A combination of Δ9-tetrahydrocannabinol and cannabidiol modulates glutamate dynamics in the hippocampus of an animal model of Alzheimer's disease.

A combination of Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) at non-psychoactive doses was previously demonstrated to reduce cognitive decline in APP/PS1 mice, an animal model of Alzheimer's disease (AD). However, the neurobiological substrates underlying these therapeutic properties of Δ9-THC and CBD are not fully understood. Considering that dysregulation of glutamatergic activity contributes to cognitive impairment in AD, the present study evaluates the hypothesis that the combination of these two natural cannabinoids might reverse the alterations in glutamate dynamics within the hippocampus of this animal model of AD. Interestingly, our findings reveal that chronic treatment with Δ9-THC and CBD, but not with any of them alone, reduces extracellular glutamate levels and the basal excitability of the hippocampus in APP/PS1 mice. These effects are not related to significant changes in the function and structure of glutamate synapses, as no relevant changes in synaptic plasticity, glutamate signaling or in the levels of key components of these synapses were observed in cannabinoid-treated mice. Our data instead indicate that these cannabinoid effects are associated with the control of glutamate uptake and/or to the regulation of the hippocampal network. Taken together, these results support the potential therapeutic properties of combining these natural cannabinoids against the excitotoxicity that occurs in AD brains.

Stretch triggers microtubule stabilization and MARCKS-dependent membrane incorporation in the shaft of embryonic axons.

Neurons have a unique polarized nature that must adapt to environmental changes throughout their lifespan. During embryonic development, axon elongation is led by the growth cone,1 culminating in the formation of a presynaptic terminal. After synapses are formed, axons elongate in a growth cone-independent manner to accompany body growth while maintaining their ultrastructure and function.2,3,4,5,6 To further understand mechanical strains on the axon shaft, we developed a computer-controlled stretchable microfluidic platform compatible with multi-omics and live imaging. Our data show that sensory embryonic dorsal root ganglia (DRGs) neurons have high plasticity, with axon shaft microtubules decreasing polymerization rates, aligning with the direction of tension, and undergoing stabilization. Moreover, in embryonic DRGs, stretch triggers yes-associated protein (YAP) nuclear translocation, supporting its participation in the regulatory network that enables tension-driven axon growth. Other than cytoskeleton remodeling, stretch prompted MARCKS-dependent formation of plasmalemmal precursor vesicles (PPVs), resulting in new membrane incorporation throughout the axon shaft. In contrast, adolescent DRGs showed a less robust adaptation, with axonal microtubules being less responsive to stretch. Also, while adolescent DRGs were still amenable to strain-induced PPV formation at higher stretch rates, new membrane incorporation in the axon shaft failed to occur. In summary, we developed a new resource to study the biology of axon stretch growth. By unraveling cytoskeleton adaptation and membrane remodeling in the axon shaft of stretched neurons, we are moving forward in understanding axon growth.

Adenosine A2A receptors control generalization of contextual fear in rats.

Fear learning is essential to survival, but traumatic events may lead to abnormal fear consolidation and overgeneralization, triggering fear responses in safe environments, as occurs in post-traumatic stress disorder (PTSD). Adenosine A2A receptors (A2AR) control emotional memory and fear conditioning, but it is not known if they affect the consolidation and generalization of fear, which was now investigated. We now report that A2AR blockade through systemic administration of the A2AR antagonist SCH58261 immediately after contextual fear conditioning (within the consolidation window), accelerated fear generalization. Conversely, A2AR activation with CGS21680 decreased fear generalization. Ex vivo electrophysiological recordings of field excitatory post-synaptic potentials (fEPSPs) in CA3-CA1 synapses and of population spikes in the lateral amygdala (LA), showed that the effect of SCH58261 is associated with a reversion of fear conditioning-induced decrease of long-term potentiation (LTP) in the dorsal hippocampus (DH) and with increased amplitude of LA LTP in conditioned animals. These data suggest that A2AR are engaged during contextual fear consolidation, controlling long-term potentiation mechanisms in both DH and LA during fear consolidation, impacting on fear generalization; this supports targeting A2AR during fear consolidation to control aberrant fear processing in PTSD and other fear-related disorders.

