Postsynaptic Mechanisms Render Syn I/II/III Mice Highly Responsive to Psychostimulants.

BACKGROUND: Synapsins are encoded by SYN I, SYN II, and SYN III, and they regulate neurotransmitter release by maintaining a reserve pool of synaptic vesicles. METHODS: Presynaptic dopamine responses to cocaine were examined by microdialysis, and postsynaptic responses were evaluated to various dopamine receptor agonists in the open field with SynI/SynII/SynIII triple knockout mice. RESULTS: Triple knockout mice showed enhanced spontaneous locomotion in a novel environment and were hyper-responsive to indirect and direct D1 and D2 dopamine agonists. Triple knockout animals appeared sensitized to cocaine upon first open field exposure; sensitization developed across days in wild-type controls. When mutants were preexposed to a novel environment before injection, cocaine-stimulated locomotion was reduced and behavioral sensitization retarded. Baseline dopamine turnover was enhanced in mutants and novel open field exposure increased their striatal dopamine synthesis rates. As KCl-depolarization stimulated comparable dopamine release in both genotypes, their readily releasable pools appeared indistinguishable. Similarly, cocaine-induced hyperlocomotion was indifferent to blockade of newly synthesized dopamine and depletion of releasable dopamine pools. Extracellular dopamine release was similar in wild-type and triple knockout mice preexposed to the open field and given cocaine or placed immediately into the arena following injection. Since motor effects to novelty and psychostimulants depend upon frontocortical-striatal inputs, we inhibited triple knockout medial frontal cortex with GABA agonists. Locomotion was transiently increased in cocaine-injected mutants, while their supersensitive cocaine response to novelty was lost. CONCLUSIONS: These results reveal presynaptic dopamine release is not indicative of agonist-induced triple knockout hyperlocomotion. Instead, their novelty response occurs primarily through postsynaptic mechanisms and network effects.

Growth and excitability at synapsin II deficient hippocampal neurons.

Synapsins are neuronal phosphoproteins that fine-tune synaptic transmission and suppress seizure activity. Synapsin II (SynII) deletion produces epileptic seizures and overexcitability in neuronal networks. Early studies in primary neuronal cultures have shown that SynII deletion results in a delay in synapse formation. More recent studies at hippocampal slices have revealed increased spontaneous activity in SynII knockout (SynII(-)) mice. To reconcile these observations, we systematically re-examined synaptic transmission, synapse formation, and neurite growth in primary hippocampal neuronal cultures. We find that spontaneous glutamatergic synaptic activity was suppressed in SynII(-) neurons during the initial developmental epoch (7 days in vitro, DIV) but was enhanced at later times (12 and18 DIV). The density of synapses, transmission between connected pairs of neurons, and the number of docked synaptic vesicles were not affected by SynII deletion. However, we found that neurite outgrowth in SynII(-) neurons was suppressed during the initial developmental epoch (7 DIV) but enhanced at subsequent developmental stages (12 and18 DIV). This finding can account for the observed effect of SynII deletion on synaptic activity. To test whether the observed phenotype resulted directly from the deletion of SynII we expressed SynII in SynII(-) cultures using an adeno-associated virus (AAV). Expression of SynII at 2 DIV rescued the SynII deletion-dependent alterations in both synaptic activity and neuronal growth. To test whether the increased neurite outgrowth in SynII(-) observed at DIV 12 and18 represents an overcompensation for the initial developmental delay or results directly from SynII deletion we performed "late expression" experiments, transfecting SynII(-) cultures with AAV at 7 DIV. The late SynII expression suppressed neurite outgrowth at 12 and 18 DIV to the levels observed in control neurons, suggesting that these phenotypes directly depend on SynII. These results reveal a novel developmentally regulated role for SynII function in the control of neurite growth.

Synapsin III deficiency hampers α-synuclein aggregation, striatal synaptic damage and nigral cell loss in an AAV-based mouse model of Parkinson's disease.

