Ole Paulsen

Long‐term electrophysiological monitoring of human cerebral organoids remains challenging, as 3D tissues rapidly develop necrotic cores once diffusion limits are exceeded. Air–liquid interface cerebral organoids (ALI‐COs) overcome this by maintaining thin slices with continuous metabolic exchange, preserving viability, yet no system allows stable, in situ recordings over months. Here, we introduce NeuroSuite, a modular platform for long‐term, spatially resolved neural monitoring of ALI‐COs. Its core, Neuroweb, is an ultrathin, perforated microelectrode array that preserves cellular diversity, evidenced by immunofluorescence and RNA sequencing, while enabling low‐impedance (∼25 kΩ at 1 kHz) recordings. Complementing this, NeuroMaps, our open‐source analysis suite, streamlines signal quality control, spike and field potential analysis, and integration with imaging. NeuroSuite is suitable for capturing maturation‐dependent network dynamics, including potential excitation‐inhibition shifts and oscillatory activity, beyond 180 days. Acute ex vivo rat brain slice recordings further demonstrate its versatility, establishing NeuroSuite as a robust platform for developmental neuroscience, disease modelling, and drug screening.

γ-Aminobutyric acid type-A (GABA _A ) receptors are the principal mediators of inhibitory neurotransmission in the human central nervous system. The α ₂ - and α ₃ -containing subtypes have tightly controlled spatial expression profiles, which influence anxiety, nociception, epilepsy, and autism. α ₂ /α ₃ -Selective small molecules compromise on strength of effect (efficacy) to avoid off-subtype modulation. To break this pharmacological deadlock, we study here a panel of nanobodies (NBs) raised against α ₂ - and α ₃ -containing GABA _A receptors. We identify subtype selective silent binders, positive allosteric modulators (PAMs), and inhibitors. Cryo–electron microscopy structures explain the binding modes and molecular mechanisms of action of representative NBs. Modulators exhibit distinct synaptic and extrasynaptic functional profiles in brain slices and neuronal networks and can reduce anxiety in vivo. These selective and efficacious NBs (whether inhibitors or positive modulators) enable strong yet precise pharmacological control of α ₂ /α ₃ -containing subtypes to advance basic research and as potential therapeutic leads to treat neuropsychiatric disorders.

Summary: Rett syndrome is a debilitating neurodevelopmental disorder with cerebral processing impairments caused by MECP2 loss-of-function mutations. Mecp2-deficient mouse models reveal disruptions of microscale cortical circuits. Yet how cellular-scale information processing is altered in Mecp2-deficient microscale functional networks is unknown. We investigated the development of functional connectivity, network topology, and dynamics in microelectrode array (MEA) recordings of primary cortical cultures from Mecp2-deficient and wild-type mice. Mecp2-deficient cortical networks developed more slowly and showed decreased functional connectivity compared to wild-type, leading to smaller network size, density, and strength of connectivity. Altered network topological features in Mecp2-deficient microscale circuits predicted decreased efficiency and information-sharing capacity. This reveals developmental deficits in microscale functional networks, which may in turn underlie the cortical decline and severe cognitive disability in Rett syndrome. These findings also offer circuit-level targets and an in-vitro approach for evaluating new therapeutic products for restoring microscale network function.

Non-rapid eye movement (NREM) sleep facilitates memory consolidation by transferring information from the hippocampus to the neocortex. This transfer is thought to occur primarily when hippocampal sharp-wave ripples (SWRs) and thalamocortical spindles are synchronized. However, the mechanisms underlying this synchronization remain unknown. In this study, we investigated the role of cortical layer 1 neuron-derived neurotrophic factor (NDNF)-expressing (L1 NDNF) interneurons in gating information transfer during SWR-spindle synchronization in NREM sleep. Using simultaneous cell-type specific calcium imaging with a head-mounted microscope and local field potential recordings in freely moving mice, we compared the activity of L1 NDNF and L2/3 neurons across vigilance states and during NREM-specific oscillations. Our findings reveal that L1 NDNF neurons form three distinct populations, assembling into cell networks tuned to specific sleep stages. REM active L1 NDNF and L2/3 neurons exhibit opposite activation patterns during spindles. While L2/3 cells are mostly inactive during SWR, NREM and REM active L1 NDNF cells inhibit the network upon SWR onset depending on their coupling with spindles. L1 NDNF neurons mediate slow inhibition primarily via GABA_B receptors. Systemic application of a GABA_B receptor antagonist resulted in decreased neuronal coupling of pyramidal cells but did not change the responses during SWRs. Overall, these findings highlight the potential role of L1 NDNF neuron-mediated inhibition in the response to synchronized sleep oscillations, with possible implications for memory consolidation.