Modification of astrocytic Cx43 hemichannel activity in animal models of AD: modulation by adenosine A2A receptors.

Increasing evidence implicates astrocytic dysfunction in Alzheimer's disease (AD), a neurodegenerative disorder characterised by progressive cognitive loss. The accumulation of amyloid-ß (Aß) plaques is a histopathological hallmark of AD and associated with increased astrocyte reactivity. In APP/PS1 mice modelling established AD (9 months), we now show an altered astrocytic morphology and enhanced activity of astrocytic hemichannels, mainly composed by connexin 43 (Cx43). Hemichannel activity in hippocampal astrocytes is also increased in two models of early AD: (1) mice with intracerebroventricular (icv) administration of Aß1-42, and (2) hippocampal slices superfused with Aß1-42 peptides. In hippocampal gliosomes of APP/PS1 mice, Cx43 levels were increased, whereas mice administered icv with Aß1-42 only displayed increased Cx43 phosphorylation levels. This suggests that hemichannel activity might be differentially modulated throughout AD progression. Additionally, we tested if adenosine A2A receptor (A2AR) blockade reversed alterations of astrocytic hemichannel activity and found that the pharmacological blockade or genetic silencing (global and astrocytic) of A2AR prevented Aß-induced hemichannel dysregulation in hippocampal slices, although A2AR genetic silencing increased the activity of astroglial hemichannels in control conditions. In primary cultures of astrocytes, A2AR-related protective effect was shown to occur through a protein kinase C (PKC) pathway. Our results indicate that the dysfunction of hemichannel activity in hippocampal astrocytes is an early event in AD, which is modulated by A2AR.

Adenosine A2A Receptor Up-Regulation Pre-Dates Deficits of Synaptic Plasticity and of Memory in Mice Exposed to Aß1-42 to Model Early Alzheimer's Disease.

The intracerebroventricular (icv) injection of amyloid peptides (Aß) models Alzheimer's disease (AD) in mice, as typified by the onset within 15 days of deficits of memory and of hippocampal long-term potentiation (LTP) that are prevented by the blockade of adenosine A2A receptors (A2AR). Since A2AR overfunction is sufficient to trigger memory deficits, we tested if A2AR were upregulated in hippocampal synapses before the onset of memory deficits to support the hypothesis that A2AR overfunction could be a trigger of AD. Six to eight days after Aß-icv injection, mice displayed no alterations of hippocampal dependent memory; however, they presented an increased excitability of hippocampal synapses, a slight increase in LTP magnitude in Schaffer fiber-CA1 pyramid synapses and an increased density of A2AR in hippocampal synapses. A2AR blockade with SCH58261 (50 nM) normalized excitability and LTP in hippocampal slices from mice sacrificed 7-8 days after Aß-icv injection. Fifteen days after Aß-icv injection, mice displayed evident deficits of hippocampal-dependent memory deterioration, with reduced hippocampal CA1 LTP but no hyperexcitability and a sustained increase in synaptic A2AR, which blockade restored LTP magnitude. This shows that the upregulation of synaptic A2AR precedes the onset of deterioration of memory and of hippocampal synaptic plasticity, supporting the hypothesis that the overfunction of synaptic A2AR could be a trigger of memory deterioration in AD.

Adenosine A2A Receptors Shut Down Adenosine A1 Receptor-Mediated Presynaptic Inhibition to Promote Implementation of Hippocampal Long-Term Potentiation.

