Molecular analysis of axonal target specificity and synapse formation.

The development of neuronal connectivity requires the growth of axons to their target region and the formation of dendritic trees that extend into specific layers. Within the target region growth cones, the tips of extending axons are guided to finer target fields including specific subcellular compartments where they form synapses. In this article we highlight recent progress on molecular aspects of axonal subcellular target selection such as the axon initial segment or specific sublaminae of the vertebrate retina. We then discuss the very recent progress on the molecular analysis of synapse formation in the central nervous system, including the direction of differentiation into an inhibitory or excitatory synapse. Apparently, initial synaptic contacts are structurally and functionally modulated by neuronal activity, raising the question how neuronal activity can modify synaptic circuits. We therefore also focus on neural proteins that are up-regulated, secreted or converted by synaptic activity and, thus, might represent molecular candidates for experience-driven refinement or remodeling of synaptic connections.

Slow IPSC kinetics, low levels of alpha1 subunit expression and paired-pulse depression are distinct properties of neonatal inhibitory GABAergic synaptic connections in the mouse superior colliculus.

Remodelling of visual maps in the superior colliculus (SC) depends on neuronal activity. Synaptic inhibition could contribute to this process because spontaneous spike discharge in the SC was modulated by GABA(A) receptor activation at postnatal days (P) 1-3. To investigate the functional capacity of GABAergic synaptic transmission at this early stage of development, whole-cell patch-clamp recordings were made from wide field neurons (WFNs) in horizontal slices comprising the superficial grey layer of the SC. Focal stimulation in the vicinity of WFNs evoked tetrodotoxin-sensitive stimulus-locked inhibitory postsynaptic currents (eIPSCs). The failure rate of eIPSCs was low ( approximately 0.2), and the maximal amplitude of evoked unitary eIPSCs exceeded the amplitude of average miniature IPSCs (mIPSCs) by a factor of 4-5, suggesting that action potential-mediated GABA release was more effective than spontaneous release. Some of the properties of GABAergic synaptic transmission in the neonatal SC were age-specific. In contrast with eIPSCs in the more mature SC at P20-22, neonatal eIPSCs decayed more slowly, preferentially fluctuated in duration, not amplitude, and mostly lacked temporal summation, due to depression at shorter intervals. The paired-pulse ratio (eIPSC2 : eIPSC1) was inversely related to the duration of eIPSCs. PCR analysis showed, in addition, that the ratio of alpha1 : alpha3 subunit expression was lower in the neonatal SC. Together, these results suggest that, at a young age, efficacy of GABAergic synaptic transmission is primarily constrained by the slow kinetics and the saturation of postsynaptic GABA(A) receptors.

Early onset of glutamatergic and GABAergic synaptic activity in the visual layers of the rodent superior colliculus.

During postnatal development, the retinocollicular pathway undergoes activity-dependent refinement, resulting in the precise retinotopic map seen in adults. Previous studies established that retinal efferents reach the mouse superior colliculus (SC) by embryonic day 16. Morphologically, synapses were found in the rat SC before birth. As part of an extended project aimed at understanding the development of synaptic transmission in the visual layers of the SC, we report here the presence of functionally active synapses immediately after birth. Circuit activity in mouse SC neurons was detected in horizontal slices of the visual layers using cell-attached voltage clamp. The spontaneous discharge of action potentials was abolished by glutamatergic blockers and facilitated by bicuculline, showing that circuit activity is based on synaptic transmission and that the action of gamma-aminobutyric acid is inhibitory. Using whole-cell voltage clamp, spontaneous glutamatergic postsynaptic currents as well as miniature GABAergic postsynaptic currents were recorded on postnatal day 1. Excitatory and inhibitory postsynaptic currents could also be evoked by electrical stimulation. Glutamatergic postsynaptic currents comprised both (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate receptor-mediated components. The early function of glutamatergic and GABAergic synaptic transmission in the visual layers of SC suggests that SC neurons are able to process information originating from retinal axons immediately after birth.

Ca2+-permeable P2X receptor channels in cultured rat retinal ganglion cells.

