The Association between Hypoxia-Induced Low Activity and Apoptosis Strongly Resembles That between TTX-Induced Silencing and Apoptosis.

In the penumbra of a brain infarct, neurons initially remain structurally intact, but perfusion is insufficient to maintain neuronal activity at physiological levels. Improving neuronal recovery in the penumbra has large potential to advance recovery of stroke patients, but penumbral pathology is incompletely understood, and treatments are scarce. We hypothesize that low activity in the penumbra is associated with apoptosis and thus contributes to irreversible neuronal damage. We explored the putative relationship between low neuronal activity and apoptosis in cultured neurons exposed to variable durations of hypoxia or TTX. We combined electrophysiology and live apoptosis staining in 42 cultures, and compared effects of hypoxia and TTX silencing in terms of network activity and apoptosis. Hypoxia rapidly reduced network activity, but cultures showed limited apoptosis during the first 12 h. After 24 h, widespread apoptosis had occurred. This was associated with full activity recovery observed upon reoxygenation within 12 h, but not after 24 h. Similarly, TTX exposure strongly reduced activity, with full recovery upon washout within 12 h, but not after 24 h. Mean temporal evolution of apoptosis in TTX-treated cultures was the same as in hypoxic cultures. These results suggest that prolonged low activity may be a common factor in the pathways towards apoptosis.

Loss and recovery of functional connectivity in cultured cortical networks exposed to hypoxia.

In the core of a brain infarct, loss of neuronal function is followed by neuronal death within minutes. In an area surrounding the core (penumbra), some perfusion remains. Here, neurons initially remain structurally intact, but massive synaptic failure strongly reduces neural activity. Activity in the penumbra may eventually recover or further deteriorate toward massive cell death. Besides activity recovery, return of brain functioning requires restoration of connectivity. However, low activity has been shown to initiate compensatory mechanisms that affect network connectivity. We investigated the effect of transient hypoxia and compensatory mechanisms on activity and functional connectivity using cultured cortical networks on multielectrode arrays. Networks were exposed to hypoxia of controlled depth (10-90% of normoxia) and duration (6-48 h). First, we determined how hypoxic depth and duration govern activity recovery. Then, we investigated connectivity changes during and after hypoxic incidents, mild enough for activity to recover. Shortly after hypoxia onset, activity and connectivity decreased. Following 4-6 h of ongoing hypoxia, we observed partial recovery. Only if the hypoxic burden was limited did connectivity show further recovery upon return to normoxia. Partial recovery during hypoxia was dominated by restored baseline connections, rather than newly formed ones. Baseline strengths of surviving (persisting or recovered) and lost connections did not differ nor did baseline activity at their "presynaptic" electrodes. However, "postsynaptic" electrodes of surviving connections were significantly more active during baseline than those of lost connections. This implies that recovery during hypoxia reflects an effective mechanism to restore network activity, which does not necessarily conserve prehypoxia connectivity.NEW & NOTEWORTHY Hypoxia reduced the firing rates of cultured neurons. Depending on hypoxic depth and duration, activity recovered during hypoxia and upon return to normoxia. Recovery (partial) during hypoxia was associated with restored baseline connections rather than newly formed ones. Predominantly, baseline connections with most active postsynaptic electrodes recovered, supporting the notion of effective activity homeostasis. This compensatory mechanism remained effective during ~20 h of hypoxia. Beyond 20 h of compensation, loss of activity and connectivity became irreversible.

Repeated stimulation of cultured networks of rat cortical neurons induces parallel memory traces.

During systems consolidation, memories are spontaneously replayed favoring information transfer from hippocampus to neocortex. However, at present no empirically supported mechanism to accomplish a transfer of memory from hippocampal to extra-hippocampal sites has been offered. We used cultured neuronal networks on multielectrode arrays and small-scale computational models to study the effect of memory replay on the formation of memory traces. We show that input-deprived networks develop an activity⇔connectivity balance where dominant activity patterns support current connectivity. Electrical stimulation at one electrode disturbs this balance and induces connectivity changes. Intrinsic forces in recurrent networks lead to a new equilibrium with activity patterns that include the stimulus response. The new connectivity is no longer disrupted by this stimulus, indicating that networks memorize it. A different stimulus again induces connectivity changes upon first application but not subsequently, demonstrating the formation of a second memory trace. Returning to the first stimulus does not affect connectivity, indicating parallel storage of both traces. A computer model robustly reproduced experimental results, suggesting that spike-timing-dependent plasticity and short time depression suffice to store parallel memory traces, even in networks without particular circuitry constraints.

