Cortical Slices Research Articles

M-current modulation of cortical slow oscillations: Network dynamics and computational modeling.

The slow oscillation is a synchronized network activity expressed by the cortical network in slow wave sleep and under anesthesia. Waking up requires a transition from this synchronized brain state to a desynchronized one. Cholinergic innervation is critical for the transition from slow-wave-sleep to wakefulness, and muscarinic action is largely exerted through the muscarinic-sensitive potassium current (M-current) block. We investigated the dynamical impact of blocking the M-current on slow oscillations, both in cortical slices and in a cortical network computational model. Blocking M-current resulted in an elongation of Up states (by four times) and in a significant firing rate increase, reflecting an increased network excitability, albeit no epileptiform discharges occurred. These effects were replicated in a biophysical cortical model, where a parametric reduction of the M-current resulted in a progressive elongation of Up states and firing rate. All neurons, and not only those modeled with M-current, increased their firing rates due to network recurrency. Further increases in excitability induced even longer Up states, approaching the microarousals described in the transition towards wakefulness. Our results bridge an ionic current with network modulation, providing a mechanistic insight into network dynamics of awakening.

Early developmental deletion of forebrain Ank2 causes seizure-related phenotypes by reshaping the synaptic proteome

Rare genetic variants in ANK2, which encodes ankyrin-B, are associated with neurodevelopmental disorders (NDDs); however, their pathogenesis is poorly understood. We find that mice with prenatal deletion in cortical excitatory neurons and oligodendrocytes (Ank2-/-:Emx1-Cre), but not with adolescent deletion in forebrain excitatory neurons (Ank2-/-:CaMKIIα-Cre), display severe spontaneous seizures, increased mortality, hyperactivity, and social deficits. Calcium imaging of cortical slices from Ank2-/-:Emx1-Cre mice shows increased neuronal calcium event amplitude and frequency, along with network hyperexcitability and hypersynchrony. Quantitative proteomic analysis of cortical synaptic membranes reveals upregulation of dendritic spine plasticity-regulatory proteins and downregulation of intermediate filaments. Characterization of the ankyrin-B interactome identifies interactors associated with autism and epilepsy risk factors and synaptic proteins. The AMPA receptor antagonist, perampanel, restores cortical neuronal activity and partially rescues survival in Ank2-/-:Emx1-Cre mice. Our findings suggest that synaptic proteome alterations resulting from Ank2 deletion impair neuronal activity and synchrony, leading to NDDs-related behavioral impairments.

Involvement of P2Y1, P2Y6, A1 and A2A Receptors in the Purinergic Inhibition of NMDA-Evoked Noradrenaline Release in the Rat Brain Cortex.

In the cerebral cortex, glutamate activates NMDA receptors (NMDARs), localized in noradrenergic neurons, inducing noradrenaline release that may have a permissive effect on glutamatergic transmission, and therefore, on the modulation of long-term plasticity. ATP is co-released with noradrenaline, and with its metabolites (ADP and adenosine) is involved in the purinergic modulation of electrically-evoked noradrenaline release. However, it is not known if noradrenaline release evoked by activation of NMDARs is also under purinergic modulation. The present study aimed to investigate and to characterize the purinergic modulation of noradrenaline release evoked by NMDARs. Stimulation of rat cortical slices with 30 µM NMDA increased noradrenaline release, which was inhibited by ATP upon metabolization into ADP and adenosine and by the selective agonists of A1 and A2A receptors, CPA and CGS2680, respectively. It was also inhibited by UTP and UDP, which are mainly released under pathophysiological situations. Characterization of the effects mediated by these compounds indicated the involvement of P2Y1, P2Y6, A1 and A2A receptors. It is concluded that, in the rat brain cortex, NMDA-evoked noradrenaline release is modulated by several purinergic receptors that may represent a relevant mechanism to regulate the permissive effect of noradrenaline on NMDA-induced neuroplasticity.

The ferroptosis inducer RSL3 triggers interictal epileptiform activity in mice cortical neurons.

