Lennart Niels Cornelisse

Divisive normalization describes how neuronal responses are scaled by the activity of a broader pool of inputs and accounts for several nonlinear response properties in primary visual cortex (V1). It accurately describes contrast gain control, as well as the response to overlapping gratings. Nevertheless, how this computation is performed by the brain is unclear. One mechanism suggested to underlie divisive normalization in V1 is short-term depression (STD) at thalamocortical synapses. Here, we used a Vglut2-Cre dependent heterozygous deletion of Stxbp1 in mice as an in vivo model for increased STD. This enabled us to test whether STD is responsible for divisive normalization in V1. As expected, responses were shorter in the mutant mice compared to wild-type siblings. Although we did find other differences in visual responses, notably in direction selectivity and response linearity, neither the contrast response function, nor the amount of cross orientation suppression differed between the two groups. We conclude that STD is likely not involved in divisive normalization of visual responses in mouse V1. Instead, short-term plasticity shapes the temporal integration through which selective cortical responses are constructed.

Human induced pluripotent stem cells (iPSCs) present a powerful approach to study human brain physiology and disease, yet robust, pure GABAergic induction has remained difficult. Here we present improved, single-step, transposon-based GABAergic induction with Ascl1/Dlx2, which yields, unlike lentiviral approaches, exclusively GABAergic neurons and was validated across three independent iPSC lines. Co-seeding with Ngn2-induced excitatory neurons created stable networks of predefined excitation/inhibition ratios, with corresponding synapse ratios. Proteomic and electrophysiological characterization at different developmental time points showed that the single-step induced GABAergic neurons gain a proteomic profile that maps to different cortical interneuron subtypes and display typical GABAergic synaptic properties, producing large, synchronous and picrotoxin-sensitive currents. During early development, synaptic strength increased threefold, which was accompanied by an increase in expression of proteins exclusively enriched for presynaptic SYNGO terms. Synaptic strength continued to increase during late development but with only minor proteomic changes. Taken together, transposon-based GABAergic induction yields exclusively mature GABAergic neurons suitable for studying genes involved in synaptic maturation and to build excitation/inhibition networks for disease modelling.

Synaptotagmin-1 (Syt1) is a presynaptic calcium sensor with two calcium binding domains, C2A and C2B, that triggers action potential-induced synchronous neurotransmitter release, while suppressing asynchronous and spontaneous release. We identified a de novo missense mutation (P401L) in the C2B domain in a patient with developmental delay and autistic symptoms. Expressing the orthologous mouse mutant (P400L) in cultured Syt1 null mutant neurons revealed a reduction in dendrite outgrowth with a proportional reduction in synapses. This was not observed in single Syt1^PL-rescued neurons that received normal synaptic input when cultured in a control network. Patch-clamp recordings showed that spontaneous miniature release events per synapse were increased more than 500% in Syt1^PL-rescued neurons, even beyond the increased rates in Syt1 KO neurons. Furthermore, action potential-induced asynchronous release was increased more than 100%, while synchronous release was unaffected. A similar shift to more asynchronous release was observed during train stimulations. These cellular phenotypes were also observed when Syt1^PL was overexpressed in wild type neurons. Our findings show that Syt1^PL desynchronizes neurotransmission by increasing the readily releasable pool for asynchronous release and reducing the suppression of spontaneous and asynchronous release. Neurons respond to this by shortening their dendrites, possibly to counteract the increased synaptic input. Syt1^PL acts in a dominant-negative manner supporting a causative role for the mutation in the heterozygous patient. We propose that the substitution of a rigid proline to a more flexible leucine at the bottom of the C2B domain impairs clamping of release by interfering with Syt1’s primary interface with the SNARE complex. This is a novel cellular phenotype, distinct from what was previously found for other SYT1 disease variants, and points to a role for spontaneous and asynchronous release in SYT1-associated neurodevelopmental disorder.

Synaptotagmin-1 (Syt1) is a presynaptic calcium sensor with two calcium binding domains, C2A and C2B, that triggers action potential-induced synchronous neurotransmitter release, while suppressing asynchronous and spontaneous release. We identified a de novo missense mutation (P401L) in the C2B domain in a patient with developmental delay and autistic symptoms. Expressing the orthologous mouse mutant (P400L) in cultured Syt1 null mutant neurons revealed a reduction in dendrite outgrowth with a proportional reduction in synapses. This was not observed in single Syt1^PL-expressing neurons that received normal synaptic input when cultured in a control network. Patch-clamp recordings showed that spontaneous neurotransmitter release per synapse was increased more than 500% in Syt1^PL-expressing neurons, even beyond the increased rates in Syt1 KO neurons. Furthermore, action potential induced asynchronous release was increased more than 100%, while synchronous release was not changed. A similar shift to more asynchronous release was observed during train stimulations. These cellular phenotypes were also observed when Syt1^PL was expressed in wild type neurons. Our findings show that Syt1^PL desynchronizes neurotransmission by reducing the suppression spontaneous and asynchronous release. Neurons respond to this by shortening their dendrites, possibly to counteract the increase in release. Syt1^PL acts in a dominant-negative manner supporting a causative role for the mutation in the heterozygous patient. We propose that the substitution of a rigid proline to a more flexible leucine at the bottom of the C2B domain impairs clamping of release by interfering with Syt1’s primary interface with the SNARE complex. This is a novel cellular phenotype, distinct from what was previously found for other Syt1 disease variants, and points to a role for spontaneous and asynchronous release in SYT1-associated neurodevelopmental disorder.

