Neurotrophin-4/5-depletion induces a delay in maturation of the periodontal Ruffini endings in mice

Neurotrophin-4/5 (NT-4/5) - a member of the neurotrophic factors - is a ligand for TrkB, which has been reported to be expressed in the mechanoreceptive Ruffini endings of the periodontal ligament. The present study examined developmental changes in the terminal morphology and neural density in homozygous mice with a targeted disruption of the nt-4/5 gene and wild-type mice by immunohistochemistry for protein gene product 9.5 (PGP 9.5), a general neuronal marker, and by quantitative analysis using an image analyzer. Postnatal development of terminal Schwann cells was also investigated by enzymatic histochemistry for non-specific cholinesterase activity (ChE). Furthermore, the immuno-expression of TrkB and low affinity nerve growth factor receptor (p75-NGFR) was surveyed in the periodontal Ruffini endings as well as trigeminal ganglion. At postnatal 1 week, the lingual periodontal ligament of both types of mice contained PGP 9.5-positive nerve fibers showing a tree-like ramification with axonal swellings in their course. In both types of mice at 2 weeks of age, comparatively thick nerve fibers with a smooth outline increased in number, and frequently ramified to form nerve terminals with dendritic profiles. However, no typical Ruffini endings with irregular outlines observed in the adult wild-type mice were found in the periodontal ligament at this stage. At postnatal 3 weeks, typical Ruffini endings with irregular outlines were discernable in the periodontal ligament of the wild-type mice while the dendritic endings showing smooth outlines were restricted to the homozygous mice. After postnatal 8 weeks, both types of mice showed an increase in the number of Ruffini endings, but the morphology differed between the wild-type and NT-4/5 homozygous mice. In the wild-type mice, a major population of the Ruffini endings expanded their axonal branches and developed many microprojections, resulting in a reduction of endings with smooth outlines. In contrast, we failed to find such typical Ruffini endings in the periodontal ligament of the homozygous mice: A majority of the periodontal Ruffini endings continued to show smooth outlines at postnatal 12 weeks. Quantitative analysis on neural density demonstrated a reduction in the homozygous mice with a significant difference by postnatal 8 weeks. Enzymatic histochemistry for non-specific ChE did not exhibit a distinct difference in the distribution and density of terminal Schwann cells between wild-type and homozygous mice. Furthermore, TrkB and p75-NGFR mRNA and proteins did not differ in the trigeminal ganglion between the two types. The periodontal Ruffini endings also displayed immunoreactivities for TrkB and p75- NGFR in both phenotypes. These findings suggest that the nt-4/5 gene depletion caused a delay in the formation and maturation of the periodontal Ruffini endings in the mice by inhibiting the growth of the periodontal nerves at an early stage, and indicate that multiple neurotrophins such as NT- 4/5 and BDNF might play roles in the development and/or maturation of the periodontal Ruffini endings.     

Morphological and cytochemical characteristics of periodontal Ruffini ending under normal and regeneration processes

Current knowledge on the Ruffini endings, primary mechanoreceptors in the periodontal ligament is reviewed with special reference to their cytochemical features and regeneration process. Morphologically, they are characterized by extensive ramifications of expanded axonal terminals and an association with specialized Schwann cells, called lamellar or terminal Schwann cells, which are categorized, based on their histochemical properties, as non-myelin-forming Schwann cells. Following nerve injury, the periodontal Ruffini endings of the rat incisor ligament can regenerate more rapidly than Ruffini endings in other tissues. During regeneration, terminal Schwann cells associated with the periodontal Ruffini endings migrate into regions where they are never found under normal conditions. Also during regeneration, alterations in the expression level of various bioactive substances occur in both axonal and Schwann cell elements in the periodontal Ruffini endings. Neuropeptide Y, which is not detected in intact periodontal Ruffini endings, is transiently expressed in their regenerating axons. Growth-associated protein-43 (GAP-43) is expressed transiently in both axonal and Schwann cell elements during regeneration, while this protein is localized in the Schwann sheath of periodontal Ruffini endings under normal conditions. The expression of calbindin D28k and calretinin, both belonging to the buffering type of calcium-binding proteins, was delayed in periodontal Ruffini endings, compared to their morphological regeneration. As the importance of axon-Schwann cell interactions has been proposed, further investigations are needed to elucidate their molecular mechanism particularly the contribution of growth factors during the regeneration as well as development of the periodontal Ruffini endings.     

