Multielectrode Array Recordings Research Articles

A new role for excitation in the retinal direction-selective circuit.

A key feature of the receptive field of neurons in the visual system is their centre-surround antagonism, whereby the centre and the surround exhibit responses of opposite polarity. This organization is thought to enhance visual acuity, but whether and how such antagonism plays a role in more complex processing remains poorly understood. Here, we investigate the role of centre and surround receptive fields in retinal direction selectivity by exposing posterior-preferring On-Off direction-selective ganglion cells (pDSGCs) to adaptive light and recording their response to globally moving objects. We reveal that light adaptation leads to surround expansion in pDSGCs. The pDSGCs maintain their original directional tuning in the centre receptive field, but present the oppositely tuned response in their surround. Notably, although inhibition is the main substrate for retinal direction selectivity, we found that following light adaptation, both the centre- and surround-mediated responses originate from directionally tuned excitatory inputs. Multi-electrode array recordings show similar oppositely tuned responses in other DSGC subtypes. Together, these data attribute a new role for excitation in the direction-selective circuit. This excitation carries an antagonistic centre-surround property, possibly designed to sharpen the detection of motion direction in the retina. KEY POINTS: Receptive fields of direction-selective retinal ganglion cells expand asymmetrically following light adaptation. The increase in the surround receptive field generates a delayed spiking phase that is tuned to the null direction and is mediated by excitation. Following light adaptation, excitation rules the computation in the centre receptive field and is tuned to the preferred direction. GABAergic and glycinergic inputs modulate the null-tuned delayed response differentially. Null-tuned delayed spiking phases can be detected in all types of direction-selective retinal ganglion cells. Light adaptation exposes a hidden directional excitation in the circuit, which is tuned to opposite directions in the centre and surround receptive fields.

Host-Graft Synapses Form Functional Microstructures and Shape the Host Light Responses After Stem Cell-Derived Retinal Sheet Transplantation.

Retinitis pigmentosa represents a leading cause of blindness in developed countries, yet effective treatments for the disease remain unestablished. Previous studies have demonstrated the potential of stem cell-derived retinal organoid (SC-RO) sheet transplantation to form host-graft synapses and to improve light responsiveness in animal models of retinal degeneration. However, the detailed microstructures of these de novo synapses and their functional contribution have not been well elucidated. This study aims to (1) elucidate the microstructures of the host-graft synapse, and (2) investigate the overall distribution and contribution of these synapses to host retinal light responses. We identified host-graft synapses using a reporter system in mouse SC-RO and rd1 mice, a well-established model of end-stage retinal degeneration. Correlative array tomography was used to reveal the microstructure of host-graft synapses. Furthermore, we developed a semi-automated algorithm that robustly detects the host-graft photoreceptor synapses in the overall grafted area using the same reporter system in flat-mount retinas. We then integrated the spatial distribution of the host-graft synapses with light responses detected by multi-electrode array recording. Correlative array tomography revealed that host-graft synapses recapitulate the developmental process of photoreceptor synapse formation involving horizontal cells first and then rod bipolar cells. By integrating the spatial distribution of host-graft synapse and multi-electrode array recording, we showed that the number of light-responsive host retinal ganglion cells is positively correlated with the local density of host-graft synapses. De novo host-graft synapses recapitulate the developmental microstructure of the photoreceptor synapse, and their formation contributes to the light responsiveness after SC-RO transplantation.

APOE4 impacts cortical neurodevelopment and alters network formation in human brain organoids.

Apolipoprotein E4 ( APOE4 ) is the leading genetic risk factor for Alzheimer's disease. While most studies examine the role of APOE4 in aging, imaging, and cognitive assessments reveal that APOE4 influences brain structure and function as early as infancy. Here, we examined human-relevant cellular phenotypes across neurodevelopment using induced pluripotent stem cell (iPSC) derived cortical and ganglionic eminence organoids (COs and GEOs). In COs, we showed that APOE4 decreased BRN2+ and SATB2+ cortical neurons, increased astrocytes and outer radial glia, and was associated with increased cell death and dysregulated GABA-related gene expression. In GEOs, APOE4 accelerated maturation of neural progenitors and neurons. Multi-electrode array recordings in assembloids revealed that APOE4 disrupted network formation and altered response to GABA, resulting in heightened excitability and synchronicity. Together, our data provides new insights into how APOE4 may influence cortical neurodevelopmental processes and network formation in the human brain.