Parkinson's disease (PD), the most common neurodegenerative movement disorder, is characterized by the progressive loss of nigral dopamine neurons. The deposition of fibrillary aggregated α-synuclein in Lewy bodies (LB), that is considered to play a causative role in the disease, constitutes another key neuropathological hallmark of PD. We have recently described that synapsin III (Syn III), a synaptic phosphoprotein that regulates dopamine release in cooperation with α-synuclein, is present in the α-synuclein insoluble fibrils composing the LB of patients affected by PD. Moreover, we observed that silencing of Syn III gene could prevent α-synuclein fibrillary aggregation in vitro. This evidence suggests that Syn III might be crucially involved in α-synuclein pathological deposition. To test this hypothesis, we studied whether mice knock-out (ko) for Syn III might be protected from α-synuclein aggregation and nigrostriatal neuron degeneration resulting from the unilateral injection of adeno-associated viral vectors (AAV)-mediating human wild-type (wt) α-synuclein overexpression (AAV-hαsyn). We found that Syn III ko mice injected with AAV-hαsyn did not develop fibrillary insoluble α-synuclein aggregates, showed reduced amount of α-synuclein oligomers detected by in situ proximity ligation assay (PLA) and lower levels of Ser129-phosphorylated α-synuclein. Moreover, the nigrostriatal neurons of Syn III ko mice were protected from both synaptic damage and degeneration triggered by the AAV-hαsyn injection. Our observations indicate that Syn III constitutes a crucial mediator of α-synuclein aggregation and toxicity and identify Syn III as a novel therapeutic target for PD.

The Knockout of Synapsin II in Mice Impairs Social Behavior and Functional Connectivity Generating an ASD-like Phenotype.

Autism spectrum disorders (ASD) and epilepsy are neurodevelopmental conditions that appear with high rate of co-occurrence, suggesting the possibility of a common genetic basis. Mutations in Synapsin (SYN) genes, particularly SYN1 and SYN2, have been recently associated with ASD and epilepsy in humans. Accordingly, mice lacking Syn1 or Syn2, but not Syn3, experience epileptic seizures and display autistic-like traits that precede the onset of seizures. Here, we analyzed social behavior and ultrasonic vocalizations emitted in 2 social contexts by SynI, SynII, or SynIII mutants and show that SynII mutants display the most severe ASD-like phenotype. We also show that the behavioral SynII phenotype correlates with a significant decrease in auditory and hippocampal functional connectivity as measured with resting state functional magnetic resonance imaging (rsfMRI). Taken together, our results reveal a permissive contribution of Syn2 to the expression of normal socio-communicative behavior, and suggest that Syn2-mediated synaptic dysfunction can lead to ASD-like behavior through dysregulation of cortical connectivity.

Hsc70 chaperone activity is required for the cytosolic slow axonal transport of synapsin.

Soluble cytosolic proteins vital to axonal and presynaptic function are synthesized in the neuronal soma and conveyed via slow axonal transport. Our previous studies suggest that the overall slow transport of synapsin is mediated by dynamic assembly/disassembly of cargo complexes followed by short-range vectorial transit (the "dynamic recruitment" model). However, neither the composition of these complexes nor the mechanistic basis for the dynamic behavior is understood. In this study, we first examined putative cargo complexes associated with synapsin using coimmunoprecipitation and multidimensional protein identification technology mass spectrometry (MS). MS data indicate that synapsin is part of a multiprotein complex enriched in chaperones/cochaperones including Hsc70. Axonal synapsin-Hsc70 coclusters are also visualized by two-color superresolution microscopy. Inhibition of Hsc70 ATPase activity blocked the slow transport of synapsin, disrupted axonal synapsin organization, and attenuated Hsc70-synapsin associations, advocating a model where Hsc70 activity dynamically clusters cytosolic proteins into cargo complexes, allowing transport. Collectively, our study offers insight into the molecular organization of cytosolic transport complexes and identifies a novel regulator of slow transport.

Subconvulsant doses of pentylenetetrazol uncover the epileptic phenotype of cultured synapsin-deficient Helix serotonergic neurons in the absence of excitatory and inhibitory inputs.

Synapsins are a family of presynaptic proteins related to several processes of synaptic functioning. A variety of reports have linked mutations in synapsin genes with the development of epilepsy. Among the proposed mechanisms, a main one is based on the synapsin-mediated imbalance towards network hyperexcitability due to differential effects on neurotransmitter release in GABAergic and glutamatergic synapses. Along this line, a non-synaptic effect of synapsin depletion increasing neuronal excitability has recently been described in Helix neurons. To further investigate this issue, we examined the effect of synapsin knock-down on the development of pentylenetetrazol (PTZ)-induced epileptic-like activity using single neurons or isolated monosynaptic circuits reconstructed on microelectrode arrays (MEAs). Compared to control neurons, synapsin-silenced neurons showed a lower threshold for the development of epileptic-like activity and prolonged periods of activity, together with the occurrence of spontaneous firing after recurrent PTZ-induced epileptic-like activity. These findings highlight the crucial role of synapsin on neuronal excitability regulation in the absence of inhibitory or excitatory inputs.