Calcium regulation in individual peripheral sensory nerve terminals of the rat

Ca²⁺ is vital for release of neurotransmitters and trophic factors from peripheral sensory nerve terminals (PSNTs), yet Ca²⁺ regulation in PSNTs remains unexplored. To elucidate the Ca²⁺ regulatory mechanisms in PSNTs, we determined the effects of a panel of pharmacological agents on electrically evoked Ca²⁺ transients in rat corneal nerve terminals (CNTs) in vitro that had been loaded with the fluorescent Ca²⁺ indicator, Oregon Green 488 BAPTA-1 dextran or fura-2 dextran in vivo . Inhibition of the sarco(endo)plasmic reticulum Ca²⁺-ATPase, disruption of mitochondrial Ca²⁺ uptake, or inhibition of the Na⁺–Ca²⁺ exchanger did not measurably alter the amplitude or decay kinetics of the electrically evoked Ca²⁺ transients in CNTs. By contrast, inhibition of the plasma membrane Ca²⁺-ATPase (PMCA) by increasing the pH slowed the decay of the Ca²⁺ transient by 2-fold. Surprisingly, the energy for ion transport across the plasma membrane of CNTs is predominantly from glycolysis rather than mitochondrial respiration, as evidenced by the observation that Ca²⁺ transients were suppressed by iodoacetate but unaffected by mitochondrial inhibitors. These observations indicate that, following electrical activity, the PMCA is the predominant mechanism of Ca²⁺ clearance from the cytosol of CNTs and glycolysis is the predominant source of energy.     

Alterations in in vitro function and protein oxidation of rat sarcoplasmic reticulum Ca2+-ATPase during recovery from high-intensity exercise

The hypothesis tested in this study was that the extent to which sarcoplasmic reticulum (SR) Ca²⁺-ATPase is oxidized would correlate with a decline in its activity. For this purpose, changes in the SR Ca²⁺-sequestering ability and the contents of carbonyl and sulfhydryl groups during recovery after exercise were examined in the superficial portions of vastus lateralis muscles from rats subjected to 5 min running at an intensity corresponding to maximal oxygen uptake (50 m min⁻¹, 10% gradient). A single bout of exercise elicited a 22.4% reduction ( P < 0.05) in SR Ca²⁺-ATPase activity. The decreased activity progressively reverted to normal levels during recovery after exercise, reaching normal levels after 60 min of recovery. This change was paralleled by a depressed SR Ca²⁺-uptake rate, and the proportional alteration in these two variables resulted in no change in the ratio of Ca²⁺-uptake rate to Ca²⁺-ATPase activity. The contents of SR Ca²⁺-ATPase protein and sulfhydryl groups in microsomes were unchanged after exercise and during recovery periods. In contrast, the content of carbonyl groups in SR Ca²⁺-ATPase behaved in an opposite manner to that of SR Ca²⁺-ATPase activity. An approximately 80% augmentation ( P < 0.05) in the carbonyl group content occurred immediately after exercise. The elevated carbonyl content decreased towards normal levels during 60 min of recovery. These results are strongly suggestive that oxidation of SR Ca²⁺-ATPase is responsible, at least in part, for a decay in the SR Ca²⁺-pumping function produced by high-intensity exercise and imply that oxidized proteins may be repaired during recovery from exercise.     

