Human 3D cellular model of hypoxic brain injury of prematurity.

Owing to recent medical and technological advances in neonatal care, infants born extremely premature have increased survival rates1,2. After birth, these infants are at high risk of hypoxic episodes because of lung immaturity, hypotension and lack of cerebral-flow regulation, and can develop a severe condition called encephalopathy of prematurity3. Over 80% of infants born before post-conception week 25 have moderate-to-severe long-term neurodevelopmental impairments4. The susceptible cell types in the cerebral cortex and the molecular mechanisms underlying associated gray-matter defects in premature infants remain unknown. Here we used human three-dimensional brain-region-specific organoids to study the effect of oxygen deprivation on corticogenesis. We identified specific defects in intermediate progenitors, a cortical cell type associated with the expansion of the human cerebral cortex, and showed that these are related to the unfolded protein response and changes. Moreover, we verified these findings in human primary cortical tissue and demonstrated that a small-molecule modulator of the unfolded protein response pathway can prevent the reduction in intermediate progenitors following hypoxia. We anticipate that this human cellular platform will be valuable for studying the environmental and genetic factors underlying injury in the developing human brain

Questioning Glutamate Excitotoxicity in Acute Brain Damage: The Importance of Spreading Depolarization

Background Within 2 min of severe ischemia, spreading depolarization (SD) propagates like a wave through compromised gray matter of the higher brain. More SDs arise over hours in adjacent tissue, expanding the neuronal damage. This period represents a therapeutic window to inhibit SD and so reduce impending tissue injury. Yet most neuroscientists assume that the course of early brain injury can be explained by glutamate excitotoxicity, the concept that immediate glutamate release promotes early and downstream brain injury. There are many problems with glutamate release being the unseen culprit, the most practical being that the concept has yielded zero therapeutics over the past 30 years. But the basic science is also flawed, arising from dubious foundational observations beginning in the 1950s Methods Literature pertaining to excitotoxicity and to SD over the past 60 years is critiqued. Results Excitotoxicity theory centers on the immediate and excessive release of glutamate with resulting neuronal hyperexcitation. This instigates poststroke cascades with subsequent secondary neuronal injury. By contrast, SD theory argues that although SD evokes some brief glutamate release, acute neuronal damage and the subsequent cascade of injury to neurons are elicited by the metabolic stress of SD, not by excessive glutamate release. The challenge we present here is to find new clinical targets based on more informed basic science. This is motivated by the continuing failure by neuroscientists and by industry to develop drugs that can reduce brain injury following ischemic stroke, traumatic brain injury, or sudden cardiac arrest. One important step is to recognize that SD plays a central role in promoting early neuronal damage. We argue that uncovering the molecular biology of SD initiation and propagation is essential because ischemic neurons are usually not acutely injured unless SD propagates through them. The role of glutamate excitotoxicity theory and how it has shaped SD research is then addressed, followed by a critique of its fading relevance to the study of brain injury. Conclusions Spreading depolarizations better account for the acute neuronal injury arising from brain ischemia than does the early and excessive release of glutamate

The Critical Role of Spreading Depolarizations in Early Brain Injury: Consensus and Contention

Background: When a patient arrives in the emergency department following a stroke, a traumatic brain injury, or sudden cardiac arrest, there is no therapeutic drug available to help protect their jeopardized neurons. One crucial reason is that we have not identified the molecular mechanisms leading to electrical failure, neuronal swelling, and blood vessel constriction in newly injured gray matter. All three result from a process termed spreading depolarization (SD). Because we only partially understand SD, we lack molecular targets and biomarkers to help neurons survive after losing their blood flow and then undergoing recurrent SD. Methods: In this review, we introduce SD as a single or recurring event, generated in gray matter following lost blood flow, which compromises the Na+/K+ pump. Electrical recovery from each SD event requires so much energy that neurons often die over minutes and hours following initial injury, independent of extracellular glutamate. Results: We discuss how SD has been investigated with various pitfalls in numerous experimental preparations, how overtaxing the Na+/K+ ATPase elicits SD. Elevated K+ or glutamate are unlikely natural activators of SD. We then turn to the properties of SD itself, focusing on its initiation and propagation as well as on computer modeling. Conclusions: Finally, we summarize points of consensus and contention among the authors as well as where SD research may be heading. In an accompanying review, we critique the role of the glutamate excitotoxicity theory, how it has shaped SD research, and its questionable importance to the study of early brain injury as compared with SD theory. © 2022, The Author(s)