Adenosine operates a modulation system fine-tuning the efficiency of synaptic transmission and plasticity through A1 and A2A receptors (A1R, A2AR), respectively. Supramaximal activation of A1R can block hippocampal synaptic transmission, and the tonic engagement of A1R-mediated inhibition is increased with increased frequency of nerve stimulation. This is compatible with an activity-dependent increase in extracellular adenosine in hippocampal excitatory synapses, which can reach levels sufficient to block synaptic transmission. We now report that A2AR activation decreases A1R-medated inhibition of synaptic transmission, with particular relevance during high-frequency-induced long-term potentiation (LTP). Thus, whereas the A1R antagonist DPCPX (50 nM) was devoid of effects on LTP magnitude, the addition of an A2AR antagonist SCH58261 (50 nM) allowed a facilitatory effect of DPCPX on LTP to be revealed. Additionally, the activation of A2AR with CGS21680 (30 nM) decreased the potency of the A1R agonist CPA (6-60 nM) to inhibit hippocampal synaptic transmission in a manner prevented by SCH58261. These observations show that A2AR play a key role in dampening A1R during high-frequency induction of hippocampal LTP. This provides a new framework for understanding how the powerful adenosine A1R-mediated inhibition of excitatory transmission can be controlled to allow the implementation of hippocampal LTP.

Astrocytic A2A receptors silencing negatively impacts hippocampal synaptic plasticity and memory of adult mice.

Astrocytes are wired to bidirectionally communicate with neurons namely with synapses, thus shaping synaptic plasticity, which in the hippocampus is considered to underlie learning and memory. Adenosine A2A receptors (A2A R) are a potential candidate to modulate this bidirectional communication, since A2A R regulate synaptic plasticity and memory and also control key astrocytic functions. Nonetheless, little is known about the role of astrocytic A2A R in synaptic plasticity and hippocampal-dependent memory. Here, we investigated the impact of genetic silencing astrocytic A2A R on hippocampal synaptic plasticity and memory of adult mice. The genetic A2A R silencing in astrocytes was accomplished by a bilateral injection into the CA1 hippocampal area of a viral construct (AAV5-GFAP-GFP-Cre) that inactivate A2A R expression in astrocytes of male adult mice carrying "floxed" A2A R gene, as confirmed by A2A R binding assays. Astrocytic A2A R silencing alters astrocytic morphology, typified by an increment of astrocytic arbor complexity, and led to deficits in spatial reference memory and compromised hippocampal synaptic plasticity, typified by a reduction of LTP magnitude and a shift of synaptic long-term depression (LTD) toward LTP. These data indicate that astrocytic A2A R control astrocytic morphology and influence hippocampal synaptic plasticity and memory of adult mice in a manner different from neuronal A2A R.

Downregulation of Sirtuin 1 Does Not Account for the Impaired Long-Term Potentiation in the Prefrontal Cortex of Female APPswe/PS1dE9 Mice Modelling Alzheimer's Disease.

Alzheimer's disease (AD), which predominantly affects women, involves at its onset a metabolic deregulation associated with a synaptic failure. Here, we performed a behavioral, neurophysiological and neurochemical characterization of 9-month-old female APPswe/PS1dE9 (APP/PS1) mice as a model of early AD. These animals showed learning and memory deficits in the Morris water maze, increased thigmotaxis and anxiety-like behavior and showed signs of fear generalization. Long-term potentiation (LTP) was decreased in the prefrontal cortex (PFC), but not in the CA1 hippocampus or amygdala. This was associated with a decreased density of sirtuin-1 in cerebrocortical synaptosomes and a decreased density of sirtuin-1 and sestrin-2 in total cerebrocortical extracts, without alterations of sirtuin-3 levels or of synaptic markers (syntaxin, synaptophysin, SNAP25, PSD95). However, activation of sirtuin-1 did not affect or recover PFC-LTP deficit in APP/PS1 female mice; instead, inhibition of sirtuin-1 increased PFC-LTP magnitude. It is concluded that mood and memory dysfunction in 9-month-old female APP/PS1 mice is associated with a parallel decrease in synaptic plasticity and in synaptic sirtuin-1 levels in the prefrontal cortex, although sirtiun1 activation failed to restore abnormal cortical plasticity.

Effects of Chronic Caffeine Consumption on Synaptic Function, Metabolism and Adenosine Modulation in Different Brain Areas.

