Long-term potentiation (LTP) is a cellular mechanism that potentially underlies learning and memory. To test the hypothesis that LTP is involved in activity-dependent synapse formation, we combined whole-cell recordings and confocal microscopy to investigate hippocampal glutamatergic synapses at their earliest stages of development. Here we report that, during the first postnatal week, the hippocampal glutamatergic network becomes gradually functional owing to the transformation of precursor, pure NMDA (N-methyl-D-aspartate)-receptor-based synaptic contacts into conducting AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionate)/NMDA-re cep tor-type synapses. This functional synapse induction is caused by an associative form of LTP, so it is input-specific and easily triggered experimentally by pairing presynaptic stimulation with postsynaptic depolarization. Our results challenge previous views that LTP occurs in the hippocampus only at later stages of development and that its induction requires dendritic spines. They also provide direct evidence that LTP is important for the activity-dependent formation of conducting glutamatergic synapses in the developing mammalian brain.

The N-methyl-D-aspartate (NMDA) receptor NR1 gene encodes RNA that is alternatively spliced to generate at least seven variants. The variants arise from splicing in or out of three exons; one encodes a 21-amino acid insert in the N-terminal domain, and two encode adjacent sequences of 37 and 38 amino acids in the C-terminal domain. Splicing out of the second C-terminal exon deletes a stop codon and results in an additional open reading frame encoding an unrelated sequence of 22 amino acids before arriving at a second stop codon. We denote the NRI variants by the presence or absence of the three alternatively spliced exons (from 5' to 3'); thus, NR1111 has all three exons, NRIoo has none, and NR11o has only the N-terminal exon. We report here electrophysiological characterization of six splice variants of the NRI receptor expressed in Xenopus oocytes. NRI receptors that lacked the N-terminal exon (NRIo1o, NRIolo and NR1o1i) exhibited a relatively high affinity for NMDA (ECso 13 ,uM) and marked potentiation by spermine. In contrast, those receptor variants with the N-terminal insert (NR11,oo NR11o1, and NRI111) showed a lower agonist affinity and little or no spermine potentiation at saturating glycine. AlU six variants showed spermine potentiation at low glycine and inhibition by spermine at more negative potentials. Variants differing only in the C-terminal domain differed little in agonist affinity and spermine potentiation. These findings indicate that the N-terminal insert either participates in agonist and polyamine binding domains or indirectly modifies their conformations. The splice variants differed in the extent to which they could be potentiated by activators of protein kinase C (PKC) from 3-to 20-fold. Presence of the N-terminal insert and absence of the C-terminal sequences increased potentiation by PKC. These findings identify the contributions of the separate polypeptide domains to modulation by polyamines and PKC and provide further support for the concept that subunit composition determines functional properties of NMDA receptors.The N-methyl-D-aspartate (NMDA)-type ofglutamate receptor is thought to play a role in long-term potentiation, memory formation, and control of brain development (1-3). NMDA receptor-mediated neurotoxicity is implicated in the neurodegeneration associated with epilepsy, ischemia, Huntington chorea, Alzheimer disease, and AIDS encephalopathy (4-6).To date, two gene families encoding NMDA receptor subunits have been identified in rat brain. One family is composed of the NR1 gene. NRI encodes RNA that undergoes alternate splicing to yield at least seven receptor variants (7-10). These variants arise from splicing in or out of three alternative exons, which we designate (from 5' to 3') a, (3, and y (see Fig. 1). Exon a encodes 21 amino acids that can be inserted into the N-terminal domain. Exons (3 and 'y are adjacent and encode the last portion of the C-terminal do-The publication costs of this article were defrayed in part by page charge payment. This article must ...

