ATP-induced non-neuronal cell permeabilization in the rat inner retina.

The P2X7 subtype holds a special position among P2X receptors because of its ability to act both as a classical, ligand-gated ion channel, and as a permeabilization pore that can induce cell death under prolonged activation by ATP. We have shown previously that, in rat retina, P2X7 receptors are located in the inner nuclear layer and ganglion cell layer (GCL). The present study was aimed at finding whether retinal P2X7 receptors can act as a mediator of cell permeabilization and, if so, at identifying the cellular target(s) of this effect. As an indicator of cell permeabilization, we used the fluorescent dye YO-PRO-1 (molecular weight, 375 Da), which enters cells only through large pores like those opened by prolonged or sustained stimulation of P2X(7) receptors and binds to DNA, providing a stable labeling of the activated cells. Different agonists for P2 receptors were tested for their ability to cause cell permeabilization in flat-mounted rat retinas. Among them, only high concentrations of ATP (500 microM) and BzATP (2',3'-O-(4-benzoyl-benzoyl)-ATP triethylammonium) (100 microM) were able to induce accumulation of YO-PRO-1 in the GCL and in the nerve fiber layer, suggesting that different cell types were responding to P2X7 stimulation. This effect was blocked by the P2 antagonists suramin and PPADS (pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid) and by the P2X7-selective inhibitor Brilliant Blue G. To identify the retinal cell types affected by ATP-induced permeabilization, we used in vivo labeling techniques. Our data clearly reveal that prolonged stimulation of P2X7 receptors elicits permeabilization exclusively in microglial cells but not in neurons of the inner retina.

Mechanisms contributing to tonic release at the cone photoreceptor ribbon synapse.

Time-resolved capacitance measurements in combination with fluorescence measurements of internal calcium suggested three kinetic components of release in acutely isolated cone photoreceptors of the tiger salamander. A 45-fF releasable pool, corresponding to about 1,000 vesicles, was identified. This pool could be depleted with a time constant of a few hundred milliseconds and its recovery from depletion was quite rapid (tau approximately 1 s). The fusion of vesicles in this pool was blocked by low-millimolar EGTA. Endocytosis was sufficiently slow that it is likely that refilling of the releasable pool occurred from preformed vesicles. A second, slower component of release (tau(depletion) approximately 3 s) was identified that was approximately twice the size of the releasable pool. This pool may serve as a first reserve pool that replenishes the releasable pool. Computer simulations indicate that the properties of the releasable and first reserve pools are sufficient to maintain synaptic signaling for several seconds in the face of near-maximal stimulations and in the absence of other sources of vesicles. Along with lower rates of depletion, additional mechanisms, such as replenishment from distal reserve pools and the fast recycling of vesicles, may further contribute to the maintenance of graded, tonic release from cone photoreceptors.

Vaccination against hepatitis A during outbreaks starting in schools: what can we learn from experiences in central Italy?

Two outbreaks of hepatitis A started almost simultaneously in a maternal school and in a day care centre located at opposite sides of Florence, Italy, at the end of 2002. Both of them originated from immigrant children, and in both cases, hepatitis A was initially not recognised due to aspecific symptoms. While vaccination of contacts started with delay in the first outbreak, the same intervention was organised and performed in 3 days in the other. The outbreak starting in the maternal school caused 30 notified cases, plus 7 cases diagnosed retrospectively. Nine of them were in a secondary school, where vaccination (in accordance with the Italian national guidelines on hepatitis A (HA) vaccination) had been started only after a secondary case occurred. Only three cases occurred overall in the other outbreak starting in the day care centre, where >80% of infants, children and personnel were immunised. Although few asymptomatic infections probably occurred, no source of contagion existed any longer 2 months after immunisation. A rapid vaccination of school and family contacts of hepatitis A cases after the first case (irrespective of school grade) seems to play an important role to shorten outbreak duration.