Light Activation of Calcein Inhibits Vesicle Release of Catecholamines.

Calcein, a fluorescent fluid phase marker, has been used to track and visualize cellular processes such as synaptic vesicle fusion. It is also the fluorophore for live cells in the commonly used Live/Dead viability assay. In pilot studies designed to determine fusion pore open size and vesicle movement in secretory cells, imaging analysis revealed that calcein reduced the number of vesicles released from the cells when stimulated with nicotine. Using amperometry to detect individual vesicle release events, we show that when calcein is present in the media, the number of vesicles that fuse with the cellular membrane is reduced when cells are stimulated with either nicotine or high K+. Experimentally, amperometric electrodes are not undergoing fouling in the presence of calcein. We hypothesized that calcein, when activated by light, releases reactive oxygen species that cause a reduction in secreted vesicles. We show that when calcein is protected from light during experimentation, little to no reduction of vesicle secretion occurred. Therefore, photoactivated calcein can cause deleterious results for measurements of cellular processes, likely to be the result of release of reactive oxygen species.

Validation of electrical stimulation models: intracellular calcium measurement in three-dimensional scaffolds.

Peripheral nerve injury can be disabling. Regeneration is limited by the rate of axonal extension, and proximal injury to peripheral nerves can take over a year to reach target organs. Electrical stimulation (ES) has been shown to increase the rate of neurite growth, though the mechanism is not yet well understood. In our prior manuscript, we developed a computational model that demonstrates how ES can functionally elevate intracellular calcium concentration ([Ca2+]i) based on ES intensity and duration. In this article, we validate the computation model for the [Ca2+]i changes in neuron soma. Embryonic chicken dorsal root ganglion cells were suspended in 3-dimensional collagen scaffolds. Fura-2 was used to measure [Ca2+]i in response to biphasic ES pulses ranging from 70 to 60,000 V/m in intensity and from 10 µs to 100 ms in duration. The computational model most closely matched the experimental data of the neurons with the highest [Ca2+]i elevation for ES pulses 100 µs or greater in duration. Nickel (200 µM) and cadmium (200 µM) blocked 98-99% of the [Ca2+]i rise, indicating that the rise in [Ca2+]i in response to ES is via voltage-dependent calcium channels. The average [Ca2+]i rise in response to ES was about one-tenth of the peak rise. Therefore, the computational model is validated for elevating [Ca2+]i of neurons and can be used as a tool for designing efficacious ES protocols for improving neuronal regeneration.NEW & NOTEWORTHY Electrical stimulation is used to enhance neuron growth, and the role of neuronal intracellular calcium concentration ([Ca2+]i) is an area of research interest. Widely varying stimulation parameters in the literature make it difficult to compare stimulation protocols. The results in this manuscript are the first to show neuronal [Ca2+]i in response to a broad and defined range of electrical pulse durations and intensities. These results validate our previously published novel computational model of [Ca2+]i.

Effects of Chemically Doped Bioactive Borate Glass on Neuron Regrowth and Regeneration.

Peripheral nerve injuries present challenges to regeneration. Currently, the gold standard for nerve repair is an autograft that results in another region of the body suffering nerve damage. Previously, bioactive borate glass (BBG) has been studied in clinical trials to treat patients with non-healing wounds, and we have reported that BBG is conducive for soft tissue repair. BBG provides structural support, degrades in a non-cytotoxic manner, and can be chemically doped. Here, we tested a wide range of chemical compounds that are reported to have neuroprotective characteristics to promote regeneration of peripheral neurons after traumatic injury. We hypothesized that chemical dopants added in trace amounts to BBG would improve neuronal survival and neurite outgrowth from dorsal root ganglion (DRG) explants. We measured neurite outgrowth from whole DRG explants, and survival rates of dissociated neurons and support cells that comprise the DRG. Results show that chemically doped BBGs have differentially variable effects on neuronal survival and outgrowth, with iron, gallium, and zinc improving outgrowth of neurons, and iodine causing the most detriment to neurons. Because chemically doped BBGs support increased nerve regrowth and survival, they show promise for use in peripheral nerve regeneration.

Computational modeling of neurons: intensity-duration relationship of extracellular electrical stimulation for changes in intracellular calcium.

