Dexamethasone and levetiracetam reduce hetero-cellular gap-junctional coupling between F98 glioma cells and glial cells in vitro.

Gap junctions (GJs) in astrocytes and glioma cells are important channels for cell-to-cell communication that contribute to homo- and heterocellular coupling. According to recent studies, heterocellular gap-junctional communication (H-GJC) between glioma cells and their surrounding environment enhances glioma progression. Therefore, we developed a new in vitro model to examine H-GJC between glioma cells, astrocytes and microglia. Consequently, F98 rat glioma cells were double-labeled with GJ-impermeable (CM-DiI) and GJ-permeable dye (calcein AM) and were seeded on unlabeled astrocyte-microglia co-cultures. Dual whole cell voltage clamp recordings were carried out on selected cell pairs to characterize the functional properties of H-GJC in vitro. The expression of four types of connexins (Cxs), including Cx32, Cx36, Cx43 and Cx45, and microglial phenotypes were analyzed by immunocytochemistry. The H-GJC between glioma cells and astrocytes/microglia increased after a longer incubation period with a higher number of glioma cells. We provided evidence for the direct GJ coupling of microglia and glioma cells under native in vitro conditions. In addition, we exploited this model to evaluate H-GJC after incubation with levetiracetam (LEV) and/or dexamethasone (DEX). Previous in vitro studies suggest that LEV and DEX are frequently used to control seizure and edema in glioma. Our findings showed that LEV and/or DEX decrease the number of heterocellular coupled cells significantly. In conclusion, our newly developed model demonstrated H-GJC between glioma cells and both astrocytes and microglia. The reduced H-GJC by LEV and DEX suggests a potential effect of both drugs on glioma progression.

Internal ribosomal entry site (IRES) activity generates endogenous carboxyl-terminal domains of Cx43 and is responsive to hypoxic conditions.

Connexin43 (Cx43) is the most abundant gap junction protein in higher vertebrate organisms and has been shown to be involved in junctional and non-junctional functions. In addition to the expression of full-length Cx43, endogenously produced carboxyl-terminal segments of Cx43 have been described and have been suggested to be involved in manifold biological functions, such as hypoxic preconditioning and neuronal migration. Molecular aspects, however, behind the separate generation of carboxyl-terminal segments of Cx43 have remained elusive. Here we report on a mechanism that may play a key role in the separate production of these domains. First, stringent evidence derived from siRNA treatment and specific knockouts revealed significant loss of the low molecular weight fragments of Cx43. By applying a dicistronic vector strategy on transfected cell lines, we were able to identify putative IRES activity (nucleotides 442­637) in the coding region of Cx43, which resides upstream from the nucleotide sequence encoding the carboxyl terminus (nucleotides 637­1149). Functional responsiveness of the endogenous expression of Cx43 fragments to hypoxic/ischemic treatment was evaluated in in vitro and in vivo models, which led to a significant increase of the fastest migrating form (20 kDa) under conditions of metabolic deprivation. By nano-MS spectrometry, we achieved stringent evidence of the identity of the 20-kDa segment as part of the carboxyl-terminal domain of full-length Cx43. Our data prove the existence of endogenously expressed carboxyl-terminal domains, which may serve as valuable tools for further translational application in ischemic disorders.

Glia and epilepsy: experimental investigation of antiepileptic drugs in an astroglia/microglia co-culture model of inflammation.