Knock-down of synapsin alters cell excitability and action potential waveform by potentiating BK and voltage-gated Ca(2+) currents in Helix serotonergic neurons.

Synapsins (Syns) are an evolutionarily conserved family of presynaptic proteins crucial for the fine-tuning of synaptic function. A large amount of experimental evidences has shown that Syns are involved in the development of epileptic phenotypes and several mutations in Syn genes have been associated with epilepsy in humans and animal models. Syn mutations induce alterations in circuitry and neurotransmitter release, differentially affecting excitatory and inhibitory synapses, thus causing an excitation/inhibition imbalance in network excitability toward hyperexcitability that may be a determinant with regard to the development of epilepsy. Another approach to investigate epileptogenic mechanisms is to understand how silencing Syn affects the cellular behavior of single neurons and is associated with the hyperexcitable phenotypes observed in epilepsy. Here, we examined the functional effects of antisense-RNA inhibition of Syn expression on individually identified and isolated serotonergic cells of the Helix land snail. We found that Helix synapsin silencing increases cell excitability characterized by a slightly depolarized resting membrane potential, decreases the rheobase, reduces the threshold for action potential (AP) firing and increases the mean and instantaneous firing rates, with respect to control cells. The observed increase of Ca(2+) and BK currents in Syn-silenced cells seems to be related to changes in the shape of the AP waveform. These currents sustain the faster spiking in Syn-deficient cells by increasing the after hyperpolarization and limiting the Na(+) and Ca(2+) channel inactivation during repetitive firing. This in turn speeds up the depolarization phase by reaching the AP threshold faster. Our results provide evidence that Syn silencing increases intrinsic cell excitability associated with increased Ca(2+) and Ca(2+)-dependent BK currents in the absence of excitatory or inhibitory inputs.

Alterations in Brain Inflammation, Synaptic Proteins, and Adult Hippocampal Neurogenesis during Epileptogenesis in Mice Lacking Synapsin2.

Synapsins are pre-synaptic vesicle-associated proteins linked to the pathogenesis of epilepsy through genetic association studies in humans. Deletion of synapsins causes an excitatory/inhibitory imbalance, exemplified by the epileptic phenotype of synapsin knockout mice. These mice develop handling-induced tonic-clonic seizures starting at the age of about 3 months. Hence, they provide an opportunity to study epileptogenic alterations in a temporally controlled manner. Here, we evaluated brain inflammation, synaptic protein expression, and adult hippocampal neurogenesis in the epileptogenic (1 and 2 months of age) and tonic-clonic (3.5-4 months) phase of synapsin 2 knockout mice using immunohistochemical and biochemical assays. In the epileptogenic phase, region-specific microglial activation was evident, accompanied by an increase in the chemokine receptor CX3CR1, interleukin-6, and tumor necrosis factor-α, and a decrease in chemokine keratinocyte chemoattractant/ growth-related oncogene. Both post-synaptic density-95 and gephyrin, scaffolding proteins at excitatory and inhibitory synapses, respectively, showed a significant up-regulation primarily in the cortex. Furthermore, we observed an increase in the inhibitory adhesion molecules neuroligin-2 and neurofascin and potassium chloride co-transporter KCC2. Decreased expression of γ-aminobutyric acid receptor-δ subunit and cholecystokinin was also evident. Surprisingly, hippocampal neurogenesis was reduced in the epileptogenic phase. Taken together, we report molecular alterations in brain inflammation and excitatory/inhibitory balance that could serve as potential targets for therapeutics and diagnostic biomarkers. In addition, the regional differences in brain inflammation and synaptic protein expression indicate an epileptogenic zone from where the generalized seizures in synapsin 2 knockout mice may be initiated or spread.

Phosphorylation of synapsin I by cyclin-dependent kinase-5 sets the ratio between the resting and recycling pools of synaptic vesicles at hippocampal synapses.

