Synaptic regulation of protein synthesis and the fragile X protein

Protein synthesis occurs in neuronal dendrites, often near synapses. Polyribosomal aggregates often appear in dendritic spines, particularly during development. Polyribosomal aggregates in spines increase during experience-dependent synaptogenesis, e.g., in rats in a complex environment. Some protein synthesis appears to be regulated directly by synaptic activity. We use “synaptoneurosomes,” a preparation highly enriched in pinched-off, resealed presynaptic processes attached to resealed postsynaptic processes that retain normal functions of neurotransmitter release, receptor activation, and various postsynaptic responses including signaling pathways and protein synthesis. We have found that, when synaptoneurosomes are stimulated with glutamate or group I metabotropic glutamate receptor agonists such as dihydroxyphenylglycine, mRNA is rapidly taken up into polyribosomal aggregates, and labeled methionine is incorporated into protein. One of the proteins synthesized is FMRP, the protein that is reduced or absent in fragile X mental retardation syndrome. FMRP has three RNA-binding domains and reportedly binds to a significant number of mRNAs. We have found that dihydroxyphenylglycine-activated protein synthesis in synaptoneurosomes is dramatically reduced in a knockout mouse model of fragile X syndrome, which cannot produce full-length FMRP, suggesting that FMRP is involved in or required for this process. Studies of autopsy samples from patients with fragile X syndrome have indicated that dendritic spines may fail to assume a normal mature size and shape and that there are more spines per unit dendrite length in the patient samples. Similar findings on spine size and shape have come from studies of the knockout mouse. Study of the development of the somatosensory cortical region containing the barrel-like cell arrangements that process whisker information suggests that normal dendritic regression is impaired in the knockout mouse. This finding suggests that FMRP may be required for the normal processes of maturation and elimination to occur in cerebral cortical development.

Synaptic sprouting increases the uptake capacities of motoneurons in amyotrophic lateral sclerosis mice

Using adenoviruses encoding reporter genes as retrograde tracers, we assessed the capacity of motoneurons to take up and retrogradely transport adenoviral particles injected into the muscles of transgenic mice expressing the G93A human superoxide dismutase mutation, a model of amyotrophic lateral sclerosis. Surprisingly, transgene expression in the motoneurons was significantly higher in symptomatic mice than in control or presymptomatic mice. Using botulinum toxin to induce nerve sprouting at neuromuscular junctions, we showed that the unexpectedly high level of motoneurons retrograde transduction results, at least in part, from newly acquired uptake properties of the sprouts. These findings demonstrate the remarkable uptake properties of amyotrophic lateral sclerosis motoneurons in response to denervation and the rationale of using intramuscular injections of adenoviruses to overexpress therapeutic proteins in motor neuron diseases.

BDNF but not NT-4 is required for normal flexion reflex plasticity and function

Neurotrophins can directly modulate the function of diverse types of central nervous system synapses. Brain-derived neurotrophic factor (BDNF) might be released by nociceptors onto spinal neurons and mediate central sensitization associated with chronic pain. We have studied the role of BDNF and neurotrophin-4 (NT-4), both ligands of the trkB tyrosine kinase receptor, in synaptic transmission and reflex plasticity in the mouse spinal cord. We used an in vitro spinal cord preparation to measure monosynaptic and polysynaptic reflexes evoked by primary afferents in BDNF- and NT-4-deficient mice. In situ hybridization studies show that both these neurotrophins are synthesized by sensory neurons, and NT-4, but not BDNF, also is expressed by spinal neurons. BDNF null mutants display selective deficits in the ventral root potential (VRP) evoked by stimulating nociceptive primary afferents whereas the non-nociceptive portion of the VRP remained unaltered. In addition, activity-dependent plasticity of the VRP evoked by repetitive (1 Hz) stimulation of nociceptive primary afferents (termed wind-up) was substantially reduced in BDNF-deficient mice. This plasticity also was reduced in a reversible manner by the protein kinase inhibitor K252a. Although the trkB ligand NT-4 is normally present, reflex properties in NT-4 null mutant mice were normal. Pharmacological studies also indicated that spinal N-methyl-d-aspartate receptor function was unaltered in BDNF-deficient mice. Using immunocytochemistry for markers of nociceptive neurons we found no evidence that their number or connectivity was substantially altered in BDNF-deficient mice. Our data therefore are consistent with a direct role for presynaptic BDNF release from sensory neurons in the modulation of pain-related neurotransmission.

