/Ca2+ exchanger (NCX) in hepatopancreatic and antennal gland basolateral membrane vesicles isolated from the freshwater crayfish Procambarus clarkii

The purpose of this study was to physiologically characterize the basolateral Na(+)/Ca(2+) exchanger (NCX) in basolateral membrane vesicles (BLMVs) of hepatopancreas and antennal gland of intermolt crayfish. Conditions were optimized to measure Na(+)-dependent Ca(2+) uptake and retention in the BLMV including use of intravesicular (IV) oxalate and measuring initial uptake rates at 20 s. Na(+)-dependent Ca(2+) uptake rate into BLMV was temperature insensitive. Na(+)-dependent Ca(2+) uptake rate was dependent upon free Ca(2+) with saturable Michaelis-Menten kinetics determined as follows: hepatopancreas, maximal uptake rate (J(max))=2.45 nmol/mg per min, concentration at which carrier operates at half-maximal uptake rate (K(m))=0.69 microM Ca(2+); antennal gland, J(max)=13.2 nmol/mg per min, K(m)=0.59 microM Ca(2+). The two vesicle populations exhibited different sensitivity to putative NCX inhibitors. Benzamil had no effect on Na(+)-dependent Ca(2+) uptake rate in hepatopancreas; in antennal gland it was inhibitory at concentrations up to 30 microM and was stimulatory at higher concentrations. Conversely the inhibitor quinacrine was inhibitory at 10 microM in hepatopancreas and was stimulatory at 1000 microM; meanwhile it was ineffective in antennal gland BLMV. Short circuiting the BLMV had no effect on Na(+)-dependent Ca(2+) uptake rate suggesting that the process may be electroneutral. Compared with another prominent basolateral transporter in hepatopancreas the plasma membrane Ca(2+) ATPase (PMCA), the NCX has 70-fold greater J(max) (at comparable temperature) and a lower affinity. In antennal gland the NCX has 40-fold greater J(max) and a lower affinity. In hepatopancreas and antennal gland BLMV NCX appears to determine the rate of basolateral Ca(2+) efflux in intermolt.

A method for reliable voluntary oral administration of a fixed dosage (mg/kg) of chronic daily medication to rats.

Stress can influence a number of physiological processes including adult neurogenesis, metabolism, cardiovascular function, immune function, neurophysiological function, endocrine function and inflammatory processes following injury. In testing drugs which may be used to treat various diseases or injuries, reducing stress associated with chronic drug delivery to animal models should then be an imperative, which led us to design a reliable voluntary oral drug delivery method. Various drug combinations were tested versus vehicle controls in four different rat stocks or strains (Wistar, Fisher, Long Evans and Sprague Dawley) with our voluntary oral delivery system. Oral medications were placed into a store-bought sugar cookie dough ball (∼4 g), thoroughly integrating the dry drugs with the dough. This method has worked consistently to deliver the medication (complete ingestion) in four different stocks or strains of rats, with reliabilities ranging from 98.6% to 100%. The percentage of rats in each stock or strain that have at any time during the study had incomplete ingestion of the drugs ranged from 1% in Sprague Dawley, approximately 4% in Wistar and Fisher, to approximately 16% in Long Evans. Both serum and brain samples were analysed for high-performance liquid chromatography (HPLC) detection of one of our administered drugs: 5 mg/kg fluoxetine. HPLC analysis shows that serum levels are detectable 2–4 h after ingestion, but not 24 h after ingestion. Brain samples however, showed detectable levels of both fluoxetine and norfluoxetine more than a week following ingestion of a single dose, with higher norfluoxetine levels seen following a month of daily administered drugs.