Ghrelin accelerates synapse formation and activity development in cultured cortical networks.

BACKGROUND: While ghrelin was initially related to appetite stimulation and growth hormone secretion, it also has a neuroprotective effect in neurodegenerative diseases and regulates cognitive function. The cellular basis of those processes is related to synaptic efficacy and plasticity. Previous studies have shown that ghrelin not only stimulates synapse formation in cultured cortical neurons and hippocampal slices, but also alters some of the electrophysiological properties of neurons in the hypothalamus, amygdala and other subcortical areas. However, direct evidence for ghrelin's ability to modulate the activity in cortical neurons is not available yet. In this study, we investigated the effect of acylated ghrelin on the development of the activity level and activity patterns in cortical neurons, in relation to its effect on synaptogenesis. Additionally, we quantitatively evaluated the expression of the receptor for acylated ghrelin--growth hormone secretagogue receptor-1a (GHSR-1a) during development. RESULTS: We performed electrophysiology and immunohistochemistry on dissociated cortical cultures from neonates, treated chronically with acylated ghrelin. On average 76±4.6% of the cortical neurons expressed GHSR-1a. Synapse density was found to be much higher in ghrelin treated cultures than in controls across all age groups (1, 2 or 3 weeks). In all cultures (control and ghrelin treated), network activity gradually increased until it reached a maximum after approximately 3 weeks, followed by a slight decrease towards a plateau. During early developmental stages (1-2 weeks), the activity was much higher in ghrelin treated cultures and consequently, they reached the plateau value almost a week earlier than controls. CONCLUSIONS: Acylated ghrelin leads to earlier network formation and activation in cultured cortical neuronal networks, the latter being a possibly consequence of accelerated synaptogenesis.

Connectivity, excitability and activity patterns in neuronal networks.

Extremely synchronized firing patterns such as those observed in brain diseases like epilepsy may result from excessive network excitability. Although network excitability is closely related to (excitatory) connectivity, a direct measure for network excitability remains unavailable. Several methods currently exist for estimating network connectivity, most of which are related to cross-correlation. An example is the conditional firing probability (CFP) analysis which calculates the pairwise probability (CFPi,j) that electrode j records an action potential at time t = τ, given that electrode i recorded a spike at t = 0. However, electrode i often records multiple spikes within the analysis interval, and CFP values are biased by the on-going dynamic state of the network. Here we show that in a linear approximation this bias may be removed by deconvoluting CFPi,j with the autocorrelation of i (i.e. CFPi,i), to obtain the single pulse response (SPRi,j)-the average response at electrode j to a single spike at electrode i. Thus, in a linear system SPRs would be independent of the dynamic network state. Nonlinear components of synaptic transmission, such as facilitation and short term depression, will however still affect SPRs. Therefore SPRs provide a clean measure of network excitability. We used carbachol and ghrelin to moderately activate cultured cortical networks to affect their dynamic state. Both neuromodulators transformed the bursting firing patterns of the isolated networks into more dispersed firing. We show that the influence of the dynamic state on SPRs is much smaller than the effect on CFPs, but not zero. The remaining difference reflects the alteration in network excitability. We conclude that SPRs are less contaminated by the dynamic network state and that mild excitation may decrease network excitability, possibly through short term synaptic depression.

Ghrelin for Neuroprotection in Post-Cardiac Arrest Coma: A Randomized Clinical Trial.