In many instances of extensive nerve damage, the injured nerve never adequately heals, leaving lack of nerve function. Electrical stimulation (ES) has been shown to increase the rate and orient the direction of neurite growth, and is a promising therapy. However, the mechanism in which ES affects neuronal growth is not understood, making it difficult to compare existing ES protocols or to design and optimize new protocols. We hypothesize that ES acts by elevating intracellular calcium concentration ([Ca(2+)]i) via opening voltage-dependent Ca(2+) channels (VDCCs). In this work, we have created a computer model to estimate the ES Ca(2+) relationship. Using COMSOL Multiphysics, we modeled a small dorsal root ganglion (DRG) neuron that includes one Na(+) channel, two K(+) channels, and three VDCCs to estimate [Ca(2+)]i in the soma and growth cone. As expected, the results show that an ES that generates action potentials (APs) can efficiently raise the [Ca(2+)]i of neurons. More interestingly, our simulation results show that sub-AP ES can efficiently raise neuronal [Ca(2+)]i and that specific high-voltage ES can preferentially raise [Ca(2+)]i in the growth cone. The intensities and durations of ES on modeled growth cone calcium rise are consistent with directionality and orientation of growth cones experimentally shown by others. Finally, this model provides a basis to design experimental ES pulse parameters, including duration, intensity, pulse-train frequency, and pulse-train duration to efficiently raise [Ca(2+)]i in neuronal somas or growth cones.

Stimulation Frequency Alters the Dorsal Root Ganglion Neurite Growth and Directionality In Vitro.

OBJECTIVE: To improve peripheral nerve repair, new techniques to increase the speed of regeneration are required. Studies have shown that the electrical stimulation can enhance nerve regeneration; however, stimulation parameters that regulate the growth increases are unknown. The objective of this study was to examine dorsal root ganglion (DRG) neurite extension, directionality, and density after using methods to specifically control ac electrical field intensity and frequency exposure. METHODS: Chick DRG explants were exposed to 20-Hz, 200-Hz, 1-MHz, and 20-MHz sinusoidal electric field of 17.86 V/m, and tissue parameters were measured. RESULTS: Results show that neurite extension and directionality were influenced by frequency; however, the ratio of support cell emigration with respect to neurite extension from the DRG body was not. These results were further verified through finite-element modeling of intracellular calcium, which show that higher frequencies have minimal effect on intracellular calcium. CONCLUSION: In conclusion, these results demonstrate that 1) directional growth of neurites within EFs can be achieved, 2) high-frequency stimulation in megahertz does not enhance or impair the neurite growth, and 3) low-frequency stimulation affects the growth and directionality. SIGNIFICANCE: The significance of this study is the direct comparison of neurite extension after high stimulation frequencies (megahertz) with typical low-frequency fields (20 and 200 Hz), and modeling the results with finite-element modeling.

Fabrication and characterization of poly-(ε)-caprolactone and bioactive glass composites for tissue engineering applications.

Much work has focused on developing synthetic materials that have tailored degradation profiles and physical properties that may prove useful in developing biomaterials for tissue engineering applications. In the present study, three different composite sheets consisting of biodegradable poly-ε-caprolactone (PCL) and varying types of bioactive glass were investigated. The three composites were composed of 50wt.% PCL and (1) 50wt.% 13-93 B3 borate glass particles, (2) 50wt.% 45S5 silicate glass particles, or (3) a blend of 25wt.% 13-93 B3 and 25wt.% 45S5 glass particles. Degradation profiles determined for each composite showed the composite that contained only 13-93 B3 borate glass had a higher degradation rate compared to the composite containing only 45S5 silicate glass. Uniaxial tensile tests were performed on the composites to determine the effect of adding glass to the polymer on mechanical properties. The peak stress of all of the composites was lower than that of PCL alone, but 100% PCL had a higher stiffness when pre-reacted in cell media for 6weeks, whereas composite sheets did not. Finally, to determine whether the composite sheets would maintain neuronal growth, dorsal root ganglia isolated from embryonic chicks were cultured on composite sheets, and neurite outgrowth was measured. The bioactive glass particles added to the composites showed no negative effects on neurite extension, and neurite extension increased on PCL:45S5 PCL:13-93 B3 when pre-reacted in media for 24h. This work shows that composite sheets of PCL and bioactive glass particles provide a flexible biomaterial for neural tissue engineering applications.

Short-term electrical stimulation to promote nerve repair and functional recovery in a rat model.