Genetic Barcoding of Marine Leeches ( Ozobranchus spp.) from Florida Sea Turtles and Their Divergence in Host Specificity

Ozobranchus margoi and Ozobranchus branchiatus are the only two species of marine turtle leeches (Ozobranchus spp.) known to inhabit the Atlantic coast of the United States and the Gulf of Mexico. In early reports of fibropapillomatosis (FP) in green turtles (Chelonia mydas), O. branchiatus was implicated as a vector in the transmission of Fibropapilloma‐associated turtle herpesvirus (FPTHV). It is imperative that the leech species be identified to elucidate the role Ozobranchus spp. may play in disease transmission. In this study, Ozobranchus branchiatus has been identified for the first time on a loggerhead (Caretta caretta) turtle, and the molecular data for this species is now available for the first time in GenBank. Both species of leeches were also found infecting a single C. mydas. Using morphological taxonomy combined with distance‐ and character‐based genetic sequence analyses, this study has established a DNA barcode for both species of Ozobranchus spp. leech and has shown it can be applied successfully to the identification of leeches at earlier stages of development when morphological taxonomy cannot be employed. The results suggest a different haplotype may exist for O. branchiatus leeches found on C. caretta versus C. mydas. Leech cocoon residue collected from a C. mydas was identified using the new method.

Depolarization-Induced Calcium Release from Isolated Triads Measured With Impermeant Fura-2

SummaryDepolarization-induced Ca2+ release was studied in a mixture of triads and terminal cisternae isolated from rabbit skeletal muscle. The vesicles were actively loaded with known amounts of Ca2+ in the absence of precipitating anions in a solution containing 100 mm K propionate buffer. Changes in extravesicular Ca2+ were monitored with 10 μm Fura-2 (membrane impermeant form). Ca2+ release was initiated by diluting an aliquot of the loaded vesicles into a TEACl release solution designed to maintain a constant [K+] · [Cl−] product. Fast release, defined as the percentage of total Ca2+ loaded which released in less than 10 sec, occurred when extravesicular free Ca2+ was in the submicromolar range and was unaffected by 5 mm caffeine under depolarizing conditions, change in external pH to 6.5, and an increase in external Mg2+ concentration from 0.1 to 0.2 mm. Thus, the Ca2+ release measured in these studies is distinct from Ca2+-induced Ca2+ release. The fast release more than doubled when a greater dilution (1 ∶ 20 versus 1 ∶ 10) of the loaded vesicles into the release solution, which would produce a larger depolarization, was used. The percentage of loaded Ca2+ which released rapidly in a particular triad preparation was similar to the percentage of vesicles structurally coupled as visualized by electron microscopy.

CNS voltage-dependent Na+ channel expression and distribution in an undifferentiated and differentiated CNS cell line

Upon serum removal, CAD-R1 cells undergo neurite outgrowth and an increase in voltage-dependent Na(+) current (VDNaC) density without changing their activation and inactivation properties. Insulin and endothelial cell growth supplement inhibited the increase in VDNaC density but not the neurite outgrowth. RI, RII, RIII Na(+) channel proteins were expressed in CAD-R1 cells. These proteins exhibited both similar and different distribution and clustering patterns which suggested the channels structural differences play a role in channel distribution.

High titer antibody to mammalian neuronal sodium channels produces sustained channel block

Antibodies specific for neuronal sodium channels recognized the alpha and beta subunits of the voltage-gated sodium channel on immunoblots of crude rat and cat brain membranes and purified rat brain sodium channels. These antibodies did not recognize channels from rat cardiac or rabbit skeletal muscle. Antibody binding to blots of crude rat brain membranes was blocked by preabsorption of the antibody with purified rat brain sodium channels. Staining of the sodium channel alpha subunit on immunoblots of crude rat brain membranes was easily visualized at antibody dilutions of up to 1:150,000, which is at least 15-fold higher than that reported in previous studies. Addition of antibody produced in one chicken to the extracellular face of batrachotoxin-activated rat brain sodium channels in planar lipid bilayers produced a sustained block of the channel at either hyperpolarized (-65 to -45 mV) or depolarized potentials (+45 to +75 mV). This block was not produced when the antibody was added to the cytoplasmic face of the channel, or if preimmune antibody was added to the extracellular face of rat brain sodium channels.