Importance: Out-of-hospital cardiac arrest survival rates have markedly risen in the last decades, but neurological outcome only improved marginally. Despite research on more than 20 neuroprotective strategies involving patients in comas after cardiac arrest, none have demonstrated unequivocal evidence of efficacy; however, treatment with acyl-ghrelin has shown improved functional and histological brain recovery in experimental models of cardiac arrest and was safe in a wide variety of human study populations. Objective: To determine safety and potential efficacy of intravenous acyl-ghrelin to improve neurological outcome in patients in a coma after cardiac arrest. Design, Setting, and Participants: A phase 2, double-blind, placebo-controlled, multicenter, randomized clinical trial, Ghrelin Treatment of Comatose Patients After Cardiac Arrest: A Clinical Trial to Promote Cerebral Recovery (GRECO), was conducted between January 18, 2019, and October 17, 2022. Adult patients 18 years or older who were in a comatose state after cardiac arrest were assessed for eligibility; patients were from 3 intensive care units in the Netherlands. Expected death within 48 hours or unfeasibility of treatment initiation within 12 hours were exclusion criteria. Interventions: Patients were randomized to receive intravenous acyl-ghrelin, 600 µg (intervention group), or placebo (control group) within 12 hours after cardiac arrest, continued for 7 days, twice daily, in addition to standard care. Main Outcomes and Measures: Primary outcome was the score on the Cerebral Performance Categories (CPC) scale at 6 months. Safety outcomes included any serious adverse events. Secondary outcomes were mortality and neuron-specific enolase (NSE) levels on days 1 and 3. Results: A total of 783 adult patients in a coma after cardiac arrest were assessed for eligibility, and 160 patients (median [IQR] age, 68 [57-75] years; 120 male [75%]) were enrolled. A total of 81 patients (51%) were assigned to the intervention group, and 79 (49%) were assigned to the control group. The common odds ratio (OR) for any CPC improvement in the intervention group was 1.78 (95% CI, 0.98-3.22; P = .06). This was consistent over all CPC categories. Mean (SD) NSE levels on day 1 after cardiac arrest were significantly lower in the intervention group (34 [6] µg/L vs 56 [13] µg/L; P = .04) and on day 3 (28 [6] µg/L vs 52 [14] µg/L; P = .08). Serious adverse events were comparable in incidence and type between the groups. Mortality was 37% (30 of 81) in the intervention group vs 51% (40 of 79) in the control group (absolute risk reduction, 14%; 95% CI, -2% to 29%; P = .08). Conclusions and Relevance: In patients in a coma after cardiac arrest, intravenous treatment with acyl-ghrelin was safe and potentially effective to improve neurological outcome. Phase 3 trials are needed for conclusive evidence. Trial Registration: Clinicaltrialsregister.eu: EUCTR2018-000005-23-NL.

Oxygen gradient generator to improve in vitro modeling of ischemic stroke.

Introduction: In the core of a brain infarct, perfusion is severely impeded, and neuronal death occurs within minutes. In the penumbra, an area near the core with more remaining perfusion, cells initially remain viable, but activity is significantly reduced. In principle, the penumbra can be saved if reperfusion is established on time, making it a promising target for treatment. In vitro models with cultured neurons on microelectrode arrays (MEAs) provide a useful tool to investigate how ischemic stroke affects neuronal functioning. These models tend to be uniform, focusing on the isolated penumbra, and typically lack adjacent regions such as a core and unaffected regions (normal perfusion). However, processes in these regions may affect neuronal functioning and survival in the penumbra. Materials and methods: Here, we designed, fabricated, and characterized a cytocompatible device that generates an oxygen gradient across in vitro neuronal cultures to expose cells to hypoxia of various depths from near anoxia to near normoxia. This marks a step in the path to mimic core, penumbra, and healthy tissue, and will facilitate better in vitro modeling of ischemic stroke. Results: The generator forms a stable and reproducible gradient within 30 min. Oxygen concentrations at the extremes are adjustable in a physiologically relevant range. Application of the generator did not negatively affect electrophysiological recordings or the viability of cultures, thus confirming the cytocompatibility of the device. Discussion: The developed device is able to impose an oxygen gradient on neuronal cultures and may enrich in vitro stroke models.

Prediction in cultured cortical neural networks.

Theory suggest that networks of neurons may predict their input. Prediction may underlie most aspects of information processing and is believed to be involved in motor and cognitive control and decision-making. Retinal cells have been shown to be capable of predicting visual stimuli, and there is some evidence for prediction of input in the visual cortex and hippocampus. However, there is no proof that the ability to predict is a generic feature of neural networks. We investigated whether random in vitro neuronal networks can predict stimulation, and how prediction is related to short- and long-term memory. To answer these questions, we applied two different stimulation modalities. Focal electrical stimulation has been shown to induce long-term memory traces, whereas global optogenetic stimulation did not. We used mutual information to quantify how much activity recorded from these networks reduces the uncertainty of upcoming stimuli (prediction) or recent past stimuli (short-term memory). Cortical neural networks did predict future stimuli, with the majority of all predictive information provided by the immediate network response to the stimulus. Interestingly, prediction strongly depended on short-term memory of recent sensory inputs during focal as well as global stimulation. However, prediction required less short-term memory during focal stimulation. Furthermore, the dependency on short-term memory decreased during 20 h of focal stimulation, when long-term connectivity changes were induced. These changes are fundamental for long-term memory formation, suggesting that besides short-term memory the formation of long-term memory traces may play a role in efficient prediction.

