Synapsin-dependent development of glutamatergic synaptic vesicles and presynaptic plasticity in postnatal mouse brain.

Inactivation of the genes encoding the neuronal, synaptic vesicle-associated proteins synapsin I and II leads to severe reductions in the number of synaptic vesicles in the CNS. We here define the postnatal developmental period during which the synapsin I and/or II proteins modulate synaptic vesicle number and function in excitatory glutamatergic synapses in mouse brain. In wild-type mice, brain levels of both synapsin I and synapsin IIb showed developmental increases during synaptogenesis from postnatal days 5-20, while synapsin IIa showed a protracted increase during postnatal days 20-30. The vesicular glutamate transporters (VGLUT) 1 and VGLUT2 showed synapsin-independent development during postnatal days 5-10, following which significant reductions were seen when synapsin-deficient brains were compared with wild-type brains following postnatal day 20. A similar, synapsin-dependent developmental profile of vesicular glutamate uptake occurred during the same age periods. Physiological analysis of the development of excitatory glutamatergic synapses, performed in the CA1 stratum radiatum of the hippocampus from the two genotypes, showed that both the synapsin-dependent part of the frequency facilitation and the synapsin-dependent delayed response enhancement were restricted to the period after postnatal day 10. Our data demonstrate that while both synaptic vesicle number and presynaptic short-term plasticity are essentially independent of synapsin I and II prior to postnatal day 10, maturation and function of excitatory synapses appear to be strongly dependent on synapsin I and II from postnatal day 20.

N-methyl-D-aspartate receptor subunit dysfunction at hippocampal glutamatergic synapses in an animal model of attention-deficit/hyperactivity disorder.

Attention-deficit/hyperactivity disorder (ADHD) is the most common neurobehavioural disorder among children. ADHD children are hyperactive, impulsive and have problems with sustained attention. These cardinal features are also present in the best validated animal model of ADHD, the spontaneously hypertensive rat (SHR), which is derived from the Wistar Kyoto rat (WKY). Current theories of ADHD relate symptom development to factors that alter learning. N-methyl-D-aspartate receptor (NMDAR) dependent long term changes in synaptic efficacy in the mammalian CNS are thought to represent underlying cellular mechanisms for some forms of learning. We therefore hypothesized that synaptic abnormality in excitatory, glutamatergic synaptic transmission might contribute to the altered behavior in SHRs. We studied physiological and anatomical aspects of hippocampal CA3-to-CA1 synapses in age-matched SHR and WKY (controls). Electrophysiological analysis of these synapses showed reduced synaptic transmission (reduced field excitatory postsynaptic potential for a defined fiber volley size) in SHR, whereas short-term forms of synaptic plasticity, like paired-pulse facilitation, frequency facilitation, and delayed response enhancement were comparable in the two genotypes, and long-term potentiation (LTP) of synaptic transmission was of similar magnitude. However, LTP in SHR was significantly reduced (by 50%) by the NR2B specific blocker CP-101,606 (10 microM), whereas the blocker had no effect on LTP magnitude in the control rats. This indicates that the SHR has a functional predominance of NR2B, a feature characteristic of early developmental stages in these synapses. Quantitative immunofluorescence and electron microscopic postembedding immunogold cytochemistry of the three major NMDAR subunits (NR1, NR2A; and NR2B) in stratum radiatum spine synapses revealed no differences between SHR and WKY. The results indicate that functional impairments in glutamatergic synaptic transmission may be one of the underlying mechanisms leading to the abnormal behavior in SHR, and possibly in human ADHD.

Conditional restoration of hippocampal synaptic potentiation in Glur-A-deficient mice.