The synaptic activity of HsDmc1, a human recombination protein specific to meiosis

Human Dmc1 protein, a meiosis-specific homolog of Escherichia coli RecA protein, has previously been shown to promote DNA homologous pairing and strand-exchange reactions that are qualitatively similar to those of RecA protein and Rad51. Human and yeast Rad51 proteins each form a nucleoprotein filament that is very similar to the filament formed by RecA protein. However, recent studies failed to find a similar filament made by Dmc1 but showed instead that this protein forms octameric rings and stacks of rings. These observations stimulated further efforts to elucidate the mechanism by which Dmc1 promotes the recognition of homology. Dmc1, purified to a state in which nuclease and helicase activities were undetectable, promoted homologous pairing and strand exchange as measured by fluorescence resonance energy transfer (FRET). Observations on the intermediates and products, which can be distinguished by FRET assays, provided direct evidence of a three-stranded synaptic intermediate. The effects of helix stability and mismatched base pairs on the recognition of homology revealed further that human Dmc1, like human Rad51, requires the preferential breathing of A⋅T base pairs for recognition of homology. We conclude that Dmc1, like human Rad51 and E. coli RecA protein, promotes homologous pairing and strand exchange by a “synaptic pathway” involving a three-stranded nucleoprotein intermediate, rather than by a “helicase pathway” involving the separation and reannealing of DNA strands.

A genetic selection for Caenorhabditis elegans synaptic transmission mutants.

We have isolated 165 Caenorhabditis elegans mutants, representing 21 genes, that are resistant to inhibitors of cholinesterase (Ric mutants). Since mutations in 20 of the genes appear not to affect acetylcholine reception, we suggest that reduced acetylcholine release contributes to the Ric phenotype of most Ric mutants. Mutations in 15 of the genes lead to defects in a gamma-aminobutyric acid-dependent behavior; these genes are likely to encode proteins with general, rather than cholinergic-specific, roles in synaptic transmission. Ten of the genes have been cloned. Seven encode homologs of proteins that function in the synaptic vesicle cycle: two encode cholinergic-specific proteins, while five encode general presynaptic proteins. Two other Ric genes encode homologs of G-protein signaling molecules. Our assessment of synaptic function in Ric mutants, combined with the homologies of some Ric mutants to presynaptic proteins, suggests that the analysis of Ric genes will continue to yield insights into the regulation and functioning of synapses.

Differential synaptic localization of two major gamma-aminobutyric acid type A receptor alpha subunits on hippocampal pyramidal cells.

Hippocampal pyramidal cells, receiving domain specific GABAergic inputs, express up to 10 different subunits of the gamma-aminobutyric acid type A (GABAA) receptor, but only 3 different subunits are needed to form a functional pentameric channel. We have tested the hypothesis that some subunits are selectively located at subsets of GABAergic synapses. The alpha 1 subunit has been found in most GABAergic synapses on all postsynaptic domains of pyramidal cells. In contrast, the alpha 2 subunit was located only in a subset of synapses on the somata and dendrites, but in most synapses on axon initial segments innervated by axo-axonic cells. The results demonstrate that molecular specialization in the composition of postsynaptic GABAA receptor subunits parallels GABAergic cell specialization in targeting synapses to a specific domain of postsynaptic cortical neurons.

Phosphorylation of synaptic vesicle proteins: modulation of the alpha SNAP interaction with the core complex.

We analyzed whether synaptic membrane trafficking proteins are substrates for casein kinase II, calcium/calmodulin-dependent protein kinase II, and cAMP-dependent protein kinase (PKA), three kinases implicated in the modulation of synaptic transmission. Each kinase phosphorylates a specific set of the vesicle proteins syntaxin 1A, N-ethylmaleimide-sensitive factor (NSF), vesicle-associated membrane protein (VAMP), synaptosome-associated 25-kDa protein (SNAP-25), n-sec1, alpha soluble NSF attachment protein (alpha SNAP), and synaptotagmin. VAMP is phosphorylated by calcium/calmodulin-dependent protein kinase II on serine 61. alpha SNAP is phosphorylated by PKA; however, the beta SNAP isoform is phosphorylated only 20% as efficiently. alpha SNAP phosphorylated by PKA binds to the core docking and fusion complex 10 times weaker than the dephosphorylated form. These studies provide a first glimpse at regulatory events that may be important in modulating neurotransmitter release during learning and memory.

Cooperative interactions in the induction of long-term potentiation and depression of synaptic excitation between hippocampal CA3-CA1 cell pairs in vitro.