Pharmacological options for neurodevelopmental disorders are limited to symptom suppressing agents that do not target underlying pathophysiological mechanisms. Studies on specific genetic disorders causing neurodevelopmental disorders have elucidated pathophysiological mechanisms to develop more rational treatments. Here, we present our concerted multi-level strategy ‘BRAINMODEL’, focusing on excitation/inhibition ratio homeostasis across different levels of neuroscientific interrogation. The aim is to develop personalized treatment strategies by linking iPSC-based models and novel EEG measurements to patient report outcome measures in individual patients. We focus our strategy on chromatin- and SNAREopathies as examples of severe genetic neurodevelopmental disorders with an unmet need for rational interventions.

Previously, we showed that modulation of the energy barrier for synaptic vesicle fusion boosts release rates supralinearly (Schotten, 2015). Here we show that mouse hippocampal synapses employ this principle to trigger Ca²⁺-dependent vesicle release and post-tetanic potentiation (PTP). We assess energy barrier changes by fitting release kinetics in response to hypertonic sucrose. Mimicking activation of the C2A domain of the Ca²⁺-sensor Synaptotagmin-1 (Syt1), by adding a positive charge (Syt1^D232N) or increasing its hydrophobicity (Syt1^4W), lowers the energy barrier. Removing Syt1 or impairing its release inhibitory function (Syt1^9Pro) increases spontaneous release without affecting the fusion barrier. Both phorbol esters and tetanic stimulation potentiate synaptic strength, and lower the energy barrier equally well in the presence and absence of Syt1. We propose a model where tetanic stimulation activates Syt1-independent mechanisms that lower the energy barrier and act additively with Syt1-dependent mechanisms to produce PTP by exerting multiplicative effects on release rates.

Previously, we showed that modulation of the energy barrier for synaptic vesicle fusion boosts release rates supralinearly (Schotten, 2015). Here we show that mouse hippocampal synapses employ this principle to trigger Ca ²⁺ -dependent vesicle release and post-tetanic potentiation (PTP). We assess energy barrier changes by fitting release kinetics in response to hypertonic sucrose. Mimicking activation of the C2A domain of the Ca ²⁺ -sensor Synaptotagmin-1 (Syt1), by adding a positive charge (Syt1 ^D232N ) or increasing its hydrophobicity (Syt1 ^4W ), lowers the energy barrier. Removing Syt1 or impairing its release inhibitory function (Syt1 ^9Pro ) increases spontaneous release without affecting the fusion barrier. Both phorbol esters and tetanic stimulation potentiate synaptic strength, and lower the energy barrier equally well in the presence and absence of Syt1. We propose a model where tetanic stimulation activates Syt1 dependent and independent mechanisms that lower the energy barrier independently in an additive manner to produce PTP by multiplication of release rates.

Regulated secretion is controlled by Ca²⁺sensors with different affinities and subcellular distributions. Inactivation ofSyt1(synaptotagmin-1), the main Ca²⁺sensor for synchronous neurotransmission in many neurons, enhances asynchronous and spontaneous release rates, suggesting that Syt1 inhibits other sensors with higher Ca²⁺affinities and/or lower cooperativities. Such sensors could include Doc2a and Doc2b, which have been implicated in spontaneous and asynchronous neurotransmitter release and compete with Syt1 for binding SNARE complexes. Here, we tested this hypothesis using triple-knock-out mice. Inactivation of Doc2a and Doc2b in Syt1-deficient neurons did not reduce the high spontaneous release rate. Overexpression of Doc2b variants in triple-knock-out neurons reduced spontaneous release but did not rescue synchronous release. A chimeric construct in which the C2AB domain of Syt1 was substituted by that of Doc2b did not support synchronous release either. Conversely, the soluble C2AB domain of Syt1 did not affect spontaneous release. We conclude that the high spontaneous release rate in synaptotagmin-deficient neurons does not involve the binding of Doc2 proteins to Syt1 binding sites in the SNARE complex. Instead, our results suggest that the C2AB domains of Syt1 and Doc2b specifically support synchronous and spontaneous release by separate mechanisms. (Both male and female neurons were studied without sex determination.)SIGNIFICANCE STATEMENTNeurotransmission in the brain is regulated by presynaptic Ca²⁺concentrations. Multiple Ca²⁺sensor proteins contribute to synchronous (Syt1, Syt2), asynchronous (Syt7), and spontaneous (Doc2a/Doc2b) phases of neurotransmitter release. Genetic ablation of synchronous release was previously shown to affect other release phases, suggesting that multiple sensors may compete for similar release sites, together encoding stimulus–secretion coupling over a large range of synaptic Ca²⁺concentrations. Here, we investigated the extent of functional overlap between Syt1, Doc2a, and Doc2b by reintroducing wild-type and mutant proteins in triple-knock-out neurons, and conclude that the sensors are highly specialized for different phases of release.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

To support frequency-coded information transfer, mammalian synapses tightly synchronize neurotransmitter release to action potentials (APs). However, release desynchronizes during AP trains, especially at room temperature. Here we show that suppression of asynchronous release by Synaptotagmin-1 (Syt1), but not release triggering, is highly temperature sensitive, and enhances synchronous release during high-frequency stimulation. In Syt1-deficient synapses, asynchronous release increased with temperature, opposite to wildtype synapses. Mutations in Syt1 C2B-domain polybasic stretch (Syt1 K326Q,K327Q,K331Q) did not affect synchronization during sustained activity, while the previously observed reduced synchronous response to a single AP was confirmed. However, an inflexible linker between the C2-domains (Syt1 9Pro) reduced suppression, without affecting synchronous release upon a single AP. Syt1 9Pro expressing synapses showed impaired synchronization during AP trains, which was rescued by buffering global Ca²⁺ to prevent asynchronous release. Hence, frequency coding relies on Syt1’s temperature sensitive suppression of asynchronous release, an aspect distinct from its known vesicle recruitment and triggering functions.