Orexin a in cortical cultures: expression and effect on synaptogenesis during development.

Orexin A (OXA) is an excitatory hypothalamic neurotransmitter and ligand for Orexin Receptor-1 (OR1), isolated from a small group of hypothalamic neurons. OXA orchestrates different brain functions, and at the cognitive level some of the effects of insufficiency of OXA are well-known, for example in Parkinson's disease. It is widely assumed that deteriorated cognitive processes are related to impaired network connectivity. However, little is known about the effects of OXA in network connectivity and synaptogenesis. Therefore, to obtain insight into this problem we designed experiments with two groups of networks of dissociated cortical neurons: one group incubated in a plain medium and another chronically treated with OXA. After 1, 2, 3 or 4 weeks in vitro we applied immunocytochemistry for detection of OXA, OR1, and synaptic marker synaptophysin. Shortly after plating, 91 ± 8% of the neurons cultivated in a plain medium expressed OXA-immunoreactivity, which does normally not occur in vivo indicating that neurons may change their phenotype under non-natural culture conditions to develop synaptically coupled networks. The fraction of orexinergic neurons decreased to 33 ± 21% after 4 weeks in vitro. OXA expression was highest in the first week of network formation, the period of maximum synaptogenesis, and then decreased and stabilized in the weeks thereafter. Our hypothesis that OXA plays a role in the network development as a synaptogenic factor was supported by higher levels, earlier onset, and sustained increase of synaptophysin expression in experiments with chronic OXA application to the culture medium.

Maximum entropy models provide functional connectivity estimates in neural networks.

Tools to estimate brain connectivity offer the potential to enhance our understanding of brain functioning. The behavior of neuronal networks, including functional connectivity and induced connectivity changes by external stimuli, can be studied using models of cultured neurons. Cultured neurons tend to be active in groups, and pairs of neurons are said to be functionally connected when their firing patterns show significant synchronicity. Methods to infer functional connections are often based on pair-wise cross-correlation between activity patterns of (small groups of) neurons. However, these methods are not very sensitive to detect inhibitory connections, and they were not designed for use during stimulation. Maximum Entropy (MaxEnt) models may provide a conceptually different method to infer functional connectivity. They have the potential benefit to estimate functional connectivity during stimulation, and to infer excitatory as well as inhibitory connections. MaxEnt models do not involve pairwise comparison, but aim to capture probability distributions of sets of neurons that are synchronously active in discrete time bins. We used electrophysiological recordings from in vitro neuronal cultures on micro electrode arrays to investigate the ability of MaxEnt models to infer functional connectivity. Connectivity estimates provided by MaxEnt models correlated well with those obtained by conditional firing probabilities (CFP), an established cross-correlation based method. In addition, stimulus-induced connectivity changes were detected by MaxEnt models, and were of the same magnitude as those detected by CFP. Thus, MaxEnt models provide a potentially powerful new tool to study functional connectivity in neuronal networks.

MEA-ToolBox: an Open Source Toolbox for Standardized Analysis of Multi-Electrode Array Data.

Functional assessment of in vitro neuronal networks-of relevance for disease modelling and drug testing-can be performed using multi-electrode array (MEA) technology. However, the handling and processing of the large amount of data typically generated in MEA experiments remains a huge hurdle for researchers. Various software packages have been developed to tackle this issue, but to date, most are either not accessible through the links provided by the authors or only tackle parts of the analysis. Here, we present ''MEA-ToolBox'', a free open-source general MEA analytical toolbox that uses a variety of literature-based algorithms to process the data, detect spikes from raw recordings, and extract information at both the single-channel and array-wide network level. MEA-ToolBox extracts information about spike trains, burst-related analysis and connectivity metrics without the need of manual intervention. MEA-ToolBox is tailored for comparing different sets of measurements and will analyze data from multiple recorded files placed in the same folder sequentially, thus considerably streamlining the analysis pipeline. MEA-ToolBox is available with a graphic user interface (GUI) thus eliminating the need for any coding expertise while offering functionality to inspect, explore and post-process the data. As proof-of-concept, MEA-ToolBox was tested on earlier-published MEA recordings from neuronal networks derived from human induced pluripotent stem cells (hiPSCs) obtained from healthy subjects and patients with neurodevelopmental disorders. Neuronal networks derived from patient's hiPSCs showed a clear phenotype compared to those from healthy subjects, demonstrating that the toolbox could extract useful parameters and assess differences between normal and diseased profiles.