Epilepsy is a neurological disorder characterized by recurrent seizures, which result from excessive, synchronous discharges of neurons in different brain areas. In about 30% of cases, epileptic discharges, which vary in their etiology and symptomatology, are difficult to treat with conventional drugs. Ferroptosis is a newly defined iron-dependent programmed cell death, characterized by excessive accumulation of lipid peroxides and reactive oxygen species. Evidence has been provided that ferroptosis is involved in epilepsy, and in particular in those forms resistant to drugs. Here, whole cell patch clamp recordings, in current and voltage clamp configurations, were performed from layer IV principal neurons in cortical slices obtained from adult mouse brain. Application of the ferroptosis inducer RAS-selective lethal 3 (RSL3) induced interictal epileptiform discharges which started at RSL3 concentrations of 2 μM and reached a plateau at 10 μM. This effect was not due to changes in active or passive membrane properties of the cells, but relied on alterations in synaptic transmission. In particular, interictal discharges were dependent on the excessive excitatory drive to layer IV principal cells, as suggested by the increase in frequency and amplitude of spontaneously occurring excitatory glutamatergic currents, possibly dependent on the reduction of inhibitory GABAergic ones. This led to an excitatory/inhibitory unbalance in cortical circuits. Interictal bursts could be prevented or reduced in frequency by the lipophilic antioxidant Vitamin E (30 μM). This study allows identifying new targets of ferroptosis-mediated epileptic discharges opening new avenues for the treatment of drug-resistant forms of epilepsy.

Source-Based EEG Neurofeedback for Sustained Motor Imagery of a Single Leg

The aim of the study was to test the feasibility of visual-neurofeedback-guided motor imagery (MI) of the dominant leg, based on source analysis with real-time sLORETA derived from 44 EEG channels. Ten able-bodied participants took part in two sessions: session 1 sustained MI without feedback and session 2 sustained MI of a single leg with neurofeedback. MI was performed in 20 s on and 20 s off intervals to mimic functional magnetic resonance imaging. Neurofeedback in the form of a cortical slice presenting the motor cortex was provided from a frequency band with the strongest activity during real movements. The sLORETA processing delay was 250 ms. Session 1 resulted in bilateral/contralateral activity in the 8–15 Hz band dominantly over the prefrontal cortex while session 2 resulted in ipsi/bilateral activity over the primary motor cortex, covering similar areas as during motor execution. Different frequency bands and spatial distributions in sessions with and without neurofeedback may reflect different motor strategies, most notably a larger proprioception in session 1 and operant conditioning in session 2. Single-leg MI might be used in the early phases of rehabilitation of stroke patients. Simpler visual feedback and motor cueing rather than sustained MI might further increase the intensity of cortical activation.

Spontaneous and multifaceted ATP release from astrocytes at the scale of hundreds of synapses.

Astrocytes participate in information processing by releasing neuroactive substances termed gliotransmitters, including ATP. Individual astrocytes come into contact with thousands of synapses with their ramified structure, but the spatiotemporal dynamics of ATP gliotransmission remains unclear, especially in physiological brain tissue. Using a genetically encoded fluorescent sensor, GRABATP1.0 , we discovered that extracellular ATP increased locally and transiently in absence of stimuli in neuron-glia co-cultures, cortical slices, and the anesthetized mouse brain. Spontaneous ATP release events were tetrodotoxin-insensitive but suppressed by gliotoxin, fluorocitrate, and typically spread over 50-250 μm2 area at concentrations capable of activating purinergic receptors. Besides, most ATP events did not coincide with Ca2+ transients, and intracellular Ca2+ buffering with BAPTA-AM did not affect ATP event frequency. Clustering analysis revealed that these events followed multiple distinct kinetics, and blockade of exocytosis only decreased a minor group of slow events. Overall, astrocytes spontaneously release ATP through multiple mechanisms, mainly in non-vesicular and Ca2+ -independent manners, thus potentially regulating hundreds of synapses all together.

Casting a Wider Net When Fishing for Mechanisms of Anesthesia.

Casting a Wider Net When Fishing for Mechanisms of Anesthesia.