Adenosine receptors mainly control synaptic function, and excessive activation of adenosine receptors may worsen the onset of many neurological disorders. Accordingly, the regular intake of moderate doses of caffeine antagonizes adenosine receptors and affords robust neuroprotection. Although caffeine intake alters brain functional connectivity and multi-omics analyses indicate that caffeine intake modifies synaptic and metabolic processes, it is unclear how caffeine intake affects behavior, synaptic plasticity and its modulation by adenosine. We now report that male mice drinking caffeinated water (0.3 g/L) for 2 weeks were behaviorally indistinguishable (locomotion, mood, memory) from control mice (drinking water) and displayed superimposable synaptic plasticity (long-term potentiation) in different brain areas (hippocampus, prefrontal cortex, amygdala). Moreover, there was a general preservation of the efficiency of adenosine A1 and A2A receptors to control synaptic transmission and plasticity, although there was a tendency for lower levels of endogenous adenosine ensuring A1 receptor-mediated inhibition. In spite of similar behavioral and neurophysiological function, caffeine intake increased the energy charge and redox state of cortical synaptosomes. This increased metabolic competence likely involved a putative increase in the glycolytic rate in synapses and a prospective greater astrocyte-synapse lactate shuttling. It was concluded that caffeine intake does not trigger evident alterations of behavior or of synaptic plasticity but increases the metabolic competence of synapses, which might be related with the previously described better ability of animals consuming caffeine to cope with deleterious stimuli triggering brain dysfunction.

The impact of inosine on hippocampal synaptic transmission and plasticity involves the release of adenosine through equilibrative nucleoside transporters rather than the direct activation of adenosine receptors.

Inosine has robust neuroprotective effects, but it is unclear if inosine acts as direct ligand of adenosine receptors or if it triggers metabolic effects indirectly modifying the activity of adenosine receptors. We now combined radioligand binding studies with electrophysiological recordings in hippocampal slices to test how inosine controls synaptic transmission and plasticity. Inosine was without effect at 30 µM and decreased field excitatory post-synaptic potentials by 14% and 33% at 100 and 300 µM, respectively. These effects were prevented by the adenosine A1 receptor antagonist DPCPX. Inosine at 300 (but not 100) µM also decreased the magnitude of long-term potentiation (LTP), an effect prevented by DPCPX and by the adenosine A2A receptor antagonist SCH58261. Inosine showed low affinity towards human and rat adenosine receptor subtypes with Ki values of > 300 µM; only at the human and rat A1 receptor slightly higher affinities with Ki values of around 100 µM were observed. Affinity of inosine at the rat A3 receptor was higher (Ki of 1.37 µM), while it showed no interaction with the human orthologue. Notably, the effects of inosine on synaptic transmission and plasticity were abrogated by adenosine deaminase and by inhibiting equilibrative nucleoside transporters (ENT) with dipyridamole and NBTI. This shows that the impact of inosine on hippocampal synaptic transmission and plasticity is not due to a direct activation of adenosine receptors but is instead due to an indirect modification of the tonic activation of these adenosine receptors through an ENT-mediated modification of the extracellular levels of adenosine.

Neuron-Microglia Contact-Dependent Mechanisms Attenuate Methamphetamine-Induced Microglia Reactivity and Enhance Neuronal Plasticity.

Exposure to methamphetamine (Meth) has been classically associated with damage to neuronal terminals. However, it is now becoming clear that addiction may also result from the interplay between glial cells and neurons. Recently, we demonstrated that binge Meth administration promotes microgliosis and microglia pro-inflammation via astrocytic glutamate release in a TNF/IP3R2-Ca2+-dependent manner. Here, we investigated the contribution of neuronal cells to this process. As the crosstalk between microglia and neurons may occur by contact-dependent and/or contact-independent mechanisms, we developed co-cultures of primary neurons and microglia in microfluidic devices to investigate how their interaction affects Meth-induced microglia activation. Our results show that neurons exposed to Meth do not activate microglia in a cell-autonomous way but require astrocyte mediation. Importantly, we found that neurons can partially prevent Meth-induced microglia activation via astrocytes, which seems to be achieved by increasing arginase 1 expression and strengthening the CD200/CD200r pathway. We also observed an increase in synaptic individual area, as determined by co-localization of pre- and post-synaptic markers. The present study provides evidence that contact-dependent mechanisms between neurons and microglia can attenuate pro-inflammatory events such as Meth-induced microglia activation.

