Francesca Gullo

ERG K+ channel blockade enhances firing and epinephrine secretion in rat chromaffin cells: the missing link to LQT2-related sudden death?

The ether‐a‐go‐go‐related genes (erg) are expressed in tissues other than heart and brain, in which human erg (HERG) K+ channels are known to regulate the repolarization of heart action potentials and neuronal spike‐frequency accommodation. We provide evidence that erg1 transcripts and ERG proteins are present in rat chromaffin cells in which we could isolate a K+ current that was biophysically and pharmacologically similar to the ERG current. Firing frequency and catecholamine release were analyzed at the single‐cell level by means of perforated patch‐clamp and carbon fiber electrochemical detection. It was found that the blocking of ERG, KATP, and KCa channels led to hyperexcitability and an increase in catecholamine release. Combined immunocytochemical experiments with antibodies directed against phenylethanolamine N‐methyltransferase and ERG channels suggested expression of these channels in epinephrine‐ but not in norepinephrine‐containing cells. It is concluded that, in addition to being crucial in regulating the QT period in the heart, ERG channels play a role in modulating epinephrine, a fundamental neurotransmitter shaping cardiac function. This finding suggests that the sudden death phenotype associated with LQT2 syndrome mutations may be the result of an emotionally triggered increase in epinephrine in a long‐QT running heart.

Exact distinction of excitatory and inhibitory neurons in neural networks: a study with GFP-GAD67 neurons optically and electrophysiologically recognized on multielectrode arrays.

Distinguishing excitatory from inhibitory neurons with multielectrode array (MEA) recordings is a serious experimental challenge. The current methods, developed in vitro, mostly rely on spike waveform analysis. These however often display poor resolution and may produce errors caused by the variability of spike amplitudes and neuron shapes. Recent recordings in human brain suggest that the spike waveform features correlate with time-domain statistics such as spiking rate, autocorrelation, and coefficient of variation. However, no precise criteria are available to exactly assign identified units to specific neuronal types, either in vivo or in vitro. To solve this problem, we combined MEA recording with fluorescence imaging of neocortical cultures from mice expressing green fluorescent protein (GFP) in GABAergic cells. In this way, we could sort out “authentic excitatory neurons” (AENs) and “authentic inhibitory neurons” (AINs). We thus characterized 1275 units (from 405 electrodes, n = 10 experiments), based on autocorrelation, burst length, spike number (SN), spiking rate, squared coefficient of variation, and Fano factor (FF) (the ratio between spike-count variance and mean). These metrics differed by about one order of magnitude between AINs and AENs. In particular, the FF turned out to provide a firing code which exactly (no overlap) recognizes excitatory and inhibitory units. The difference in FF between all of the identified AEN and AIN groups was highly significant (p < 10−8, ANOVA post-hoc Tukey test). Our results indicate a statistical metric-based approach to distinguish excitatory from inhibitory neurons independently from the spike width.

Orchestration of “Presto” and “Largo” Synchrony in Up-Down Activity of Cortical Networks

It has been demonstrated using single-cell and multiunit electrophysiology in layer III entorhinal cortex and disinhibited hippocampal CA3 slices that the balancing of the up-down activity is characterized by both GABAA and GABAB mechanisms. Here we report novel results obtained using multi-electrode array (60 electrodes) simultaneous recordings from reverberating postnatal neocortical networks containing 19.2 ± 1.4% GABAergic neurons, typical of intact tissue. We observed that in each spontaneous active-state the total number of spikes in identified clusters of excitatory and inhibitory neurons is almost equal, thus suggesting a balanced average activity. Interestingly, in the active-state, the early phase is sustained by only 10% of the total spikes and the firing rate follows a sigmoidal regenerative mode up to peak at 35 ms with the number of excitatory spikes greater than inhibitory, therefore indicating an early unbalance. Concentration-response pharmacology of up- and down-state lifetimes in clusters of excitatory (n = 1067) and inhibitory (n = 305) cells suggests that, besides the GABAA and GABAB mechanisms, others such as GAT-1-mediated uptake, Ih, INaP and IM ion channel activity, robustly govern both up- and down-activity. Some drugs resulted to affect up- and/or down-states with different IC50s, providing evidence that various mechanisms are involved. These results should reinforce not only the role of synchrony in CNS networks, but also the recognized analogies between the Hodgkin–Huxley action potential and the population bursts as basic mechanisms for originating membrane excitability and CNS network synchronization, respectively.

