Enrico Cherubini

In recent years, significant advances in the field of Cellular Neurosciences have contributed to translating basic science into ways to ameliorate diseases affecting the nervous system that carry high economic and social burdens such as neurodevelopmental and neurodegenerative disorders. This has been made possible by emerging technologies such as machine learning and artificial intelligence, humanized mouse and human iPSC models, imaging innovations, brain-computer interfaces, non-invasive brain stimulation, gene editing, identification of biomarkers for drug discovery, etc.The aim of this Research Topic is to understand the paradigm shifts that have shaped and continue to shape Cellular Neuroscience. This collection includes six Reviews, one Opinion and four Research Articles.According to the seminal work at the neuromuscular junction by Del Castillo and Katz (1954) , transmitter release occurs in packets of relatively constant size (quanta), which are equal to the content of a single vesicle fused to the presynaptic membrane. Fusion is favored by a complex network of mutually interacting proteins including synaptotagmin. Riccardo Fesce reports how, with the advent of optogenetics, it has been possible to demonstrate that transmitter release can occur also through the transient opening of a pore between the vesicle and the plasma membrane, without the need for the vesicle to completely fuse with the latter (Harata et al.2006), as already suggested by Bruno Ceccarelli, who called this event "kiss and run". Long lasting, activity-dependent changes in synaptic strength such as those occurring in Long Term Potentation (LTP) are thought to be the cellular correlates of learning and memory (Bliss and Lomo, 1973). Here, Hansel and Yuste suggest that activity-dependent increases in neuronal excitability can recruit neurons into ensembles and maintain them active. They propose a permissive gate model by which the enhanced excitability facilitates ensemble integration by converting subthreshold into supra-threshold connections and by promoting the propagation of dendritic potentials towards the soma, thus allowing to enhance the EPSP/spike coupling. This cellular plasticity mechanism can take place in the absence of LTP and thus leaves the synaptic weight distribution unchanged. Learning and memory processes are associated with morphological modifications of dendritic spines (Bourne and Harris, 2007) which are recognized as the loci of synaptic plasticity expression. Wu et al highlight recent findings on the functional role of synaptopodin, an actin-associated protein found in a subset of dendritic spines of telencephalic neurons, in various forms of Hebbian synaptic plasticity where it plays a central role in regulating postsynaptic calcium dynamics. Of particular interest are synaptic plasticity processes occurring in the primary visual cortex (V1), which, as demonstrated by anatomical and molecular studies, develop over multiple time windows, from the first trimester to aging (Siu et al. 2018). Murphy and Monteiro provide an overview of human primary visual cortex development, highlighting the molecular mechanisms regulating the expression of glutamatergic and GABAergic receptors involved in V1 Excitatory/Inhibitory balance and experience-dependent plasticity, including the late shift of GluN2A/GluN2B balance, consequent to the loss of GluN2B subunits in adulthood (Siu et al. 2017). Emerging technologies such as human induced pluripotent stem cell (hiPSCs) are poised to play a crucial role in identifying the cellular and molecular mechanisms underlying genetic neuropathologies such as neurodegenerative diseases and epilepsy. Towards a precision/personalized medicine, Farahani et al. highlights how hiPSCs derived from somatic cells can produce various neuronal cell types in which non-neuronal immune cell types like microglia can be incorporated to develop new therapeutic tools to prevent and treat these disorders. The use of machine learning/ artificial intelligence and quantitative neuroimaging representations would enhance precision by integrating hiPSC-neuronal models with patients' biophysical data (Vo et al. 2024) Molecular analysis of hiPSC from pediatric patients affected by Focal Cortical Dysplasia Type II and Tuberous Sclerosis has facilitated the identification of several dysregulated genes involved in neuronal migration and differentiation which are responsible for cortical malformations and drugresistant forms of epilepsy (Afshar Saber and Sahin,2020;Lu et al. 2024). The review by Zhang et al. focuses on balloon/giant cells (BC/GC), commonly found in these malformations, which are unable to generate action potentials, with special emphasis to their electrophysiological and morphological glial-like properties similar to astrocytes. BC/GC express a range of glial markers, such as GFAP, vimentin, and nestin, indicating a heterogeneous population of cells with mixed neuronal and glial characteristics.In different brain areas, distinct synaptic input converging onto Pyramidal neurons (Pn) show a macroscale distribution across large dendritic compartments. In this Opinion paper, Cupolillo et al. discuss how spatially distributed excitatory and inhibitory signals converge and integrate onto Pn to shape the neuronal output. The authors provide experimental and computational evidence that these events closely rely on the ability of neurons to generate different forms of local dendritic spikes. The impact of clustering and cooperative plasticity among glutamatergic synapses and the specific spatial organization of GABAergic inputs on dendritic branches are key determinants for shaping dendritic excitability.Transient elevations of intracellular calcium by voltage-gated calcium channels (VGCCs) activated by back-propagating action potentials play a crucial role in dendritic integration (Stuart and Sakmann, 1994). Using ultrafast membrane potential recordings and calcium imaging...