A Multi-Electrode Array Platform for Modeling Epilepsy Using Human Pluripotent Stem Cell-Derived Brain Assembloids.

Human brain organoids are three-dimensional (3D) structures derived from human pluripotent stem cells (hPSCs) that recapitulateaspects of fetal brain development. The fusion of dorsal with ventral regionally specified brain organoids in vitro generates assembloids, which have functionally integrated microcircuits with excitatory and inhibitory neurons. Due to their structural complexity and diverse population of neurons, assembloids have become a useful in vitro tool for studying aberrant network activity. Multi-electrode array (MEA) recordings serve as a method for capturing electrical field potentials, spikes, and longitudinal network dynamics from a population of neurons without compromising cell membrane integrity. However, adhering assembloids onto the electrodes for long-term recordings can be challenging due to their large size and limited contact surface area with the electrodes. Here, we demonstrate a method to plate assembloids onto MEA plates for recording electrophysiological activity over a 2-month span. Although the current protocol utilizes human cortical organoids, it can be broadly adapted to organoids differentiated to model other brain regions. This protocol establishes a robust, longitudinal, electrophysiological assay for studying the development of a neuronal network, and this platform has the potential to be used in drug screening for therapeutic development in epilepsy.

Functional efficacy of the MAO-B inhibitor safinamide in murine substantia nigra pars compacta dopaminergic neurons in vitro: A comparative study with tranylcypromine

Safinamide (SAF) is currently used to treat Parkinson's disease (PD) symptoms based on its theoretical ability to potentiate the dopamine (DA) signal, blocking monoamine oxidase (MAO) B. The present work aims to highlight the functional relevance of SAF as an enhancer of the DA signal, by evaluating its ability to prolong recovery from DA-mediated firing inhibition of DAergic neurons of the substantia nigra pars compacta (SNpc), compared to another MAO antagonist, tranylcypromine (TCP). Using multielectrode array (MEA) and single electrode extracellular recordings of spontaneous spikes from presumed SNpc DAergic cells in vitro, we show that SAF (30 μM) mildly prolongs the DA-mediated firing inhibition, as opposed to the profound effect of TCP (10 μM). In patch-clamp recordings, we found that SAF (30 μM) significantly reduced the number of spikes evoked by depolarizing currents in SNpc DAergic neurons, in a sulpiride (1 μM) independent manner. According to our results, SAF marginally potentiates the DA signal in SNpc DAergic neurons, while exerting an inhibitory effect on the postsynaptic excitability acting on membrane conductances. Thus, we propose that the therapeutic effects of SAF in PD patients partially depends on MAO inhibition, while other MAO-independent sites of action could be more relevant.

Human Pluripotent Stem Cell-Derived Astrocyte Functionality Compares Favorably with Primary Rat Astrocytes.

