Search

Objectives: Visual expertise is essential for image-based tasks that rely on visual cues, such as in radiology or histology. Studies suggest that eye movements are related to visual expertise and can be measured by near-infrared eye-tracking. With the popularity of device-embedded webcam eye-tracking technology, cost-effective use in educational contexts has recently become amenable. This study investigated the feasibility of such methodology in a curricular online-only histology course during the 2021 summer term.Methods: At two timepoints (t1 and t2), third-semester medical students were asked to diagnose a series of histological slides while their eye movements were recorded. Students’ eye metrics, performance and behavioral measures were analyzed using variance analyses and multiple regression models.Results: First, webcam-eye tracking provided eye movement data with satisfactory quality (=115.7 px±31.1). Second, the eye movement metrics reflected the students’ proficiency in finding relevant image sections (=6.96±1.56 vs. irrelevant areas=4.50±1.25). Third, students’ eye movement metrics successfully predicted their performance (R=0.39, p

Paroxysmal nonkinesigenic dyskinesia (PNKD) is an autosomal dominant episodic movement disorder precipitated by coffee, alcohol, and stress. We previously identified the causative gene but the function of the encoded protein remains unknown. We also generated a PNKD mouse model that revealed dysregulated dopamine signaling in vivo. Here, we show that PNKD interacts with synaptic active zone proteins Rab3-interacting molecule (RIM)1 and RIM2, localizes to synapses, and modulates neurotransmitter release. Overexpressed PNKD protein suppresses release, and mutant PNKD protein is less effective than wild-type at inhibiting exocytosis. In PNKD KO mice, RIM1/2 protein levels are reduced and synaptic strength is impaired. Thus, PNKD is a novel synaptic protein with a regulatory role in neurotransmitter release.

Presynaptic Ca2+ influx through voltage-gated Ca2+ channels (VGCCs) is a key signal for synaptic vesicle release. Synaptic neurexins can partially determine the strength of transmission by regulating VGCCs. However, it is unknown whether neurexins modulate Ca2+ influx via all VGCC subtypes similarly. Here, we performed live cell imaging of synaptic boutons from primary hippocampal neurons with a Ca2+ indicator. We used the expression of inactive and active Cre recombinase to compare control to conditional knockout neurons lacking either all or selected neurexin variants. We found that reduced total presynaptic Ca2+ transients caused by the deletion of all neurexins were primarily due to the reduced contribution of P/Q-type VGCCs. The deletion of neurexin1α alone also reduced the total presynaptic Ca2+ influx but increased Ca2+ influx via N-type VGCCs. Moreover, we tested whether the decrease in Ca2+ influx induced by activation of cannabinoid receptor 1 (CB1-receptor) is modulated by neurexins. Unlike earlier observations emphasizing a role for β-neurexins, we found that the decrease in presynaptic Ca2+ transients induced by CB1-receptor activation depended more strongly on the presence of α-neurexins in hippocampal neurons. Together, our results suggest that neurexins have unique roles in the modulation of presynaptic Ca2+ influx through VGCC subtypes and that different neurexin variants may affect specific VGCCs. [ABSTRACT FROM AUTHOR]

The α2δ auxiliary subunits of voltage-gated calcium channels (VGCC) were traditionally regarded as modulators of biophysical channel properties. In recent years, channel-independent functions of these subunits, such as involvement in synapse formation, have been identified. In the central nervous system, α2δ isoforms 1, 2, and 3 are strongly expressed, regulating glutamatergic synapse formation by a presynaptic mechanism. Although the α2δ-4 isoform is predominantly found in the retina with very little expression in the brain, it was recently linked to brain functions. In contrast, Cachd1, a novel α2δ-like protein, shows strong expression in brain, but its function in neurons is not yet known. Therefore, we aimed to investigate the presynaptic functions of α2δ-4 and Cachd1 by expressing individual proteins in cultured hippocampal neurons. Both α2δ-4 and Cachd1 are expressed in the presynaptic membrane and could rescue a severe synaptic defect present in triple knockout/knockdown neurons that lacked the α2δ-1-3 isoforms (α2δ TKO/KD). This observation suggests that presynaptic localization and the regulation of synapse formation in glutamatergic neurons is a general feature of α2δ proteins. In contrast to this redundant presynaptic function, α2δ-4 and Cachd1 differentially regulate the abundance of presynaptic calcium channels and the amplitude of presynaptic calcium transients. These functional differences may be caused by subtle isoform-specific differences in α1-α2δ protein–protein interactions, as revealed by structural homology modelling. Taken together, our study identifies both α2δ-4 and Cachd1 as presynaptic regulators of synapse formation, differentiation, and calcium channel functions that can at least partially compensate for the loss of α2δ-1-3. Moreover, we show that regulating glutamatergic synapse formation and differentiation is a critical and surprisingly redundant function of α2δ and Cachd1. [ABSTRACT FROM AUTHOR]