Orai, RyR, and IP3R channels cooperatively regulate calcium signaling in brain mid-capillary pericytes

Pericytes are multifunctional cells of the vasculature that are vital to brain homeostasis, yet many of their fundamental physiological properties, such as Ca2+ signaling pathways, remain unexplored. We performed pharmacological and ion substitution experiments to investigate the mechanisms underlying pericyte Ca2+ signaling in acute cortical brain slices of PDGFRβ-Cre::GCaMP6f mice. We report that mid-capillary pericyte Ca2+ signalling differs from ensheathing type pericytes in that it is largely independent of L- and T-type voltage-gated calcium channels. Instead, Ca2+ signals in mid-capillary pericytes were inhibited by multiple Orai channel blockers, which also inhibited Ca2+ entry triggered by endoplasmic reticulum (ER) store depletion. An investigation into store release pathways indicated that Ca2+ transients in mid-capillary pericytes occur through a combination of IP3R and RyR activation, and that Orai store-operated calcium entry (SOCE) is required to sustain and amplify intracellular Ca2+ increases evoked by the GqGPCR agonist endothelin-1. These results suggest that Ca2+ influx via Orai channels reciprocally regulates IP3R and RyR release pathways in the ER, which together generate spontaneous Ca2+ transients and amplify Gq-coupled Ca2+ elevations in mid-capillary pericytes. Thus, SOCE is a major regulator of pericyte Ca2+ and a target for manipulating their function in health and disease.

Neocortical and medial temporal seizures have distinct impacts on brain responsiveness.

Focal epileptic seizures are characterized by abnormal neuronal discharges that can spread to other cortical areas and interfere with brain activity, thereby altering the patient's experience and behavior. The origin of these pathological neuronal discharges encompasses various mechanisms that converge toward similar clinical manifestations. Recent studies have suggested that medial temporal lobe (MTL) and neocortical (NC) seizures are often underpinned by two characteristic onset patterns, which, respectively, affect and spare synaptic transmission in cortical slices. However, these synaptic alterations and their effects have never been confirmed or studied in intact human brains. To fill this gap, we here evaluate whether responsiveness of MTL and NC are differentially affected by focal seizures, using a unique data set of cortico-cortical evoked potentials (CCEPs) collected during seizures triggered by single-pulse electrical stimulation (SPES). We find that responsiveness is abruptly reduced by the onset of MTL seizures, despite increased spontaneous activity, whereas it is preserved in the case of NC seizures. The present results provide an extreme example of dissociation between responsiveness and activity and show that brain networks are diversely affected by the onset of MTL and NC seizures, thus extending at the whole brain level the evidence of synaptic alteration found in vitro.

Deletion of CaMKIIα disrupts glucose metabolism, glutamate uptake, and synaptic energetics in the cerebral cortex.

Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) is a key regulator of neuronal signaling and synaptic plasticity. Synaptic activity and neurotransmitter homeostasis are closely coupled to the energy metabolism of both neurons and astrocytes. However, whether CaMKIIα function is implicated in brain energy and neurotransmitter metabolism remains unclear. Here, we explored the metabolic consequences of CaMKIIα deletion in the cerebral cortex using a genetic CaMKIIα knockout (KO) mouse. Energy and neurotransmitter metabolism was functionally investigated in acutely isolated cerebral cortical slices using stable 13C isotope tracing, whereas the metabolic function of synaptosomes was assessed by the rates of glycolytic activity and mitochondrial respiration. The oxidative metabolism of [U-13C]glucose was extensively reduced in cerebral cortical slices of the CaMKIIα KO mice. In contrast, metabolism of [1,2-13C]acetate, primarily reflecting astrocyte metabolism, was unaffected. Cellular uptake, and subsequent metabolism, of [U-13C]glutamate was decreased in cerebral cortical slices of CaMKIIα KO mice, whereas uptake and metabolism of [U-13C]GABA were unaffected, suggesting selective metabolic impairments of the excitatory system. Synaptic metabolic function was maintained during resting conditions in isolated synaptosomes from CaMKIIα KO mice, but both the glycolytic and mitochondrial capacities became insufficient when the synaptosomes were metabolically challenged. Collectively, this study shows that global deletion of CaMKIIα significantly impairs cellular energy and neurotransmitter metabolism, particularly of neurons, suggesting a metabolic role of CaMKIIα signaling in the brain.

AMP-activated protein kinase activators have compound and concentration-specific effects on brain metabolism.