Voltage-dependent Nav1.7 sodium channels: multiple roles in adrenal chromaffin cells and peripheral nervous system

Voltage‐dependent Na+ channels consist of the principal α‐subunit (∼260 kDa), without or with auxiliary β‐subunit (∼38 kDa). Nine α‐subunit isoforms (Nav1.1–Nav1.9) are encoded in nine different genes (SCN1A–SCN5A and SCN8A–SCN11A). Besides initiating and propagating action potentials in established neuronal circuit, Na+ channels engrave, maintain and repair neuronal network in the brain throughout the life. Adrenal chromaffin cells express Nav1.7 encoded in SCN9A, which is widely distributed among peripheral autonomic and sensory ganglia, neuroendocrine cells, as well as prostate cancer cell lines. In chromaffin cells, Nav1.7‐specific biophysical properties have been characterized; physiological stimulation by acetylcholine produces muscarinic receptor‐mediated hyperpolarization followed by nicotinic receptor‐mediated depolarization. In human patients with Nav1.7 channelopathies, gain‐of‐pathological function mutants (i.e. erythermalgia and paroxysmal extreme pain disorder) or loss‐of‐physiological function mutant (channelopathy‐associated insensivity to pain) proved the causal involvement of mutant Nav1.7 in generating intolerable pain syndrome, Nav1.7 being the first molecular target convincingly identified for pain treatment. Importantly, aberrant upregulation/hyperactivity of even the native Nav1.7 produces pain associated with inflammation, nerve injury and diabetic neuropathy in rodents. Various extra‐ and intracellular signals, as well as therapeutic drugs modulate the activity of Nav1.7, and also cause up‐ and downregulation of Nav1.7. Nav1.7 seems to play an increasing number of crucial roles in health, disease and therapeutics.

Novel modulatory effects of neurosteroids and benzodiazepines on excitatory and inhibitory neurons excitability: a multi-electrode array recording study

The balance between glutamate- and GABA-mediated neurotransmission in the brain is fundamental in the nervous system, but it is regulated by the “tonic” release of a variety of endogenous factors. One such important group of molecules are the neurosteroids (NSs) which, similarly to benzodiazepines (BDZs), enhance GABAergic neurotransmission. The purpose of our work was to investigate, at in vivo physiologically relevant concentrations, the effects of NSs and BDZs as GABA modulators on dissociated neocortical neuron networks grown in long-term culture. We used a multi-electrode array (MEA) recording technique and a novel analysis that was able to both identify the action potentials of engaged excitatory and inhibitory neurons and to detect drug-induced network up-states (burst). We found that the NSs tetrahydrodeoxycorticosterone (THDOC) and allopregnanolone (ALLO) applied at low nanomolar concentrations, produced different modulatory effects on the two neuronal clusters. Conversely, at high concentrations (1 μM), both NSs, decreased excitatory and inhibitory neuron cluster excitability; however, even several hours after wash-out, the excitability of inhibitory neurons continued to be depressed, leading to a network long-term depression (LTD). The BDZs clonazepam (CLZ) and midazolam (MDZ) also decreased the network excitability, but only MDZ caused LTD of inhibitory neuron cluster. To investigate the origin of the LTD after MDZ application, we tested finasteride (FIN), an inhibitor of endogenous NSs synthesis. FIN did not prevent the LTD induced by MDZ, but surprisingly induced it after application of CLZ. The significance and possible mechanisms underlying these LTD effects of NSs and BDZs are discussed. Taken together, our results not only demonstrate that ex vivo networks show a sensitivity to NSs and BDZs comparable to that expressed in vivo, but also provide a new global in vitro description that can help in understanding their activity in more complex systems.