PURPOSE: To evaluate the effect of duration of electrical stimulation on peripheral nerve regeneration and functional recovery. Based on previous work, we hypothesized that applying 10 minutes of electrical stimulation to a 10-mm rat sciatic nerve defect would significantly improve nerve regeneration and functional recovery compared with the non-electrical stimulation group. METHODS: A silicone tube filled with a collagen gel was used to bridge a 10-mm nerve defect in rats, and either 10 minutes or 60 minutes of electrical stimulation was applied to the nerve during surgery. Controls consisted of a silicone tube with collagen gel and no electrical stimulation or an isograft. We analyzed recovery over a 12-week period, measuring sciatic functional index and extensor postural thrust scores and concluding with histological examination of the nerve. RESULTS: Functional assessment scores at week 12 increased 24% in the 10-minute group as compared to the no stimulation control group. Electrical stimulation of either 10 or 60 minutes improved the number of nerve fibers over no stimulation. Additionally, the electrical stimulation group's histomorphometric analysis was not different from the isograft group. CONCLUSIONS: Several previous studies have demonstrated the effectiveness of 60-minute stimulations on peripheral nerve regeneration. This study demonstrated that an electrical stimulation of 10 minutes enhanced several functional and histomorphometric outcomes of nerve regeneration and was overall similar to a 60-minute stimulation over 12 weeks. CLINICAL RELEVANCE: Decreasing the electrical stimulation time from 60 minutes to 10 minutes provided a potential clinically feasible and safe method to enhance nerve regeneration and functional recovery.

PC12 cells that lack synaptotagmin I exhibit loss of a subpool of small dense core vesicles.

Neurons communicate by releasing neurotransmitters that are stored in intracellular vesicular compartments. PC12 cells are frequently used as a model secretory cell line that is described to have two subpools of vesicles: small clear vesicles and dense core vesicles. We measured transmitter molecules released from vesicles in NGF-differentiated PC12 cells using carbon-fiber amperometry, and relative diameters of individual vesicles using electron microscopy. Both amperometry and electron micrograph data were analyzed by statistical and machine learning methods for Gaussian mixture models. An electron microscopy size correction algorithm was used to predict and correct for observation bias of vesicle size due to tangential slices through some vesicles. Expectation maximization algorithms were used to perform maximum likelihood estimation for the Gaussian parameters of different populations of vesicles, and were shown to be better than histogram and cumulative distribution function methods for analyzing mixed populations. The Bayesian information criterion was used to determine the most likely number of vesicle subpools observed in the amperometric and electron microscopy data. From this analysis, we show that there are three major subpools, not two, of vesicles stored and released from PC12 cells. The three subpools of vesicles include small clear vesicles and two subpools of dense core vesicles, a small and a large dense core vesicle subpool. Using PC12 cells stably transfected with short-hairpin RNA targeted to synaptotagmin I, an exocytotic Ca(2+) sensor, we show that the presence and release of the small dense core vesicle subpool is dependent on synaptotagmin I. Furthermore, synaptotagmin I also plays a role in the formation and/or maintenance of the small dense core vesicle subpool in PC12 cells.

Electrical and neurotrophin enhancement of neurite outgrowth within a 3D collagen scaffold.

Electrical and chemical stimulation have been studied as potent mechanisms of enhancing nerve regeneration and wound healing. However, it remains unclear how electrical stimuli affect nerve growth, particularly in the presence of neurotrophic factors. The objective of this study was to explore (1) the effect of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) supplementation to support neurite outgrowth in a 3D scaffold, and (2) the effect of brief, low voltage, electrical stimulation (ES) on neurite outgrowth prior to neurotrophin supplementation. Dissociated E11 chick dorsal root ganglia (DRG) were seeded within a 1.5 mg/mL type-I collagen scaffold. For neurotrophin treatments, scaffolds were incubated for 24 h in culture media containing NGF (10 ng/mL) or BDNF (200 ng/mL), or both. For ES groups, scaffolds containing neurons were stimulated for 10 min at 8-10 V/m DC, then incubated for 24 h with neurotrophin. Fixed and labeled neurons were imaged to measure neurite growth and directionality. BDNF supplementation was not as effective as NGF at supporting DRG neurite outgrowth. ES prior to NGF supplementation improved DRG neurite outgrowth compared to NGF alone. This combination of brief ES with NGF treatment was the most effective treatment compared to NGF or BDNF alone. Brief ES had no impact on neurite directionality in the 3D scaffolds. These results demonstrate that ES improves neurite outgrowth in the presence of neurotrophins, and could provide a potential therapeutic approach to improve nerve regeneration when coupled with neurotrophin treatment.

Effects of borate-based bioactive glass on neuron viability and neurite extension.