PURPOSE: The contribution of glial cells, mainly astrocytes and microglia, to the pathophysiology of epilepsy is increasingly appreciated. Glia play a pivotal role in the initiation and maintenance of the central nervous system (CNS) immune response and neuronal metabolic and trophic supply. Recent clinical and experimental evidence suggests a direct relationship between epileptic activity and CNS inflammation, which is characterized by accumulation, activation, and proliferation of microglia and astrocytes. Concomitant glia-mediated mechanisms of action of several antiepileptic drugs (AEDs) have been proposed. However, their direct effects on glial cells have been rarely investigated. We aimed to investigate the effect of commonly used AEDs on glial viability, the gap junctional network, the microglial activation, and cytokine expression in an in vitro astroglia/microglia co-culture model. METHODS: Primary astrocytic cultures were prepared from brains of postnatal (P0-P2) Wistar rats and co-cultured with a physiologic amount of 5%, as well as 30% microglia in order to mimic inflammatory conditions. Co-cultures were treated with valproic acid (VPA), carbamazepine (CBZ), phenytoin (PHE), and gabapentin (GBT). Viability and proliferation were measured using the tetrazolium (MTT) assay. The microglial activation state was determined by immunocytochemical labeling. The astroglial connexin 43 (Cx43) expression was measured by Western blot analysis. The transforming growth factor-ß1 (TGF-ß1) and tumor necrosis factor-α (TNF-α) cytokine levels were measured by the quantitative sandwich enzyme immunosorbent assay (ELISA). KEY FINDINGS: Astrocytes, co-cultured with 5% microglia (M5 co-cultures), showed a dose-dependent, significant reduction in glial viability after incubation with PHE and CBZ. Furthermore, VPA led to highly significant microglial activation at all doses examined. The antiinflammatory cytokine TGF-ß1 release was induced by high doses of GBT and PHE. Astrocytes co-cultured with 30% microglia (M30 co-cultures) revealed a dose-dependent significant reduction in glial viability after incubation with PHE, accompanied by increased TGF-ß1 and TNF-α levels. However, CBZ significantly reduced the amount of activated microglial cells and increased the total number of inactivated microglia. Finally, CBZ resulted in reduced viability at all doses examined. SIGNIFICANCE: CNS inflammation is characterized by a disturbance of glial cell functions. Strong microglial activation, a typical hallmark of inflammation, was induced by VPA in M5 and continued in M30 co-cultures. With regard to the direct relation between CNS inflammation and seizures, VPA seems to be unsuitable for reducing inflammatory conditions. The reverse effect was achieved after CBZ. We noticed significant microglial inactivation, after incubation of the M30 co-cultures. In conclusion, we suggest that AEDs with antiinflammatory glial features are beneficial for seizures caused by persistent brain inflammation.

Material attrition and bone micromorphology after conventional and ultrasonic implant site preparation.

OBJECTIVES: Little is known about the recently introduced ultrasonic implant site preparation. The purpose of this study was to compare material attrition and micromorphological changes after ultrasonic and conventional implant site preparations. MATERIAL AND METHODS: Implant site preparations were performed on fresh bovine ribs using one conventional (Straumann, Freiburg, Germany) and two ultrasonic (Piezosurgery; Mectron Medical Technology, Carasco, Italy and Variosurg; NSK, Tochigi, Japan) systems with sufficient saline irrigation. Sections were examined by environmental scanning electron microscopy (ESEM). Energy-dispersive X-ray spectroscopy (EDX) was performed to evaluate the metal attrition within the bone and the irrigation fluid. RESULTS ESEM: After conventional osteotomy, partially destroyed trabecular structures of the cancellous bone that were loaded with debris were observed, whereas after ultrasonic implant site preparations, the anatomic structures were preserved. EDX: None of the implant site preparation methods resulted in metal deposits in the adjacent bone structures. However, within the irrigation liquid, there was significantly higher metal attrition with ultrasonic osteotomy (P < 0.0001 and P < 0.0001 for Mectron and NSK, respectively). Whereas for Straumann system used, 15.5% of the SEM/EDX findings were drill-origin metals, this percentage increased to 37.3% and 37.9% with the application of Mectron and NSK, respectively. CONCLUSIONS: Ultrasonic implant site preparation is associated with the preservation of bone microarchitecture and with the increased attrition of metal particles. Therefore, copious irrigation seems to be even more essential for ultrasonic implant site preparation than for the conventional method.

Micromorphometrical analyses of five different ultrasonic osteotomy devices at the rabbit skull.