Cyclin-dependent kinase-5 (Cdk5) was reported to downscale neurotransmission by sequestering synaptic vesicles (SVs) in the release-reluctant resting pool, but the molecular targets mediating this activity remain unknown. Synapsin I (SynI), a major SV phosphoprotein involved in the regulation of SV trafficking and neurotransmitter release, is one of the presynaptic substrates of Cdk5, which phosphorylates it in its C-terminal region at Ser(549) (site 6) and Ser(551) (site 7). Here we demonstrate that Cdk5 phosphorylation of SynI fine tunes the recruitment of SVs to the active recycling pool and contributes to the Cdk5-mediated homeostatic responses. Phosphorylation of SynI by Cdk5 is physiologically regulated and enhances its binding to F-actin. The effects of Cdk5 inhibition on the size and depletion kinetics of the recycling pool, as well as on SV distribution within the nerve terminal, are virtually abolished in mouse SynI knock-out (KO) neurons or in KO neurons expressing the dephosphomimetic SynI mutants at sites 6,7 or site 7 only. The observation that the single site-7 mutant phenocopies the effects of the deletion of SynI identifies this site as the central switch in mediating the synaptic effects of Cdk5 and demonstrates that SynI is necessary and sufficient for achieving the effects of the kinase on SV trafficking. The phosphorylation state of SynI by Cdk5 at site 7 is regulated during chronic modification of neuronal activity and is an essential downstream effector for the Cdk5-mediated homeostatic scaling.

Epileptic synapsin triple knockout mice exhibit progressive long-term aberrant plasticity in the entorhinal cortex.

Studying epileptogenesis in a genetic model can facilitate the identification of factors that promote the conversion of a normal brain into one chronically prone to seizures. Synapsin triple-knockout (TKO) mice exhibit adult-onset epilepsy, thus allowing the characterization of events as preceding or following seizure onset. Although it has been proposed that a congenital reduction in inhibitory transmission is the underlying cause for epilepsy in these mice, young TKO mice are asymptomatic. We report that the genetic lesion exerts long-term progressive effects that extend well into adulthood. Although inhibitory transmission is initially reduced, it is subsequently strengthened relative to its magnitude in control mice, so that the excitation to inhibition balance in adult TKOs is inverted in favor of inhibition. In parallel, we observed long-term alterations in synaptic depression kinetics of excitatory transmission and in the extent of tonic inhibition, illustrating adaptations in synaptic properties. Moreover, age-dependent acceleration of the action potential did not occur in TKO cortical pyramidal neurons, suggesting wide-ranging secondary changes in brain excitability. In conclusion, although congenital impairments in inhibitory transmission may initiate epileptogenesis in the synapsin TKO mice, we suggest that secondary adaptations are crucial for the establishment of this epileptic network.

Synapsin II and Rab3a cooperate in the regulation of epileptic and synaptic activity in the CA1 region of the hippocampus.

Some forms of idiopathic epilepsy in animals and humans are associated with deficiency of synapsin, a phosphoprotein that reversibly associates with synaptic vesicles. We have previously shown that the epileptic phenotype seen in synapsin II knock-out mice (SynII(-)) can be rescued by the genetic deletion of the Rab3a protein. Here we have examined the cellular basis for this rescue using whole-cell recordings from CA1 hippocampal pyramidal cells in brain slices. We find that SynII(-) neurons have increased spontaneous activity and a reduced threshold for the induction of epileptiform activity by 4-aminopyridine (4-AP). Using selective recordings of glutamatergic and GABAergic activity we show that in wild-type neurons low concentrations of 4-AP facilitate glutamatergic and GABAergic transmission in a balanced way, whereas in SynII(-) neurons this balance is shifted toward excitation. This imbalance reflects a deficit in inhibitory synaptic transmission that appears to be secondary to reduced Ca(2+) sensitivity in SynII(-) neurons. This suggestion is supported by our finding that synaptic and epileptiform activity at SynII(-) and wild-type synapses is similar when GABAergic transmission is blocked. Deletion of Rab3a results in glutamatergic synapses that have a compromised responsiveness to either low 4-AP concentrations or elevated extracellular Ca(2+). These changes mitigate the overexcitable phenotype observed in SynII(-) neurons. Thus, Rab3a deletion appears to restore the excitatory/inhibitory imbalance observed in SynII(-) hippocampal slices indirectly, not by correcting the deficit in GABAergic synaptic transmission but rather by impairing excitatory glutamatergic synaptic transmission.