Plasticity of mature hippocampal CA1 synapses is dependent on l-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors containing the glutamate receptor A (GluR-A) subunit. In GluR-A-deficient mice, plasticity could be restored by controlled expression of green fluorescent protein (GFP)-tagged GluR-A, which contributes to channel formation and displayed the developmental redistribution of AMPA receptors in CA1 pyramidal neurons. Long-term potentiation (LTP) induced by pairing or tetanic stimulation was rescued in adult GluR-A(-/-) mice when (GFP)GluR-A expression was constitutive or induced in already fully developed pyramidal cells. This shows that GluR-A-independent forms of synaptic plasticity can mediate the establishment of mature hippocampal circuits that are prebuilt to express GluR-A-dependent LTP.

Importance of AMPA receptors for hippocampal synaptic plasticity but not for spatial learning.

Gene-targeted mice lacking the L-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunit GluR-A exhibited normal development, life expectancy, and fine structure of neuronal dendrites and synapses. In hippocampal CA1 pyramidal neurons, GluR-A-/- mice showed a reduction in functional AMPA receptors, with the remaining receptors preferentially targeted to synapses. Thus, the CA1 soma-patch currents were strongly reduced, but glutamatergic synaptic currents were unaltered; and evoked dendritic and spinous Ca2+ transients, Ca2+-dependent gene activation, and hippocampal field potentials were as in the wild type. In adult GluR-A-/- mice, associative long-term potentiation (LTP) was absent in CA3 to CA1 synapses, but spatial learning in the water maze was not impaired. The results suggest that CA1 hippocampal LTP is controlled by the number or subunit composition of AMPA receptors and show a dichotomy between LTP in CA1 and acquisition of spatial memory.

A possible postsynaptic role for SNAP-25 in hippocampal synapses.

The SNARE protein SNAP-25 is well documented as regulator of presynaptic vesicle exocytosis. Increasing evidence suggests roles for SNARE proteins in postsynaptic trafficking of glutamate receptors as a basic mechanism in synaptic plasticity. Despite these indications, detailed quantitative subsynaptic localization studies of SNAP-25 have never been performed. Here, we provide novel electron microscopic data of SNAP-25 localization in postsynaptic spines. In addition to its expected presynaptic localization, we show that the protein is also present in the postsynaptic density (PSD), the postsynaptic lateral membrane and on small vesicles in the postsynaptic cytoplasm. We further investigated possible changes in synaptic SNAP-25 protein expression after hippocampal long-term potentiation (LTP). Quantitative analysis of immunogold-labeled electron microscopy sections did not show statistically significant changes of SNAP-25 gold particle densities 1 h after LTP induction, indicating that local trafficking of SNAP-25 does not play a role in the early phases of LTP. However, the strong expression of SNAP-25 in postsynaptic plasma membranes suggests a function of the protein in postsynaptic vesicle exocytosis and a possible role in hippocampal synaptic plasticity.

Correction to: A possible postsynaptic role for SNAP-25 in hippocampal synapses.

In the original publication of the article the author name M. Schupps was incorrect.

Neurological dysfunctions in mice expressing different levels of the Q/R site-unedited AMPAR subunit GluR-B.

We generated mouse mutants with targeted AMPA receptor (AMPAR) GluR-B subunit alleles, functionally expressed at different levels and deficient in Q/R-site editing. All mutant lines had increased AMPAR calcium permeabilities in pyramidal neurons, and one showed elevated macroscopic conductances of these channels. The AMPAR-mediated calcium influx induced NMDA-receptor-independent long-term potentiation (LTP) in hippocampal pyramidal cell connections. Calcium-triggered neuronal death was not observed, but mutants had mild to severe neurological dysfunctions, including epilepsy and deficits in dendritic architecture. The seizure-prone phenotype correlated with an increase in the macroscopic conductance, as independently revealed by the effect of a transgene for a Q/R-site-altered GluR-B subunit. Thus, changes in GluR-B gene expression and Q/R site editing can affect critical architectural and functional aspects of excitatory principal neurons.

Dysfunctions in mice by NMDA receptor point mutations NR1(N598Q) and NR1(N598R).