OBJECTIVES: The recently introduced ultrasonic osteotome procedure is an alternative to conventional methods of osteotomy. The aim of the present study was to establish the differences between five recently introduced ultrasonic osteotomes and to perform micromorphological and quantitative roughness analyses of osteotomized bone surfaces. MATERIALS AND METHODS: Fresh, standard-sized bony samples were taken from a rabbit skull using the following ultrasonic osteotomes: the Piezosurgery 3 with insert tip OT7, Piezosurgery Medical with insert tip MT1-10, Piezon Master Surgery with insert tip SL1, VarioSurg with inert tip SG1, and Piezotome 2 with insert tip BS1 II. The required duration of time for each osteotomy was recorded. The prepared surfaces were examined via light microscopy, environmental surface electron microscopy (ESEM), and confocal laser scanning microscopy (CLSM). RESULTS: All of the investigated piezoelectric osteotomes preserved the anatomical structure of bone. The mean roughness values of the osteotomized bone edge obtained using the investigated piezoelectric osteotomes were as follows: 2.47 µm (Piezosurgery 3), 9.79 µm (Piezosurgery Medical), 4.66 µm (Piezon Master Surgery), 6.38 µm (VarioSurg), and 6.06 µm (Piezotome 2). Significantly higher roughness values were observed when using the Piezosurgery Medical in comparison with those achieved by the Piezosurgery 3 (P<0.0001) and Piezon Master Surgery (P=0.002). Different osteotomy durations were achieved using the different piezoelectric osteotomes: 144 s (Piezosurgery 3), 126 s (Piezosurgery Medical), 142 s (Piezon Master Surgery), 149 s (VarioSurg), and 137 s (Piezotome 2). CONCLUSIONS: In the present study, micromorphological differences following the use of various ultrasonic devices were clearly identified. According to this study, it can be concluded that the power and the composition of the teeth of the insert tip might impact procedure duration and cutting qualities.

Intracellular cysteine 346 is essentially involved in regulating Panx1 channel activity.

Pannexins constitute a family of proteins exhibiting predominantly hemichannel activity. Pannexin channels have been suggested to participate in a wide spectrum of biological functions such as propagation of calcium waves, release of IL-1ß, and responses to ischemic conditions. At present, the molecular mechanisms regulating pannexin hemichannel activity are essentially unknown. Because cysteines have been shown to constitute key elements in regulating hemichannel properties of the connexin-type we performed site-directed mutagenesis of intracellular cysteine residues of Panx1. Cysteine to serine exchange (Cys → Ser) at the C-terminal position amino acid 346 led to a constitutively leaky hemichannel and subsequently to cell death. Increased channel activity was demonstrated by dye uptake and electrophysiological profiling in injected Xenopus laevis oocytes and transfected N2A cells. Mutations of the remaining intracellular cysteines did not result in major changes of Panx1 channel properties. From these data we conclude that the Cys-346 residue is important for proper functioning of the Panx1 channel.

Protection from diabetes development by single-chain antibody-mediated delivery of a NF-κB inhibitor specifically to ß-cells in vivo.

Recently, we reported the generation of single-chain antibodies (SCAs) highly specific for rodent and human ß-cells. Our current report describes the generation of a fusion protein of one of these SCAs (SCA B1) with a NF-κB essential modifier (NEMO)-binding domain (NBD) peptide, thereby creating a selective inhibitor of NF-κB activation in ß-cells. The SCA B1-NBD fusion protein was cloned in the pIRES-EGFP, expressed in bacteria, and purified by metal affinity chromatography; the newly generated complex was then administered intravenously to rodents and evaluated for its ability to protect ß-cells against cytokines in vitro and diabetogenic agents in vivo. First, it was shown clearly that our SCA B1-NBD fusion protein binds highly selective to CD rat ß-cells in vivo. Second, we observed that SCA B1-mediated in vivo delivery of the NBD peptide completely blocked IL-1ß + IFNγ- and TNFα + IFNγ-mediated induction of NF-κB as well as islet dysfunction in culture. Finally, repeated intravenous injection of SCA B1-NBD prior to multiple low-dose administration of streptozotocin in CD mice not only induced a striking resistance to diabetes development but also preserved ß-cell mass. In conclusion, our data show for the first time that a SCA B1-NBD fusion peptide reliably protects ß-cells against cytokines in vitro and allows protection from diabetes development in CD mice in vivo.

Heat production during different ultrasonic and conventional osteotomy preparations for dental implants.

