After homologous recombination in embryonic stem (ES) cells, the targeted allele contained a 10.3-kb EcoRV fragment, whereas the wild-type allele had a 13.4-kb EcoRV fragment (Fig. A targeting vector was constructed to replace a 4.2-kb fragment containing the first exon of spinophilin with a neomycin-resistance selection marker. Standard gene-targeting methods were used to generate mice that did not express spinophilin.
Convulsant-Induced Seizures and Neuronal Apoptosis. Experiments were performed blind to genotype. Paired-pulse facilitation of the response at various interpulse intervals (25–400 msec) also was measured. LTP was induced by tetanic stimulation (100 Hz twice for 1 sec with a 20-sec interval). Homosynaptic LTD was induced by prolonged low-frequency stimulation (1 Hz for 15 min). Stimulus intensity was adjusted to produce a response of approximately 1 mV amplitude, with an initial slope of approximately −0.5 mV/msec.
A bipolar tungsten stimulating electrode was placed in the stratum radiatum in the CA1 region, and extracellular field potentials were recorded by using a glass microelectrode (3–12 MΩ, filled with artificial cerebrospinal fluid) also in the stratum radiatum. Long-term depression (LTD) and long-term potentiation (LTP) studies were carried out in 4- to 6-week-old wild-type and knockout littermates as described previously ( 32).
#DENDRITE FUNCTIONS SOFTWARE#
Data were collected with pclamp software and analyzed with axograph, kaleidograph, and statview. N-methyl- d-aspartate (NMDA 100 μM) was applied in the same manner to evoke NMDA receptor currents in Mg 2+-free solution. Kainate (200 μM) was applied briefly (2–4 sec) every 30 sec to evoke α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor currents. Whole-cell patch–clamp recording was performed on acutely dissociated striatal and hippocampal neurons from 3- to 4-week-old wild-type and knockout littermates as described previously ( 26). Hippocampal Cultures and Immunocytochemistry. In total, more than 8,000 spines were counted. Spines were counted on 58 segments of secondary dendrites (total dendritic length of 4,152 μm) for P15 mice and 54 segments of secondary dendrites (total dendritic length of 4,225 μm) for adult mice. A group of four knockouts and four wild-type littermates was analyzed at postnatal day 15 (P15), and a second group of four knockouts and four wild-type littermates was analyzed at 2–3 months. Secondary dendrites were drawn with a camera lucida, and the number of spines per unit length of dendrite was counted. An insufficient number of neurons was impregnated in the hippocampus, and we therefore examined dendritic spine density in the caudatoputamen. The slides were fastened together at the top with electrical tape and immersed in silver nitrate (1% aqueous) overnight. In brief, sections were incubated in osmium tetroxide (1% in PBS) for 30 min, rinsed in PBS (three times for 10 min each), incubated in potassium dichromate (3.5% aqueous) for 1.5 h, and mounted between two microscope slides. Vibratome sections (100 μm) were stained with a single-section Golgi technique ( 30). After 1 h of postfixation in situ, brains were removed and stored overnight in fixative at 4☌. Mice were perfused transcardially with 4% formaldehyde in PBS. However, with the exception of evidence that localization of glutamate receptors is altered with neuronal activity ( 20, 21), little is known about the molecular mechanisms underlying the linkage between synaptic activity and the dynamic morphological changes of dendritic spines. Conversely, morphological changes in dendritic spines have profound effects on their electrical and biochemical properties ( 15– 17), thereby regulating the efficacy of synaptic transmission ( 13, 18, 19). It is increasingly evident that alterations in synaptic activity can cause morphological changes of dendritic spines ( 2, 11– 14): high-intensity stimulation of CA1 neurons induces rapid formation of spine-like protrusions (or filopodia) ( 12), and decreased synaptic activity results in loss of dendritic spines ( 14). Dynamic changes in the number, size, and shape of dendritic spines have been associated with learning ( 2, 3), electrophysiological ( 4– 6), developmental ( 7, 8), and hormonal changes ( 9, 10). Dendritic spines are specialized protrusions from dendritic shafts that receive the vast majority of excitatory input in the central nervous system ( 1).
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