Heterogeneous localizations of Trk B among individual periodontal Ruffini endings in the rat incisor

The present immunocytochemical study examined the localization of Trk B, a high affinity neurotrophin receptor, in the neural elements of the periodontal ligament of the rat incisor. In light microscopy, the immunoreactivity was demonstrated in dendritic profiles in the alveolar half of the periodontal ligament. Their location and morphological features indicated that they were periodontal Ruffini endings. Occasional rounded cells associated with periodontal Ruffini endings, which had immunonegative kidney-shaped nuclei, were immunoreactive; these were judged to be terminal Schwann cells. Immunoelectron microscopy revealed the heterogeneous localization of Trk B among individual Ruffini endings. Some terminal Schwann cells contained immunoreactive products for Trk B in the cytoplasm, while others did not. Similarly, a part of the Schwann sheaths covering the axon terminals showed Trk B immunoreactivity. Most axon terminals associated with periodontal Ruffini endings were immunopositive for Trk B, though a few of them were immunonegative. The ordinary Schwann cells did not contain Trk B immunoreactive products. These findings imply that Trk B is required for the maintenance of periodontal Ruffini endings. The different expression pattern of Trk B suggests that neuronal and glial elements comprising individual periodontal Ruffini endings are subject to heterogeneous conditions with regard to the requirement of Trk B.     

Junctin and the histidine-rich Ca2+ binding protein: potential roles in heart failure and arrhythmogenesis

Contractile dysfunction and ventricular arrhythmias associated with heart failure have been attributed to aberrant sarcoplasmic reticulum (SR) Ca²⁺ cycling. The study of junctin (JCN) and histidine-rich Ca²⁺ binding protein (HRC) becomes of particular importance since these proteins have been shown to be critical regulators of Ca²⁺ cycling. Specifically, JCN is a SR membrane protein, which is part of the SR Ca²⁺ release quaternary structure that also includes the ryanodine receptor, triadin and calsequestrin. Functionally, JCN serves as a bridge between calsequestrin and the Ca²⁺ release channel, ryanodine receptor. HRC is a SR luminal Ca²⁺ binding protein known to associate with both triadin and the sarcoplasmic reticulum Ca²⁺-ATPase, and may thus mediate the crosstalk between SR Ca²⁺ uptake and release. Indeed, evidence from genetic models of JCN and HRC indicate that they are important in cardiophysiology as alterations in these proteins affect SR Ca²⁺ handling and cardiac function. In addition, downregulation of JCN and HRC may contribute to Ca²⁺ cycling perturbations manifest in the failing heart, where their protein levels are significantly reduced. This review examines the roles of JCN and HRC in SR Ca²⁺ cycling and their potential significance in heart failure.     

Involvement of GDNF and its receptors in the maturation of the periodontal Ruffini endings

Our recent study revealed an intense immunoreaction for GDNF and its receptors in the Ruffini endings, primary mechanoreceptors in the periodontal ligament, of young rats. However, no information is available for the expression of GDNF and its receptors during their development. The present study aimed to reveal postnatal changes in the immuno-expression of GDNF, GFRalpha1 and RET in the periodontal Ruffini endings of the rat incisors by double immunofluorescent staining. At postnatal day 3 (PO 3d), no structure with GDNF-, GFRalpha1-, or RET-immunoreaction existed in the periodontal ligament. The PGP 9.5-positive nerve fibers without GDNF- and RET-immunoreaction displayed a dendritic fashion at PO 1w, with a GFRalpha1-reaction found around these nerves. At PO 2w, GDNF-positive terminal Schwann cells occurred near the thick and dendritic axons, a part of which showed a RET-reaction, with no reactive cells near the thin nerves. The terminal Schwann cells became positive for GFRalpha1, but lacked RET-immunoreaction. At PO 3w, when the formation of the periodontal Ruffini endings had proceeded, GDNF-positive terminal Schwann cells began to increase in number. This stage-specific immuno-expression pattern suggests that GDNF is a key molecule for the maturation and maintenance of the periodontal Ruffini endings.     