NMDA receptors in mice were mutated by gene targeting to substitute asparagine (N) in position 598 of the NR1 subunit to glutamine (Q) or arginine (R). Animals expressing exclusively the mutated NR1 alleles, NR1(Q/Q) and NR1(-/R) mice, developed a perinatally lethal phenotype mainly characterized by respiratory failure. The dysfunctions were partially rescued in heterozygous mice by the presence of pure wild-type receptors. Thus, NR1(+/Q) mice exhibited reduced life expectancy, with females being impaired in nurturing; NR1(+/R) mice displayed signs of underdevelopment such as growth retardation and impaired righting reflex, and died before weaning. We analyzed the key properties of NMDA receptors, high Ca(2+) permeability, and voltage-dependent Mg(2+) block, in the mutant mice. Comparison of the complex physiological and phenotypical changes observed in the different mutants indicates that properties controlled by NR1 subunit residue N598 are important for autonomic brain functions at birth and during postnatal development. We conclude that disturbed NMDA receptor signaling mediates a variety of neurological phenotypes.

Protein phosphatase-1 regulation in the induction of long-term potentiation: heterogeneous molecular mechanisms.

Protein phosphatase inhibitor-1 (I-1) has been proposed as a regulatory element in the signal transduction cascade that couples postsynaptic calcium influx to long-term changes in synaptic strength. We have evaluated this model using mice lacking I-1. Recordings made in slices prepared from mutant animals and also in anesthetized mutant animals indicated that long-term potentiation (LTP) is deficient at perforant path-dentate granule cell synapses. In vitro, this deficit was restricted to synapses of the lateral perforant path. LTP at Schaffer collateral-CA1 pyramidal cell synapses remained normal. Thus, protein phosphatase-1-mediated regulation of NMDA receptor-dependent synaptic plasticity involves heterogeneous molecular mechanisms, in both different dendritic subregions and different neuronal subtypes. Examination of the performance of I-1 mutants in spatial learning tests indicated that intact LTP at lateral perforant path-granule cell synapses is either redundant or is not involved in this form of learning.

Dendritic excitation by glutamate in CA1 hippocampal cells.

In order to reveal properties and effects of glutamate excitation, CA1 pyramidal cells in rat hippocampal slices were impaled and responses to iontophoresis of glutamate onto sensitive spots in the dendrites were analyzed. The glutamate-elicited response consisted of a steady depolarization; its amplitude was dose-dependent. The cellular response to repeated applications of glutamate showed a striking degree of stability. Both dendritic and somatic depolarization, induced by glutamate and current, respectively, elicited similar discharge patterns. The sensitivity to glutamate was highly localized, corresponding to the dendritic tree of a given cell. Short, repeated glutamate pulses did not interfere with an orthodromic test response, whereas longer glutamate ejections often depressed the EPSP. Combined temporal and spatial pairing of glutamate and orthodromic activation was followed by a lasting increase in synaptic efficiency, similar to LTP.

Impaired spatial working memory but spared spatial reference memory following functional loss of NMDA receptors in the dentate gyrus.

Novel spatially restricted genetic manipulations can be used to assess contributions made by synaptic plasticity to learning and memory, not just selectively within the hippocampus, but even within specific hippocampal subfields. Here we generated genetically modified mice (NR1(deltaDG) mice) exhibiting complete loss of the NR1 subunit of the N-methyl-D-aspartate receptor specifically in the granule cells of the dentate gyrus. There was no evidence of any reduction in NR1 subunit levels in any of the other hippocampal subfields, or elsewhere in the brain. NR1(deltaDG) mice displayed severely impaired long-term potentiation (LTP) in both medial and lateral perforant path inputs to the dentate gyrus, whereas LTP was unchanged in CA3-to-CA1 cell synapses in hippocampal slices. Behavioural assessment of NR1(deltaDG) mice revealed a spatial working memory impairment on a three-from-six radial arm maze task despite normal hippocampus-dependent spatial reference memory acquisition and performance of the same task. This behavioural phenotype resembles that of NR1(deltaCA3) mice but differs from that of NR1(deltaCA1) mice which do show a spatial reference memory deficit, consistent with the idea of subfield-specific contributions to hippocampal information processing. Furthermore, this pattern of selective functional loss and sparing is the same as previously observed with the global GluR-A L-alpha-amino-3-hydroxy-5-methyl-4-isoxazelopropionate receptor subunit knockout, a mutation which blocks the expression of hippocampal LTP. The present results show that dissociations between spatial working memory and spatial reference memory can be induced by disrupting synaptic plasticity specifically and exclusively within the dentate gyrus subfield of the hippocampal formation.