The requirement for cooperative interactions between multiple synaptic inputs in the induction of long-term potentiation (LTP) and long-term depression (LTD) has been tested at Schaffer collateral synapses with paired recordings from monosynaptically coupled CA3-CA1 cell pairs in rat hippocampal slice cultures. Tetanization of single presynaptic neurons at 50 Hz (repeated 5-7 times for 300-500 ms each) induced only a transient potentiation (< 3 min) of excitatory postsynaptic potentials (EPSPs). Persistent potentiation (> 15 min) was induced only when single presynaptic action potentials were synchronously paired with directly induced postsynaptic depolarizing pulses (repeated 50-100 times). Tetanus-induced potentiation of extracellularly evoked EPSPs lasting > 4 min could only be obtained if the EPSP was > 4 mV. Because unitary EPSP amplitudes average approximately 1 mV, we conclude that high-frequency discharge must occur synchronously] in 4-5 CA3 cells for LTP to be induced in a common postsynaptic CA1 cell. Asynchronous pairing of presynaptic action potentials with postsynaptic depolarizing current pulses (preceding each EPSP by 800 ms) depressed both naive and previously potentiated unitary EPSPs. Likewise, homosynaptic LTD of unitary EPSPs was induced when the presynaptic cell was tetanized at 3 Hz for 3 min, regardless of their amplitude (0.3-3.2 mV). Homosynaptic LTD of extracellularly evoked Schaffer collateral EPSPs < 4 mV could be induced if no inhibitory postsynaptic potential was apparent, but was prevented by eliciting a large inhibitory postsynaptic potential or by injection of hyperpolarizing current in the postsynaptic cell. We conclude that cooperative interactions among multiple excitatory inputs are not required for induction of homosynaptic LTD of unitary EPSPs.

Operant conditioning of H-reflex changes synaptic terminals on primate motoneurons.

Operant conditioning of the primate triceps surae H-reflex, the electrical analog of the spinal stretch reflex, creates a memory trace that includes changes in the spinal cord. To define the morphological correlates of this plasticity, we analyzed the synaptic terminal coverage of triceps surae motoneurons from animals in which the triceps surae H-reflex in one leg had been increased (HRup mode) or decreased (HRdown mode) by conditioning and compared them to each other and to motoneurons from unconditioned animals. Motoneurons were labeled by intramuscular injection of cholera toxin-horseradish peroxidase. A total of 5055 terminals on the cell bodies and proximal dendrites of 114 motoneurons from 14 animals were studied by electron microscopy. Significant differences were found between HRup and HRdown animals and between HRup and naive (i.e., unconditioned) animals. F terminals (i.e., putative inhibitory terminals) were smaller and their active zone coverage on the cell body was lower on motoneurons from the conditioned side of HRup animals than on motoneurons from the conditioned side of HRdown animals. C terminals (i.e., terminals associated with postsynaptic cisterns and rough endoplasmic reticulum) were smaller and the number of C terminals in each C complex (i.e., a group of contiguous C terminals) was larger on motoneurons from the conditioned side of HRup animals than on motoneurons either from the conditioned side of HRdown animals or from naive animals. Because the treatment of HRup and HRdown animals differed only in the reward contingency, the results imply that the two contingencies had different effects on motoneuron synaptic terminals. In combination with other recent data, they show that H-reflex conditioning produces a complex pattern of spinal cord plasticity that includes changes in motoneuron physiological properties as well as in synaptic terminals. Further delineation of this pattern should reveal the contribution of the structural changes described here to the learned change in behavior.

Long-term modifications of synaptic efficacy in the human inferior and middle temporal cortex.

The primate temporal cortex has been demonstrated to play an important role in visual memory and pattern recognition. It is of particular interest to investigate whether activity-dependent modification of synaptic efficacy, a presumptive mechanism for learning and memory, is present in this cortical region. Here we address this issue by examining the induction of synaptic plasticity in surgically resected human inferior and middle temporal cortex. The results show that synaptic strength in the human temporal cortex could undergo bidirectional modifications, depending on the pattern of conditioning stimulation. High frequency stimulation (100 or 40 Hz) in layer IV induced long-term potentiation (LTP) of both intracellular excitatory postsynaptic potentials and evoked field potentials in layers II/III. The LTP induced by 100 Hz tetanus was blocked by 50-100 microM DL-2-amino-5-phosphonovaleric acid, suggesting that N-methyl-D-aspartate receptors were responsible for its induction. Long-term depression (LTD) was elicited by prolonged low frequency stimulation (1 Hz, 15 min). It was reduced, but not completely blocked, by DL-2-amino-5-phosphonovaleric acid, implying that some other mechanisms in addition to N-methyl-DL-aspartate receptors were involved in LTD induction. LTD was input-specific, i.e., low frequency stimulation of one pathway produced LTD of synaptic transmission in that pathway only. Finally, the LTP and LTD could reverse each other, suggesting that they can act cooperatively to modify the functional state of cortical network. These results suggest that LTP and LTD are possible mechanisms for the visual memory and pattern recognition functions performed in the human temporal cortex.

Miniature endplate current rise times less than 100 microseconds from improved dual recordings can be modeled with passive acetylcholine diffusion from a synaptic vesicle.