Astrocytes are essential for the formation and maintenance of neural networks. However, a major technical challenge for investigating astrocyte function and disease-related pathophysiology has been the limited ability to obtain functional human astrocytes. Despite recent advances in human pluripotent stem cell (hPSC) techniques, primary rodent astrocytes remain the gold standard in coculture with human neurons. We demonstrate that a combination of leukemia inhibitory factor (LIF) and bone morphogenetic protein-4 (BMP4) directs hPSC-derived neural precursor cells to a highly pure population of astroglia in 28 d. Using single-cell RNA sequencing, we confirm the astroglial identity of these cells and highlight profound transcriptional adaptations in cocultured hPSC-derived astrocytes and neurons, consistent with their further maturation. In coculture with human neurons, multielectrode array recordings revealed robust network activity of human neurons in a coculture with hPSC-derived or rat astrocytes [3.63 ± 0.44 min-1 (hPSC-derived), 2.86 ± 0.64 min-1 (rat); p = 0.19]. In comparison, we found increased spike frequency within network bursts of human neurons cocultured with hPSC-derived astrocytes [56.31 ± 8.56 Hz (hPSC-derived), 24.77 ± 4.04 Hz (rat); p < 0.01], and whole-cell patch-clamp recordings revealed an increase of postsynaptic currents [2.76 ± 0.39 Hz (hPSC-derived), 1.07 ± 0.14 Hz (rat); p < 0.001], consistent with a corresponding increase in synapse density [14.90 ± 1.27/100 μm2 (hPSC-derived), 8.39 ± 0.63/100 μm2 (rat); p < 0.001]. Taken together, we show that hPSC-derived astrocytes compare favorably with rat astrocytes in supporting human neural network activity and maturation, providing a fully human platform for investigating astrocyte function and neuronal-glial interactions.

A high-density multi-electrode platform examining the effects of radiation on in vitro cortical networks

Radiation therapy and stereotactic radiosurgery are common treatments for brain malignancies. However, the impact of radiation on underlying neuronal circuits is poorly understood. In the prefrontal cortex (PFC), neurons communicate via action potentials that control cognitive processes, thus it is important to understand the impact of radiation on these circuits. Here we present a novel protocol to investigate the effect of radiation on the activity and survival of PFC networks in vitro. Escalating doses of radiation were applied to PFC slices using a robotic radiosurgery platform at a standard dose rate of 10 Gy/min. High-density multielectrode array recordings of radiated slices were collected to capture extracellular activity across 4,096 channels. Radiated slices showed an increase in firing rate, functional connectivity, and complexity. Graph-theoretic measures of functional connectivity were altered following radiation. These results were compared to pharmacologically induced epileptic slices where neural complexity was markedly elevated, and functional connections were strong but remained spatially focused. Finally, propidium iodide staining revealed a dose-dependent effect of radiation on apoptosis. These findings provide a novel assay to investigate the impacts of clinically relevant doses of radiation on brain circuits and highlight the acute effects of escalating radiation doses on PFC neurons.

Epilepsy-associated Kv1.1 channel subunits regulate intrinsic cardiac pacemaking in mice.

The heartbeat originates from spontaneous action potentials in specialized pacemaker cells within the sinoatrial node (SAN) of the right atrium. Voltage-gated potassium channels in SAN myocytes mediate outward K+ currents that regulate cardiac pacemaking by controlling action potential repolarization, influencing the time between heartbeats. Gene expression studies have identified transcripts for many types of voltage-gated potassium channels in the SAN, but most remain of unknown functional significance. One such gene is Kcna1, which encodes epilepsy-associated voltage-gated Kv1.1 K+ channel α-subunits that are important for regulating action potential firing in neurons and cardiomyocytes. Here, we investigated the functional contribution of Kv1.1 to cardiac pacemaking at the whole heart, SAN, and SAN myocyte levels by performing Langendorff-perfused isolated heart preparations, multielectrode array recordings, patch clamp electrophysiology, and immunocytochemistry using Kcna1 knockout (KO) and wild-type (WT) mice. Our results showed that either genetic or pharmacological ablation of Kv1.1 significantly decreased the SAN firing rate, primarily by impairing SAN myocyte action potential repolarization. Voltage-clamp electrophysiology and immunocytochemistry revealed that Kv1.1 exerts its effects despite contributing only a small outward K+ current component, which we term IKv1.1, and despite apparently being present in low abundance at the protein level in SAN myocytes. These findings establish Kv1.1 as the first identified member of the Kv1 channel family to play a role in sinoatrial function, thereby rendering it a potential candidate and therapeutic targeting of sinus node dysfunction. Furthermore, our results demonstrate that small currents generated via low-abundance channels can still have significant impacts on cardiac pacemaking.