Economic efficiency has been a popular explanation for how networks self-organize within the developing nervous system. However, the precise nature of the economic negotiations governing this putative organizational principle remains unclear. Here, we address this question further by combining large-scale electrophysiological recordings to characterize the functional connectivity of developing neuronal networks in vitro, with a generative modeling approach capable of simulating network formation. We find that the best fitting model uses a homophilic generative wiring principle in which neurons form connections to other neurons which are spatially proximal and have similar connectivity patterns to themselves. Homophilic generative models outperform more canonical models in which neurons wire depending upon their spatial proximity either alone or in combination with the extent of their local connectivity. This homophily-based mechanism for neuronal network emergence accounts for a wide range of observations that are described, but not sufficiently explained, by traditional analyses of network topology. Using rodent and human neuronal cultures, we show that homophilic generative mechanisms can accurately recapitulate the topology of emerging cellular functional connectivity, representing an important wiring principle and determining factor of neuronal network formation in vitro.

Microelectrode arrays (MEAs) permit recordings with high electrode counts, thus generating complex datasets that would benefit from precise neuronal spike sorting for meaningful data extraction. Nevertheless, conventional spike sorting methods face limitations in recognizing diverse spike shapes. Here, we introduce PseudoSorter, which uses self-supervised learning techniques, a density-based pseudolabeling strategy, and an iterative fine-tuning process to enhance spike sorting accuracy. Through benchmarking, we demonstrate the superior performance of PseudoSorter compared to other spike sorting algorithms before applying PseudoSorter on MEA recordings from hippocampal neurons exposed to subneuronal concentrations of monomeric Tau as a model for Alzheimer’s disease. Our results unveil that Tau diminishes the firing rate of a subset of neurons, which complement our findings observed using conventional electrophysiology analysis, and demonstrate that PseudoSorter’s high accuracy and throughput make it a valuable tool for studying neurodegenerative diseases, enhancing our understanding of their underlying mechanisms, as well as for therapeutic drug screening.

The reward and novelty-related neuromodulator dopamine plays an important role in hippocampal long-term memory, which is thought to involve protein-synthesis-dependent synaptic plasticity. However, the direct effects of dopamine on protein synthesis, and the functional implications of newly synthesised proteins for synaptic plasticity, have not yet been investigated. We have previously reported that timing-dependent synaptic depression (t-LTD) can be converted into potentiation by dopamine application during synaptic stimulation (Brzosko et al., 2015) or postsynaptic burst activation (Fuchsberger et al., 2022). Here, we show that dopamine increases protein synthesis in mouse hippocampal CA1 neurons, enabling dopamine-dependent long-term potentiation (DA-LTP), which is mediated via the Ca²⁺-sensitive adenylate cyclase (AC) subtypes 1/8, cAMP, and cAMP-dependent protein kinase (PKA). We found that neuronal activity is required for the dopamine-induced increase in protein synthesis. Furthermore, dopamine induced a protein-synthesis-dependent increase in the AMPA receptor subunit GluA1, but not GluA2. We found that DA-LTP is absent in GluA1 knock-out mice and that it requires calcium-permeable AMPA receptors. Taken together, our results suggest that dopamine together with neuronal activity controls synthesis of plasticity-related proteins, including GluA1, which enable DA-LTP via a signalling pathway distinct from that of conventional LTP.

