GABA has a presynaptic inhibitory effect at Lumbricus terrestris body wall muscle synapses.

Earthworm body wall muscle synapses have been suggested to contain both excitatory and inhibitory inputs, and therefore allow for investigation of excitatory/inhibitory signaling in an easily accessible model system. While previous studies have focused on postsynaptic GABAergic inhibitory mechanisms, this study investigated the hypothesis that GABAergic signaling also has presynaptic inhibitory function. This hypothesis was tested by loading synaptogreen C4 dye (also called FM1-43) into presynaptic vesicles in the presence of GABA at Lumbricus terrestris longitudinal muscle synapses. GABA treatment significantly reduced the fluorescence intensity observed at these synapses, suggesting that GABAergic signaling does indeed have a presynaptic inhibitory mechanism.

Functional distribution of synapsin I in human sperm.

Proteins known to function during cell-cell communication and exocytosis in neurons and other secretory cells have recently been reported in human sperm. Synapsins are a group of proteins that have been very well characterized in neurons, but little is known about synapsin function in other cell types. Based upon previous findings and the known function of synapsin, we tested the hypothesis that synapsin I was present in human sperm. Washed, capacitated, and acrosome induced sperm preparations were used to evaluate the functional distribution of synapsin I using immunocytochemistry. Protein extracts from mouse brain, mouse testis/epididymis, and human semen were used for protein blotting techniques. Immunolocalization revealed synapsin I was enriched in the sperm equatorial segment. Protein extracts from mouse brain, mouse testis/epididymis, and human semen were positive for synapsin I using several different antibodies, and dot blot results were confirmed by Western blot analyses. Finally, treatment of capacitated and acrosome reaction induced samples with anti-synapsin antibodies significantly reduced sperm motility. Localization of synapsin I in human sperm is a novel finding. The association of synapsin I with the sperm equatorial segment and effects on motility are suggestive of a role associated with capacitation and/or acrosome reaction, processes that render sperm capable of fertilizing an oocyte.

Tonotopic organization of the superior olivary nucleus in the chicken auditory brainstem.

Topographic maps are salient features of neuronal organization in sensory systems. Inhibitory components of neuronal circuitry are often embedded within this organization, making them difficult to isolate experimentally. The auditory system provides opportunities to study the topographic organization of inhibitory long-range projection nuclei, such as the superior olivary nucleus (SON). We analyzed the topographic organization of response features of neurons in the SON of chickens. Quantitative methods were developed to assess and communicate this organization. These analyses led to three main conclusions: 1) sound frequency is linearly arranged from dorsal (low frequencies) to ventral (high frequencies) in SON; 2) this tonotopic organization is less precise than the organization of the excitatory nuclei in the chicken auditory brainstem; and 3) neurons with different response patterns to pure tone stimuli are interspersed throughout the SON and show similar tonotopic organizations. This work provides a predictive model to determine the optimal stimulus frequency for a neuron from its spatial location in the SON.

Cooperative regulation of neurotransmitter release by Rab3a and synapsin II.

To understand how the presynaptic proteins synapsin and Rab3a may interact in the regulation of the synaptic vesicle cycle and the release process, we derived a double knockout (DKO) mouse lacking both synapsin II and Rab3a. We found that Rab3a deletion rescued epileptic-like seizures typical for synapsin II gene deleted animals (Syn II(-)). Furthermore, action potential evoked release was drastically reduced in DKO synapses, although spontaneous release remained normal. At low Ca2+ conditions, quantal content was equally reduced in Rab3a(-) and DKO synapses, but as Ca2+ concentration increased, the increase in quantal content was more prominent in Rab3a(-). Electron microscopy analysis revealed that DKO synapses have a combined phenotype, with docked vesicles being reduced similar to Rab3a(-), and intraterminal vesicles being depleted similar to Syn II(-). Consistently, both Syn II(-) and DKO terminals had increased synaptic depression and incomplete recovery. Taken together, our results suggest that synapsin II and Rab3a have separate roles in maintaining the total store of synaptic vesicles and cooperate in promoting the latest steps of neuronal secretion.

Rab3a-mediated vesicle recruitment regulates short-term plasticity at the mouse diaphragm synapse.