Impact of blunting astrocyte activity on hippocampal synaptic plasticity in a mouse model of early Alzheimer's disease based on amyloid-ß peptide exposure.

Amyloid-ß peptides (Aß) accumulate in the brain since early Alzheimer's disease (AD) and dysregulate hippocampal synaptic plasticity, the neurophysiological basis of memory. Although the relationship between long-term potentiation (LTP) and memory processes is well established, there is also evidence that long-term depression (LTD) may be crucial for learning and memory. Alterations in synaptic plasticity, namely in LTP, can be due to communication failures between astrocytes and neurons; however, little is known about astrocytes' ability to control hippocampal LTD, particularly in AD-like conditions. We now aimed to test the involvement of astrocytes in changes of hippocampal LTP and LTD triggered by Aß1-42 , taking advantage of L-α-aminoadipate (L-AA), a gliotoxin that blunts astrocytic function. The effects of Aß1-42 exposure were tested in two different experimental paradigms: ex vivo (hippocampal slices superfusion) and in vivo (intracerebroventricular injection), which were previously validated to impair memory and hippocampal synaptic plasticity, two features of early AD. Blunting astrocytic function with L-AA reduced LTP and LTD amplitude in hippocampal slices from control mice, but the effect on LTD was less evident, suggesting that astrocytes have a greater influence on LTP than on LTD under non-pathological conditions. However, under AD conditions, blunting astrocytes did not consistently alter the reduction of LTP magnitude, but reverted the LTD-to-LTP shift caused by both ex vivo and in vivo Aß1-42 exposure. This shows that astrocytes were responsible for the hippocampal LTD-to-LTP shift observed in early AD conditions, reinforcing the interest of strategies targeting astrocytes to restore memory and synaptic plasticity deficits present in early AD.

l-α-aminoadipate causes astrocyte pathology with negative impact on mouse hippocampal synaptic plasticity and memory.

Increasing evidence shows that astrocytes, by releasing and uptaking neuroactive molecules, regulate synaptic plasticity, considered the neurophysiological basis of memory. This study investigated the impact of l-α-aminoadipate (l-AA) on astrocytes which sense and respond to stimuli at the synaptic level and modulate hippocampal long-term potentiation (LTP) and memory. l-AA selectivity toward astrocytes was proposed in the early 70's and further tested in different systems. Although it has been used for impairing the astrocytic function, its effects appear to be variable in different brain regions. To test the effects of l-AA in the hippocampus of male C57Bl/6 mice we performed two different treatments (ex vivo and in vivo) and took advantage of other compounds that were reported to affect astrocytes. l-AA superfusion did not affect the basal synaptic transmission but decreased LTP magnitude. Likewise, trifluoroacetate and dihydrokainate decreased LTP magnitude and occluded the effect of l-AA on synaptic plasticity, confirming l-AA selectivity. l-AA superfusion altered astrocyte morphology, increasing the length and complexity of their processes. In vivo, l-AA intracerebroventricular injection not only reduced the astrocytic markers but also LTP magnitude and impaired hippocampal-dependent memory in mice. Interestingly, d-serine administration recovered hippocampal LTP reduction triggered by l-AA (2 h exposure in hippocampal slices), whereas in mice injected with l-AA, the superfusion of d-serine did not fully rescue LTP magnitude. Overall, these data show that both l-AA treatments affect astrocytes differently, astrocytic activation or loss, with similar negative outcomes on hippocampal LTP, implying that opposite astrocytic adaptive alterations are equally detrimental for synaptic plasticity.

Astrocytes and Adenosine A2A Receptors: Active Players in Alzheimer's Disease.