AMP-activated protein kinase (AMPK) is a key sensor of energy balance playing important roles in the balancing of anabolic and catabolic activities. The high energy demands of the brain and its limited capacity to store energy indicate that AMPK may play a significant role in brain metabolism. Here, we activated AMPK in guinea pig cortical tissue slices, both directly with A769662 and PF 06409577 and indirectly with AICAR and metformin. We studied the resultant metabolism of [1-13C]glucose and [1,2-13C]acetate using NMR spectroscopy. We found distinct activator concentration-dependent effects on metabolism, which ranged from decreased metabolic pool sizes at EC50 activator concentrations with no expected stimulation in glycolytic flux to increased aerobic glycolysis and decreased pyruvate metabolism with certain activators. Further, activation with direct versus indirect activators produced distinct metabolic outcomes at both low (EC50) and higher (EC50 × 10) concentrations. Specific direct activation of β1-containing AMPK isoforms with PF 06409577 resulted in increased Krebs cycle activity, restoring pyruvate metabolism while A769662 increased lactate and alanine production, as well as labelling of citrate and glutamine. These results reveal a complex metabolic response to AMPK activators in brain beyond increased aerobic glycolysis and indicate that further research is warranted into their concentration- and mechanism-dependent impact.

The protective effects of astaxanthin on pre-antral follicle degeneration in ovine vitrified/warmed ovarian tissue

This study assesses the protective effects of astaxanthin (AST) against vitrification/warming-induced cryoinjuries of ovarian tissue slices in sheep. Cortical slices of slaughterhouse acquired-ovine ovaries were randomly distributed in different groups: fresh, toxicity, and five vitrification groups including vitrification in presence of 0 (control group), 1, 10 and 100 μM astaxanthin or 100 μM vitamin E. After vitrification/warming and 24 h culturing, the samples were subjected to histological studies, antioxidant evaluation by TAC and TBAR assays, and assessment of relative expression of BMP4, BMP15, GDF9 and KITLG genes related to folliculogenesis and follicular growth regulation. According to the results, vitrification reduced the percentage of morphologically intact follicles compared to the fresh and toxicity groups (p < 0.05). In vitrification groups, vitamin E and all three concentrations of AST increased the percentage of intact pre-antral follicles and antioxidant activity relative to the vitrified control (p < 0.05). This enhancement significantly occurred in 10 μM AST group more than vitamin E (p < 0.05). Also, 10 μM concentration of AST enhanced the expression of all the examined genes compared to the control (p < 0.05), while the expression of BMP4, BMP15 and KITLG was higher in the AST than vitamin E (p < 0.05). The latter could increase only the expression of GDF9 compared to the control group (p = 0.011). In conclusion, AST is a highly effective antioxidant for maintaining the survival of pre-antral follicles, retaining cell density, increasing total antioxidant capacity, and increasing the expression of some genes related to follicular development after short-term culture of vitrified/warmed ovarian tissue slices.

Deep isoflurane anesthesia is associated with alterations in ion homeostasis and specific Na+/K+-ATPase impairment in the rat brain.

Maintenance of ion homeostasis is essential for normal brain function. Inhalational anesthetics are known to act on various receptors but their effects on ion homeostatic systems, such as the Na+/K+-ATPase, remain largely unexplored. Based on reports demonstrating global network activity and wakefulness modulation by interstitial ions, we hypothesized that deep isoflurane anesthesia affects ion homeostasis and the key mechanism for clearing extracellular K+, the Na+/K+-ATPase. Using ion-selective microelectrodes, we assessed isoflurane-induced extracellular ion dynamics in cortical slices of male and female Wistar rats in the absence of synaptic activity, the presence of two-pore-domain K+ channel antagonists, during seizures, and spreading depolarizations. We measured specific isoflurane effects on Na+/K+-ATPase function using a coupled enzyme assay and studied the relevance of our findings in vivo and in silico. Isoflurane concentrations clinically relevant for burst suppression anesthesia increased baseline [K+]o (mean±SD: 3.0 ± 0.0 versus 3.9 ± 0.5mM, p<0.001, n=39) and lowered [Na+]o (153.4 ± 0.8 versus 145.2 ± 6.0mM, p<0.001, n=28). Similar changes in [K+]o, [Na+]o, and a substantial drop in [Ca2+]o (1.5 ± 0.0 versus 1.2 ± 0.1mM, p=0.001, n=16) during inhibition of synaptic activity and two-pore-domain K+ suggested a different underlying mechanism. Following seizure-like events and spreading depolarization, isoflurane greatly slowed [K+]o clearance (63.4 ± 18.2 versus 196.2 ± 82.4s, p<0.001, n=14). Na+/K+-ATPase activity was markedly reduced after isoflurane exposure (>25%), affecting specifically the α2/3 activity fraction. In vivo, isoflurane-induced burst suppression resulted in impaired [K+]o clearance and interstitial K+ accumulation. A computational biophysical model reproduced the observed effects on [K+]o and displayed intensified bursting when Na+/K+-ATPase activity was reduced by 35%. Finally, Na+/K+-ATPase inhibition with ouabain induced burst-like activity during light anesthesia in vivo. Our results demonstrate cortical ion homeostasis perturbation and specific Na+/K+-ATPase impairment during deep isoflurane anesthesia. Slowed K+ clearance and extracellular accumulation might modulate cortical excitability during burst suppression generation while prolonged Na+/K+-ATPase impairment could contribute to neuronal dysfunction after deep anesthesia.