Repetitive firing of CA1 hippocampal pyramidal cells elicited by dendritic glutamate: slow prepotentials and burst-pause pattern.

In order to compare responses to dendritic vs. somatic depolarization, CA1 pyramidal cells in rat hippocampal slices were stimulated by iontophoresis of glutamate to sensitive spots in the dendrites, and by somatic current injection. Low intensities of either stimulus elicited slow repetitive firing. Each action potential was preceded by a slow depolarizing prepotential (SPP), lasting 50-300 ms and was followed by fast (3-5 ms) and slow (more than 100 ms) afterhyperpolarizations (AHPs). The SPPs, and AHPs were indistinguishable for the two types of stimuli. In response to strong depolarizations, most cells showed an initial burst of spikes, followed by a pause before the steady discharge. This pattern was elicited by both glutamate and current. The input resistance usually increased 5-20% during subthreshold depolarizations by glutamate or current. In contrast, large doses of glutamate caused a slow decline in the resistance (up to 40%), which was larger than during comparable current-induced discharge, and the response was followed by a longer AHP. It is concluded that both dendritic and somatic depolarization, induced by glutamate and current, respectively, can elicit sustained repetitive firing with SPPs, fast and slow AHPs and burst-pause pattern, thus, increasing the likelihood that these phenomena play a role during natural activation of CA1 cells.

Intracellular analysis of potentiation of CA1 hippocampal synaptic transmission by phorbol ester application.

(1) The effect of active and inactive phorbol esters on synaptic transmission and on membrane properties of CA1 pyramidal cells in hippocampus have been analyzed by intracellular recording. (2) 4 beta-phorbol-12,13 dibutyrate (beta PDBu), but not the alpha-isomer, increased the firing probability, reduced the spike latency and enhanced the EPSP amplitude in response to synaptic activation. The effect was similar to the changes seen in long term potentiation. After alpha PDBu addition it was possible to elicit further enhancement by tetanization, but not after beta PDBu administration. (3) A slowly developing hyperpolarization was seen after active phorbol ester application without apparent changes in the soma input resistance. (4) Active phorbol esters reduced the slow afterhyperpolarization (AHP) in these cells without affecting the intermediate AHP.

Glutamate-induced action potentials are preceded by regenerative prepotentials in rat hippocampal pyramidal cells in vitro.

(1) The responses of CA1 pyramidal cells to short glutamate pulses (10-50 ms) delivered at sensitive spots in the apical dendrites have been analysed by intracellular recording. (2) The glutamate pulses elicited stable depolarizing responses in a dose- and frequency-dependent manner. (3) When a single action potential with a firing probability around 0.5 was elicited, a subtraction procedure showed that a slow depolarizing ramp preceded each spike. We call this ramp the glutamate-induced prepotential (GluPP). (4) In contrast to the upward convex subthreshold depolarization the GluPP was upward concave. (5) The GluPP amplitude and time course increased with depolarization of the membrane, a phenomenon which appears to be connected to the elevation of action potential threshold. (6) The GluPP was regenerative since once started, it ended in an action potential. (7) A specific N-methyl-D-aspartate receptor antagonist, DL-2-amino-5-phosphonovaleric acid (50 microM) reduced the glutamate-induced depolarization, but did not affect the form or amplitude of GluPP, once the latter was induced. (8) It is concluded that short glutamate pulses elicited action potentials through a prepotential mechanism, similar to the slow prepotentials induced by long depolarizing current pulses across the soma membrane. (9) A possible physiological role for the GluPP is discussed.