Rab3a is a small GTP-binding protein associated with presynaptic vesicles. We have measured the releasable pool in the neuromuscular junction of Rab3a(-) mice by recordings of the asynchronous release activity produced by local applications of hypertonic solutions and demonstrated that the releasable pool is significantly reduced in Rab3a(-) synapses. We found that the activity-dependent vesicle recruitment, as well as the synaptic enhancement associated with it, is disrupted in Rab3a(-) synapses. We employed Ca2+ chelators and disruption of Ca2+ sensitivity of fusion machinery by botulinum neurotoxin type-A microinjections, and demonstrated that local Ca2+ elevation may overcome the Rab3a deficiency in maintaining the releasable pool. Rab3a(-) terminals demonstrated a small but significant low-frequency depression, probably due to insufficient refilling of the releasable pool. Our results, taken together, support the hypothesis that Rab3a maintains the pool of fusion competent vesicles tightly coupled to Ca2+ channels.

Synapsin II and calcium regulate vesicle docking and the cross-talk between vesicle pools at the mouse motor terminals.

The synapsins, an abundant and highly conserved family of proteins that associate with synaptic vesicles, have been implicated in regulating the synaptic vesicle cycle. However, it has not been determined whether synapsin directly regulates the number of docked vesicles. Here we document that reducing Ca(2+) concentration [Ca(2+)](o) in the extracellular medium from 2 to 0.5 mm led to an approximately 40% decrease in both docked and undocked synaptic vesicles in wild-type nerve terminals of the mouse diaphragm. The same treatment reduced the number of undocked vesicles in nerve terminals derived from synapsin II gene deleted animals, but surprisingly it did not decrease vesicle docking, indicating that synapsin II inhibits docking of synaptic vesicles at reduced [Ca(2+)](o). In accordance with the morphological findings, at reduced [Ca(2+)](o) synapsin II (-) terminals had a higher rate of quantal neurotransmitter release. Microinjection of a recombinant synapsin II protein into synapsin II (-) terminals reduced vesicular docking and inhibited quantal release, indicating a direct and selective synapsin II effect for regulating vesicle docking and, in turn, quantal release. To understand why [Ca(2+)](o) has a prominent effect on synapsin function, we investigated the effect of [Ca(2+)](o) on the distribution of synaptic vesicles and on the concentration of intraterminal Ca(2+). We found that reduced [Ca(2+)](o) conditions produce a decrease in intracellular Ca(2+) and overall vesicle depletion. To explore why at these conditions the role of synapsin II in vesicle docking becomes more prominent, we developed a quantitative model of the vesicle cycle, with a two step synapsin action in stabilizing the vesicle store and regulating vesicle docking. The results of the modelling were in a good agreement with the observed dependence of vesicle distribution on synapsin II and calcium deficiency.

Regulation of transmitter release by synapsin II in mouse motor terminals.

We investigated quantal release and ultrastructure in the neuromuscular junctions of synapsin II knockout (Syn II KO) mice. Synaptic responses were recorded focally from the diaphragm synapses during electrical stimulation of the phrenic nerve. We found that synapsin II affects transmitter release in a Ca(2+)-dependent manner. At reduced extracellular Ca(2+) (0.5 mM), Syn II KO mice demonstrated a significant increase in evoked and spontaneous quantal release, while at the physiological Ca(2+) concentration (2 mM), quantal release in Syn II KO synapses was unaffected. Protein kinase inhibitor H7 (100 microM) suppressed quantal release significantly stronger in Syn II KO synapses than in wild type (WT), indicating that Syn II KO synapses may compensate for the lack of synapsin II via a phosphorylation-dependent pathway. Electron microscopy analysis demonstrated that the lack of synapsin II results in an approximately 40% decrease in the density of synaptic vesicles in the reserve pool, while the number of vesicles docked to the presynaptic membrane remained unchanged. Synaptic depression in Syn II KO synapses was slightly increased, which is consistent with the depleted vesicle store in these synapses. At reduced Ca(2+) frequency facilitation of synchronous release was significantly increased in Syn II KO, while facilitation of asynchronous release was unaffected. Thus, at the reduced Ca(2+) concentration, synapsin II suppressed transmitter release and facilitation. These results demonstrate that synapsin II can regulate vesicle clustering, transmitter release, and facilitation.

Fathers and pediatricians: enhancing men's roles in the care and development of their children.

Research substantiates that fathers' interactions with their children can exert a positive influence on their children's development. This report suggests ways pediatricians can enhance fathers' caregiving involvement by offering specific, culturally sensitive advice and how pediatricians might change their office practices to support and increase fathers' active involvement in their children's care and development.