Astrocytes, through their numerous processes, establish a bidirectional communication with neurons that is crucial to regulate synaptic plasticity, the purported neurophysiological basis of memory. This evidence contributed to change the classic "neurocentric" view of Alzheimer's disease (AD), being astrocytes increasingly considered a key player in this neurodegenerative disease. AD, the most common form of dementia in the elderly, is characterized by a deterioration of memory and of other cognitive functions. Although, early cognitive deficits have been associated with synaptic loss and dysfunction caused by amyloid-ß peptides (Aß), accumulating evidences support a role of astrocytes in AD. Astrocyte atrophy and reactivity occurring at early and later stages of AD, respectively, involve morphological alterations that translate into functional changes. However, the main signals responsible for astrocytic alterations in AD and their impact on synaptic function remain to be defined. One possible candidate is adenosine, which can be formed upon extracellular catabolism of ATP released by astrocytes. Adenosine can act as a homeostatic modulator and also as a neuromodulator at the synaptic level, through the activation of adenosine receptors, mainly of A1R and A2A R subtypes. These receptors are also present in astrocytes, being particularly relevant in pathological conditions, to control the morphofunctional responses of astrocytes. Here, we will focus on the role of A2A R, since they are particularly associated with neurodegeneration and also with memory processes. Furthermore, A2A R levels are increased in the AD brain, namely in astrocytes where they can control key astrocytic functions. Thus, unveiling the role of A2A R in astrocytes function might shed light on novel therapeutic strategies for AD.

Crosstalk Between ATP-P2X7 and Adenosine A2A Receptors Controlling Neuroinflammation in Rats Subject to Repeated Restraint Stress.

Depressive conditions precipitated by repeated stress are a major socio-economical burden in Western countries. Previous studies showed that ATP-P2X7 receptors (P2X7R) and adenosine A2A receptors (A2AR) antagonists attenuate behavioral modifications upon exposure to repeated stress. Since it is unknown if these two purinergic modulation systems work independently, we now investigated a putative interplay between P2X7R and A2AR. Adult rats exposed to restraint stress for 14 days displayed an anxious (thigmotaxis, elevated plus maze), depressive (anhedonia, increased immobility), and amnesic (modified Y maze, object displacement) profile, together with increased expression of Iba-1 (a marker of microglia "activation") and interleukin-1ß (IL1ß) and tumor necrosis factor α (TNFα; proinflammatory cytokines) and an up-regulation of P2X7R (mRNA) and A2AR (receptor binding) in the hippocampus and prefrontal cortex. All these features were attenuated by the P2X7R-preferring antagonist brilliant blue G (BBG, 45 mg/kg, i.p.) or by caffeine (0.3 g/L, p.o.), which affords neuroprotection through A2AR blockade. Notably, BBG attenuated A2AR upregulation and caffeine attenuated P2X7R upregulation. In microglial N9 cells, the P2X7R agonist BzATP (100 µM) or the A2AR agonist CGS26180 (100 nM) increased calcium levels, which was abrogated by the P2X7R antagonist JNJ47965567 (1 µM) and by the A2AR antagonist SCH58261 (50 nM), respectively; notably JNJ47965567 prevented the effect of CGS21680 and the effect of BzATP was attenuated by SCH58261 and increased by CGS21680. These results provide the first demonstration of a functional interaction between P2X7R and A2AR controlling microglia reactivity likely involved in behavioral adaptive responses to stress and are illustrative of a cooperation between the two arms of the purinergic system in the control of brain function.

Use of knockout mice to explore CNS effects of adenosine.

The initial exploration using pharmacological tools of the role of adenosine receptors in the brain, concluded that adenosine released as such acted on A1R to inhibit excitability and glutamate release from principal neurons throughout the brain and that adenosine A2A receptors (A2AR) were striatal-'specific' receptors controlling dopamine D2R. This indicted A1R as potential controllers of neurodegeneration and A2AR of psychiatric conditions. Global knockout of these two receptors questioned the key role of A1R and instead identified extra-striatal A2AR as robust controllers of neurodegeneration. Furthermore, transgenic lines with altered metabolic sources of adenosine revealed a coupling of ATP-derived adenosine to activate A2AR and a role of A1R as a hurdle to initiate neurodegeneration. Additionally, cell-selective knockout of A2AR unveiled the different roles of A2AR in different cell types (neurons/astrocytes) in different portions of the striatal circuits (dorsal versus lateral) and in different brain areas (hippocampus/striatum). Finally, a new transgenic mouse line with deletion of all adenosine receptors seems to indicate a major allostatic rather than homeostatic role of adenosine and may allow isolating P2R-mediated responses to unravel their role in the brain, a goal close to heart of Geoffrey Burnstock, to whom we affectionately dedicate this review.