Sensitive and critical periods in the development of handling induced seizures in mice lacking synapsins: differences between synapsin I and synapsin II knockouts.

Mice lacking either synapsin I or synapsin II develop handling induced seizures from around two months of age. In mice lacking synapsin I (synapsin 1 knock-out mice, Syn1KO mice) such seizures can either consist of mild myoclonic jerks or of fully developed generalized tonic-clonic seizures, and the two seizure types are quite evenly distributed. In mice lacking synapsin II (synapsin 2 knock-out mice, Syn2KO mice) all seizures are in the form of generalized tonic-clonic seizures. Through the use of specialized animal rearing procedures whereby human-animal interaction was minimized (minimal handling procedures), this study investigated effects of handling also prior to the emergence of actual seizures. The effect of minimal handling procedures was significant in both genotypes, but most pronounced in Syn1KO mice. In this genotype, minimal handling reduced the frequency of mild seizures, and completely eliminated generalized tonic-clonic seizures when the animals were tested with regular handling at 4 1/2 months of age. Neither seizure frequency nor generalized tonic-clonic seizures could be re-established through regular handling from 4 1/2 to 8 months. This suggests that the period up to 4 1/2 months constitute a sensitive period for seizures in general, and a critical period for generalized tonic-clonic seizures in this genotype. In Syn2KO mice minimal handling did not remove generalized tonic-clonic seizures, as such seizures were present when handling was introduced at 4 1/2 months. We found an initial reduction of seizure frequency, but the seizure frequency eventually reached levels seen in mice kept under regular handling regimes. Thus, it is unlikely that the period up to 4 1/2 months is a sensitive period in the Syn2KO genotype.

Synaptic and extrasynaptic origin of the excitation/inhibition imbalance in the hippocampus of synapsin I/II/III knockout mice.

Synapsins (Syn I, Syn II, and Syn III) are a family of synaptic vesicle phosphoproteins regulating synaptic transmission and plasticity. SYN1/2 genes have been identified as major epilepsy susceptibility genes in humans and synapsin I/II/III triple knockout (TKO) mice are epileptic. However, excitatory and inhibitory synaptic transmission and short-term plasticity have never been analyzed in intact neuronal circuits of TKO mice. To clarify the generation and expression of the epileptic phenotype, we performed patch-clamp recordings in the CA1 region of acute hippocampal slices from 1-month-old presymptomatic and 6-month-old epileptic TKO mice and age-matched controls. We found a strong imbalance between basal glutamatergic and γ-aminobutyric acid (GABA)ergic transmission with increased evoked excitatory postsynaptic current and impaired evoked inhibitory postsynaptic current amplitude. This imbalance was accompanied by a parallel derangement of short-term plasticity paradigms, with enhanced facilitation of glutamatergic transmission in the presymptomatic phase and milder depression of inhibitory synapses in the symptomatic phase. Interestingly, a lower tonic GABA(A) current due to the impaired GABA release is responsible for the more depolarized resting potential found in TKO CA1 neurons, which makes them more susceptible to fire. All these changes preceded the appearance of epilepsy, indicating that the distinct changes in excitatory and inhibitory transmission due to the absence of Syns initiate the epileptogenic process.

Temporal evolution of neurophysiological and behavioral features of synapsin I/II/III triple knock-out mice.

Deletion of one or more synapsin genes in mice results in a spontaneous epilepsy. In these animals, seizures can be evoked by opening or moving the cage. Aim of the present study was to characterize the evolution of the epileptic phenotype by neurophysiological examination and behavioral observation in synapsin triple knock-out (Syn-TKO) mice. Syn-TKO mice were studied from 20 postnatal days (PND) up to 6 months of age by video-EEG recording and behavioral observation. Background EEG spectral analysis was performed and data were compared to WT animals. Syn-TKO revealed rare spontaneous seizures and increased susceptibility to evoked seizures in mice from 60 to 100 PND. Spontaneous and evoked seizures presented similar duration and morphology. At times, seizures were followed by a post-ictal phase characterized by a 4 Hz rhythmic activity and immobility of the animal. Spectral analysis of background EEG evidenced a slowing of the theta-alpha peak in Syn-TKO mice compared to WT mice within the period from PND 40 to 100. These data indicate that Syn-TKO mice do not exhibit a linear progression of the epileptic phenotype, with the period corresponding to a higher susceptibility to evoked seizures characterized by background EEG slowing. This aspect might be connected to brain dysfunction often associated to epilepsy in the interictal period.