Inducing oscillations in positive end-expiratory pressure improves assessment of cerebrovascular pressure reactivity in patients with traumatic brain injury.

The cerebral pressure reactivity index (PRx), through intracranial pressure (ICP) measurements, informs clinicians about the cerebral autoregulation (CA) status in adult-sedated patients with traumatic brain injury (TBI). Using PRx in clinical practice is currently limited by variability over shorter monitoring periods. We applied an innovative method to reduce the PRx variability by ventilator-induced slow (1/min) positive end-expiratory pressure (PEEP) oscillations. We hypothesized that, as seen in a previous animal model, the PRx variability would be reduced by inducing slow arterial blood pressure (ABP) and ICP oscillations without other clinically relevant physiological changes. Patients with TBI were ventilated with a static PEEP for 30 min (PRx period) followed by a 30-min period of slow [1/min (0.0167 Hz)] +5 cmH2O PEEP oscillations (induced (iPRx period). Ten patients with TBI were included. No clinical monitoring was discontinued and no additional interventions were required during the iPRx period. The PRx variability [measured as the standard deviation (SD) of PRx] decreased significantly during the iPRx period from 0.25 (0.22-0.30) to 0.14 (0.09-0.17) (P = 0.006). There was a power increase around the induced frequency (1/min) for both ABP and ICP (P = 0.002). In conclusion, 1/min PEEP-induced oscillations reduced the PRx variability in patients with TBI with ICP levels <22 mmHg. No other clinically relevant physiological changes were observed. Reduced PRx variability might improve CA-guided perfusion management by reducing the time to find "optimal" perfusion pressure targets. Larger studies with prolonged periods of PEEP-induced oscillations are required to take it to routine use.NEW & NOTEWORTHY Cerebral autoregulation assessment requires sufficient slow arterial blood pressure (ABP) waves. However, spontaneous ABP waves may be insufficient for reliable cerebral autoregulation estimations. Therefore, we applied a ventilator "sigh-function" to generate positive end-expiratory pressure oscillations that induce slow ABP waves. This method demonstrated a reduced variability of the pressure reactivity index, commonly used as continuous cerebral autoregulation measure in a traumatic brain injury population.

Neuroprotective Treatment of Postanoxic Encephalopathy: A Review of Clinical Evidence.

Postanoxic encephalopathy is the key determinant of death or disability after successful cardiopulmonary resuscitation. Animal studies have provided proof-of-principle evidence of efficacy of divergent classes of neuroprotective treatments to promote brain recovery. However, apart from targeted temperature management (TTM), neuroprotective treatments are not included in current care of patients with postanoxic encephalopathy after cardiac arrest. We aimed to review the clinical evidence of efficacy of neuroprotective strategies to improve recovery of comatose patients after cardiac arrest and to propose future directions. We performed a systematic search of the literature to identify prospective, comparative clinical trials on interventions to improve neurological outcome of comatose patients after cardiac arrest. We included 53 studies on 21 interventions. None showed unequivocal benefit. TTM at 33 or 36°C and adrenaline (epinephrine) are studied most, followed by xenon, erythropoietin, and calcium antagonists. Lack of efficacy is associated with heterogeneity of patient groups and limited specificity of outcome measures. Ongoing and future trials will benefit from systematic collection of measures of baseline encephalopathy and sufficiently powered predefined subgroup analyses. Outcome measurement should include comprehensive neuropsychological follow-up, to show treatment effects that are not detectable by gross measures of functional recovery. To enhance translation from animal models to patients, studies under experimental conditions should adhere to strict methodological and publication guidelines.

Consolidation of memory traces in cultured cortical networks requires low cholinergic tone, synchronized activity and high network excitability.