The membrane periodic skeleton is an actomyosin network that regulates axonal diameter and conduction.

Neurons have a membrane periodic skeleton (MPS) composed of actin rings interconnected by spectrin. Here, combining chemical and genetic gain- and loss-of-function assays, we show that in rat hippocampal neurons the MPS is an actomyosin network that controls axonal expansion and contraction. Using super-resolution microscopy, we analyzed the localization of axonal non-muscle myosin II (NMII). We show that active NMII light chains are colocalized with actin rings and organized in a circular periodic manner throughout the axon shaft. In contrast, NMII heavy chains are mostly positioned along the longitudinal axonal axis, being able to crosslink adjacent rings. NMII filaments can play contractile or scaffolding roles determined by their position relative to actin rings and activation state. We also show that MPS destabilization through NMII inactivation affects axonal electrophysiology, increasing action potential conduction velocity. In summary, our findings open new perspectives on axon diameter regulation, with important implications in neuronal biology.

µSpikeHunter: An advanced computational tool for the analysis of neuronal communication and action potential propagation in microfluidic platforms.

Understanding neuronal communication is fundamental in neuroscience, but there are few methodologies offering detailed analysis for well-controlled conditions. By interfacing microElectrode arrays with microFluidics (µEF devices), it is possible to compartmentalize neuronal cultures with a specified alignment of axons and microelectrodes. This setup allows the extracellular recording of spike propagation with a high signal-to-noise ratio over the course of several weeks. Addressing these µEF devices, we developed an advanced yet easy-to-use publically available computational tool, µSpikeHunter, which provides a detailed quantification of several communication-related properties such as propagation velocity, conduction failure, spike timings, and coding mechanisms. The combination of µEF devices and µSpikeHunter can be used in the context of standard neuronal cultures or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. The ability to analyze axonal signals (in a user-friendly, time-efficient, high-throughput manner) opens the door to new approaches in studies of peripheral innervation, neural coding, and neuroregeneration, among many others. We demonstrate the use of µSpikeHunter in dorsal root ganglion neurons where we analyze the presence of both anterograde and retrograde signals in µEF devices. A fully functional version of µSpikeHunter is publically available for download from https://github.com/uSpikeHunter .

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation.

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of electrophysiological activity for long periods. However, the property of sensing signals from all sources around every microelectrode can become unfavorable when trying to understand communication and signal propagation in neuronal circuits. In a neuronal network, several neurons can be simultaneously activated and can generate overlapping action potentials, making it difficult to discriminate and track signal propagation. Considering this limitation, we have established an in vitro setup focused on assessing electrophysiological communication, which is able to isolate and amplify axonal signals with high spatial and temporal resolution. By interfacing microfluidic devices and MEAs, we are able to compartmentalize neuronal cultures with a well-controlled alignment of the axons and microelectrodes. This setup allows recordings of spike propagation with a high signal-to-noise ratio over the course of several weeks. Combined with specialized data analysis algorithms, it provides detailed quantification of several communication related properties such as propagation velocity, conduction failure, firing rate, anterograde spikes, and coding mechanisms. This protocol demonstrates how to create a compartmentalized neuronal culture setup over substrate-integrated MEAs, how to culture neurons in this setup, and how to successfully record, analyze and interpret the results from such experiments. Here, we show how the established setup simplifies the understanding of neuronal communication and axonal signal propagation. These platforms pave the way for new in vitro models with engineered and controllable neuronal network topographies. They can be used in the context of homogeneous neuronal cultures, or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. This setup provides very interesting conditions to study, for example, neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration approaches.