The voltage dependence of depolarization-induced calcium release in isolated skeletal muscle triads

We demonstrate for the first time in this study that triadic vesicles derived from skeletal muscle display a voltage dependence of depolarization-induced calcium release similar to that found in intact muscle. We confirm previous studies by Dunn (1989) which demonstrated that changes in extravesicular potassium induced membrane potential changes in isolated transverse tubules with the voltage sensitive dye DiSC(3)-5. Depolarization-induced calcium release was studied in isolated triadic vesicles through similar changes in extravesicular [K] while clamping extravesicular Ca++ to submicromolar concentrations. The amplitude of fast phase of calcium release, identified as depolarizationinduced calcium release, varied with the percentage of transverse tubules in the preparation (determined through 3 H-PN200-110 specific activity) and different levels of depolarization. Threshold activation of calcium release was obtained with a 40.5 mV potential change; maximal calcium release was obtained with a 75 to 81 mV potential change. Boltzmann fits to the normalized depolarization induced calcium release plotted against the membrane potential change yielded a voltage dependence (k = 4.5 mV per e-fold change) very similar to that found in intact muscle (k = 3–4 mV per e-fold change; Baylor, Chandler & Marshall 1978, 1983; Miledi et al., 1981). Substitution of methanesulfonate for propionate as the impermeant ion or addition of valinomycin in the depolarizing solutions had little effect on the voltage dependence of calcium release.

Enrichment of Triadic and Terminal Cisternae Vesicles from Rabbit Skeletal Muscle

An enriched triad and terminal cisternae preparation was achieved from skeletal muscle through alterations of the differential centrifugation and muscle homogenization protocols. Both yield and specific activity (pmoles of radioligand binding per mg protein) were optimized for 3H-PN200-l10 (transverse tubule marker) and 3H-ryanodine (terminal cisternae marker) binding sites. By pelleting crude microsomes between 2,000 an 12,000 × g without any rehomogenizations, we improved both the yield and specific activity of transverse tubule and terminal cisternae markers in crude microsomes by approximately 4-fold to 1000–3000 pmoles binding sites (starting material: approximately 400 grams wet weight fast twitch skeletal muscle), with 10–15 pmoles/mg. Rehomogenization of the 1,000 × g pellet, which is typically discarded, allowed recovery of an additional 5000 pmoles PN200-110 binding sites and an additional 8000 pmoles ryanodine binding sites. Crude microsomes from the rehomogenized 1,000 × g pellets typically displayed specific activities of 20–25 pmoles binding/mg for both 3H-PN200-110 and 3H-ryanodine. Separation of crude microsomes on a sucrose gradient increased specific activity up to a maximum of 50 pmoles/mg in a specific fraction, a five- to ten-fold increase over standard triadic or terminal cisternae preparations. The mean specific activity for enriched triads was 30–40 pmoles/mg for both PN200-110 and ryanodine in pooled fractions, while pooled fractions of enriched terminal cisternae displayed low 3H-PN200-110 binding (3–5 pmoles/mg) and high 3H-ryanodine-specific activity (30–40 pmoles/mg).

Examining the reversibility of long-term behavioral disruptions in progeny of maternal SSRI exposure

Visual Abstract Serotonergic dysregulation is implicated in numerous psychiatric disorders. Serotonin plays widespread trophic roles during neurodevelopment; thus perturbations to this system during development may increase risk for neurodevelopmental disorders. Epidemiological studies have examined association between selective serotonin reuptake inhibitor (SSRI) treatment during pregnancy and increased autism spectrum disorder (ASD) risk in offspring. It is unclear from these studies whether ASD susceptibility is purely related to maternal psychiatric diagnosis, or if treatment poses additional risk. We sought to determine whether maternal SSRI treatment alone or in combination with genetically vulnerable background was sufficient to induce offspring behavior disruptions relevant to ASD. We exposed C57BL/6J or Celf6 +/- mouse dams to fluoxetine (FLX) during different periods of gestation and lactation and characterized offspring on tasks assessing social communicative interaction and repetitive behavior patterns including sensory sensitivities. We demonstrate robust reductions in pup ultrasonic vocalizations (USVs) and alterations in social hierarchy behaviors, as well as perseverative behaviors and tactile hypersensitivity. Celf6 mutant mice demonstrate social communicative deficits and perseverative behaviors, without further interaction with FLX. FLX re-exposure in adulthood ameliorates the tactile hypersensitivity yet exacerbates the dominance phenotype. This suggests acute deficiencies in serotonin levels likely underlie the abnormal responses to sensory stimuli, while the social alterations are instead due to altered development of social circuits. These findings indicate maternal FLX treatment, independent of maternal stress, can induce behavioral disruptions in mammalian offspring, thus contributing to our understanding of the developmental role of the serotonin system and the possible risks to offspring of SSRI treatment during pregnancy.