Modulation of large rhythmic depolarizations in human large basket cells by norepinephrine and acetylcholine

Rhythmic brain activity is critical to many brain functions and is sensitive to neuromodulation, but so far very few studies have investigated this activity on the cellular level in vitro in human brain tissue samples. This study reveals and characterizes a novel rhythmic network activity in the human neocortex. Using intracellular patch-clamp recordings of human cortical neurons, we identify large rhythmic depolarizations (LRDs) driven by glutamate release but not by GABA. These LRDs are intricate events made up of multiple depolarizing phases, occurring at ~0.3 Hz, have large amplitudes and long decay times. Unlike human tissue, rat neocortex layers 2/3 exhibit no such activity under identical conditions. LRDs are mainly observed in a subset of L2/3 interneurons that receive substantial excitatory inputs and are likely large basket cells based on their morphology. LRDs are highly sensitive to norepinephrine (NE) and acetylcholine (ACh), two neuromodulators that affect network dynamics. NE increases LRD frequency through β-adrenergic receptor activity while ACh decreases it via M4 muscarinic receptor activation. Multi-electrode array recordings show that NE enhances and synchronizes oscillatory network activity, whereas ACh causes desynchronization. Thus, NE and ACh distinctly modulate LRDs, exerting specific control over human neocortical activity.

Topical administration of GLP-1 eyedrops improves retinal ganglion cell function by facilitating presynaptic GABA release in early experimental diabetes.

Diabetic retinopathy is a prominent cause of blindness in adults, with early retinal ganglion cell (RGC) loss contributing to visual dysfunction or blindness. In the brain, defects in y-aminobutyric acid (GABA) synaptic transmission are associated with pathophysiological and neurodegenerative disorders, whereas glucagon-like peptide-1 (GLP-1) has demonstrated neuroprotective effects. However, it is not yet clear whether diabetes causes alterations in inhibitory input to RGCs and whether and how GLP-1 protects against neurodegeneration in the diabetic retina through regulating inhibitory synaptic transmission to RGCs. In the present study, we used the patch-clamp technique to record GABA subtype A receptor-mediated miniature inhibitory postsynaptic currents (mIPSCs) in RGCs from streptozotocin-induced diabetes model rats. We found that early diabetes (4 weeks of hyperglycemia) decreased the frequency of GABAergic mIPSCs in RGCs without altering their amplitude, suggesting a reduction in the spontaneous release of GABA to RGCs. Topical administration of GLP-1 eyedrops over a period of 2 weeks effectively countered the hyperglycemia-induced downregulation of GABAergic mIPSC frequency, subsequently enhancing the survival of RGCs. Concurrently, the protective effects of GLP-1 on RGCs in diabetic rats were eliminated by topical administration of exendin-9-39, a specific GLP-1 receptor antagonist, or SR95531, a specific antagonist of the GABA subtype A receptor. Furthermore, extracellular perfusion of GLP-1 was found to elevate the frequencies of GABAergic mIPSCs in both ON- and OFF-type RGCs. This elevation was shown to be mediated by activation of the phosphatidylinositol-phospholipase C/inositol 1,4,5-trisphosphate receptor/Ca2+/protein kinase C signaling pathway downstream of GLP-1 receptor activation. Moreover, multielectrode array recordings revealed that GLP-1 functionally augmented the photoresponses of ON-type RGCs. Optomotor response tests demonstrated that diabetic rats exhibited reductions in visual acuity and contrast sensitivity that were significantly ameliorated by topical administration of GLP-1. These results suggest that GLP-1 facilitates the release of GABA onto RGCs through the activation of GLP-1 receptor, leading to the de-excitation of RGC circuits and the inhibition of excitotoxic processes associated with diabetic retinopathy. Collectively, our findings indicate that the GABA system has potential as a therapeutic target for mitigating early-stage diabetic retinopathy. Furthermore, the topical administration of GLP-1 eyedrops represents a non-invasive and effective treatment approach for managing early-stage diabetic retinopathy.