Sensory receptors in the periodontal ligament of hamster incisors with special reference to the distribution, ultrastructure and three-dimensional reconstruction of Ruffini endings

The innervation of the periodontal ligament in hamster incisors was investigated by means of immunohistochemistry for nervous-specific proteins and electron microscopy. The lingual periodontal ligament was found to be exclusively innervated by Ruffini endings which appeared to be most developed in this species among rodents; the labial periodontal ligament lacked them. The Ruffini endings occupied the alveolar half of the periodontal ligament, being intertwined with transverse collagen fibers. In electron microscopy, the Ruffini endings displayed expanded axon terminals filled with large-sized mitochondria. Three-dimensional reconstructions of the Ruffini endings at the electron microscopic level revealed complicated shapes for the axon terminals and a characteristic relationship with the associated terminal Schwann cells. The axon terminals were plate- or knob-shaped, the former being predominant. Each axon terminal was covered by thick Schwann sheaths derived from more than two terminal Schwann cells whose cell bodies were located apart from the axon terminals and contained a developed Golgi apparatus and rough endoplasmic reticulum. On the other hand, each terminal Schwann cell simultaneously extended their cytoplasmic processes to several axon terminals just like astrocytes. The thick Schwann sheath, for the most part, was covered by a multiple layer of basal lamina. These findings have aided us in understanding the entire structure of the periodontal Ruffini endings.     

Subunit composition and role of Na+,K+-ATPases in adrenal chromaffin cells

Adrenal medullary (AM) cells are exposed to high concentrations of cortical hormones, one of which is a ouabain-like substance. Thus, the effects of ouabain on catecholamine secretion and distribution of Na⁺,K⁺-ATPase α and β subunits in rat and guinea-pig AM cells were examined using amperometry and immunological techniques. While exposure to 1 μ m ouabain did not have a marked effect on resting secretion, it induced an increase in secretion due to mobilization of Ca²⁺ ions that were stored during a 4 min interval between muscarine applications. Immunocytochemistry revealed that Na⁺,K⁺-ATPase α1 subunit-like and β3 subunit-like immunoreactive (IR) materials were distributed ubiquitously at the cell periphery, whereas α2- and β2-like IR materials were present in restricted parts of the cell periphery. The α1 and α2 subunits were mainly immunoprecipitated from AM preparations by anti-β3 and anti-β2 antisera, respectively. Peripheral BODIPY-FL-InsP₃ binding sites were localized below membrane domains with α2- and β2-like IR materials. The results indicate that in AM cells, α1β3 isozymes of Na⁺,K⁺-ATPase were present ubiquitously in the plasma membrane, while α2β2 isozymes were in the membrane domain closely associated with peripheral Ca²⁺ store sites. This close association of the α2β2 isozyme with peripheral Ca²⁺ store sites may account for the facilitation of mobilization-dependent secretion in the presence of 1 μ m ouabain.     

Interplay between SERCA and sarcolemmal Ca2+ efflux pathways controls spontaneous release of Ca2+ from the sarcoplasmic reticulum in rat ventricular myocytes

Waves of calcium-induced calcium release occur in a variety of cell types and have been implicated in the origin of cardiac arrhythmias. We have investigated the effects of inhibiting the SR Ca²⁺-ATPase (SERCA) with the reversible inhibitor 2',5'-di(tert-butyl)-1,4-benzohydroquinone (TBQ) on the properties of these waves. Cardiac myocytes were voltage clamped at a constant potential between −65 and −40 mV and spontaneous waves evoked by increasing external Ca²⁺ concentration to 4 m m . Application of 100 μ m TBQ decreased the frequency of waves. This was associated with increases of resting [Ca²⁺]_i, the time constant of decay of [Ca²⁺]_i and the integral of the accompanying Na⁺–Ca²⁺ exchange current. There was also a decrease in propagation velocity of the waves. There was an increase of the calculated Ca²⁺ efflux per wave. The SR Ca²⁺ content when a wave was about to propagate decreased to 91.7 ± 3.2%. The period between waves increased in direct proportion to the Ca²⁺ efflux per wave meaning that TBQ had no effect on the Ca²⁺ efflux per unit time. We conclude that (i) decreased wave frequency is not a direct consequence of decreased Ca²⁺ pumping by SERCA between waves but, rather, to more Ca²⁺ loss on each wave; (ii) inhibiting SERCA increases the chance of spontaneous Ca²⁺ release propagating at a given SR content.     