In systems consolidation, encoded memories are replayed by the hippocampus during slow-wave sleep (SWS), and permanently stored in the neocortex. Declarative memory consolidation is believed to benefit from the oscillatory rhythms and low cholinergic tone observed in this sleep stage, but underlying mechanisms remain unclear. To clarify the role of cholinergic modulation and synchronized activity in memory consolidation, we applied repeated electrical stimulation in mature cultures of dissociated rat cortical neurons with high or low cholinergic tone, mimicking the cue replay observed during systems consolidation under distinct cholinergic concentrations. In the absence of cholinergic input, these cultures display activity patterns hallmarked by network bursts, synchronized events reminiscent of the low frequency oscillations observed during SWS. They display stable activity and connectivity, which mutually interact and achieve an equilibrium. Electrical stimulation reforms the equilibrium to include the stimulus response, a phenomenon interpreted as memory trace formation. Without cholinergic input, activity was burst-dominated. First application of a stimulus induced significant connectivity changes, while subsequent repetition no longer affected connectivity. Presenting a second stimulus at a different electrode had the same effect, whereas returning to the initial stimuli did not induce further connectivity alterations, indicating that the second stimulus did not erase the 'memory trace' of the first. Distinctively, cultures with high cholinergic tone displayed reduced network excitability and dispersed firing, and electrical stimulation did not induce significant connectivity changes. We conclude that low cholinergic tone facilitates memory formation and consolidation, possibly through enhanced network excitability. Network bursts or SWS oscillations may merely reflect high network excitability.

Neuroprotective effect of hypoxic preconditioning and neuronal activation in a in vitro human model of the ischemic penumbra.

OBJECTIVE: In ischemic stroke, treatments to protect neurons from irreversible damage are urgently needed. Studies in animal models have shown that neuroprotective treatments targeting neuronal silencing improve brain recovery, but in clinical trials none of these were effective in patients. This failure of translation poses doubts on the real efficacy of treatments tested and on the validity of animal models for human stroke. Here, we established a human neuronal model of the ischemic penumbra by using human induced pluripotent stem cells and we provided an in-depth characterization of neuronal responses to hypoxia and treatment strategies at the network level. APPROACH: We generated neurons from induced pluripotent stem cells derived from healthy donor and we cultured them on micro-electrode arrays. We measured the electrophysiological activity of human neuronal networks under controlled hypoxic conditions. We tested the effect of different treatment strategies on neuronal network functionality. MAIN RESULTS: Human neuronal networks are vulnerable to hypoxia reflected by a decrease in activity and synchronicity under low oxygen conditions. We observe that full, partial or absent recovery depend on the timing of re-oxygenation and we provide a critical time threshold that, if crossed, is associated with irreversible impairments. We found that hypoxic preconditioning improves resistance to a second hypoxic insult. Finally, in contrast to previously tested, ineffective treatments, we show that stimulatory treatments counteracting neuronal silencing during hypoxia, such as optogenetic stimulation, are neuroprotective. SIGNIFICANCE: We presented a human neuronal model of the ischemic penumbra and we provided insights that may offer the basis for novel therapeutic approaches for patients after stroke. The use of human neurons might improve drug discovery and translation of findings to patients and might open new perspectives for personalized investigations.

Phase-dependent effects of stimuli locked to oscillatory activity in cultured cortical networks.

The archetypal activity pattern in cultures of dissociated neurons is spontaneous network-wide bursting. Bursts may interfere with controlled activation of synaptic plasticity, but can be suppressed by the application of stimuli at a sufficient rate. We sinusoidally modulated (4 Hz) the pulse rate of random background stimulation (RBS) and found that cultures were more active, burst less frequently, and expressed oscillatory activity. Next, we studied the effect of phase-locked tetani (four pulses, 200 s(-1)) on network activity. Tetani were applied to one electrode at the peak or trough of mRBS stimulation. We found that when tetani were applied at the peak of modulated RBS (mRBS), a significant potentiation of poststimulus histograms (PSTHs) occurred. Conversely, tetani applied at the trough resulted in a small but insignificant depression of PSTHs. In addition to PSTHs, electrode-specific firing rate profiles within spontaneous bursts before and after mRBS were analyzed. Here, significant changes in firing rate profiles were found only for stimulation at the peak of mRBS. Our study shows that rhythmic activity in culture is possible, and that the network responds differentially to strong stimuli depending on the phase at which they are delivered. This suggests that plasticity mechanisms may be differentially accessible in an oscillatory state.

Orexin-A and orexin-B during the postnatal development of the rat brain.

