Dario A. Protti

Transmitter release and presynaptic Ca2+ currents blocked by the spider toxin ω-Aga-IVA

Mammalian neuromuscular transmission is resistant to L and N type calcium channel blockers but very sensitive to a low molecular weight funnel web spider venom toxin, FTX, which selectively blocks P type calcium channels. To further characterize the calcium channels involved in neuromuscular transmission we studied the effect of omega Agatoxin (omega-Aga-IVA) a polypeptide P type channel blocker from the same spider venom. We show that omega-Aga-IVA is a potent and irreversible inhibitor of the presynaptic Ca2+ currents and of acetylcholine release induced by electrical stimulation or by K+ depolarization. This provides further evidences that transmitter release at the mammalian neuromuscular junction is mediated by P type Ca2+ channels.

Light evokes Ca2+ spikes in the axon terminal of a retinal bipolar cell.

Bipolar cells in the vertebrate retina have been characterized as nonspiking interneurons. Using patch-clamp recordings from goldfish retinal slices, we find, however, that the morphologically well-defined Mb1 bipolar cell is capable of generating spikes. Surprisingly, in dark-adapted retina, spikes were reliably evoked by light flashes and had a long (1-2 s) refractory period. In light-adapted retina, most Mb1 cells did not spike. However, an L-type Ca2+ channel agonist could induce periodic spiking in these cells. Spikes were determined to be Ca2+ action potentials triggered at the axon terminal and were abolished by 2-amino-4-phosphonobutyric acid (APB), an agonist that mimics glutamate. Signaling via spikes in a specific class of bipolar cells may serve to accelerate and amplify small photo-receptor signals, thereby securing the synaptic transmission of dim and rapidly changing visual input.

Calcium channel blockers and transmitter release at the normal human neurornuscular junction

Transmitter release evoked by nerve stimulation is highly dependent on Ca2+ entry through voltage-activated plasma membrane channels. Calcium influx may be modified in some neuromuscular diseases like Lambert-Eaton syndrome and amyotrophic lateral sclerosis. We studied the pharmacologic sensitivity of the transmitter release process to different calcium channel blockers in normal human muscles and found that funnel web toxin and Ω-Agatoxin-IVA, both P-type calcium channel blockers, blocked nerve-elicited muscle action potentials and inhibited evoked synaptic transmission. The transmitter release was not affected either by nitrendipine, an L-type channel blocker, or Ω-Conotoxin-GVIA, an N-type channel blocker. The pharmacologic profile of neuromuscular transmission observed in normal human muscles indicates that P-like channels mediate transmitter release at the motor nerve terminals.

Effects of Ca2+ channel blocker neurotoxins on transmitter release and presynaptic currents at the mouse neuromuscular junction

1 The effects of the voltage‐dependent calcium channel (VDCC) blockers ω‐agatoxin IVA (ω‐AgaIVA), ω‐conotoxin GVIA (ω‐CgTx), ω‐conotoxin MVIIC (ω‐MVIIC) and ω‐conotoxin MVIID (ω‐MVIID) were evaluated on transmitter release in the mouse diaphragm preparation. The effects of ω‐AgaIVA and ω‐MVIIC were also evaluated on the perineurial calcium and calcium‐dependent potassium currents, ICa and IK(Ca), respectively, in the mouse levator auris preparation. 2 The P‐ and Q‐type VDCC blocker ω‐AgaIVA (100 nM) and P‐ Q‐ and N‐type channel blockers ω‐MVIIC (1 μM) and ω‐MVIID (3 μM) strongly reduced transmitter release (>80–90% blockade) whereas the selective N‐type channel blocker ω‐CgTx (5 μM) was ineffective. 3 The process of release was much more sensitive to ω‐MVIIC (IC50=39 nM) than to ω‐MVIID (IC50=1.4 μM). After almost completely blocking transmitter release (quantal content ∼0.3% of its control value) with 3 μMω‐MVIIC, elevating the external [Ca2+] from 2 to 10 mM induced an increase of ∼20 fold on the quantal content of the endplate potential (e.p.p.) (from 0.2±0.04 to 4.8±1.4). 4 Nerve‐evoked transmitter release in a low Ca2+‐high Mg2+ medium (low release probability, quantal content = 2±0.1) had the same sensitivity to ω‐AgaIVA (IC50=16.8 nM) as that in normal saline solutions. In addition, K+‐evoked transmitter release was also highly sensitive to the action of this toxin (IC50=11.5 nM; 100 nM >95% blockade). The action of ω‐AgaIVA on transmitter release could be reversed by toxin washout if the experiments were carried out at 31–33°C. Conversely, the effect of ω‐AgaIVA persisted even after two hours of toxin washout at room temperature. 5 Both the calcium and calcium‐dependent potassium presynaptic currents, ICa and IK(Ca), respectively, were highly sensitive to low concentrations (10–30 nM) of ω‐AgaIVA. The ICa and the IK(Ca) were also strongly reduced by 1 μMω‐MVIIC. The most marked difference between the action of these two toxins was the long incubation times required to achieve maximal effects with ω‐MVIIC. 6 In summary these results provide more evidence that synaptic transmission at the mammalian neuromuscular junction is mediated by Ca2+ entry through P‐ and/or Q‐type calcium channels.