Synapsins contribute to the dynamic spatial organization of synaptic vesicles in an activity-dependent manner.

The precise subcellular organization of synaptic vesicles (SVs) at presynaptic sites allows for rapid and spatially restricted exocytotic release of neurotransmitter. The synapsins (Syns) are a family of presynaptic proteins that control the availability of SVs for exocytosis by reversibly tethering them to each other and to the actin cytoskeleton in a phosphorylation-dependent manner. Syn ablation leads to reduction in the density of SV proteins in nerve terminals and increased synaptic fatigue under high-frequency stimulation, accompanied by the development of an epileptic phenotype. We analyzed cultured neurons from wild-type and Syn I,II,III(-/-) triple knock-out (TKO) mice and found that SVs were severely dispersed in the absence of Syns. Vesicle dispersion did not affect the readily releasable pool of SVs, whereas the total number of SVs was considerably reduced at synapses of TKO mice. Interestingly, dispersion apparently involved exocytosis-competent SVs as well; it was not affected by stimulation but was reversed by chronic neuronal activity blockade. Altogether, these findings indicate that Syns are essential to maintain the dynamic structural organization of synapses and the size of the reserve pool of SVs during intense SV recycling, whereas an additional Syn-independent mechanism, whose molecular substrate remains to be clarified, targets SVs to synaptic boutons at rest and might be outpaced by activity.

Site-specific synapsin I phosphorylation participates in the expression of post-tetanic potentiation and its enhancement by BDNF.

A large amount of experimental evidence has highlighted the rapid changes in synaptic efficacy induced by high-frequency stimulation and BDNF at central excitatory synapses. We clarified the quantal mechanisms and the involvement of Synapsin I (SynI) phosphorylation in the expression of post-tetanic potentiation (PTP) and in its modulation by BDNF in mouse glutamatergic autapses. We found that PTP is associated with an elevation in the probability of release and a concomitant increase in the size of the readily releasable pool (RRP). The latter component was virtually absent in SynI knock-out (KO) neurons, which indeed displayed impaired PTP. PTP was fully rescued by the expression of wild-type SynI, but not of its dephosphomimetic mutants in the phosphorylation sites for cAMP-dependent protein kinase and Ca²âº/calmodulin-dependent protein kinases I/II. BDNF potently enhanced PTP through a further increase in the RRP size, which was missing in SynI KO neurons. In these neurons, the BDNF-induced PTP enhancement was rescued by the expression of wild-type SynI, but not of its dephosphomimetic mutant at the mitogen-dependent protein kinase sites. The results indicate that the increase in RRP size necessary for the full expression of PTP, and its sensitivity to BDNF, involve phosphorylation of SynI at distinct sites, thus implicating SynI as an essential downstream effector for the expression of PTP and for its enhancement by BDNF.

Synapsin selectively controls the mobility of resting pool vesicles at hippocampal terminals.

Presynaptic terminals are specialized sites for information transmission where vesicles fuse with the plasma membrane and are locally recycled. Recent work has extended this classical view, with the observation that a subset of functional vesicles is dynamically shared between adjacent terminals by lateral axonal transport. Conceptually, such transport would be expected to disrupt vesicle retention around the active zone, yet terminals are characterized by a high-density vesicle cluster, suggesting that counteracting stabilizing mechanisms must operate against this tendency. The synapsins are a family of proteins that associate with synaptic vesicles and determine vesicle numbers at the terminal, but their specific function remains controversial. Here, using multiple quantitative fluorescence-based approaches and electron microscopy, we show that synapsin is instrumental for resisting vesicle dispersion and serves as a regulatory element for controlling lateral vesicle sharing between synapses. Deleting synapsin disrupts the organization of presynaptic vesicle clusters, making their boundaries hard to define. Concurrently, the fraction of vesicles amenable to transport is increased, and more vesicles are translocated to the axon. Importantly, in neurons from synapsin knock-out mice the resting and recycling pools are equally mobile. Synapsin, when present, specifically restricts the mobility of resting pool vesicles without affecting the division of vesicles between these pools. Specific expression of synapsin IIa, the sole isoform affecting synaptic depression, rescues the knock-out phenotype. Together, our results show that synapsin is pivotal for maintaining synaptic vesicle cluster integrity and that it contributes to the regulated sharing of vesicles between terminals.