Molecular cloning identified complementary DNA species, from a rat ventral midbrain library, encoding apparent splice variants of the N-methyl-D-aspartate (NMDA) receptor NMDAR1 (which we now term NR1a). Sequencing revealed that one variant, NRlb, differs from NRla by the presence of a 21-amino acid insert near the amino end of the N-terminal domain and by an alternate C-terminal domain in which the last 75 amino acids are replaced by an unrelated sequence of 22 amino acids. NRlb is virtually identical to NRla in the remainder of the N-and C-terminal domains, at the 5' and 3' noncoding ends, and within the predicted transmembrane domains and extracellular and cytoplasmic loops. These findings suggest that the two forms of the receptor arise by differential splicing of a transcript from the same gene. Sequencing of other clones indicates the existence of a third variant, NR1c, identical to NRlb in its C terminus but lacking the N-terminal insert. NRlb RNA injected into Xenopus oocytes generated functional homomeric NMDA channels with electrophysiological properties distinct from those of NRla homomeric channels. NRlb channels exhibited a lower apparent affinity for NMDA and for glutamate. NRlb channels exhibited a lower affmiity for D-2-amino-5-phosphonovaleric acid and a higher affinity for Zn2+. The two receptor variants showed nearly identical affmities for glycine, Mg2+, and phencyclidine. Spermine potentiation of NMDA responses, prominent in oocytes injected with rat forebrain message, was also prominent for NRla receptors, but was greatly reduced or absent for NR1b receptors. Treatment with the protein kinase C activator phorbol 12-myristate 13-acetate potentiated NMDA responses in NR1b-injected oocytes by about 20-fold; potentiation of NMDA responses in NRla-iajected oocytes was much less, about 4-fold. These findins support a role for alternate splicing in generating NMDA channels with different functional properties.Glutamate is the primary excitatory neurotransmitter in the central nervous system. Channel-forming glutamate receptors in rat are encoded by about 20 identified genes in at least four gene subfamilies, GluRi to GluR4 [encoding kainate/aamino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid receptors (1-4)], GluRS, GluR6, and KA [kainate receptors (5-7)], and NMDAR [N-methyl-D-asparate (NMDA) receptors (8, 9)]. In recent years the NMDA receptor has attracted considerable interest due to its proposed roles in long-term potentiation (e.g., ref. 10), synaptogenesis, and developmental structuring (11). Moreover, NMDA receptor-mediated toxicity is thought to represent a final common pathway for the neurodegeneration associated with a wide range of neurological insults including trauma, hypoglycemia, ischemia, and epilepsy, as well as Alzheimer disease and Huntington disease (12).The recently cloned NMDA receptors from rat, NMDAR1 (8) and NMDAR2A-C (9), belong to the large superfamily of ligand-gated ion channels. Analyses of the predicted protein sequences reveal characteristic structural m...

G-protein-coupled metabotropic glutamate group I receptors (mGluR1s) mediate synaptic transmission and plasticity in Purkinje cells and, therefore, critically determine cerebellar motor control and learning. Purkinje cells express two members of the G-protein G q family, namely G q and G 11 . Although in vitro coexpression of mGluR1 with either G␣ 11 or G␣ q produces equally well functioning signaling cascades, G␣ q -and G␣ 11 -deficient mice exhibit distinct alterations in motor coordination. By using whole-cell recordings and Ca 2ϩ imaging in Purkinje cells, we show that G␣ q is required for mGluR-dependent synaptic transmission and for long-term depression (LTD). G␣ 11 has no detectable contribution for synaptic transmission but also contributes to LTD. Quantitative single-cell RT-PCR analyses in Purkinje cells demonstrate a more than 10-fold stronger expression of G␣ q versus G␣ 11 . Our findings suggest an expression leveldependent action of G␣ q and G␣ 11 for Purkinje cell signaling and assign specific roles of these two G q isoforms for motor coordination.

Functionally diverse kainate/alpha-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA) receptors are generated by assembly of glutamate receptor (GluR)1, 2, and 3 subunits into homomeric and heteromeric channels. We examined GluR1, 2, and 3 gene expression in embryonic, neonatal, and adult rat brain by northern analysis under conditions of high stringency. In the adult, hybridization to a GluR1 riboprobe revealed the presence of an abundant RNA species, 5.2 kb in size, and minor bands of 3.2 and 3.9 kb. GluR2 hybridized to two species, 3.9 and 5.9 kb, of comparable abundance, presumably attributable to alternate splice products. Hybridization to the GluR3 riboprobe showed a major species of 5.2 kb. This pattern of RNA species was invariant over all the brain regions examined. Examination of GluR expression in development revealed that in the postnatal period, GluR1, 2, and 3 mRNAs are regulated as a function of age. In adult rat brain, GluR1 and 2 mRNA expression was highest in hippocampus; GluR3 was expressed at highest density in hippocampus and frontal cortex. The three transcripts were first detected at embryonic day 16 and then exhibited changes in expression levels in a region-specific manner. In hippocampus, all three transcripts exhibited elevated expression in the late neonatal period; in frontal cortex, elevated expression was observed for GluR2 and 3 only. In striatum, all three transcripts were expressed at relatively low levels throughout development, with a modest peak at postnatal day 14. In cerebellum, the GluR1 mRNA level was high from postnatal day 28 to adult.(ABSTRACT TRUNCATED AT 250 WORDS)