A grin is a motor neuron-derived factor that directs formation of the postsynaptic apparatus of the neuromuscular junction. Agrin is also expressed in the brain, raising the possibility that it might serve a related function at neuron-neuron synapses. Previously, we identified an agrin signaling pathway in central nervous system (CNS) neurons, establishing the existence of a neural receptor that mediates responses to agrin. As a step toward identifying this agrin receptor, we have characterized the minimal domains in agrin that bind and activate it. Structures required for agrin signaling in CNS neurons are contained within a 20-kD COOH-terminal fragment of the protein. Agrin signaling is independent of alternative splicing at the z site, but requires sequences that flank it because their deletion results in a 15-kD fragment that acts as an agrin antagonist. Thus, distinct regions within agrin are responsible for receptor binding and activation. Using the minimal agrin fragments as affinity probes, we also studied the expression of the agrin receptor on CNS neurons. Our results show that both agrin and its receptor are concentrated at neuron-neuron synapses. These data support the hypothesis that agrin plays a role in formation and/or function of CNS synapses.

Previous studies have described maturational changes in GABAergic inhibitory synaptic transmission in the rodent somatosensory cortex during the early postnatal period. To determine whether alterations in the functional properties of synaptically localized GABA(A) receptors (GABA(A)Rs) contribute to development of inhibitory transmission, we used the whole cell recording technique to examine GABAergic miniature postsynaptic currents (mPSCs) in developing cortical neurons. Neurons harvested from somatosensory cortices of newborn mice showed a progressive, eightfold increase in GABAergic mPSC frequency during the first 4 wk of development in dissociated cell culture. A twofold decrease in the decay time of the GABAergic mPSCs, between 1 and 4 wk, demonstrates a functional change in the properties of GABA(A)Rs mediating synaptic transmission in cortical neurons during development in culture. A similar maturational profile observed in GABAergic mPSC frequency and decay time in cortical neurons developing in vivo (assessed in slices), suggests that these changes in synaptically localized GABA(A)Rs contribute to development of inhibition in the rodent neocortex. Pharmacological and reverse transcription-polymerase chain reaction (RT-PCR) studies were conducted to determine whether changes in subunit expression might contribute to the observed developmental alterations in synaptic GABA(A)Rs. Zolpidem (300 nM), a subunit-selective benzodiazepine agonist with high affinity for alpha1-subunits, caused a reversible slowing of the mPSC decay kinetics in cultured cortical neurons. Development was characterized by an increase in the potency of zolpidem in modulating the mPSC decay, suggesting a maturational increase in percentage of functionally active GABA(A)Rs containing alpha1 subunits. The relative expression of alpha1 versus alpha5 GABA(A)R subunit mRNA in cortical tissue, both in vivo and in vitro, also increased during this same period. Furthermore, single-cell RT-multiplex PCR analysis revealed more rapidly decaying mPSCs in individual neurons in which alpha1 versus alpha5 mRNA was amplified. Together these data suggest that changes in alpha-subunit composition of GABA(A)Rs contribute to the maturation of GABAergic mPSCs mediating inhibition in developing cortical neurons.