The reward and novelty related neuromodulator dopamine plays an important role in hippocampal long- term memory, which is thought to involve protein synthesis-dependent synaptic plasticity. However, the direct effects of dopamine on protein synthesis, and the functional implications of newly synthesized proteins for synaptic plasticity, have not yet been investigated. We have previously reported that timing- dependent synaptic depression (t-LTD) can be converted into potentiation by dopamine application during synaptic stimulation (Brzosko et al., 2015) or postsynaptic burst activation (Fuchsberger et al., 2022). Here we show that dopamine increases protein synthesis in mouse hippocampal CA1 neurons, enabling dopamine-dependent long-term potentiation (DA-LTP), which is mediated via the Ca2+-sensitive adenylate cyclase (AC) subtypes 1/8, cAMP, and cAMP-dependent protein kinase (PKA). We found that neuronal activity is required for the dopamine-induced increase in protein synthesis. Furthermore, dopamine induced a protein synthesis-dependent increase in the AMPA receptor subunit GluA1, but not GluA2. We found that DA-LTP is absent in GluA1 knock-out mice and that it requires calcium-permeable AMPA receptors. Taken together, our results suggest that dopamine together with neuronal activity controls synthesis of plasticity-related proteins, including GluA1, which enable DA-LTP via a signalling pathway distinct from that of conventional LTP.

Non-rapid eye movement (NREM) sleep facilitates memory consolidation by transferring information from the hippocampus to the neocortex. This transfer is thought to occur primarily when hippocampal sharp-wave ripples (SWRs) and thalamocortical spindles are synchronized. However, the mechanisms underlying this synchronization remain unknown. In this study, we investigated the role of cortical layer 1 NDNF-expressing (L1 NDNF) interneurons in gating information transfer during SWR-spindle synchronization in NREM sleep. Using simultaneous cell-specific calcium imaging with a head-mounted microscope and local field potential recordings in freely moving mice, we compared the activity of L1 NDNF and L2/3 neurons across vigilance states and during NREM-specific oscillations. Our findings reveal that L1 NDNF neurons form three distinct populations, assembling into cell networks tuned to specific sleep stages. REM active L1 NDNF and L2/3 neurons exhibit opposite activation patterns during spindles. While L2/3 cells are mostly inactive during SWR, NREM, and REM active L1 NDNF cells inhibit the network upon SWR onset depending on their coupling with spindles. L1 NDNF neurons mediate slow inhibition primarily via GABA _B receptors. Systemic application of a GABA _B receptor antagonist resulted in decreased neuronal coupling of pyramidal cells but did not change the inhibitory responses during SWRs. Overall, these findings highlight the potential role of L1 NDNF neuron-mediated inhibition in the response to synchronized sleep oscillations, with potential implications for memory consolidation. Significance Statement: This study highlights how a specific type of inhibitory neuron, L1 NDNF-expressing interneurons, is activated during deep sleep. We identified three different subpopulations of L1 NDNF neurons based on their activity during vigilance states. These subpopulations show specific and complementary activation patterns during two key sleep oscillations-sharp-wave ripples (SWRs) and spindles-which are crucial for transferring memories from short-term to long-term storage. This research provides new insights into how brain oscillations synchronize and how this phenomenon correlates with local neuronal activity, with potential implications for understanding learning and memory mechanisms.

The reward and novelty related neuromodulator dopamine plays an important role in hippocampal long- term memory, which is thought to involve protein synthesis-dependent synaptic plasticity. However, the direct effects of dopamine on protein synthesis, and the functional implications of newly synthesized proteins for synaptic plasticity, have not yet been investigated. We have previously reported that timing- dependent synaptic depression (t-LTD) can be converted into potentiation by dopamine application during synaptic stimulation (Brzosko et al., 2015) or postsynaptic burst activation (Fuchsberger et al., 2022). Here we show that dopamine increases protein synthesis in mouse hippocampal CA1 neurons, enabling dopamine-dependent long-term potentiation (DA-LTP), which is mediated via the Ca ²⁺ -sensitive adenylate cyclase (AC) subtypes 1/8, cAMP, and cAMP-dependent protein kinase (PKA). We found that neuronal activity is required for the dopamine-induced increase in protein synthesis. Furthermore, dopamine induced a protein synthesis-dependent increase in the AMPA receptor subunit GluA1, but not GluA2. We found that DA-LTP is absent in GluA1 knock-out mice and that it requires calcium-permeable AMPA receptors. Taken together, our results suggest that dopamine together with neuronal activity controls synthesis of plasticity-related proteins, including GluA1, which enable DA-LTP via a signalling pathway distinct from that of conventional LTP.