Dynamic gain decomposition reveals functional effects of dendrites, ion channels and input statistics in population coding

Modern, high-density neuronal recordings reveal at ever higher precision how information is represented by neural populations. Still, we lack the tools to understand these processes bottom-up, emerging from the biophysical properties of neurons, synapses, and network structure. The concept of the dynamic gain function, a spectrally resolved approximation of a population's coding capability, has the potential to link cell-level properties to network-level performance. However, the concept is not only useful but also very complex because the dynamic gain's shape is co-determined by axonal and somatodendritic parameters and the population's operating regime. Previously, this complexity precluded an understanding of any individual parameter's impact. Here, we decomposed the dynamic gain function into three components corresponding to separate signal transformations. This allowed attribution of network-level encoding features to specific cell-level parameters. Applying the method to data from real neurons and biophysically plausible models, we found: 1. The encoding bandwidth of real neurons, approximately 400 Hz, is constrained by the volt­age dependence of axonal currents during early action potential initiation. 2. State-of-the-art models only achieve encoding bandwidths around 100 Hz and are limited mainly by subthreshold processes instead. 3. Large dendrites and low-threshold potassium currents modulate the bandwidth by shaping the subthresh­old stimulus-to-voltage transformation. Our decomposition provides physiological interpretations when the dynamic gain curve changes, for instance during spectrinopathies and neurodegeneration. By pinpointing shortcomings of current models, it also guides inference of neuron models best suited for large-scale network simulations. Significant Statement The dynamic gain function quantifies how neurons can engage in collective, network-level activity, shape brain rhythms and information encoding. Its shape results from a complex interaction between properties of different molecules (ion channels) and cell compartments (morphology, resistance), and is so far only understood for the simplest neuron models. Here we provide an interpretable analysis, decomposing the dynamic gain based on the stimulus transformation steps in individual neurons. We apply the decomposition to data from real neurons and complex models, and attribute changes of the dynamic gain to specific sub-and suprathreshold processes. Using this decomposition method, we reveal the relevance of subthreshold potassium channels for ultrafast information encoding and expose the shortcomings of even the state-of-the art neuron model

The Outwardly Rectifying Current of Layer 5 Neocortical Neurons that was Originally Identified as "Non-Specific Cationic" Is Essentially a Potassium Current.

In whole-cell patch clamp recordings from layer 5 neocortical neurons, blockade of voltage gated sodium and calcium channels leaves a cesium current that is outward rectifying. This current was originally identified as a "non-specific cationic current", and subsequently it was hypothesized that it is mediated by TRP channels. In order to test this hypothesis, we used fluorescence imaging of intracellular sodium and calcium indicators, and found no evidence to suggest that it is associated with influx of either of these ions to the cell body or dendrites. Moreover, the current is still prominent in neurons from TRPC1-/- and TRPC5-/- mice. The effects on the current of various blocking agents, and especially its sensitivity to intracellular tetraethylammonium, suggest that it is not a non-specific cationic current, but rather that it is generated by cesium-permeable delayed rectifier potassium channels

Pharmacological sensitivity of I_cs.