Bioactive glasses have recently been shown to promote regeneration of soft tissues by positively influencing tissue remodeling during wound healing. We were interested to determine whether bioactive glasses have the potential for use in the treatment of peripheral nerve injury. In these experiments, degradable bioactive borate glass was fabricated into rods and microfibers. To study the compatibility with neurons, embryonic chick dorsal root ganglia (DRG) were cultured with different forms of bioactive borate glass. Cell viability was measured with no media exchange (static condition) or routine media exchange (transient condition). Neurite extension was measured within fibrin scaffolds with embedded glass microfibers or aligned rod sheets. Mixed cultures of neurons, glia, and fibroblasts growing in static conditions with glass rods and microfibers resulted in decreased cell viability. However, the percentage of neurons compared with all cell types increased by the end of the culture protocol compared with culture without glass. Furthermore, bioactive glass and fibrin composite scaffolds promoted neurite extension similar to that of control fibrin scaffolds, suggesting that glass does not have a significant detrimental effect on neuronal health. Aligned glass scaffolds guided neurite extension in an oriented manner. Together these findings suggest that bioactive glass can provide alignment to support directed axon growth.

The impact of laminin on 3D neurite extension in collagen gels.

The primary goal of this research was to characterize the effect of laminin on three-dimensional (3D) neurite growth. Gels were formed using type I collagen at concentrations of 0.4-2.0 mg mL(-1) supplemented with laminin at concentrations of 0, 1, 10, or 100 µg mL(-1). When imaged with confocal microscopy, laminin was shown to follow the collagen fibers; however, the addition of laminin had minimal effect on the stiffness of the scaffolds at any concentration of collagen. Individual neurons dissociated from E9 chick dorsal root ganglia were cultured in the gels for 24 h, and neurite lengths were measured. For collagen gels without laminin, a typical bimodal response of neurite outgrowth was observed, with increased growth at lower concentrations of collagen gel. However, alteration of the chemical nature of the collagen gel by the laminin additive shifted, or completely mitigated, the bimodal neurite growth response seen in gels without laminin. Expression of integrin subunits, α1, α3, α6 and ß1, were confirmed by PCR and immunolabeling in the 3D scaffolds. These results provide insight into the interplay between mechanical and chemical environment to support neurite outgrowth in 3D. Understanding the relative impact of environmental factors on 3D nerve growth may improve biomaterial design for nerve cell regeneration.

Upregulation of synaptotagmin IV inhibits transmitter release in PC12 cells with targeted synaptotagmin I knockdown.

BACKGROUND: The function of synaptotagmins (syt) in Ca2+-dependent transmitter release has been attributed primarily to Ca2+-dependent isoforms such as syt I. Recently, syt IV, an inducible Ca2+-independent isoform has been implicated in transmitter release. We postulated that the effects of syt IV on transmitter release are dependent on the expression of syt I. RESULTS: To test this, we increased syt IV expression in PC12 cells by either upregulation with forskolin treatment or overexpression with transfection. Two separately generated stable PC12 cell lines with syt I expression abolished by RNAi targeting were used and compared to control cells. We measured catecholamine release from single vesicles by amperometry and neuropeptide Y release from populations of cells by an immunoassay. In syt I targeted cells with forskolin-induced syt IV upregulation, amperometry measurements showed a reduction in the number of release events and the total amount of transmitter molecules released per cell. In cells with syt IV overexpressed, similar amperometry results were obtained, except that the rate of expansion for full fusion was slowed. Neuropeptide Y (NPY) release from syt I knockdown cells was decreased, and overexpression of syt IV did not rescue this effect. CONCLUSIONS: These data support an inhibitory effect of syt IV on release of vesicles and their transmitter content. The effect became more pronounced when syt I expression was abolished.

Fabrication of amperometric electrodes.

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements. Here, we describe the techniques needed to generate low noise (<0.5 pA) electrodes. The techniques have been modified from published descriptions by previous researchers (1,2). Electrodes are made by preparing carbon fibers and threading them individually into each capillary tube by using a vacuum with a filter to aspirate the fiber. Next, the capillary tube with fiber is pulled by an electrode puller, creating two halves, each with a fine-pointed tip. The electrodes are dipped in hot, liquid epoxy mixed with hardener to create an epoxy-glass seal. Lastly, the electrodes are placed in an oven to cure the epoxy. Careful handling of the electrodes is critical to ensure that they are made consistently and without damage. This protocol shows how to fabricate and cut amperometric electrodes for recording from single cells.

Native and recombinant ASIC1a receptors conduct negligible Ca2+ entry.