Neuroethologically delineated differences in the seizure behavior of synapsin 1 and synapsin 2 knock-out mice.

The highly homologous nerve terminal phosphoproteins synapsin I and synapsin II have been linked to the pathogenesis of epilepsy through associations between synapsin gene mutations and epileptic disease in humans and to the observation of handling induced seizures in mice genetically depleted of one or both of these proteins. Whereas seizure behavior in mice lacking both synapsin I and synapsin II is well characterized, the seizure behavior in mice lacking either is less well studied. Through so called neuroethologically based analyses of fully established seizure behavior in Synapsin 1 and 2 knock-out mice (Syn1KO and Syn2KO mice) aged 4 1/2 months, this study reveals significant differences in the seizure behavior of the two genotypes: whereas Syn1KO mice show both partial and generalized forebrain seizure activity, Syn2KO mice show only fully generalized forebrain seizures. Analysis of seizure behavior at earlier stages shows that the mature seizure pattern in Syn2KO mice establishes rapidly from the age of ∼2 months, when Syn1KO partial seizures are rare, and Syn1KO generalized seizures are almost absent. The specific behavioral phenotypes of the two strains suggest that the slight differences in structure, function and expression of these highly related proteins could be important factors during seizure generating neural activity.

A new kinetic framework for synaptic vesicle trafficking tested in synapsin knock-outs.

At least two rate-limiting mechanisms in vesicle trafficking operate at mouse Schaffer collateral synapses, but their molecular/physical identities are unknown. The first mechanism determines the baseline rate at which reserve vesicles are supplied to a readily releasable pool. The second causes the supply rate to depress threefold when synaptic transmission is driven hard for extended periods. Previous models invoked depletion of a reserve vesicle pool to explain the reductions in the supply rate, but the mass-action assumption at their core is not compatible with kinetic measurements of neurotransmission under maximal-use conditions. Here we develop a new theoretical model of rate-limiting steps in vesicle trafficking that is compatible with previous and new measurements. A physical interpretation is proposed where local reserve pools consisting of four vesicles are tethered to individual release sites and are replenished stochastically in an all-or-none fashion. We then show that the supply rate depresses more rapidly in synapsin knock-outs and that the phenotype can be fully explained by changing the value of the single parameter in the model that would specify the size of the local reserve pools. Vesicle-trafficking rates between pools were not affected. Finally, optical imaging experiments argue against alternative interpretations of the theoretical model where vesicle trafficking is inhibited without reserve pool depletion. This new conceptual framework will be useful for distinguishing which of the multiple molecular and cell biological mechanisms involved in vesicle trafficking are rate limiting at different levels of synaptic throughput and are thus candidates for physiological and pharmacological modulation.

Electroencephalographic characterization of seizure activity in the synapsin I/II double knockout mouse.

We present a detailed comparison of the behavioral and electrophysiological development of seizure activity in mice genetically depleted of synapsin I and synapsin II (SynDKO mice), based on combined video and surface EEG recordings. SynDKO mice develop handling-induced epileptic seizures at the age of 2months. The seizures show a very regular behavioral pattern, where activity is initially dominated by truncal muscle contractions followed by various myoclonic elements. Whereas seizure behavior goes through clearly defined transitions, cortical activity as reflected by EEG recordings shows a more gradual development with respect to the emergence of different EEG components and the frequency of these components. No EEG pattern was seen to define a particular seizure behavior. However, myoclonic activity was characterized by more regular patterns of combined sharp waves and spikes. Where countable, the number of myoclonic jerks was significantly correlated to the number of such EEG complexes. Furthermore, some EEG recordings revealed epileptic regular discharges without clear behavioral seizure correlates. Our findings suggest that seizure behavior in SynDKO mice is not solely determined by cortical activity but rather reflects interplay between cortical activity and activity in other brain regions.