Loss of caveolin-3 induced by the dystrophy-associated P104L mutation impairs L-type calcium channel function in mouse skeletal muscle cells

Caveolins are membrane scaffolding proteins that associate with and regulate a variety of signalling proteins, including ion channels. A deficiency in caveolin-3 (Cav-3), the major striated muscle isoform, is responsible for skeletal muscle disorders, such as limb-girdle muscular dystrophy 1C (LGMD 1C). The molecular mechanisms leading to the muscle wasting that characterizes this pathology are poorly understood. Here we show that a loss of Cav-3 induced by the expression of the LGMD 1C-associated mutant P104L (Cav-3^P104L) provokes a reduction by half of the maximal conductance of the voltage-dependent L-type Ca²⁺ channel in mouse primary cultured myotubes and fetal skeletal muscle fibres. Confocal immunomiscrocopy indicated a colocalization of Cav-3 and Ca_v1.1, the pore-forming subunit of the L-type Ca²⁺ channel, at the surface membrane and in the developing T-tubule network in control myotubes and fetal fibres. In myotubes expressing Cav-3^P104L, the loss of Cav-3 was accompanied by a 66% reduction in Ca_v1.1 mean labelling intensity. Our results suggest that Cav-3 is involved in L-type Ca²⁺ channel membrane function and localization in skeletal muscle cells and that an alteration of L-type Ca²⁺ channels could be involved in the physiopathological mechanisms of caveolinopathies.     

Adenosine inhibition via A1 receptor of N-type Ca2+ current and peptide release from isolated neurohypophysial terminals of the rat

Effects of adenosine on voltage-gated Ca²⁺ channel currents and on arginine vasopressin (AVP) and oxytocin (OT) release from isolated neurohypophysial (NH) terminals of the rat were investigated using perforated-patch clamp recordings and hormone-specific radioimmunoassays. Adenosine, but not adenosine 5'-triphosphate (ATP), dose-dependently and reversibly inhibited the transient component of the whole-terminal Ba²⁺ currents, with an IC₅₀ of 0.875 μ m. Adenosine strongly inhibited, in a dose-dependent manner (IC₅₀= 2.67 μ m ), depolarization-triggered AVP and OT release from isolated NH terminals. Adenosine and the N-type Ca²⁺ channel blocker ω-conotoxin GVIA, but not other Ca²⁺ channel-type antagonists, inhibited the same transient component of the Ba²⁺ current. Other components such as the L-, Q- and R-type channels, however, were insensitive to adenosine. Similarly, only adenosine and ω-conotoxin GVIA were able to inhibit the same component of AVP release. A₁ receptor agonists, but not other purinoceptor-type agonists, inhibited the same transient component of the Ba²⁺ current as adenosine. Furthermore, the A₁ receptor antagonist 8-cyclopentyltheophylline (CPT), but not the A₂ receptor antagonist 3, 7-dimethyl-1-propargylxanthine (DMPGX), reversed inhibition of this current component by adenosine. The inhibition of AVP and OT release also appeared to be via the A₁ receptor, since it was reversed by CPT. We therefore conclude that adenosine, acting via A₁ receptors, specifically blocks the terminal N-type Ca²⁺ channel thus leading to inhibition of the release of both AVP and OT.     

Effects of different types of injury to the inferior alveolar nerve on the behavior of Schwann cells during the regeneration of periodontal nerve fibers of rat incisor