Acid Sensing Ion Channels (ASICs) are a family of proton-gated cation channels that play a role in the sensation of noxious stimuli. Of these, ASIC1a is the only family member that is reported to be permeable to Ca(2+), although the absolute magnitude of the Ca(2+) current is unclear. Here, we used patch-clamp photometry to determine the contribution of Ca(2+) to total current through native and recombinant ASIC1a receptors. We found that acidification of the extracellular medium evoked amiloride and psalmotoxin 1-sensitive currents in isolated chick dorsal root ganglion neurons and human embryonic kidney cells, but did not alter fura-2 fluorescence when the bath concentration of Ca(2+) was close to that found in normal physiological conditions. Further, activation of recombinant ASIC1a receptors also failed to produce measurable changes in fluorescence despite of the fact that the total cation current through the over-expressed receptor was ten-fold larger than that of the native channels. Finally, we imaged a field of intact DRG neurons loaded with the Ca(2+)-sensing dye Fluo-4, and found that acidification increased [Ca(2+)](i) in a small population of cells. Thus, although our whole-field imaging data agree with previous studies that activation of ASIC1a receptors can potentially cause elevations in intracellular free Ca(2+), our single cell data strongly challenges the view that Ca(2+) entry through the ASIC1a receptor itself contributes to this response.

Stable RNA interference of synaptotagmin I in PC12 cells results in differential regulation of transmitter release.

In sympathetic neurons, it is well-established that the neurotransmitters, norepinephrine (NE), neuropeptide Y (NPY), and ATP are differentially coreleased from the same neurons. In this study, we determined whether synaptotagmin (syt) I, the primary Ca(2+) sensor for regulated release, could function as the protein that differentially regulates release of these neurotransmitters. Plasmid-based RNA interference was used to specifically and stably silence expression of syt I in a model secretory cell line. Whereas stimulated release of NPY and purines was abolished, stimulated catecholamine (CA) release was only reduced by approximately 50%. Although expression levels of tyrosine hydroxylase, the rate-limiting enzyme in the dopamine synthesis pathway, was unaffected, expression of the vesicular monoamine transporter 1 was reduced by 50%. To evaluate whether NPY and CAs are found within the same vesicles and whether syt I is found localized to each of these NPY- and CA-containing vesicles, we used immunocytochemistry to determine that syt I colocalized with large dense core vesicles, with NPY, and with CAs. Furthermore, both CAs and NPY colocalized with one another and with large dense core vesicles. Electron micrographs show that large dense core vesicles are synthesized and available for release in cells that lack syt I. These results are consistent with syt I regulating differential release of transmitters.

Variability in RNA interference in neuroendocrine PC12 cell lines stably transfected with an shRNA plasmid.

RNA interference (RNAi) has quickly become a very powerful technique for specifically suppressing or knocking down the expression of any desired gene. Many fields of research, including neuroscience, have benefitted from RNAi methods. It has been well documented that different small interfering RNAs (siRNAs) and small hairpin RNAs (shRNAs) vary greatly in terms of their effectiveness, and much attention has been focused on guidelines and algorithms for the selection of effective siRNAs. However, it has not been widely appreciated that a single shRNA-expressing plasmid can also produce widely varying levels of knockdown in different stably transfected cell lines derived from the same transfection. Here we report that knockdown of three distinct target proteins varies from minimal to almost complete in independent, stably transfected PC12 cell lines. This variability in knockdown among cell lines emphasizes the importance of characterizing a number of cell lines when attempting to establish stable knockdown cell lines, but also offers the possibility of studying the effects of graded levels of protein expression.

Stable gene silencing of synaptotagmin I in rat PC12 cells inhibits Ca2+-evoked release of catecholamine.

Synaptotagmin (syt) I is a Ca2+-binding protein that is well accepted as a major sensor for Ca2+-regulated release of transmitter. However, controversy remains as to whether syt I is the only protein that can function in this role and whether the remaining syt family members also function as Ca2+ sensors. In this study, we generated a PC12 cell line that continuously expresses a short hairpin RNA (shRNA) to silence expression of syt I by RNA interference. Immunoblot and immunocytochemistry experiments demonstrate that expression of syt I was specifically silenced in cells that stably integrate the shRNA-syt I compared with control cells stably transfected with the empty shRNA vector. The other predominantly expressed syt isoform, syt IX, was not affected, nor was the expression of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins when syt I levels were knocked down. Resting Ca2+ and stimulated Ca2+ influx imaged with fura-2 were not altered in syt I knockdown cells. However, evoked release of catecholamine detected by carbon fiber amperometry and HPLC was significantly reduced, although not abolished. Human syt I rescued the release events in the syt I knockdown cells. The reduction of stimulated catecholamine release in the syt I knockdown cells strongly suggests that although syt I is clearly involved in catecholamine release, it is not the only protein to regulate stimulated release in PC12 cells, and another protein likely has a role as a Ca2+ sensor for regulated release of transmitter.