Functional Regeneration of the ex-vivo Reconstructed Mesocorticolimbic Dopaminergic System

CNS reparative-medicine therapeutic strategies need answers on the putative recapitulation of the basic rules leading to mammalian CNS development. To achieve this aim, we focus on the regeneration of functional connections in the mesocorticolimbic dopaminergic system. We used organotypic slice cocultures of ventral tegmental area/substantia nigra (VTA/SN) and prefrontal cortex (PFC) on a multielectrode array (MEA) platform to record spikes and local field potentials. The spontaneously growing synaptically based bidirectional bursting activity was followed from 2 to 28 days in vitro (DIV). A statistical analysis of excitatory and inhibitory neurons properties of the physiological firing activity demonstrated a remarkable, exponentially increasing maturation with a time constant of about 5-7 DIV. Immunohistochemistry demonstrated that the ratio of excitatory/inhibitory neurons (3:1) was in line with the functional results obtained. Exemplary pharmacology suggested that GABAA receptors were able to exert phasic and tonic inhibition typical of an adulthood network. Moreover, dopamine D2 receptor inactivation was equally inhibitory both on the spontaneous neuronal activity recorded by MEA and on patch-clamp electrophysiology in PFC pyramidal neurons. These results demonstrate that axon growth cones reach synaptic targets up to full functionality and that organotypic cocultures of the VTA/SN-PFC perfectly model their newly born dopaminergic, glutamatergic and GABAergic neuronal circuitries.

Multi-electrode array study of neuronal cultures expressing nicotinic β2-V287L subunits, linked to autosomal dominant nocturnal frontal lobe epilepsy. An in vitro model of spontaneous epilepsy

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a partial sleep-related epilepsy which can be caused by mutant neuronal nicotinic acetylcholine receptors (nAChR). We applied multi-electrode array (MEA) recording methods to study the spontaneous firing activity of neocortical cultures obtained from mice expressing or not (WT) an ADNFLE-linked nAChR subunit (β2-V287L). More than 100,000 up-states were recorded during experiments sampling from several thousand neurons. Data were analyzed by using a fast sliding-window procedure which computes histograms of the up-state durations. Differently from the WT, cultures expressing β2-V287L displayed long (10–32 s) synaptic-induced up-state firing events. The occurrence of such long up-states was prevented by both negative (gabazine, penicillin G) and positive (benzodiazepines) modulators of GABAA receptors. Carbamazepine (CBZ), a drug of choice in ADNFLE patients, also inhibited the long up-states at micromolar concentrations. In cultures expressing β2-V287L, no significant effect was observed on the action potential waveform either in the absence or in the presence of pharmacological treatment. Our results show that some aspects of the spontaneous hyperexcitability displayed by a murine model of a human channelopathy can be reproduced in neuronal cultures. In particular, our cultures represent an in vitro chronic model of spontaneous epileptiform activity, i.e., not requiring pre-treatment with convulsants. This opens the way to the study in vitro of the role of β2-V287L on synaptic formation. Moreover, our neocortical cultures on MEA platforms allow to determine the effects of prolonged pharmacological treatment on spontaneous network hyperexcitability (which is impossible in the short-living brain slices). Methods such as the one we illustrate in the present paper should also considerably facilitate the preliminary screening of antiepileptic drugs (AEDs), thereby reducing the number of in vivo experiments.