The present study reports on different regeneration patterns of axons and Schwann cells in the periodontal ligament of the rat incisor using immunohistochemistry of protein gene product 9.5 (PGP 9.5) and S-100 protein. Three kinds of injury (transection, crush and segmental resection) were applied to the inferior alveolar nerve. In normal animals, PGP 9.5- and S-100-immunoreactivities were detected in the axons and Schwann cell elements of periodontal Ruffini endings, respectively. They were restricted to the alveolus-related part, occurring only rarely in the tooth-related part and in the shear zone (the border between the alveolus-related and tooth-related parts). Both transection and segmental resection caused the complete disappearance of PGP 9.5-immunoreactive nerve fibers in the periodontal ligament, while a small number of them could be found following the crush injury. Regenerating PGP 9.5-reactive nerve fibers appeared at 5 days and 21 days following the transection and segmental resection, respectively. The regeneration of periodontal nerve fibers completed in a period of 21-28 days and 14-21 days following the transection and crush, respectively, but was not completed even at 56 days following the segmental resection. The behavior of Schwann cells during regeneration was similar after the different nerve injuries; spindle-shaped S-100-immunoreactive cells, presumably Schwann cells, appeared in the shear zone and the tooth-related part. These cells disappeared 5-7 days prior to the completion of the regeneration of axonal elements of the periodontal ligament following the transection and crush. Following the segmental resection, in contrast, spindle-shaped S-100-positive cells disappeared from the tooth-related part at 42 days, although the axonal regeneration of periodontal Ruffini endings proceeded even until 56 days. We thus conclude that the duration of the migration of Schwann cells depends on the state of the regeneration of axons.     

Visualization of localized store-operated calcium entry in mouse astrocytes. Close proximity to the endoplasmic reticulum

Unloading of endoplasmic reticulum (ER) Ca²⁺ stores activates influx of extracellular Ca²⁺ through 'store-operated' Ca²⁺ channels (SOCs) in the plasma membrane (PM) of most cells, including astrocytes. A key unresolved issue concerning SOC function is their spatial relationship to ER Ca²⁺ stores. Here, using high resolution imaging with the membrane-associated Ca²⁺ indicator, FFP-18, it is shown that store-operated Ca²⁺ entry (SOCE) in primary cultured mouse cortical astrocytes occurs at plasma membrane–ER junctions. In the absence of extracellular Ca²⁺, depletion of ER Ca²⁺ stores using cyclopiazonic acid, an ER Ca²⁺-ATPase inhibitor, and caffeine transiently increases the sub-plasma-membrane Ca²⁺ concentration ([Ca²⁺]_SPM) within a restricted space between the plasma membrane and adjacent ER. Restoration of extracellular Ca²⁺ causes localized Ca²⁺ influx that first increases [Ca²⁺]_SPM in the same restricted regions and then, with a delay, in ER-free regions. Antisense knockdown of the TRPC1 gene, proposed to encode endogenous SOCs , markedly reduces SOCE measured with Fura-2. High resolution immunocytochemistry with anti-TRPC1 antibody reveals that these TRPC-encoded SOCs are confined to the PM microdomains adjacent to the underlying 'junctional' ER. Thus, Ca²⁺ entry through TRPC-encoded SOCs is closely linked, not only functionally, but also structurally, to the ER Ca²⁺ stores.     

Postnatal expression of calretinin-immunoreactivity in periodontal Ruffini endings in the rat incisor: a comparison with protein gene product 9.5 (PGP 9.5)-immunoreactivity

The postnatal expression of immunoreactivity for calretinin, one of the calcium binding proteins, and for protein gene product 9.5 (PGP 9.5), a general neuronal marker, was investigated in mechanoreceptive Ruffini endings in the periodontal ligament of the rat incisor. Age-related changes in the expression of these two proteins in periodontal nerves were further quantified with a computerized image analysis. At 1 day after birth, a few PGP 9.5-immunoreactive nerve fibers and a still smaller number of calretinin-positive fibers were found in the periodontal ligament: they were thin and beaded in appearance and no specialized nerve terminals were recognized. Tree-like terminals, reminiscent of immature Ruffini endings, were recognizable in 4-day-old rats by PGP 9.5-immunohistochemistry, while calretinin-immunostaining failed to reveal these specialized endings. At postnatal 7-11 days when PGP 9.5-immunostaining could demonstrate typical Ruffini endings, calretinin-immunopositive nerve fibers merely tapered off without forming the Ruffini type endings. A small number of Ruffini endings showing calretinin-immunoreactivity began to occur in the periodontal ligament at 24-26 days after birth when the occlusion of the first molars had been established. At the functional occlusion stage (60-80 days after birth), the Ruffini endings showing calretinin-immunoreactivity drastically increased in number and density, but less so than those positive for PGP 9.5-immunoreaction. The delayed expression of calretinin suggests that the function of the periodontal Ruffini endings is established after the completion of terminal formation because Ca2+, which binds to calcium binding proteins including calretinin with high affinity, plays an important role in mechano-electric transduction.     