Standardizing a method for functional assessment of neural networks in brain organoids

During the last decade brain organoids have emerged as an attractive model system, allowing stem cells to be differentiated into complex 3D models, recapitulating many aspects of human brain development. Whilst many studies have analysed anatomical and cytoarchitectural characteristics of organoids, their functional characterisation has been limited, and highly variable between studies. Standardised, consistent methods for recording functional activity are critical to providing a functional understanding of neuronal networks at the synaptic and network level that can yield useful information about functional network phenotypes in disease and healthy states. In this study we outline a detailed methodology for calcium imaging and Multi-Electrode Array (MEA) recordings in brain organoids. To illustrate the utility of these functional interrogation techniques in uncovering induced differences in neural network activity we applied various stimulating media protocols. We demonstrate overlapping information from the two modalities, with comparable numbers of active cells in the four treatment groups and an increase in synchronous behaviour in BrainPhys treated groups. Further development of analysis pipelines to reveal network level changes in brain organoids will enrich our understanding of network formation and perturbation in these structures, and aid in the future development of drugs that target neurological disorders at the network level.

Chronic intermittent hypoxia (CIH) enhances nTS sensitivity to oxytocin (OT) and corticotropin-releasing hormone (CRH) via effects at the nTS soma and on presynaptic release

OT and CRH neurons from the hypothalamus project to nucleus tractus solitarii (nTS) to influence its activity and response to hypoxia. CIH increases sympathetic nervous system activity, blood pressure, and respiration, and the nTS contributes to these responses. Yet, the influence of these OT or CRH inputs on nTS activity during CIH is unclear. We hypothesized that OT or CRH individually increases nTS activity, and their combined influence would further enhance activity. We also expected these responses to be enhanced by CIH. Male Sprague-Dawley rats (~3 wks) were exposed to 10d normoxia (21% O2) or CIH (~30s of 6% O2, 10x/hr, 8 h/day). Recorded events were examined during exogenous OT and CRH or their receptor block (OTR-x, CRHR2-x). As a time control, aCSF only was examined, which did not alter any events over the 40-min recording period. In nTS slices, extracellular neuronal discharge from multielectrode array recording was increased by OT and its co-application with CRH throughout the nTS after normoxia. Following CIH, OT and CRH alone, and their co-application increased firing rate, and the response to OT+CRH after CIH was greater than in normoxia slices. Fura-2 calcium imaging in dissociated nTS neurons demonstrated enhanced responses to OT+ CRH following CIH vs normoxia. In second-order nTS neurons recorded via patch clamp, OT and CRH induced spontaneous action potential discharge after CIH in quiescent neurons during baseline periods. Excitatory postsynaptic currents (EPSCs) were recorded to examine synaptic transmission. Spontaneous (s)EPSCs were not altered by OT and CRH after normoxia or CIH. Yet, only after CIH OTR-x and/or CRHR2-x reduced sEPSC amplitude, indicating tonic activation of these receptors maintain current amplitude. Afferent (TS)-EPSCs evoked at 0.5 and 20 Hz were not altered by OT and CRH or their receptor blockers in normoxia. However, CIH elevated TS-EPSCs in response to CRH and CRH+OT. Block of OTR and CRHR2 after CIH did not alter TS-EPSC amplitude, indicating their minimal tonic influence on sensory integration. After a 20Hz stimulation, the peak of asynchronous EPSCs increased in presence of CRH and OT+CRH only after CIH. We examined presynaptic Ca2+ in pre-labeled GCaMP6m afferent terminals in response to a 20 Hz stimulation. In normoxia-exposed slices, OT and CRH increased Ca2+ fluorescence. However, after CIH, the application of CRH and the co-application of OT+CRH decreased the terminal Ca2+ response. The expression of OT but not CRH fibers within the nTS decreased (immunohistochemistry) while OTR and CRHR2 mRNA increased (RNA Scope). The CRH and OT neuron populations within PVN were not altered after CIH (immunohistochemistry). Altogether, our data show that following CIH, OT and CRH augment neuronal activity via postsynaptic mechanisms while CRH also increases synaptic transmission through enhancing presynaptic release. Those alterations may contribute to elevated sympathetic response and blood pressure observed after CIH. NIH R01 HL098602 &amp; R01 HL128454 (DDK). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Engineering luminopsins with improved coupling efficiencies.

