We have performed mRNA in situ hybridization studies and northern blot analysis in the mouse and human, respectively, to determine the normal gene expression patterns of FMR-1. Expression in the adult mouse was localized to several regions of the brain and the tubules of the testes, which are two of the major organs affected in fragile X syndrome. Universal and very strong expression was observed in early mouse embryos, with differentially decreasing expression during subsequent stages of embryonic development. The early embryonic onset and tissue specificity of FMR-1 gene expression is consistent with involvement in the fragile X phenotype, and also suggests additional organ systems in which clinical manifestations of reduced FMR-1 gene expression may occur.

We have identified a novel glutamate receptor subunit on the human and mouse genome. Cloning of the mouse cDNA revealed a protein consisting of 1003 amino acids encoded by at least nine exons. This protein showed the highest similarity (51%) to the NR3A subunit of the NMDA receptor and therefore was termed NR3B. NR3B has a structure typical of glutamate receptor family members with a signal peptide and four membrane-associated regions. Amino acids forming a ligand-binding pocket are conserved. When coexpressed with NR1 and NR2A in heterologous cells, NR3B suppressed glutamate-induced current similarly to NR3A. Thus members of the NR3 class of NMDA receptors act as dominant-negative subunits in the NMDA receptor complex. NR3B shows very restricted expression in somatic motoneurons of the brainstem and spinal cord. Its expression in other types of motoneurons, including autonomic motoneurons in Onuf's nucleus and oculomotor neurons, is significantly weaker. Our results indicate that NR3B is important as a regulatory subunit that controls NMDA receptor transmission in motoneurons. It may be involved in the pathogenesis of neurodegenerative diseases involving motoneurons as well.

A significant fraction of the total calcium͞calmodulin-dependent protein kinase II (CaMKII) activity in neurons is associated with synaptic connections and is present in nerve terminals, thus suggesting a role for CaMKII in neurotransmitter release. To determine whether CaMKII regulates neurotransmitter release, we generated and analyzed knockout mice in which the dominant ␣-isoform of CaMKII was specifically deleted from the presynaptic side of the CA3-CA1 hippocampal synapse. Conditional CA3 ␣-CaMKII knockout mice exhibited an unchanged basal probability of neurotransmitter release at CA3-CA1 synapses but showed a significant enhancement in the activity-dependent increase in probability of release during repetitive presynaptic stimulation, as was shown with the analysis of unitary synaptic currents. These data indicate that ␣-CaMKII serves as a negative activity-dependent regulator of neurotransmitter release at hippocampal synapses and maintains synapses in an optimal range of release probabilities necessary for normal synaptic operation. Synapses mediate interneuronal communication and display a variety of properties (1). Presynaptic neurotransmitter release is one of the first steps in the sequence of events underlying synaptic transmission. Discrete packets of neurotransmitter encased in vesicles are released at synaptic connections with a certain probability (probability of release, P r ) in a tightly regulated fashion (1, 2). Calcium͞calmodulin-dependent protein kinase II (CaMKII), a serine͞threonine protein kinase, is well positioned to serve a role in synaptic function regulation, because it is highly expressed in the brain and is known to phosphorylate multiple synaptic proteins (2). Accordingly, CaMKII has previously been implicated in the long-lasting frequency-dependent regulation of synaptic function in mammals (3-9), birds (10), frogs (11), and invertebrates (12).Although a postsynaptic role for CaMKII in synaptic function and plasticity has been well demonstrated (3,4,6,7,13), studies of the presynaptic function of this enzyme are scarce. Early microinjection experiments in the squid giant synapse showed that CaMKII injected presynaptically enhanced neurotransmitter release (14,15). This effect appeared to be mediated by phosphorylation of a synaptic vesicle protein, synapsin I, which in its dephosphorylated form inhibited synaptic transmission. However, mice lacking synapsin I do not demonstrate a major change in the basal P r (16), suggesting that certain differences in the mechanisms of release may exist between vertebrate and invertebrate synapses. It has also been shown that ␣-CaMKII can either potentiate or depress CA3-CA1 synapses in the hippocampus depending on the pattern of presynaptic activation. Pairedpulse facilitation (PPF), an index of presynaptic function, was decreased in the CA1 region of mice heterozygous for a global null mutation of ␣-CaMKII, whereas another form of presynaptic plasticity, which also depends on P r , posttetanic potentiation, was enhanced (17). A direct esti...