Failure to induce long-term depression by an anti-correlation procedure in area CA1 of the rat hippocampal slice.

We have independently in our two laboratories re-examined the report by Stanton and Sejnowski (Nature, 339, 215-218, 1989) that single stimuli to a test pathway in area CA1 of the hippocampal slice, when delivered between short bursts of stimuli to a second, convergent pathway, produce an associative long-term depression (LTD) in the test pathway. While robust associative LTP was observed when stimuli to the two inputs were correlated in time, the anti-correlation procedure failed to induce LTD; rather, a trend towards potentiation was observed. This result was obtained using both submerged and interface chambers, and in two different strains of rat. A transient depression lasting for a few minutes could usually be elicited by strong tetanic stimulation; this depression was not restricted to activated pathways.

Postsynaptic long-term potentiation follows coupling of dendritic glutamate application and synaptic activation.

Dendritic depolarization, which seems to be involved in the induction of long-term potentiation (LTP), was elicited by localized glutamate application. When paired to low frequency synaptic activation in the same area, the subsequent changes had features in common with LTP, expressed as an increased probability of firing and shorter spike latency. The EPSP was not significantly increased.

Synaptically triggered action potentials begin as a depolarizing ramp in rat hippocampal neurones in vitro.

1. During just-suprathreshold synaptic activation of CA1 pyramidal cells in rat hippocampal slices in vitro the action potential begins as a slow depolarizing ramp, superimposed on the underlying EPSP and forming an integral part of the action potential. We call this ramp a synaptic prepotential (SyPP). 2. In order to examine the SyPP, a procedure for subtraction of the underlying EPSP was necessary. Because action potentials were only elicited by a subset of EPSPs with larger than average amplitude, a subtraction of the mean subthreshold EPSP would not give valid results. Instead, an EPSP to be subtracted was selected from an assemblage of subthreshold EPSPs, so that its amplitude matched the initial part of the spike-generating EPSP. 3. Virtually all action potentials started with a SyPP. Using an amplitude criterion of 1 S.D. of the mean of the matching subthreshold EPSPs, just-suprathreshold EPSPs gave prepotentials in 72-100% of all action potentials from fifteen randomly selected cells. With a criterion of 2 S.D.S, the frequency of occurrence ranged from 36 to 100%. 4. With a constant stimulus strength, there was a certain variability of the spike latencies. Shorter latency spikes had steeper, but smaller SyPPs than later spikes, suggesting that the slope of SyPP influenced the timing of the cell discharge. 5. The SyPP was best fitted by a single, exponentially rising curve, and was both smaller and slower than the large amplitude action potential. Its amplitude was 1-6 mV and the time constant 1-5 ms, which was 10-50 times slower than that of the upstroke of the action potential. 6. A properly timed hyperpolarizing current pulse could block the large amplitude action potential, thereby unmasking the SyPP as an initial depolarizing ramp. 7. The SyPP was more sensitive than the large amplitude action potential to intracellular injection of QX-314, a lidocaine derivative. At the concentrations used (10 or 30 mM) no detectable changes were seen in the large amplitude action potential. 8. Droplet application of a specific N-methyl-D-aspartate receptor antagonist, DL-2-amino-5-phosphonovaleric acid (1 mM), reduced both the EPSP and the firing probability, but did not change the SyPP. 9. The SyPP amplitude and time course depended upon the membrane potential at which the cell was activated. Depolarization enhanced and prolonged the SyPP, while hyperpolarization gave opposite effects. In part, the depolarization-induced amplitude increase could be attributed to membrane accommodation. 10. Antidromically evoked action potentials never started with a prepotential.(ABSTRACT TRUNCATED AT 400 WORDS)

Phosphorylation of the inositol 1,4,5-trisphosphate receptor by cyclic nucleotide-dependent kinases in vitro and in rat cerebellar slices in situ.