OBJECTIVES: The purpose of this study was to evaluate the intraosseous temperature changes during ultrasonic and conventional implant site preparation in vitro with respect to the effect of load and irrigation volume. MATERIAL AND METHODS: Implant sites were prepared using two different ultrasonic devices (Piezosurgery, Mectron Medical Technology and VarioSurg, NSK) and one conventional device (Straumann) at loads of 5, 8, 15 and 20 N and with irrigation volumes of 20, 50 and 80 ml/min. During implant site preparation, temperatures were measured in fresh, equally tempered bovine ribs using two thermocouples placed at a distance of 1.5 mm around the drilling site in cortical and cancellous bone. The preparation time was recorded. RESULTS: The heat production and time required for implant site preparation using both ultrasonic devices were significantly higher than those for conventional drilling (P<0.01). Increased loading had no effect on heat production. A higher irrigation volume was associated with a diminished temperature increase in the cortical bone for ultrasonic but not for conventional drilling, which resulted in significantly lower temperatures in cortical as compared with cancellous bone during ultrasonic implant site preparation. CONCLUSIONS: Ultrasonic implant site preparation is more time consuming and generates higher bone temperatures than conventional drilling. However, with the levels of irrigation, ultrasonic implant site preparation can be an equally safe method.

Perforator Dissection Simulation: A High-Fidelity Five-Flap Porcine Training Model.

INTRODUCTION: Perforator dissection can be technically demanding with a steep learning curve. Inadvertent perforator damage during dissection can be minimized with practice and familiarity with tissue-handling techniques unique to perforator dissection. There currently lacks a simulation model that mimics the human perforator in size and course. We present a porcine training model with five consistent perforator flaps per side that can be readily harvested and is a reproducible simulation model. MATERIALS AND METHODS: Five fresh cadaveric pigs were used in this study to evaluate the feasibility and location of the perforators. Ten perforators were dissected out in each pig (five per side) by the same surgeon. The length of perforator was measured and intramuscular route was noted. The ease of dissection was graded, and its similarity to actual surgery was graded as well. RESULTS: Five consistent perforators were identified across each side of five fresh cadaveric pigs. The perforators were located, namely in the neck, anterior flank, posterior flank, rectus and hindlimb. They were fasciocutaneous and had an intramuscular course of each (average 2.5 cm length). The perforators were found to be on each side of the pig, giving ten perforators for dissection practice in total. DISCUSSION: The five perforators named in this porcine model are easily replicated and can be performed for perforator dissection simulation and practice.

Acquisition of chromosome 1q duplication in parental and genome-edited human-induced pluripotent stem cell-derived neural stem cells results in their higher proliferation rate in vitro and in vivo.

OBJECTIVES: Genetic engineering of human-induced pluripotent stem cell-derived neural stem cells (hiPSC-NSC) may increase the risk of genomic aberrations. Therefore, we asked whether genetic modification of hiPSC-NSCs exacerbates chromosomal abnormalities that may occur during passaging and whether they may cause any functional perturbations in NSCs in vitro and in vivo. MATERIALS AND METHODS: The transgenic cassette was inserted into the AAVS1 locus, and the genetic integrity of zinc-finger nuclease (ZFN)-modified hiPSC-NSCs was assessed by the SNP-based karyotyping. The hiPSC-NSC proliferation was assessed in vitro by the EdU incorporation assay and in vivo by staining of brain slices with Ki-67 antibody at 2 and 8 weeks after transplantation of ZFN-NSCs with and without chromosomal aberration into the striatum of immunodeficient rats. RESULTS: During early passages, no chromosomal abnormalities were detected in unmodified or ZFN-modified hiPSC-NSCs. However, at higher passages both cell populations acquired duplication of the entire long arm of chromosome 1, dup(1)q. ZNF-NSCs carrying dup(1)q exhibited higher proliferation rate than karyotypically intact cells, which was partly mediated by increased expression of AKT3 located on Chr1q. Compared to karyotypically normal ZNF-NSCs, cells with dup(1)q also exhibited increased proliferation in vivo 2 weeks, but not 2 months, after transplantation. CONCLUSIONS: These results demonstrate that, independently of ZFN-editing, hiPSC-NSCs have a propensity for acquiring dup(1)q and this aberration results in increased proliferation which might compromise downstream hiPSC-NSC applications.

Replacement of a single cysteine in the fourth transmembrane region of zebrafish pannexin 1 alters hemichannel gating behavior.