Long-term neuromuscular dysfunction produced by passive transfer of amyotrophic lateral sclerosis immunoglobulins

We investigated the role of the immune system in the pathogenesis of amyotrophic lateral sclerosis (ALS) by studying the long-term consequences of ALS immunoglobulin (Ig) application on the levator auris muscle of the mouse. We applied Ig from seven ALS patients, four disease controls, and a pool of normal Ig (6 mg of Ig in 2 weeks) by subcutaneous injection; removed the muscles 4 to 12 weeks after the beginning of treatment; and recorded both spontaneous and evoked release of transmitter. None of the control Ig induced changes in transmitter, whereas five of seven ALS Ig induced a significant increase in the rate of spontaneous release, and all ALS Ig produced significant changes in the quantal content of evoked release. In muscles treated with one of the ALS Igs, synaptic activity was completely absent. Cholinesterase and silver staining demonstrated intact neuromuscular junctions in the control Ig-treated muscles and also in many areas of ALS Ig-treated muscles. Axonal degeneration and denervation were present in most muscles treated with ALS Ig. There was complete denervation when no synaptic activity could be recorded. Thus, ALS Ig appears to lead to long-lasting effects at the neuromuscular junction, and such effects may be an early stage in the immune-mediated pathogenesis of ALS.

Responses of Retinal Ganglion Cells to Extracellular Electrical Stimulation, from Single Cell to Population: Model-Based Analysis

Retinal ganglion cells (RGCs), which survive in large numbers following neurodegenerative diseases, could be stimulated with extracellular electric pulses to elicit artificial percepts. How do the RGCs respond to electrical stimulation at the sub-cellular level under different stimulus configurations, and how does this influence the whole-cell response? At the population level, why have experiments yielded conflicting evidence regarding the extent of passing axon activation? We addressed these questions through simulations of morphologically and biophysically detailed computational RGC models on high performance computing clusters. We conducted the analyses on both large-field RGCs and small-field midget RGCs. The latter neurons are unique to primates. We found that at the single cell level the electric potential gradient in conjunction with neuronal element excitability, rather than the electrode center location per se, determined the response threshold and latency. In addition, stimulus positioning strongly influenced the location of RGC response initiation and subsequent activity propagation through the cellular structure. These findings were robust with respect to inhomogeneous tissue resistivity perpendicular to the electrode plane. At the population level, RGC cellular structures gave rise to low threshold hotspots, which limited axonal and multi-cell activation with threshold stimuli. Finally, due to variations in neuronal element excitability over space, following supra-threshold stimulation some locations favored localized activation of multiple cells, while others favored axonal activation of cells over extended space.

Permanent Functional Reorganization of Retinal Circuits Induced by Early Long-Term Visual Deprivation

Early sensory experience shapes the functional and anatomical connectivity of neuronal networks. Light deprivation alters synaptic transmission and modifies light response properties in the visual system, from retinal circuits to higher visual centers. These effects are more pronounced during a critical period in juvenile life and are mostly reversed by restoring normal light conditions. Here we show that complete light deprivation, from birth to periods beyond the critical period, permanently modifies the receptive field properties of retinal ganglion cells. Visual deprivation reduced both the strength of light responses in ganglion cells and their receptive field size. Light deprivation produced an imbalance in the ratio of inhibitory to excitatory inputs, with a shift toward larger inhibitory conductances. Ganglion cell receptive fields in visually deprived animals showed a spatial mismatch of inhibitory and excitatory inputs and inhibitory inputs were highly scattered over the receptive field. These results indicate that visual experience early in life is critical for the refinement of retinal circuits and for appropriate signaling of the spatiotemporal properties of visual stimuli, thus influencing the response properties of neurons in higher visual centers and their processing of visual information.