Guanfacine inhibits interictal epileptiform events and sodium currents in prefrontal cortex pyramidal neurons

BackgroundGuanfacine (an alpha-2A receptor agonist) is a commonly used drug with recognized efficacy in the treatment of attention deficit hyperactivity disorder (ADHD). This study aimed to assess the effects of guanfacine on short-lasting (interictal) epileptiform discharges in cortical neurons. Moreover, we assessed the effects of guanfacine on voltage-gated sodium currents.MethodsWe conducted patch-clamp recordings in prefrontal cortex pyramidal neurons obtained from young rats. Interictal epileptiform events were evoked in cortical slices in a zero magnesium proepileptic extracellular solution with an elevated concentration of potassium ions.ResultsInterictal epileptiform discharges were spontaneous depolarisations, which triggered action potentials. Guanfacine (10 and 100 µM) inhibited the frequency of epileptiform discharges. The effect of guanfacine on interictal events persisted in the presence of alpha-2 adrenergic receptor antagonist idazoxan. The tested drug inhibited neuronal excitability. Tonic NMDA currents were not influenced by guanfacine. Recordings from dispersed neurons showed that the tested drug (10 and 100 µM) inhibited persistent and fast inactivating voltage-gated sodium currents.ConclusionsThis study shows that guanfacine inhibits interictal discharges in cortical neurons independently of alpha-2A adrenergic receptors. This effect may be mediated by voltage-gated sodium currents. Inhibition of interictal activity by guanfacine may be of clinical importance because interictal events often occur in patients with ADHD and may contribute to symptoms of this disease.Graphical abstract

Transient receptor potential Ankyrin-1 (TRPA1) agonists suppress myelination and induce demyelination in organotypic cortical slices.

Oligodendrocytes are highly specialized glial cells characterized by their production of multilayer myelin sheaths that wrap axons to speed up action potential propagation. It is due to their specific role in supporting axons that impairment of myelin structure and function leads to debilitating symptoms in a wide range of degenerative diseases, including Multiple Sclerosis and Leukodystrophies. It is known that myelin damage can be receptor-mediated and recently oligodendrocytes have been shown to express Ca2+ -permeable Transient Receptor Potential Ankyrin-1 (TRPA1) channels, whose activation can result in myelin damage in ischemia. Here, we show, using organotypic cortical slice cultures, that TRPA1 activation, by TRPA1 agonists JT010 and Carvacrol for varying lengths of time, induces myelin damage. Although TRPA1 activation does not appear to affect oligodendrocyte progenitor cell number or proliferation, it prevents myelin formation and after myelination causes internodal shrinking and significant myelin degradation. This does not occur when the TRPA1 antagonist, A967079, is also applied. Of note is that when TRPA1 agonists are applied for either 24 h, 3 days or 7 days, axon integrity appears to be preserved while mature myelinated oligodendrocytes remain but with significantly shortened internodes. These results provide further evidence that TRPA1 inhibition could be protective in demyelination diseases and a promising therapy to prevent demyelination and promote remyelination.

A transcriptional constraint mechanism limits the homeostatic response to activity deprivation in mammalian neocortex.