Pannexin1 (Panx1) is a novel candidate for an electrical synapse protein in the retina. At present Panx1 is considered to function as a hemichannel. Since information about the gating properties of Panx1 channels to date rely on blocker pharmacology, we have begun to establish a structural context of channel function starting with site directed mutagenesis of cysteine residues in transmembrane domains of Panx1. Dye uptake and whole cell voltage clamp recordings of transfected N2a cells demonstrate that zfPanx1 forms voltage activated hemichannels with a large unitary conductance in vitro. The function of this channel was significantly reduced following mutation of a single cysteine residue (C282W) in the fourth transmembrane region. This result suggests a role of this domain in gating of the Panx1 hemichannel.

Estradiol Receptors Regulate Differential Connexin 43 Expression in F98 and C6 Glioma Cell Lines.

INTRODUCTION: Glioma is the most common malignant primary brain tumour with male preponderance and poor prognosis. Glioma cells express variable amounts of connexin 43 (Cx43) and estrogen receptors (ERs). Both, Cx43 and ERs, play important roles in cell proliferation and migration. Therefore, we investigated the effects of 17-ß estradiol (E2) on Cx43 expression in two glioma cell lines with variable native expression of Cx43. MATERIALS AND METHODS: F98 and C6 rat glioma cells were cultured for 24 h in the presence of 10 nM or 100 nM E2, and the E2-antagonist, Fulvestrant. An MTT assay was performed to evaluate cell viability. ERα, ERß and Cx43 protein expressions were analysed by western blotting and Cx43 mRNA expression was analysed by real-time polymerase chain reaction. To quantify cell migration, an exclusive zone migration assay was used. Functional coupling of cells via gap junctions was examined using whole-cell patch-clamp technique. RESULTS: E2 reduced Cx43 expression in C6 cells, but increased Cx43 expression in F98 cultures. These effects were mediated via ERs. Moreover, E2 promoted C6 cell migration, but it did not affect F98 cell migration. The expression level of ERα was found to be high in C6, but low in F98 cells. ERß was exclusively expressed in C6 cells. In addition, E2 treatment induced a significant decrease of ERß in C6 cultures, while it decreased ERα expression in F98 glioma cells. DISCUSSION: These findings show that E2 differentially modulates Cx43 expression in F98 and C6 glioma cells, likely due to the differential expression of ERs in each of these cell lines. Our findings point to the molecular mechanisms that might contribute to the gender-specific differences in the malignancy of glioma and could have implications for therapeutic strategies against glioma.

Multiple Sclerosis Patient-Specific Primary Neurons Differentiated from Urinary Renal Epithelial Cells via Induced Pluripotent Stem Cells.

As multiple sclerosis research progresses, it is pertinent to continue to develop suitable paradigms to allow for ever more sophisticated investigations. Animal models of multiple sclerosis, despite their continuing contributions to the field, may not be the most prudent for every experiment. Indeed, such may be either insufficient to reflect the functional impact of human genetic variations or unsuitable for drug screenings. Thus, we have established a cell- and patient-specific paradigm to provide an in vitro model within which to perform future genetic investigations. Renal proximal tubule epithelial cells were isolated from multiple sclerosis patients' urine and transfected with pluripotency-inducing episomal factors. Subsequent induced pluripotent stem cells were formed into embryoid bodies selective for ectodermal lineage, resulting in neural tube-like rosettes and eventually neural progenitor cells. Differentiation of these precursors into primary neurons was achieved through a regimen of neurotrophic and other factors. These patient-specific primary neurons displayed typical morphology and functionality, also staining positive for mature neuronal markers. The development of such a non-invasive procedure devoid of permanent genetic manipulation during the course of differentiation, in the context of multiple sclerosis, provides an avenue for studies with a greater cell- and human-specific focus, specifically in the context of genetic contributions to neurodegeneration and drug discovery.

Low-flow lower body perfusion for spinal protection in a frozen elephant trunk simulation model.