Glycine Receptors and Glycinergic Synaptic Input at the Axon Terminals of Mammalian Retinal Rod Bipolar Cells

We investigated the properties of glycine receptors and glycinergic synaptic inputs at the axon terminals of rod bipolar cells (RBCs) in rats by patch-clamp recording. Glycine currents recorded from isolated axon terminals were larger than those from isolated somata/dendrites; this was confirmed by puffing glycine onto these two regions in retinal slices. The current density at terminal endings was more than one order of magnitude higher than the density at somatic/dendritic regions. Glycine currents from isolated terminals and isolated somata/dendrites showed similar sensitivity to picrotoxinin blockade. Single-channel opening recorded from isolated terminals and somata/dendrites displayed a similar main-state conductance of ≈46 pS. Application of glycine effectively suppressed depolarization-evoked increases in intracellular Ca²⁺ at the terminals. In the presence of GABA_A and GABA_C antagonists, strychnine-sensitive chloride currents were evoked in RBCs in retinal slices by puffing kainate onto the inner plexiform layer. No such currents were observed if the recorded RBCs did not retain axon terminals or if Ca²⁺ was replaced by Co²⁺ in the extracellular solution. The currents displayed discrete miniature-like events, which were partially blocked by tetrodotoxin. Consistent with early studies in the rabbit and mouse, this study demonstrates that glycine receptors are highly concentrated at the axon terminals of rat RBCs. The pharmacological and physiological properties of glycine receptors located in the axon terminal and somatic/dendritic regions, however, appear to be the same. This study provides evidence for the existence of functional glycinergic synaptic input at the axon terminals of RBCs, suggesting that glycine receptors may play a role in modulating bipolar cell synaptic transmission.     

A possible role of the junctional face protein JP-45 in modulating Ca2+ release in skeletal muscle

We investigated the functional role of JP-45, a recently discovered protein of the junctional face membrane (JFM) of skeletal muscle. For this purpose, we expressed JP-45 C-terminally tagged with the fluorescent protein DsRed2 by nuclear microinjection in myotubes derived from the C2C12 skeletal muscle cell line and performed whole-cell voltage-clamp experiments. We recorded in parallel cell membrane currents and Ca²⁺ signals using fura-2 during step depolarization. It was found that properties of the voltage-activated Ca²⁺ current were not significantly changed in JP-45–DsRed2-expressing C2C12 myotubes whereas the amplitude of depolarization-induced Ca²⁺ transient was decreased compared to control myotubes expressing only DsRed2. Converting Ca²⁺ transients to Ca²⁺ input flux using a model fit approach to quantify Ca²⁺ removal, the change could be attributed to an alteration in voltage-activated Ca²⁺ permeability rather than to altered removal properties or a lower Ca²⁺ content of the sarcoplasmic reticulum (SR). Determining non-linear capacitive currents revealed a reduction of Ca²⁺ permeability per voltage-sensor charge. The results may be explained by a modulatory effect of JP-45 related to its reported in vitro interaction with the dihydropyridine receptor and the SR Ca²⁺ binding protein calsequestrin (CSQ).     