We have examined cyclic nucleotide-regulated phosphorylation of the neuronal type I inositol 1,4,5-trisphosphate (IP3) receptor immunopurified from rat cerebellar membranes in vitro and in rat cerebellar slices in situ. The isolated IP3 receptor protein was phosphorylated by both cAMP- and cGMP-dependent protein kinases on two distinct sites as determined by thermolytic phosphopeptide mapping, phosphopeptide 1, representing Ser-1589, and phosphopeptide 2, representing Ser-1756 in the rat protein (Ferris, C. D., Cameron, A. M., Bredt, D. S., Huganir, R. L., and Snyder, S. H. (1991) Biochem. Biophys. Res. Commun. 175, 192-198). Phosphopeptide maps show that cAMP-dependent protein kinase (PKA) labeled both sites with the same time course and same stoichiometry, whereas cGMP-dependent protein kinase (PKG) phosphorylated Ser-1756 with a higher velocity and a higher stoichiometry than Ser-1589. Synthetic decapeptides corresponding to the two phosphorylation sites (peptide 1, AARRDSVLAA (Ser-1589), and peptide 2, SGRRESLTSF (Ser-1756)) were used to determine kinetic constants for the phosphorylation by PKG and PKA, and the catalytic efficiencies were in agreement with the results obtained by in vitro phosphorylation of the intact protein. In cerebellar slices prelabeled with [32P]orthophosphate, activation of endogenous kinases by incubation in the presence of cAMP/cGMP analogues and specific inhibitors of PKG and PKA induced in both cases a 3-fold increase in phosphorylation of the IP3 receptor. Thermolytic phosphopeptide mapping of in situ labeled IP3 receptor by PKA showed labeling on the same sites (Ser-1589 and Ser-1756) as in vitro. In contrast to the findings in vitro, PKG preferentially phosphorylated Ser-1589 in situ. Because both PKG and the IP3 receptor are specifically enriched in cerebellar Purkinje cells, PKG may be an important IP3 receptor regulator in vivo.

Protein kinase C injection into hippocampal pyramidal cells elicits features of long term potentiation.

Long-term potentiation (LTP) in the hippocampus is an interesting example of synaptic plasticity because of its induction by physiological discharge rates and its long duration. Of the possible biochemical mechanisms that regulate prolonged changes in cell function, protein phosphorylation is a particularly attractive candidate. We have therefore examined the effect of intracellular injection of calcium/diacylglycerol-dependent protein kinase (protein kinase C (PKC] in CA1 pyramidal neurones in hippocampal slices. Injection of the active enzyme elicited long-lasting enhancement of synaptic transmission, similar to LTP, whereas inactivated kinase failed to do so. The observed changes included an increased amplitude of the excitatory post-synaptic potential (e.p.s.p.) and an increased probability of firing and a reduced latency of the associated actin potential.

Impairment of synaptic vesicle clustering and of synaptic transmission, and increased seizure propensity, in synapsin I-deficient mice.

Synapsin I has been proposed to be involved in the modulation of neurotransmitter release by controlling the availability of synaptic vesicles for exocytosis. To further understand the role of synapsin I in the function of adult nerve terminals, we studied synapsin I-deficient mice generated by homologous recombination. The organization of synaptic vesicles at presynaptic terminals of synapsin I-deficient mice was markedly altered: densely packed vesicles were only present in a narrow rim at active zones, whereas the majority of vesicles were dispersed throughout the terminal area. This was in contrast to the organized vesicle clusters present in terminals of wild-type animals. Release of glutamate from nerve endings, induced by K+,4-aminopyridine, or a Ca2+ ionophore, was markedly decreased in synapsin I mutant mice. The recovery of synaptic transmission after depletion of neurotransmitter by high-frequency stimulation was greatly delayed. Finally, synapsin I-deficient mice exhibited a strikingly increased response to electrical stimulation, as measured by electrographic and behavioral seizures. These results provide strong support for the hypothesis that synapsin I plays a key role in the regulation of nerve terminal function in mature synapses.