OBJECTIVES: The frozen elephant trunk (FET) procedure using isolated selective cerebral perfusion (SCP) at moderate hypothermia is associated with an increased risk for spinal cord ischaemia. The aim of this study was to evaluate the benefit of a combined selective cerebral and low-flow lower body perfusion (CLBP) in a porcine model. METHODS: Twenty pigs (46 ± 5 kg) were cooled on cardiopulmonary bypass (CPB) to 28°C. After aortic clamping and occlusion of the thoracic segmental arteries (TSAT4-T13), a pressure-controlled SCP (50 mmHg) was established for 90 min. Randomly, in n = 10 animals, an additional lower body perfusion (LBP) was performed with 15 ml/kg/min (CLBP). Regional spinal cord blood flow (SCBF), cerebrospinal fluid pressure (CSFP) and motor-evoked potentials (MEPs) were registered at six time points. The animals were sacrificed after 120 min of weaning from CPB, and the spinal cord was analysed histologically using a schematic scoring system (0 = normal, 8 = total necrosis). RESULTS: Isolated SCP led to an SCBF decrease from 18.5 ± 9.4 to 0.9 ± 1.4 ml/min/100 g in the L1-L5 region (P = 0.005). CLBP preserved an almost physiological lumbar SCBF of 11.3 ± 5.3 ml/min/100 g. CSFP decreased in both groups during cooling and SCP/CLBP to 70-80% and increased during reperfusion to 150%, without showing significant differences between groups. The MEP amplitude decreased in both groups, with certain regional differences: T7-T11. MEP recording revealed a more pronounced amplitude decrease in the CLBP group (52.5 ± 2.0 vs 71.3 ± 0.9%), but MEP amplitudes recovered in both groups (SCP: 73.7 ± 0.5 vs CLBP: 82.6 ± 0.1%). During selective hypothermic perfusion, SCP-treated animals showed significant lower MEP amplitudes, when compared with CLBP-treated animals: 60 ± 9 vs 90 ± 3% (P < 0.001). After weaning, CLBP animals showed a better MEP recovery, especially in the L1-L5 region (99 ± 7 vs 70 ± 13%; P < 0.001). The histological analysis did not show significant differences in the necrosis extension in the thoracic spinal cord. A different situation was seen in the L1-L5 area: all animals with isolated SCP, but only 50% of the CLBP animals presented a score of >5. A higher grade of lumbar ischaemia could be seen after isolated SCP (score: 5.9 ± 0.6 vs 3.6 ± 2.9). CONCLUSION: The prolonged SCP provides an insufficient lumbar spinal cord protection during the FET procedure at 28°C. The use of a low-flow LBP in addition to SCP may reduce functional and structural spinal damage.

Spinal cord ischemia after selective cerebral perfusion in a porcine "frozen elephant trunk" simulation model.

BACKGROUND: The "frozen elephant trunk" procedure (FET) represents the therapy of choice for extended aortic diseases. The aim of our study was to analyze whether 90 minutes of selective cerebral perfusion (SCP) at 28 °C followed by permanent occlusion of the thoracic segmental arteries (TSA) would cause spinal cord ischemia in a porcine model. METHODS: 14 pigs (41 ± 3 kg) were cooled on CPB to 28 °C. After aortic clamping, SCP was established for 90 minutes. Randomly, in 7 animals the TSA were clipped (T4-T13); the TSA of 7 animals remained untouched. After the animals were weaned from CPB, hemodynamic data were registered for 120 minutes. Regional spinal cord blood flow (SCBF) was calculated, and motor-evoked potentials (MEP) were assessed at 6 time points. After sacrifice of the animals, the spinal cord was analyzed histologically by use of a schematic grading system (0 = normal; 8 = total necrosis). RESULTS: During SCP the SCBF was maintained at baseline (5.9 ± 2.4 mL/min/100 g) in the T4-T13 region but showed a decrease (from 8.4 ± 4.3 to 1.3 ± 1.5 mL/min/100 g) in the L1-L5 region. During reperfusion it increased, with two to three times higher values in the nonclipped animals. After 90 minutes of SCP, the MEP reached lower levels in the L1-L5 region of the TSA-clipped animals: 59% ± 7% vs 84 ± 15% (vastus medialis muscle) and 48% ± 6% vs 82% ± 26% (tibialis anterior muscle). The MEP recovered only in the nonclipped group. Higher ischemia rates were seen in the L1-L5 region of the TSA-clipped animals (score: 6.0 ± 0.6 vs 2.5 ± 2.3). CONCLUSIONS: 90 minutes of SCP provided sufficient spinal cord protection during arch replacement at 28 °C. In combination with permanent TSA occlusion, the lumbar spinal cord perfusion may be altered, which causes functional and structural damage.