Presynaptic N-type and P/Q-type Ca2+ channels mediating synaptic transmission at the calyx of Held of mice

At the nerve terminal, both N- and P/Q-type Ca²⁺ channels mediate synaptic transmission, with their relative contribution varying between synapses and with postnatal age. To clarify functional significance of different presynaptic Ca²⁺ channel subtypes, we recorded N-type and P/Q-type Ca²⁺ currents directly from calyces of Held nerve terminals in α_1A-subunit-deficient mice and wild-type (WT) mice, respectively. The most prominent feature of P/Q-type Ca²⁺ currents was activity-dependent facilitation, which was absent for N-type Ca²⁺ currents. EPSCs mediated by P/Q-type Ca²⁺ currents showed less depression during high-frequency stimulation compared with those mediated by N-type Ca²⁺ currents. In addition, the maximal inhibition by the GABA_B receptor agonist baclofen was greater for EPSCs mediated by N-type channels than for those mediated by P/Q-type channels. These results suggest that the developmental switch of presynaptic Ca²⁺ channels from N- to P/Q-type may serve to increase synaptic efficacy at high frequencies of activity, securing high-fidelity synaptic transmission.     

Presynaptic plasma membrane Ca2+ ATPase isoform 2a regulates excitatory synaptic transmission in rat hippocampal CA3

Plasma membrane calcium ATPase isoforms (PMCAs) are expressed in a wide variety of tissues where cell-specific expression provides ample opportunity for functional diversity amongst these transporters. The PMCAs use energy derived from ATP to extrude submicromolar concentrations of intracellular Ca²⁺ ([Ca²⁺]_i) out of the cell. Their high affinity for Ca²⁺ and the speed with which they remove [Ca²⁺]_i depends upon splicing at their carboxy (C)-terminal site. Here we provide biochemical and functional evidence that a brain-specific, C-terminal truncated and therefore fast variant of PMCA2, PMCA2a, has a role at hippocampal CA3 synapses. PMCA2a was enriched in forebrain synaptosomes, and in hippocampal CA3 it colocalized with the presynaptic marker proteins synaptophysin and the vesicular glutamate transporter 1, but not with the postsynaptic density protein PSD-95. PMCA2a also did not colocalize with glutamic acid decarboxylase-65, a marker of GABA-ergic terminals, although it did localize to a small extent with parvalbumin-positive presumed inhibitory terminals. Pharmacological inhibition of PMCA increased the frequency but not the amplitude of mEPSCs with little effect on mIPSCs or paired-pulse depression of evoked IPSCs. However, inhibition of PMCA activity did enhance the amplitude and slowed the recovery of paired-pulse facilitation (PPF) of evoked EPSCs. These results indicated that fast PMCA2a-mediated clearance of [Ca²⁺]_i from presynaptic excitatory terminals regulated excitatory synaptic transmission within hippocampal CA3.     

Modulation of Ca2+-activated K+ currents and Ca2+-dependent action potentials by exocytosis in goldfish bipolar cell terminals

Retinal bipolar cells convey light-evoked potentials from photoreceptors to ganglion cells and mediate the initial stages of visual signal processing. They do not fire Na⁺-dependent action potentials (APs) but the Mb1 class of goldfish bipolar cell exhibits Ca²⁺-dependent APs and regenerative potentials that originate in the axon terminal. I have examined the properties of Ca²⁺-dependent APs in isolated bipolar-cell terminals in goldfish retinal slices. All recorded terminals fired spontaneous or evoked APs at frequencies of up to 15 Hz. When an AP waveform was used as a voltage stimulus, exocytosis was evoked by single APs, maintained throughout AP trains and modulated by AP frequency. Furthermore, feedback inhibition of the Ca²⁺ current ( I _Ca) by released vesicular protons reduced depression of exocytosis during AP trains. In the absence of K⁺ current inhibition, step depolarizations and AP waveforms evoked a rapidly activated outward current that was dependent on Ca²⁺ influx ( I _K(Ca)). I therefore investigated whether proton-mediated feedback inhibition of I _Ca affected the activation of I _K(Ca). A transient inhibition of I _K(Ca) was observed that was dependent on exocytosis, blocked by high-pH extracellular buffer, of similar magnitude to inhibition of I _Ca but occurred with a delay of 2.7 ms. In addition, the amplitude of APs evoked under current clamp was inhibited by the action of vesicular protons released by the APs. Protons released via exocytosis may therefore be a significant modulator of Ca²⁺-dependent currents and regenerative potentials in bipolar-cell terminals.