[alpha]-Neurexins are neuron-specific cell-surface molecules that are essential for the functional organization of presynaptic [Ca.sup.2+] channels and release sites. We have now examined postsynaptic glutamate receptor function in [alpha]-neurexin knockout (KO) mice by using whole-cell recordings in cultured neocortical slices. Unexpectedly, we find that [alpha]-neurexins are required for normal activity of N-methyl-D-aspartate (NMDA)- but not [alpha]-amino-3-hydroxy-5-methyl-4-isoxyzolepropionic acid (AMPA)-type glutamate receptors. In [alpha]-neurexin-deficient mice, the ratio of NMDA- to AMPA-receptor currents, recorded as evoked synaptic responses, was diminished [approximately equal to] 50%. Furthermore, the NMDA-receptor-dependent component of spontaneous synaptic miniature responses was reduced [approximately equal to] 50%, whereas the AMPA-receptor-dependent component was unaffected. No alterations in the levels of NMDA- or AMPA-receptor proteins were detected. These results suggest that [alpha]-neurexins are required to maintain normal postsynaptic NMDA-receptor function. The decrease in NMDA-receptor activity in [alpha]-neurexin-deficient synapses could be due to a transsynaptic effect on the postsynaptic neuron (i.e., [alpha]-neurexins on the presynaptic inputs guide postsynaptic NMDA-receptor function) or to a cell-autonomous postsynaptic effect of [alpha]-neurexins on NMDA-receptor activity. To distinguish between these two possibilities, we cocultured WT GFP-labeled neurons with neocortical slices from [alpha]-neurexin-deficient or control mice. No difference was found between WT neurons innervated by inputs that contained or lacked [alpha]-neurexins, indicating that the absence of presynaptic [alpha]-neurexins alone does not depress postsynaptic NMDA-receptor function. Our data suggest that, in addition to the previously described presynaptic impairments, loss of [alpha]-neurexins induces postsynaptic changes by a cell-autonomous mechanism.

Author(s): Markus Missler (corresponding author) [1, 2, 3]; Weiqi Zhang [3]; Astrid Rohlmann [3]; Gunnar Kattenstroth [4]; Robert E. Hammer [2, 5]; Kurt Gottmann [4]; Thomas C. Südhof (corresponding author) [...]

Complexins, which are cytosolic proteins present in high concentration in neurons, control soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAP) receptor binding during membrane fusion. Binding of SNAP receptor-core complex, containing syntaxin, synaptobrevin and SNAP-25 molecules, to complexins is very strong. Complexins do not bind to synaptobrevin and SNAP-25, and bind only weakly to syntaxin.

At the conditions of accelerated vesicle traffic, the synapsins I and II adjust the transfer of fusion-competent synaptic vesicles at the active site. The synaptic vesicles are destabilized in the absence of the regulatory function. Stimulation of mice bred without either I or II shows that the two synapsins are redundant and only when mice are bred without either does stimulation result in seizures. However, the seizures do not otherwise impair the mice nor is their life expectancy lower than mice with both synapsins.

Neuroligin 1 is a surface protein which functions as the binding site only for beta-neurexins but only if the neurexins do not have an insert in the alternatively spliced sequence of the G domain. Neuroligin 1 is present at the posterior side of the synapses and contains an extracellular domain, a transmembrane region and a cytoplasmic tail. The interactions of neuroligin 1 and beta-neurexins increase the specificity of interactions of neurons in the brain.