Healthy neuronal networks rely on homeostatic plasticity to maintain stable firing rates despite changing synaptic drive. These mechanisms, however, can themselves be destabilizing if activated inappropriately or excessively. For example, prolonged activity deprivation can lead to rebound hyperactivity and seizures. While many forms of homeostasis have been described, whether and how the magnitude of homeostatic plasticity is constrained remains unknown. Here, we uncover negative regulation of cortical network homeostasis by the PARbZIP family of transcription factors. In cortical slice cultures made from knockout mice lacking all three of these factors, the network response to prolonged activity withdrawal measured with calcium imaging is much stronger, while baseline activity is unchanged. Whole-cell recordings reveal an exaggerated increase in the frequency of miniature excitatory synaptic currents reflecting enhanced upregulation of recurrent excitatory synaptic transmission. Genetic analyses reveal that two of the factors, Hlf and Tef, are critical for constraining plasticity and for preventing life-threatening seizures. These data indicate that transcriptional activation is not only required for many forms of homeostatic plasticity but is also involved in restraint of the response to activity deprivation.

Abstract 107: Potentiating GPR68 Function With Ogerin Protects Against Ischemic Brain Injury In Mice

Introduction: Ischemic stroke leads to prolonged reduction in brain pH. We previously found that G-protein coupled receptor 68 (GPR68), a proton-sensitive GPCR, mediates a neuroprotective pathway in brain ischemia. To investigate translational potential of GPR68 targeting, we examine here the effect of Ogerin, a positive allosteric modulator of GPR68, on ischemic brain injury. Methods: Using organotypic cortical slices, we examined the effect of Ogerin on signaling. Next, we induced oxygen-glucose deprivation (OGD) in slices and analyzed delayed neuronal injury at 24 hr with propidium iodide (PI) staining. Further, we performed transient middle cerebral artery occlusion (tMCAO) in WT and GPR68 knockout mice, treated them with vehicle or Ogerin, and analyzed brain infarct with TTC staining. We then assessed the effect of Ogerin on cerebral blood flow using Laser Speckle Imaging. Lastly, we performed immunostaining to determine the effect of Ogerin on neuroinflammation. Results: Ogerin induced phosphorylation of protein kinase C substrates in wild-type but not GPR68-/- slices. Ogerin (10 μM) reduced PI intake in OGD-treated slices, suggesting a reduced neuronal injury. Following tMCAO, intraperitoneal injection of Ogerin had no significant effect on brain infarct size. In contrast, Ogerin through intracerebroventricular injection (i.c.v) (0.25 mg/kg) significantly reduced brain infarct in WT (18.5 ± 11.8% (Ogerin) vs. 37.7 ± 7.2% (vehicle); p = 0.0043) but had no significant effect in GPR68-/- mice. Intravenous (i.v.) delivery of Ogerin at 3 &amp; 1 mg/kg, but not at 0.3 mg/kg, significantly reduced brain infarct in WT. Moreover, delayed i.v delivery of Ogerin (1 mg/kg) at both 3 hours and 6 hours after reperfusion significantly reduced brain infarct. We further replicated the experiment in female mice, focusing on the metestrus stage. Ogerin through i.v (1 mg/kg) also reduced brain infarct in female mice. In parallel with the reduced brain infarct, Ogerin-treated mice exhibited a balanced (ipsilateral: contralateral) cerebral blood perfusion at 24 hour, and a reduced inflammatory response in microglia/macrophages. Conclusion: These data suggest that GPR68 potentiation with Ogerin is a promising neuroprotective reagent in brain ischemia.

Axonal connections between S1 barrel, M1, and S2 cortex in the newborn mouse.

The development of functionally interconnected networks between primary (S1), secondary somatosensory (S2), and motor (M1) cortical areas requires coherent neuronal activity via corticocortical projections. However, the anatomical substrate of functional connections between S1 and M1 or S2 during early development remains elusive. In the present study, we used ex vivo carbocyanine dye (DiI) tracing in paraformaldehyde-fixed newborn mouse brain to investigate axonal projections of neurons in different layers of S1 barrel field (S1Bf), M1, and S2 toward the subplate (SP), a hub layer for sensory information transfer in the immature cortex. In addition, we performed extracellular recordings in neocortical slices to unravel the functional connectivity between these areas. Our experiments demonstrate that already at P0 neurons from the cortical plate (CP), layer 5/6 (L5/6), and the SP of both M1 and S2 send projections through the SP of S1Bf. Reciprocally, neurons from CP to SP of S1Bf send projections through the SP of M1 and S2. Electrophysiological recordings with multi-electrode arrays in cortical slices revealed weak, but functional synaptic connections between SP and L5/6 within and between S1 and M1. An even lower functional connectivity was observed between S1 and S2. In summary, our findings demonstrate that functional connections between SP and upper cortical layers are not confined to the same cortical area, but corticocortical connection between adjacent cortical areas exist already at the day of birth. Hereby, SP can integrate early cortical activity of M1, S1, and S2 and shape the development of sensorimotor integration at an early stage.