Luminopsins (LMOs) are bioluminescent-optogenetic tools with a luciferase fused to an opsin that allow bimodal control of neurons by providing both optogenetic and chemogenetic access. Determining which design features contribute to the efficacy of LMOs will be beneficial for further improving LMOs for use in research. We investigated the relative impact of luciferase brightness, opsin sensitivity, pairing of emission and absorption wavelength, and arrangement of moieties on the function of LMOs. We quantified efficacy of LMOs through whole cell patch clamp recordings in HEK293 cells by determining coupling efficiency, the percentage of maximum LED induced photocurrent achieved with bioluminescent activation of an opsin. We confirmed key results by multielectrode array recordings in primary neurons. Luciferase brightness and opsin sensitivity had the most impact on the efficacy of LMOs, and N-terminal fusions of luciferases to opsins performed better than C-terminal and multi-terminal fusions. Precise paring of luciferase emission and opsin absorption spectra appeared to be less critical. Whole cell patch clamp recordings allowed us to quantify the impact of different characteristics of LMOs on their function. Our results suggest that coupling brighter bioluminescent sources to more sensitive opsins will improve LMO function. As bioluminescent activation of opsins is most likely based on Förster resonance energy transfer, the most effective strategy for improving LMOs further will be molecular evolution of luciferase-fluorescent protein-opsin fusions.

Functionally-selective inhibition of threshold sodium currents and excitability in dorsal root ganglion neurons by cannabinol

Cannabinol (CBN), an incompletely understood metabolite for ∆9-tetrahydrocannabinol, has been suggested as an analgesic. CBN interacts with endocannabinoid (CB) receptors, but is also reported to interact with non-CB targets, including various ion channels. We assessed CBN effects on voltage-dependent sodium (Nav) channels expressed heterologously and in native dorsal root ganglion (DRG) neurons. Our results indicate that CBN is a functionally-selective, but structurally-non-selective Nav current inhibitor. CBN’s main effect is on slow inactivation. CBN slows recovery from slow-inactivated states, and hyperpolarizes steady-state inactivation, as channels enter deeper and slower inactivated states. Multielectrode array recordings indicate that CBN attenuates DRG neuron excitability. Voltage- and current-clamp analysis of freshly isolated DRG neurons via our automated patch-clamp platform confirmed these findings. The inhibitory effects of CBN on Nav currents and on DRG neuron excitability add a new dimension to its actions and suggest that this cannabinoid may be useful for neuropathic pain.

Human organotypic brain slice cultures: a detailed and improved protocol for preparation and long-term maintenance

The investigation of the human brain at cellular and microcircuit level remains challenging due to the fragile viability of neuronal tissue, inter- and intra-variability of the samples and limited availability of human brain material. Especially brain slices have proven to be an excellent source to investigate brain physiology and disease at cellular and small network level, overcoming the temporal limits of acute slices. Here we provide a revised, detailed protocol of the production and in-depth knowledge on long-term culturing of such human organotypic brain slice cultures for research purposes. We highlight the critical pitfalls of the culturing process of the human brain tissue and present exemplary results on viral expression, single-cell Patch-Clamp recordings, as well as multi-electrode array recordings as readouts for culture viability, enabling the use of organotypic brain slice cultures of these valuable tissue samples for basic neuroscience and disease modeling (Fig. 1).

Distinctive In Vitro Phenotypes in iPSC-Derived Neurons From Patients With Gain- and Loss-of-Function SCN2A Developmental and Epileptic Encephalopathy.