Orexin-A and orexin-B are hypothalamic neuropeptides isolated from a small group of neurons in the hypothalamus, which project their axons to all major parts of the central nervous system. Despite the extensive information about orexin expression and function at different parts of the nervous system in adults, data about the development and maturation of the orexin system in the brain are a bit contradictory and insufficient. A previous study has found expression of orexins in the hypothalamus after postnatal day 15 only, while others report orexins detection at embryonic stages of brain formation. In the present study, we investigated the distribution of orexin-A and orexin-B neuronal cell bodies and fibers in the brain at three different postnatal stages: 1-week-, 2-week-old and adult rats. By means of immunohistochemical techniques, we demonstrated that a small subset of cells in the lateral hypothalamus, and the perifornical and periventricular areas were orexin-A and orexin-B positive not only in 2-week-old and adult rats but also in 1-week-old animals. In addition, orexin-A and orexin-B expressing neuronal varicosities were found in many other brain regions. These results suggest that orexin-A and orexin-B play an important role in the early postnatal brain development. The widespread distribution of orexinergic projections through all these stages may imply an involvement of the two neurotransmitters in a large variety of physiological and behavioral processes also including higher brain functions like learning and memory.

In Vitro Models of Brain Disorders.

The brain is the most complex organ of the body, and many pathological processes underlying various brain disorders are poorly understood. Limited accessibility hinders observation of such processes in the in vivo brain, and experimental freedom is often insufficient to enable informative manipulations. In vitro preparations (brain slices or cultures of dissociated neurons) offer much better accessibility and reduced complexity and have yielded valuable new insights into various brain disorders. Both types of preparations have their advantages and limitations with regard to lifespan, preservation of in vivo brain structure, composition of cell types, and the link to behavioral outcome is often unclear in in vitro models. While these limitations hamper general usage of in vitro preparations to study, e.g., brain development, in vitro preparations are very useful to study neuronal and synaptic functioning under pathologic conditions. This chapter addresses several brain disorders, focusing on neuronal and synaptic functioning, as well as network aspects. Recent progress in the fields of brain circulation disorders, excitability disorders, and memory disorders will be discussed, as well as limitations of current in vitro models.

Ischemic Stroke: Treatments to Improve Neuronal Functional Recovery in vitro.

The understanding of the neurophysiological processes that occur in the areas that surround the core of a brain infarct is crucial for the creation of new therapies and treatments to improve neuronal recovery. The present study aims to demonstrate that both rodent and human neuronal networks lose their activity under low oxygen conditions and that electrical stimulation can increase the probability of recovery. Hypoxia was induced in rodent and human neurons and the effects of electrical stimulation were assessed in the rat cultures. The results obtained show that neuronal activation, in the form of electrical stimulation, has the potential to maintain the networks at higher levels of activity and, therefore, to improve cell survival. This study will open the way for new treatment strategies based on brain-stimulation to enhance neuronal recovery and will be of large relevance for patients, families, and society.

Mild stimulation improves neuronal survival in an in vitro model of the ischemic penumbra.

OBJECTIVE: In the core of a brain infarct, characterized by severely reduced blood supply, loss of neuronal function is rapidly followed by neuronal death. In peripheral areas of the infarct, the penumbra, damage is initially reversible, and neuronal activity is typically reduced due to ischemia-induced synaptic failure. There is limited understanding of factors governing neuronal recovery or the transition to irreversible damage. Neuronal activity has been shown to be crucial for survival. Consequently, hypoxia induced neuronal inactivity may contribute to cell death, and activation of penumbral neurons possibly improves survival. Adversely, activation increases ATP demand, and a balance should be found between the available energy and sufficient activity. APPROACH: We monitored activity and viability of neurons in an in vitro model of the penumbra, consisting of (rat) neuronal networks on micro electrode arrays (MEAs) under controlled hypoxic conditions. We tested effects of optogenetic and electrical activation during hypoxia. MAIN RESULTS: Mild stimulation yielded significantly better recovery of activity immediately after re-oxygenation, compared with no stimulation, and a 60%-70% higher survival rate after 5 d. Stronger stimulation was not associated with better recovery than no stimulation, suggesting that beneficial effects depend on a delicate balance between sufficient activity and available energy. SIGNIFICANCE: We show that mild activation during hypoxia/ischemia is beneficial for cell survival in an in vitro model of the penumbra. This finding opposes the current common belief that suppression of neuronal activity is the cornerstone of neuroprotection during cerebral ischemia, and may open new possibilities for the treatment of secondary brain damage after stroke.