This Special Issue, “GABA Signaling: Therapeutic Targets for Neurodegenerative and Neurodevelopmental Disorders”, focuses on a fundamental property of the neurotransmitter γ-aminobutyric acid (GABA), namely its capacity to shift, in particular conditions, from the hyperpolarizing to the depolarizing direction

Oscillations occurring simultaneously in a given area represent a physiological unit of brain states. They allow for temporal segmentation of spikes and support distinct behaviors. To establish how multiple oscillatory components co-vary simultaneously and influence neuronal firing during sleep and wakefulness in mice, we describe a multivariate analytical framework for constructing the state space of hippocampal oscillations. Examining the co-occurrence patterns of oscillations on the state space, across species, uncovered the presence of network constraints and distinct set of cross-frequency interactions during wakefulness compared to sleep. We demonstrated how the state space can be used as a canvas to map the neural firing and found that distinct neurons during navigation were tuned to different sets of simultaneously occurring oscillations during sleep. This multivariate analytical framework provides a window to move beyond classical bivariate pipelines for investigating oscillations and neuronal firing, thereby allowing to factor-in the complexity of oscillation–population interactions.

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

Editorial: Insights in cellular neurophysiology: 2021

GABA, the main inhibitory neurotransmitter in the adult brain, depolarizes and excites immature neurons because of an initially higher intracellular chloride concentration [Cl⁻]i due to the delayed expression of the chloride exporter KCC2 at birth. Depolarization-induced calcium rise via NMDA receptors and voltage-dependent calcium channels is instrumental in shaping neuronal circuits and in controlling the excitatory (E)/inhibitory (I) balance in selective brain areas. An E/I imbalance accounts for cognitive impairment observed in several neuropsychiatric disorders. The aim of this review is to summarize recent data on the mechanisms by which alterations of GABAergic signaling alter the E/I balance in cortical and hippocampal neurons in Alzheimer’s disease (AD) and the role of cation-chloride co-transporters in this process. In particular, we discuss the NGF and AD relationship and how mice engineered to express recombinant neutralizing anti-NGF antibodies (AD11 mice), which develop a neurodegenerative pathology reminiscent of that observed in AD patients, exhibit a depolarizing action of GABA due to KCC2 impairment. Treating AD and other forms of dementia with bumetanide, a selective NKCC1 antagonist, contributes to re-establishing a proper E/I balance in selective brain areas, leading to amelioration of AD symptoms and the slowing down of disease progression.

GABA depolarizes and often excites immature neurons in all animal species and brain structures investigated due to a developmentally regulated reduction in intracellular chloride concentration ([Cl⁻]_i) levels. The control of [Cl⁻]_i levels is mediated by the chloride cotransporters NKCC1 and KCC2, the former usually importing chloride and the latter exporting it. The GABA polarity shift has been extensively validated in several experimental conditions using often the NKCC1 chloride importer antagonist bumetanide. In spite of an intrinsic heterogeneity, this shift is abolished in many experimental conditions associated with developmental disorders including autism, Rett syndrome, fragile X syndrome, or maternal immune activation. Using bumetanide, an EMA- and FDA-approved agent, many clinical trials have shown promising results with the expected side effects. Kaila et al. have repeatedly challenged these experimental and clinical observations. Here, we reply to the recent reviews by Kaila et al. stressing that the GABA polarity shift is solidly accepted by the scientific community as a major discovery to understand brain development and that bumetanide has shown promising effects in clinical trials.

The construction of the brain relies on a series of well-defined genetically and experience- or activity -dependent mechanisms which allow to adapt to the external environment. Disruption of these processes leads to neurological and psychiatric disorders, which in many cases are manifest already early in postnatal life. GABA, the main inhibitory neurotransmitter in the adult brain is one of the major players in the early assembly and formation of neuronal circuits. In the prenatal and immediate postnatal period GABA, acting on GABA_A receptors, depolarizes and excites targeted cells via an outwardly directed flux of chloride. In this way it activates NMDA receptors and voltage-dependent calcium channels contributing, through intracellular calcium rise, to shape neuronal activity and to establish, through the formation of new synapses and elimination of others, adult neuronal circuits. The direction of GABA_A-mediated neurotransmission (depolarizing or hyperpolarizing) depends on the intracellular levels of chloride [Cl⁻]_i, which in turn are maintained by the activity of the cation-chloride importer and exporter KCC2 and NKCC1, respectively. Thus, the premature hyperpolarizing action of GABA or its persistent depolarizing effect beyond the postnatal period, leads to behavioral deficits associated with morphological alterations and an excitatory (E)/inhibitory (I) imbalance in selective brain areas. The aim of this review is to summarize recent data concerning the functional role of GABAergic transmission in building up and refining neuronal circuits early in development and its dysfunction in neurodevelopmental disorders such as Autism Spectrum Disorders (ASDs), schizophrenia and epilepsy. In particular, we focus on novel information concerning the mechanisms by which alterations in cation-chloride co-transporters (CCC) generate behavioral and cognitive impairment in these diseases. We discuss also the possibility to re-establish a proper GABA_A-mediated neurotransmission and excitatory (E)/inhibitory (I) balance within selective brain areas acting on CCC.

Acetylcholine (ACh), released in the hippocampus from fibers originating in the medial septum/diagonal band of Broca (MSDB) complex, is crucial for learning and memory. The CA2 region of the hippocampus has received increasing attention in the context of social memory. However, the contribution of ACh to this process remains unclear. Here, we show that in mice, ACh controls social memory. Specifically, MSDB cholinergic neurons inhibition impairs social novelty discrimination, meaning the propensity of a mouse to interact with a novel rather than a familiar conspecific. This effect is mimicked by a selective antagonist of nicotinic AChRs delivered in CA2. Ex vivo recordings from hippocampal slices provide insight into the underlying mechanism, as activation of nAChRs by nicotine increases the excitatory drive to CA2 principal cells via disinhibition. In line with this observation, optogenetic activation of cholinergic neurons in MSDB increases the firing of CA2 principal cells in vivo. These results point to nAChRs as essential players in social novelty discrimination by controlling inhibition in the CA2 region.