P/Q-type calcium channels activate neighboring calcium-dependent potassium channels in mouse motor nerve terminals

Abstract The identity of the voltage-dependent calcium channels (VDCC), which trigger the Ca2+-gated K+ currents (IK(Ca)) in mammalian motor nerve terminals, was investigated by means of perineurial recordings. The effects of Ca2+ chelators with different binding kinetics on the activation of IK(Ca) were also examined. The calcium channel blockers of the P/Q family, ω-agatoxin IVA (ω-Aga-IVA) and funnel-web spider toxin (FTX), have been shown to exert a strong blocking effect on IK(Ca). In contrast, nitrendipine and ω-conotoxin GVIA (ω-CgTx) did not affect the Ca2+-activated K+ currents. The intracellular action of the fast Ca2+ buffers BAPTA and DM-BAPTA prevented the activation of the IK(Ca), while the slow Ca2+ buffer EGTA was ineffective at blocking it. These data indicate that P/Q-type VDCC mediate the Ca2+ influx which activates IK(Ca). The spatial association between Ca2+ and Ca2+-gated K+ channels is discussed, on the basis of the differential effects of the fast and slow Ca2+ chelators.

Cannabinoids modulate spontaneous synaptic activity in retinal ganglion cells

The endocannabinoid (ECB) system has been found throughout the central nervous system and modulates cell excitability in various forms of short-term plasticity. ECBs and their receptors have also been localized to all retinal cells, and cannabinoid receptor activation has been shown to alter voltage-dependent conductances in several different retinal cell types, suggesting a possible role for cannabinoids in retinal processing. Their effects on synaptic transmission in the mammalian retina, however, have not been previously investigated. Here, we show that exogenous cannabinoids alter spontaneous synaptic transmission onto retinal ganglion cells (RGCs). Using whole-cell voltage-clamp recordings in whole-mount retinas, we measured spontaneous postsynaptic currents (SPSCs) in RGCs in adult and young (P14-P21) mice. We found that the addition of an exogenous cannabinoid agonist, WIN55212-2 (5 μM), caused a significant reversible reduction in the frequency of SPSCs. This change, however, did not alter the kinetics of the SPSCs, indicating a presynaptic locus of action. Using blockers to isolate inhibitory or excitatory currents, we found that cannabinoids significantly reduced the release probability of both GABA and glutamate, respectively. While the addition of cannabinoids reduced the frequency of both GABAergic and glutamatergic SPSCs in both young and adult mice, we found that the largest effect was on GABA-mediated currents in young mice. These results suggest that the ECB system may potentially be involved in the modulation of signal transmission in the retina. Furthermore, they suggest that it might play a role in the developmental maturation of synaptic circuits, and that exogenous cannabinoids are likely able to disrupt retinal processing and consequently alter vision.

Ionotropic glutamate receptors of amacrine cells of the mouse retina

The mammalian retina contains approximately 30 different morphological types of amacrine cells, receiving glutamatergic input from bipolar cells. In this study, we combined electrophysiological and pharmacological techniques in order to study the glutamate receptors expressed by different types of amacrine cells. Whole-cell currents were recorded from amacrine cells in vertical slices of the mouse retina. During the recordings the cells were filled with Lucifer Yellow/Neurobiotin allowing classification as wide-field or narrow-field amacrine cells. Amacrine cell recordings were also carried out in a transgenic mouse line whose glycinergic amacrine cells express enhanced green fluorescent protein (EGFP). Agonist-induced currents were elicited by exogenous application of NMDA, AMPA, and kainate (KA) while holding cells at -75 mV. Using a variety of specific agonists and antagonists (NBQX, AP5, cyclothiazide, GYKI 52466, GYKI 53655, SYM 2081) responses mediated by AMPA, KA, and NMDA receptors could be dissected. All cells (n = 300) showed prominent responses to non-NMDA agonists. Some cells expressed AMPA receptors exclusively and some cells expressed KA receptors exclusively. In the majority of cells both receptor types could be identified. NMDA receptors were observed in about 75% of the wide-field amacrine cells and in less than half of the narrow-field amacrine cells. Our results confirm that different amacrine cell types express distinct sets of ionotropic glutamate receptors, which may be critical in conferring their unique temporal responses to this diverse neuronal class.