Atypical “seizure-like” activity in cortical reverberating networks in vitro can be caused by LPS-induced inflammation: A multi-electrode array study from a hundred neurons

We show here that a mild sterile inflammation induced by the endotoxin lipopolysaccharide (LPS), in a neuron/astrocyte/microglial cortical network, modulates neuronal excitability and can initiate long-duration burst events resembling epileptiform seizures, a recognized feature of various central nervous neurodegenerative, neurological and acute systemic diseases associated with neuroinflammation. To study this action, we simultaneously analyzed the reverberating bursting activity of a hundred neurons by using in vitro multi-electrode array methods. ∼5 h after LPS application, we observed a net increase in the average number of spikes elicited in engaged cells and within each burst, but no changes neither in spike waveforms nor in burst rate. This effect was characterized by a slow, twofold exponential increase of the burst duration and the appearance of rarely occurring long burst events that were never seen during control recordings. These changes and the time-course of microglia-released proinflammatory cytokine, tumor necrosis factor-alpha (TNF-α), were blocked by pre-treatment with 50 nM minocycline, an established anti-inflammatory agent which was inactive when applied alone. Assay experiments also revealed that application of 60 pM exogenous TNF-α after 12–15 h, produced non-washable changes of neuronal excitability, completely different from those induced by LPS, suggesting that TNF-α release alone was not responsible for our observed findings. Our results indicate that the link between neuroinflammation and hyperexcitability can be unveiled by studying the long-term activity of in vitro neuronal/astrocyte/microglial networks.

How do TTX and AP5 affect the post-recovery neuronal network activity synchronization?

A lot of methods were created in last decade for the spatio-temporal analysis of multi-electrode array (MEA) neuronal data sets. The greater part of these methods does not consider the network as a whole but performs an analysis channel by channel. In this paper we illustrate how a very simple approach that considers the total network activity, is able to show interesting neuronal network features. In particular we perform two different analyses: a connectivity examination studying networks at different days in vitro and an analysis of the long period effects of the administration of two common neuro-active drugs, i.e. TTX and AP5. Our analysis is performed considering burst topology, i.e. cataloguing network bursts as Global (if they involve more than the 25% of the MEA channels) or Local (if less that 25%). This division allows, in the first analysis, to understand the network connectivity (increasing from div 1 to 6) and decreasing till reaching a plateau (from div 6 to 10). The second analysis highlights a substantial difference between the long period effects of TTX and AP5. While TTX induces a massive Global activity explosion, sign of a prolonged inhibitory synapse depression, AP5 shows only a modest Local activity increase, mark of the low effect of NMDA receptors on a mature neuronal network without inputs.

Mesoporous silica nanoparticles trigger mitophagy in endothelial cells and perturb neuronal network activity in a size- and time-dependent manner

Purpose Mesoporous silica nanoparticles (MSNPs) are excellent candidates for biomedical applications and drug delivery to different human body areas, the brain included. Although toxicity at cellular level has been investigated, we are still far from using MSNPs in the clinic, because the mechanisms involved in the cellular responses activated by MSNPs have not yet been elucidated. Materials and methods This study used an in vitro multiparametric approach to clarify relationships among size, dose, and time of exposure of MSNPs (0.05–1 mg/mL dose range), and cellular responses by analyzing the morphology, viability, and functionality of human vascular endothelial cells and neurons. Results The results showed that 24 hours of exposure of endothelial cells to 250 nm MSNPs exerted higher toxicity in terms of mitochondrial activity and membrane integrity than 30 nm MSN at the same dose. This was due to induced cell autophagy (in particular mitophagy), probably consequent to MSNP cellular uptake (>20%). Interestingly, after 24 hours of treatment with 30 nm MSNPs, very low MSNP uptake (<1%) and an increase in nitric oxide production (30%, P<0.01) were measured. This suggests that MSNPs were able to affect endothelial functionality from outside the cells. These differences could be attributed to the different protein-corona composition of the MSNPs used, as suggested by sodium dodecyl sulfate polyacrylamide-gel electrophoresis analysis of the plasma proteins covering the MSNP surface. Moreover, doses of MSNPs up to 0.25 mg/mL perturbed network activity by increasing excitability, as detected by multielectrode-array technology, without affecting neuronal cell viability. Conclusion These results suggest that MSNPs may be low-risk if prepared with a diameter <30 nm and if they reach human tissues at doses <0.25 mg/mL. These important advances could help the rational design of NPs intended for biomedical uses, demonstrating that careful toxicity evaluation is necessary before using MSNPs in patients.