Deletion of the synaptic protein interaction site of the N-type (CaV2.2) calcium channel inhibits secretion in mouse pheochromocytoma cells.

Presynaptic N-type Ca2+ channels (CaV2.2, alpha1B) are thought to bind to SNARE (SNAP-25 receptor) complex proteins through a synaptic protein interaction (synprint) site on the intracellular loop between domains II and III of the alpha1B subunit. Whether binding of syntaxin to the N-type Ca2+ channels is required for coupling Ca2+ ion influx to rapid exocytosis has been the subject of considerable investigation. In this study, we deleted the synprint site from a recombinant alpha1B Ca2+ channel subunit and transiently transfected either the wild-type alpha1B or the synprint deletion mutant into mouse pheochromocytoma (MPC) cell line 9/3L, a cell line that has the machinery required for rapid stimulated exocytosis but lacks endogenous voltage-dependent Ca2+ channels. Secretion was elicited by activation of exogenously transfected Ca2+ channel subunits. The current-voltage relationship was similar for the wild-type and mutant alpha1B-containing Ca2+ channels. Although total Ca2+ entry was slightly larger for the synprint deletion channel, compared with the wild-type channel, when Ca2+ entry was normalized to cell size and limited to cells with similar Ca2+ entry (approximately 150 x 10(6) Ca2+ ions/pF cell size), total secretion and the rate of secretion, determined by capacitance measurements, were significantly reduced in cells expressing the synprint deletion mutant channels, compared with wild-type channels. Furthermore, the amount of endocytosis was significantly reduced in cells with the alpha1B synprint deletion mutant, compared with the wild-type subunit. These results suggest that the synprint site is necessary for efficient coupling of Ca2+ influx through alpha1B-containing Ca2+ channels to exocytosis.

Expression of recombinant calcium channels support secretion in a mouse pheochromocytoma cell line.

We have characterized a recently established mouse pheochromocytoma cell line (MPC 9/3L) as a useful model for studying neurotransmitter release and neuroendocrine secretion. MPC 9/3L cells express many of the proteins involved in Ca2+-dependent neurotransmitter release but do not express functional endogenous Ca2+-influx pathways. When transfected with recombinant N-type Ca2+ channel subunits alpha1B,beta2a,alpha2delta (Cav2.2), the cells expressed robust Ca2+ currents that were blocked by omega-conotoxin GVIA. Activation of N-type Ca2+ currents caused rapid increases in membrane capacitance of the MPC 9/3L cells, indicating that the Ca2+ influx was linked to exocytosis of vesicles similar to that reported in chromaffin or PC12 cells. Synaptic protein interaction (synprint) sites, like those found on N-type Ca2+ channels, are thought to link voltage-dependent Ca2+ channels to SNARE proteins involved in synaptic transmission. Interestingly, MPC 9/3L cells transfected with either LC-type (alpha1C, beta2a, alpha2delta, Cav1.2) or T-type (alpha1G, beta2a, alpha2delta, Cav3.1) Ca2+ channel subunits, which do not express synprint sites, expressed appropriate Ca2+ currents that supported rapid exocytosis. Thus MPC 9/3L cells provide a unique model for the study of exocytosis in cells expressing specific Ca2+ channels of defined subunit composition without complicating contributions from endogenous channels. This model may help to distinguish the roles that different Ca2+ channels play in Ca2+-dependent secretion.

Cell death in weaver mouse cerebellum.

Mice with the weaver mutation exhibit an uneven weave to their gait, ataxia, mild locomotor hyperactivity and, occasionally, tonic-clonic seizures. A single amino acid mutation in a G-protein coupled, inwardly rectifying K+ channel, GIRK2, gives rise to the symptoms seen in the weaver mice. Two areas of the brain are primarily affected. Cerebellar granule cell neurons die soon after birth and dopaminergic neurons are severely depleted in the substantia nigra. In this article we review recent studies of wild-type and mutant GIRK channels found in native cells or introduced into expression systems. We also review two models that explain some of the details leading to the neuronal cell death observed in weaver mice.