<p><i>A</i>: Changes (as compared to break-in) in current amplitude at +40 mV in response to the potential I_cs antagonists: carbenoxelone (100 μM), SKF96365 (100 μM), 2-APB (100 μM), La³⁺ (1 mM) and TEA (40 mM). La³⁺ concentration was reduced to 100 μM when co-applied with TEA. <i>B</i>: Comparison of current amplitudes at +40 mV when recording electrodes contained either Cs⁺ (135 mM) or Cs⁺ + TEA (30 mM of TEA replaced) to block K⁺ channels. <i>P</i> values represent comparison to control, <i>P</i> > 0.05; *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001). Data shown are averages and error bars represent s.e.m.</p

I_cs in cortical layer 5 pyramidal neurons.

<p><i>A</i>: Whole cell, voltage clamp recording while blocking Na⁺ currents with TTX (1μM), Ca²⁺ currents with Cd²⁺ (200μM) and Cs⁺ (135mM) in the recording pipette to block K⁺ currents. Voltage steps (10 mV increments) revealed an outward current at voltages more depolarized than -20mV. <i>B</i>: In the same cell, a slow (110 mV/s) voltage ramp (-70 to +40 mV) generated an outward rectifying instantaneous IV curve similar to the one in A (red squares). <i>C</i>: Repolarizing voltage steps from +40 mV (10 mV increments) reveal tail currents at potentials more negative than -40 mV <i>(left)</i>. <i>Right</i>: IV curve for this cell shows reversal at around -40 mV. Inset: distribution of reversal potentials of I_cs for 8 neurons.</p

Voltage-dependent activation and deactivation of I_cs.

<p><i>A</i>: Response of a representative neuron to depolarizing voltage steps from a holding potential of -70 mV. Application of voltage steps (lower panel) resulted in outward currents (upper panel, solid lines) which were fitted mono-exponentially (dashed lines). <i>B</i>: Hyperpolarizing steps (lower panel) from +20 mV, resulted in inward tail currents (upper panel, solid lines). Deactivation of I_cs was voltage-dependent and was also fitted with single exponentials (dashed lines). <i>C</i>: Time constants of activation (empty squares) and deactivation (filled squares) as a function of membrane voltage (n = 5 neurons).</p

Pharmacological sensitivity of I_cs.

<p><i>A</i>: Changes (as compared to break-in) in current amplitude at +40 mV in response to the potential I_cs antagonists: carbenoxelone (100 μM), SKF96365 (100 μM), 2-APB (100 μM), La³⁺ (1 mM) and TEA (40 mM). La³⁺ concentration was reduced to 100 μM when co-applied with TEA. <i>B</i>: Comparison of current amplitudes at +40 mV when recording electrodes contained either Cs⁺ (135 mM) or Cs⁺ + TEA (30 mM of TEA replaced) to block K⁺ channels. <i>P</i> values represent comparison to control, <i>P</i> > 0.05; *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001). Data shown are averages and error bars represent s.e.m.</p

Human 3D cellular model of hypoxic brain injury of prematurity.

Owing to recent medical and technological advances in neonatal care, infants born extremely premature have increased survival rates1,2. After birth, these infants are at high risk of hypoxic episodes because of lung immaturity, hypotension and lack of cerebral-flow regulation, and can develop a severe condition called encephalopathy of prematurity3. Over 80% of infants born before post-conception week 25 have moderate-to-severe long-term neurodevelopmental impairments4. The susceptible cell types in the cerebral cortex and the molecular mechanisms underlying associated gray-matter defects in premature infants remain unknown. Here we used human three-dimensional brain-region-specific organoids to study the effect of oxygen deprivation on corticogenesis. We identified specific defects in intermediate progenitors, a cortical cell type associated with the expansion of the human cerebral cortex, and showed that these are related to the unfolded protein response and changes. Moreover, we verified these findings in human primary cortical tissue and demonstrated that a small-molecule modulator of the unfolded protein response pathway can prevent the reduction in intermediate progenitors following hypoxia. We anticipate that this human cellular platform will be valuable for studying the environmental and genetic factors underlying injury in the developing human brain