Spontaneous activity of rat pretectal nuclear complex neurons in vitro.

BACKGROUND: Neurons in the mammalian pretectum are involved in the control of various visual and oculomotor tasks. Because functionally independent pretectal cell populations show a wide variation of response types to visual stimulation in vivo, they may also differ in their intrinsic properties when recorded in vitro. We therefore performed whole-cell patch clamp recordings from neurons in the caudal third of the pretectal nuclear complex in frontal brain slices obtained from 3 to 6 week old hooded rats and tried to classify pretectal neurons electrophysiologically. RESULTS: Pretectal neurons showed various response types to intracellular depolarizations, including bursting and regular firing behavior. One population of pretectal nuclear complex neurons could be particularly distinguished from others because they displayed spontaneous activity in vitro. These cells had more positive resting potentials and higher input resistances than cells that were not spontaneously active. The maintained firing of spontaneously active pretectal cells was characterized by only small variances in interspike intervals and thus showed a regular temporal patterning. The firing rate was directly correlated to the membrane potential. Removing excitatory inputs by blockade of AMPA and/or NMDA receptors did not change the spontaneous activity. Simultaneous blockade of excitatory and inhibitory synaptic input by a substitution of extracellular calcium with cobalt neither changed the firing rate nor its temporal patterning. Each action potential was preceeded by a depolarizing inward current which was insensitive to calcium removal but which disappeared in the presence of tetrodotoxin. CONCLUSIONS: Our results indicate that a specific subpopulation of pretectal neurons is capable of generating maintained activity in the absence of any external synaptic input. This maintained activity depends on a sodium conductance and is independent from calcium currents.

Relevance of gap junctions and large pore channels in traumatic brain injury.

In case of traumatic brain injury (TBI), occurrence of central nervous tissue damage is frequently aligned with local modulations of neuronal and glial gap junction channel expression levels. The degree of gap junctional protein expression and intercellular coupling efficiency, as well as hemichannel function has substantially impact on the course of trauma recovery and outcome. During TBI, gap junctions are especially involved in the intercellular molecule trafficking on repair of blood vessels and the regulation of vasomotor tone. Furthermore, gliosis and astrocytic swelling due to mechanical strain injury point out the consequences of derailed gap junction communication. This review addresses the outstanding role of gap junction channels in TBI pathophysiology and links the current state of results to applied clinical procedures as well as perspectives in acute and long-term treatment options.

Bisphosphonates and connexin 43: a critical review of evidence.

Bisphosphonates (BPs) are drugs commonly used in the treatment of various disease arising or affecting bone tissue. There is a standard use in bone neoplasia and metastasis, hormonal and developmental disorders as well as for compensation of adverse effects in several medical therapies. Many in-vivo and in-vitro studies have assessed the efficacy of this drug and its function in cellular scale. In this concern, BPs are described to inhibit the resorptive function of osteoclasts and to prevent apoptosis of osteoblasts and osteocytes. They can preserve the osteocytic network, reduce fracture rate, and increase the bone mineral content, which is therapeutically used. Connexin 43 (Cx43) is a crucial molecule for basal regulation of bone homeostasis, development, and differentiation. It is described for signal transduction in many physiological and pathological stimuli and recently to be involved in BP action.

An electrophysiologic approach to quantify impaired synaptic transmission and plasticity in experimental autoimmune encephalomyelitis.

Despite its various limitations, for many decades the experimental autoimmune encephalomyelitis (EAE) has been indispensable for understanding the pathology of multiple sclerosis (MS) and for establishing widely used MS therapeutics. We tested whether synaptic plasticity is a suitable measure for EAE and whether it can detect detrimental effects on supra-spinal structures that are too subtle to be captured by the motor score. Our data show functional synaptic deficits in the EAE that were beyond the measurable EAE score: long-term depression responses were strongly weakened in superior colliculus and cerebellum resulting from impaired postsynaptic transmission. In addition to further insight into neuronal deficits associated with the autoimmune disease, quantification of synaptic transmission may serve as a complementary method of EAE evaluation.