Control of G2 Phase Duration by CDC25B Modulates the Switch from Direct to Indirect Neurogenesis in the Neocortex.

During development, cortical neurons are produced in a temporally regulated sequence from apical progenitors, directly or indirectly, through the production of intermediate basal progenitors. The balance between these major progenitor types is critical for the production of the proper number and types of neurons, and it is thus important to decipher the cellular and molecular cues controlling this equilibrium. Here we address the role of a cell cycle regulator, the CDC25B phosphatase, in this process. We show that, in the developing mouse neocortex of both sex, deleting CDC25B in apical progenitors leads to a transient increase in the production of TBR1+ neurons at the expense of TBR2+ basal progenitors. This phenotype is associated with lengthening of the G2 phase of the cell cycle, the total cell cycle length being unaffected. Using in utero electroporation and cortical slice cultures, we demonstrate that the defect in TBR2+ basal progenitor production requires interaction with CDK1 and is because of the G2 phase lengthening in CDC25B mutants. Together, this study identifies a new role for CDC25B and G2 phase length in direct versus indirect neurogenesis at early stages of cortical development.SIGNIFICANCE STATEMENT This study is the first analysis of the function of CDC25B, a G2/M regulator, in the developing neocortex. We show that removing CDC25B function leads to a transient increase in neuronal differentiation at early stages, occurring simultaneously with a decrease in basal intermediate progenitors (bIPs). Conversely, a CDC25B gain of function promotes production of bIPs, and this is directly related to CDC25B's ability to regulate CDK1 activity. This imbalance of neuron/progenitor production is linked to a G2 phase lengthening in apical progenitors; and using pharmacological treatments on cortical slice cultures, we show that shortening the G2 phase is sufficient to enhance bIP production. Our results reveal the importance of G2 phase length regulation for neural progenitor fate determination.

Dynamic regulation of excitatory and inhibitory synaptic transmission by growth hormone in the developing mouse brain.

Normal sensory and cognitive function of the brain relies on its intricate and complex neural network. Synaptogenesis and synaptic plasticity are critical to neural circuit formation and maintenance, which are regulated by coordinated intracellular and extracellular signaling. Growth hormone (GH) is the most abundant anterior pituitary hormone. Its deficiencies could alter brain development and impair learning and memory, while GH replacement therapy in human patients and animal models has been shown to ameliorate cognitive deficits caused by GH deficiency. However, the underlying mechanism remains largely unknown. In this study, we investigated the neuromodulatory function of GH in young (pre-weaning) mice at two developmental time points and in two different brain regions. Neonatal mice were subcutaneously injected with recombinant human growth hormone (rhGH) on postnatal day (P) 14 or 21. Excitatory and inhibitory synaptic transmission was measured using whole-cell recordings in acute cortical slices 2 h after the injection. We showed that injection of rhGH (2 mg/kg) in P14 mice significantly increased the frequency of mEPSCs, but not that of mIPSCs, in both hippocampal CA1 pyramidal neurons and L2/3 pyramidal neurons of the barrel field of the primary somatosensory cortex (S1BF). Injection of rhGH (2 mg/kg) in P21 mice significantly increased the frequency of mEPSCs and mIPSCs in both brain regions. Perfusion of rhGH (1 μM) onto acute brain slices in P14 mice had similar effects. Consistent with the electrophysiological results, the dendritic spine density of CA1 pyramidal neurons and S1BF L2/3 pyramidal neurons increased following in vivo injection of rhGH. Furthermore, NMDA receptors and postsynaptic calcium-dependent signaling contributed to rhGH-dependent regulation of both excitatory and inhibitory synaptic transmission. Together, these results demonstrate that regulation of excitatory and inhibitory synaptic transmission by rhGH occurs in a developmentally dynamic manner, and have important implication for identifying GH treatment strategies without disturbing excitation/inhibition balance.