In nerve cells the genes encoding for α2δ subunits of voltage-gated calcium channels have been linked to synaptic functions and neurological disease. Here we show that α2δ subunits are essential for the formation and organization of glutamatergic synapses. Using a cellular α2δ subunit triple-knockout/knockdown model, we demonstrate a failure in presynaptic differentiation evidenced by defective presynaptic calcium channel clustering and calcium influx, smaller presynaptic active zones, and a strongly reduced accumulation of presynaptic vesicle-associated proteins (synapsin and vGLUT). The presynaptic defect is associated with the downscaling of postsynaptic AMPA receptors and the postsynaptic density. The role of α2δ isoforms as synaptic organizers is highly redundant, as each individual α2δ isoform can rescue presynaptic calcium channel trafficking and expression of synaptic proteins. Moreover, α2&#948-2 and α2&#948-3 with mutated metal iondependent adhesion sites can fully rescue presynaptic synapsin expression but only partially calcium channel trafficking, suggesting that the regulatory role of α2δ subunits is independent from its role as a calcium channel subunit. Our findings influence the current view on excitatory synapse formation. First, our study suggests that postsynaptic differentiation is secondary to presynaptic differentiation. Second, the dependence of presynaptic differentiation on α2δ implicates α2δ subunits as potential nucleation points for the organization of synapses. Finally, our results suggest that α2δ subunits act as transsynaptic organizers of glutamatergic synapses, thereby aligning the synaptic active zone with the postsynaptic density. [ABSTRACT FROM AUTHOR]

Presynaptic α2δ subunits of voltage-gated calcium channels regulate channel abundance and are involved in glutamatergic synapse formation. However, little is known about the specific functions of the individual α2δ isoforms and their role in GABAergic synapses. Using primary neuronal cultures of embryonic mice of both sexes, we here report that presynaptic overexpression of α2δ-2 in GABAergic synapses strongly increases clustering of postsynaptic GABAARs. Strikingly, presynaptic α2δ-2 exerts the same effect in glutamatergic synapses, leading to a mismatched localization of GABAARs. This mismatching is caused by an aberrant wiring of glutamatergic presynaptic boutons with GABAergic postsynaptic positions. The trans-synaptic effect of α2δ is independent of the prototypical cell-adhesion molecules a-neurexins (α-Nrxns); however, α-Nrxns together with α2δ can modulate postsynaptic GABAAR abundance. Finally, exclusion of the alternatively spliced exon 23 of α2δ is essential for the trans-synaptic mechanism. The novel function of α2δ identified here may explain how abnormal α2δ subunit expression can cause excitatory-inhibitory imbalance often associated with neuropsychiatric disorders. [ABSTRACT FROM AUTHOR]

Action potential-evoked neurotransmitter release is impaired in knock-out neurons lacking synaptic cell-adhesion molecules α-neurexins (αNrxns), the extracellularly longer variants of the three vertebrate Nrxn genes. Ca2+ influx through presynaptic high-voltage gated calcium channels like the ubiquitous P/Q-type (Cav2.1) triggers release of fusion-ready vesicles at many boutons. α28 Auxiliary subunits regulate trafficking and kinetic properties of Cav2.1 pore-forming subunits but it has remained unclear if this involves αNrxns. Using live cell imaging with Ca2+ indicators, we report here that the total presynaptic Ca2+ influx in primary hippocampal neurons of αNrxn triple knock-out mice of both sexes is reduced and involved lower Cav2.1-mediated transients. This defect is accompanied by lower vesicle release, reduced synaptic abundance of Cav2.1 pore-forming subunits, and elevated surface mobility of α2δ-1 on axons. Overexpression of Nrxn1 α in αNrxn triple knock-out neurons is sufficient to restore normal presynaptic Ca2+ influx and synaptic vesicle release. Moreover, coexpression of Nrxn1α together with α2δ-1 subunits facilitates Ca2+ influx further but causes little augmentation together with a different subunit, α2δ-3, suggesting remarkable specificity. Expression of defined recombinant Cav2.1 channels in heterologous cells validates and extends the findings from neurons. Whole-cell patch-damp recordings show that Nrxnlα in combination with α2δ-1, but not with α2δ-3, facilitates Ca2+ currents of recombinant Cav2.1 without altering channel kinetics. These results suggest that presynaptic Nrxnla acts as a positive regulator of Ca2+ influx through Cav2.1 channels containing α2δ-1 subunits. We propose that this regulation represents an important way for neurons to adjust synaptic strength. [ABSTRACT FROM AUTHOR]