SCN2A encodes NaV1.2, an excitatory neuron voltage-gated sodium channel and a major monogenic cause of neurodevelopmental disorders, including developmental and epileptic encephalopathies (DEE) and autism. Clinical presentation and pharmocosensitivity vary with the nature of SCN2A variant dysfunction and can be divided into gain-of-function (GoF) cases with pre- or peri-natal seizures and loss-of-function (LoF) patients typically having infantile spasms after 6 months of age. We established and assessed patient induced pluripotent stem cell (iPSC) - derived neuronal models for two recurrent SCN2A DEE variants with GoF R1882Q and LoF R853Q associated with early- and late-onset DEE, respectively. Two male patient-derived iPSC isogenic pairs were differentiated using Neurogenin-2 overexpression yielding populations of cortical-like glutamatergic neurons. Functional properties were assessed using patch clamp and multielectrode array recordings and transcriptomic profiles obtained with total mRNA sequencing after 2-4 weeks in culture. At 3 weeks of differentiation, increased neuronal activity at cellular and network levels was observed for R1882Q iPSC-derived neurons. In contrast, R853Q neurons showed only subtle changes in excitability after 4 weeks and an overall reduced network activity after 7 weeks in vitro. Consistent with the reported efficacy in some GoF SCN2A patients, phenytoin (sodium channel blocker) reduced the excitability of neurons to the control levels in R1882Q neuronal cultures. Transcriptomic alterations in neurons were detected for each variant and convergent pathways suggested potential shared mechanisms underlying SCN2A DEE. In summary, patient iPSC-derived neuronal models of SCN2A GoF and LoF pathogenic variants causing DEE show specific functional and transcriptomic in vitro phenotypes.

Low glycemic index diet restrains epileptogenesis in a gender-specific fashion

Dietary restriction, such as low glycemic index diet (LGID), have been successfully used to treat drug-resistant epilepsy. However, if such diet could also counteract antiepileptogenesis is still unclear. Here, we investigated whether the administration of LGID during the latent pre-epileptic period, prevents or delays the appearance of the overt epileptic phenotype. To this aim, we used the Synapsin II knockout (SynIIKO) mouse, a model of temporal lobe epilepsy in which seizures manifest 2–3 months after birth, offering a temporal window in which LGID may affect epileptogenesis. Pregnant SynIIKO mice were fed with either LGID or standard diet during gestation and lactation. Both diets were maintained in weaned mice up to 5 months of age. LGID delayed the seizure onset and induced a reduction of seizures severity only in female SynIIKO mice. In parallel with the epileptic phenotype, high-density multielectrode array recordings revealed a reduction of frequency, amplitude, duration, velocity of propagation and spread of interictal events by LGID in the hippocampus of SynIIKO females, but not mutant males, confirming the gender-specific effect. ELISA-based analysis revealed that LGID increased cortico-hippocampal allopregnanolone (ALLO) levels only in females, while it was unable to affect ALLO plasma concentrations in either sex. The results indicate that the gender-specific interference of LGID with the epileptogenic process can be ascribed to a gender-specific increase in cortical ALLO, a neurosteroid known to strengthen GABAergic transmission. The study highlights the possibility of developing a personalized gender-based therapy for temporal lobe epilepsy.

HIPSC model reveals M1 macrophages cause atrial arrhythmia correlated to electrophysiological remodelling of atrial cardiomyocytes in 2D and 3D engineered heart tissue

Abstract Atrial fibrillation (AF) is the most common sustained arrhythmia with an estimated prevalence of 1.5–2%. Current treatments lack effectiveness and do not prevent recurrence. Inflammation is reported in AF patients, however, a causal connection remains elusive. In this study, we investigated the arrhythmic effects of pro-inflammatory macrophages (M1) on human induced pluripotent stem cell (hiPSC)-derived atrial cardiomyocytes (aCM), to better understand their role in AF. Macrophages are known to play a role in cardiac electrophysiology, but their role in arrhythmia is unclear. Three healthy hiPSC lines were differentiated to aCM and M1. Multi-electrode array recordings of isogenic, 2D cocultures of M1 and aCM showed a significant increase in beat rate irregularity compared to control conditions (P&amp;lt;0.001). Furthermore, spike amplitude of aCM and M1 cocultures was significantly decreased (P&amp;lt;0.001). Arrhythmias occurred only after activation of macrophages in the coculture. Addition of anti-inflammatory glucocorticoids significantly reduced beat irregularity in aCM and M1 cocultures compared to vehicle (1.9-fold decrease for Hydrocortisone, P&amp;lt;0.005) further supporting macrophage mediated inflammation to be the cause of the arrhythmia. RNA sequencing of aCM and M1 cocultures revealed aberrant expression of arrhythmia associated cardiac sodium and atrial potassium ion channels (SCN5A, KCNA5). Normal expression was restored through hydrocortisone addition. In addition, inflammation-induced arrhythmia was observed in 3D engineered heart tissues containing hiPSC derived aCM, M1 and cardiac fibroblasts. Tissues showed a significant increase in beating irregularity (P&amp;lt;0.005) compared to controls and had a significant, median reduction (&amp;gt;30%) of contraction amplitude (P&amp;lt;0.05). Our findings suggest a causal relationship between M1 and the occurrence of atrial arrhythmia. The reduction of arrhythmia using glucocorticoids correlates to clinical observations, that show their use being linked to reduced post operative AF burden. These results strongly support the relevance of the proposed hiPSC coculture model and elucidate a new potential AF mechanism.