ATP has been identified as an excitatory neurotransmitter in both the CNS and peripheral nervous system; however, little is known about the functional properties of ATP-gated channels in central neurons. Here we used a culture preparation of the postnatal rat retina to test the responsiveness of identified retinal ganglion cells (RGCs) and putative amacrines to exogenous ATP and other purinoceptor agonists. Rapidly activating ATP-induced currents (IATP) were exclusively generated in a subpopulation (approximately 65%) of RGCs. The latter were identified by Thy1.1 immunostaining, repetitive firing patterns, and activation of glutamatergic autaptic currents. None of the putative amacrine cells was ATP-sensitive. IATP could be induced with ATP, ADP, and alpha,beta-mATP but not with adenosine. It was antagonized by suramin. The current-voltage relationship of IATP showed marked inward rectification. Dose-response analysis yielded an EC50 of 14.5 microM, with a Hill coefficient of 0.9. Noise analysis of IATP suggested a mean single channel conductance of 2.3 pS. Retinal P2X purinoceptor channels exhibited a high permeability for Ca2+. PCa/PCs obtained from reversal potentials of IATP under bi-ionic conditions amounted to 2. 2 +/- 0.7. In the majority of cells, the decay of IATP was biphasic. The degree of current inactivation during the first 2 sec of agonist application was highly variable. Heterogeneity was also found with respect to the sensitivity to ADP and alpha,beta-mATP and the blocking action of suramin, suggesting expression of multiple P2X receptor subtypes. Our results indicate that activation of P2X receptor channels represents an important pathway for Ca2+ influx in postnatal RGCs.

Ion conductances related to development of repetitive firing in mouse retinal ganglion neurons in situ.

In the retina, the ability to encode graded depolarizations into spike trains of variable frequency appears to be a specific property of retinal ganglion neurons (RGNs). To deduce the developmental changes in ion conductances underlying the transition from single to repetitive firing, patch-clamp recordings were performed in the isolated mouse retina between embryonic day 15 (E15) and postnatal day 5 (P5). Immature neurons of the E15 retina were selected according to their capacity to generate voltage-activated Na+ currents (I(Na)(v)). Identification of P5 RGNs was based on retrograde labeling, visualization of the axon, or the amplitude of I(Na)(v). At E15, half of the cells were excitable but none of them generated more than one spike. At P5, all cells were excitable and a majority discharged in tonic fashion. Ion conductances subserving maintenance of repetitive discharge were identified at P5 by exposure to low extracellular Ca2+, Cd2+, and charybdotoxin, all of which suppressed repetitive discharge. omega-Conotoxin GVIA and nifedipine had no effect. We compared passive membrane properties and a variety of voltage-activated ion channels at E15 and P5. It was found that the density of high voltage-activated (HVA) Ca2+ currents increased in parallel with the development of repetitive firing, while the density of Ni2+-sensitive low voltage-activated (LVA) Ca2+ currents decreased. Changes in density and activation kinetics of tetrodotoxin-sensitive Na+ currents paralleled changes in firing thresholds and size of action potentials, but seemed to be unrelated to maintenance of repetitive firing. Densities of A-type K+ currents and delayed rectifier currents did not change. The results suggest that HVA Ca2+ channels, and among them a toxin-resistant subtype, are specifically engaged in activation of Ca2+-sensitive K+ conductance and thereby account for frequency coding in postnatal RGNs.

Characterization of Mg2+ transport in brush border membrane vesicles of rabbit ileum studied with mag-fura-2.

Mg2+ transport in rabbit ileal brush border membrane vesicles (BBMV) was characterized by means of a modified mag-fura-2 technique. In the presence of an i>o Na+ gradient, BBMV showed a saturable Mg2+ uptake with a Km of 1.64 mmol l-1. There was no evidence of an overshoot. K+, Li+, and choline+ were as effective as Na+ in stimulating Mg2+ transport. In contrast, only a small amount of Mg2+ transport was observed in the presence either of an o>i Na+ gradient, or in an Na+ equilibrium or in the absence of Na+. Moreover, the findings that Na+ efflux was not stimulated but inhibited by outside Mg2+ and that the nonfluorescent amiloride-analogues DMA and EIPA did not affect Mg2+ transport do not favour the idea of an Mg2+/Na+ antiport system. At Cl- equilibrium, independent of the Na+ gradient, the rate of Mg2+ transport was markedly suppressed compared with the transport rate noted in the presence of an i>o Cl- gradient. The stimulating effect of inside anions could be enhanced by SCN- and decreased by SO2-4. Furthermore, nonfluorescent anion transport antagonist H2-DIDS stimulated Mg2+ transport. These findings indicate that Mg2+ transport can be modulated by inside anions. Mg2+ transport appeared to be electroneutral because it was not dependent on membrane potential. Mg2+ transport was neither stimulated by Bay K8644, a Ca2+ channel agonist, nor inhibited by verapamil, diltiazem, nifedipine and imipramine, the Ca2+ channel antagonists. It, therefore, seems unlikely that Mg2+ uses the Ca2+ transport system.