We recorded miniature endplate currents (mEPCs) using simultaneous voltage clamp and extracellular methods, allowing correction for time course measurement errors. We obtained a 20-80% rise time (tr) of approximately 80 micros at 22 degrees C, shorter than any previously reported values, and tr variability (SD) with an upper limit of 25-30 micros. Extracellular electrode pressure can increase tr and its variability by 2- to 3-fold. Using Monte Carlo simulations, we modeled passive acetylcholine diffusion through a vesicle fusion pore expanding radially at 25 nm x ms(-1) (rapid, from endplate omega figure appearance) or 0.275 nm x ms(-1) (slow, from mast cell exocytosis). Simulated mEPCs obtained with rapid expansion reproduced tr and the overall shape of our experimental mEPCs, and were similar to simulated mEPCs obtained with instant acetylcholine release. We conclude that passive transmitter diffusion, coupled with rapid expansion of the fusion pore, is sufficient to explain the time course of experimentally measured synaptic currents with trs of less than 100 micros.

The timing of synaptic vesicle endocytosis.

Alternative models to describe the endocytosis phase of synaptic vesicle recycling are associated with time scales of vesicle recovery ranging from milliseconds to tens of seconds. There have been suggestions that one of the major models, envisioned as a slow process that occurs only after complete fusion of the vesicle membrane with the neurolemma, might be applicable only under conditions of heavy, nonphysiological stimulation. Using FM 1-43 and similar fluorescent probes to label recycling synaptic vesicles in rat hippocampal neurons, we have measured the kinetics of endocytosis with a wide range of action-potential-driven exocytotic loads. Our results indicate that when either 5% or 25% of the vesicle pool is used, vesicles are recovered with a half-time on the order of 20 s (24 degrees C). This endocytosis rate was not influenced by operations designed to alter intracellular Ca2+ during membrane retrieval, suggesting that residual Ca2+ after strong stimuli probably does not greatly retard endocytosis. Finally, we have shown that vesicle-destaining kinetics are not strongly influenced by the substantially differing rates at which two marker dyes tested dissociate from membranes. This observation suggests that vesicles remain open long enough for essentially complete dissociation of even the slower dye (a few seconds) or, alternatively, that both dyes readily escape vesicle membrane by lateral diffusion through any exocytotic opening. These data seem most consistent with applicability of the slow-endocytosis, complete-fusion model at low as well as high levels of exocytosis.

Glutamate receptor blockade at cortical synapses disrupts development of thalamocortical and columnar organization in somatosensory cortex.

The segregation of thalamocortical inputs into eye-specific stripes in the developing cat or monkey visual cortex is prevented by manipulations that perturb or abolish neural activity in the visual pathway. Such findings show that proper development of the functional organization of visual cortex is dependent on normal patterns of neural activity. The generalisation of this conclusion to other sensory cortices has been questioned by findings that the segregation of thalamocortical afferents into a somatotopic barrel pattern in developing rodent primary somatosensory cortex (S1) is not prevented by activity blockade. We show that a temporary block of N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors in rat S1 during the critical period for barrel development disrupts the topographic refinement of thalamocortical connectivity and columnar organization. These effects are evident well after the blockade is ineffective and thus may be permanent. Our findings show that neural activity and specifically the activation of postsynaptic cortical neurons has a prominent role in establishing the primary sensory map in S1, as well as the topographic organization of higher order synaptic connections.

A presynaptic locus for long-term potentiation of elementary synaptic transmission at mossy fiber synapses in culture.

The complex circuitry of the CA3 region and the abundance of collateral connections has made it difficult to study the mossy fiber pathway in hippocampal slices and therefore to establish the site of expression of long-term potentiation at these synapses. Using a novel cell culture system, we have produced long-term potentiation of the elementary synaptic connections on single CA3 pyramidal neurons following tetanic stimulation of individual dentate gyrus granule cells. As is the case for the hippocampal slice, this potentiation was independent of N-methyl-D-aspartate receptor activation, was simulated by application of forskolin, and its induction did not require any modulatory input. The increase in synaptic strength was accompanied by a reduction in the number of failures of transmission and by an increase in the coefficient of variation of the responses and was prevented by presynaptic injection of an inhibitor of protein kinase A. These findings show that mossy fiber long-term potentiation has a presynaptic locus and that its expression is dependent on protein kinase A.

Synaptophysin, a major synaptic vesicle protein, is not essential for neurotransmitter release.

Synaptophysin (syp I) is a synaptic vesicle membrane protein that constitutes approximately 7% of the total vesicle protein. Multiple lines of evidence implicate syp I in a number of nerve terminal functions. To test these, we have disrupted the murine Syp I gene. Mutant mice lacking syp I were viable and fertile. No changes in the structure and protein composition of the mutant brains were observed except for a decrease in synaptobrevin/VAMP II. Synaptic transmission was normal with no detectable changes in synaptic plasticity or the probability of release. Our data demonstrate that one of the major synaptic vesicle membrane proteins is not essential for synaptic transmission, suggesting that its function is either redundant or that it has a more subtle function not apparent in the assays used.