A common cause for nystagmus in different congenital stationary night blindness mouse models.

In Nyxnob mice, a model for congenital nystagmus associated with congenital stationary night blindness (CSNB), synchronous oscillating retinal ganglion cells (RGCs) lead to oscillatory eye movements, i.e. nystagmus. Given the specific expression of mGluR6 and Cav 1.4 in the photoreceptor to bipolar cell synapses, as well as their clinical association with CSNB, we hypothesize that Grm6nob3 and Cav 1.4-KO mutants show, like the Nyxnob mouse, oscillations in both their RGC activity and eye movements. Using multi-electrode array recordings of RGCs and measurements of the eye movements, we demonstrate that Grm6nob3 and Cav 1.4-KO mice also show oscillations of their RGCs as well as a nystagmus. Interestingly, the preferred frequencies of RGC activity as well as the eye movement oscillations of the Grm6nob3 , Cav 1.4-KO and Nyxnob mice differ among mutants, but the neuronal activity and eye movement behaviour within a strain remain aligned in the same frequency domain. Model simulations indicate that mutations affecting the photoreceptor-bipolar cell synapse can form a common cause of the nystagmus of CSNB by driving oscillations in RGCs via AII amacrine cells. KEY POINTS: In Nyxnob mice, a model for congenital nystagmus associated with congenital stationary night blindness (CSNB), their oscillatory eye movements (i.e. nystagmus) are caused by synchronous oscillating retinal ganglion cells. Here we show that the same mechanism applies for two other CSNB mouse models - Grm6nob3 and Cav 1.4-KO mice. We propose that the retinal ganglion cell oscillations originate in the AII amacrine cells. Model simulations show that by only changing the input to ON-bipolar cells, all phenotypical differences between the various genetic mouse models can be reproduced.

Differential encoding of temporally evolving color patterns across nearby V1 neurons.

Whereas studies of the V1 cortex have focused mainly on neural line orientation preference, color inputs are also known to have a strong presence among these neurons. Individual neurons typically respond to multiple colors and nearby neurons have different combinations of preferred color inputs. However, the computations performed by V1 neurons on such color inputs have not been extensively studied. Here we aimed to address this issue by studying how different V1 neurons encode different combinations of inputs composed of four basic colors. We quantified the decoding accuracy of individual neurons from multi-electrode array recordings, comparing multiple individual neurons located within 2 mm along the vertical axis of the V1 cortex of the anesthetized rat. We found essentially all V1 neurons to be good at decoding spatiotemporal patterns of color inputs and they did so by encoding them in different ways. Quantitative analysis showed that even adjacent neurons encoded the specific input patterns differently, suggesting a local cortical circuitry organization which tends to diversify rather than unify the neuronal responses to each given input. Using different pairs of monocolor inputs, we also found that V1 neocortical neurons had a diversified and rich color opponency across the four colors, which was somewhat surprising given the fact that rodent retina express only two different types of opsins. We propose that the processing of color inputs in V1 cortex is extensively composed of multiple independent circuitry components that reflect abstract functionalities resident in the internal cortical processing rather than the raw sensory information per se.