Functional Properties Of Neurons Research Articles

Transcriptomic correlates of electrophysiological and morphological diversity within and across excitatory and inhibitory neuron classes.

In order to further our understanding of how gene expression contributes to key functional properties of neurons, we combined publicly accessible gene expression, electrophysiology, and morphology measurements to identify cross-cell type correlations between these data modalities. Building on our previous work using a similar approach, we distinguished between correlations which were “class-driven,” meaning those that could be explained by differences between excitatory and inhibitory cell classes, and those that reflected graded phenotypic differences within classes. Taking cell class identity into account increased the degree to which our results replicated in an independent dataset as well as their correspondence with known modes of ion channel function based on the literature. We also found a smaller set of genes whose relationships to electrophysiological or morphological properties appear to be specific to either excitatory or inhibitory cell types. Next, using data from PatchSeq experiments, allowing simultaneous single-cell characterization of gene expression and electrophysiology, we found that some of the gene-property correlations observed across cell types were further predictive of within-cell type heterogeneity. In summary, we have identified a number of relationships between gene expression, electrophysiology, and morphology that provide testable hypotheses for future studies.

Lessons from Worm Dendritic Patterning.

The structural and functional properties of neurons have intrigued scientists since the pioneering work of Santiago Ramón y Cajal. Since then, emerging cutting-edge technologies, including light and electron microscopy, electrophysiology, biochemistry, optogenetics, and molecular biology, have dramatically increased our understanding of dendritic properties. This advancement was also facilitated by the establishment of different animal model organisms, from flies to mammals. Here we describe the emerging model system of a Caenorhabditis elegans polymodal neuron named PVD, whose dendritic tree follows a stereotypical structure characterized by repeating candelabra-like structural units. In the past decade, progress has been made in understanding PVD's functions, morphogenesis, regeneration, and aging, yet many questions still remain.

Влияние антагонистов NMDA рецепторов разных типов на вызванные ответы пирамидных нейронов в срезах коры мозга крысы

The use of NMDA receptor antagonists for the treatment of CNS diseases is currently limited. This may be due to the lack of knowledge about the effects of antagonists on the functional properties of neurons since in vitro models do not fully reproduce the real pathophysiological processes. We studied the effects of NMDA receptor antagonists on properties of pyramidal neuron responses in the rat prefrontal cortex. A competitive antagonist APV (50 µM) and an ion channel blocker memantine (100 µM) did not produce significant effects on evoked EPSP. When the inhibitory transmission was suppressed by picrotoxin (50 µM), the extracellular stimulation caused prolonged membrane depolarization and generation a series of action potentials. APV decreased duration and amplitude of such responses. Memantine was active only when magnesium was excluded from the extracellular environment. The lack of effects of memantine can be explained by its competition with magnesium for the binding site within the ion channel. This work demonstrates that in order to predict the systemic effects of blockers on the functions of neurons and their excitability, it is necessary to use in vitro models in which the activation of NMDA receptors occurs in the presence of physiological concentrations of magnesium and without clamping of membrane potential.

Human Dental Pulp Stem Cells and Gingival Mesenchymal Stem Cells Display Action Potential Capacity In Vitro after Neuronogenic Differentiation.

The potential of human mesenchymal stromal/stem cells (MSCs) including oral stem cells (OSCs) as a cell source to derive functional neurons has been inconclusive. Here we tested a number of human OSCs for their neurogenic potential compared to non-OSCs and employed various neurogenic induction methods. OSCs including dental pulp stem cells (DPSCs), gingiva-derived mesenchymal stem cells (GMSCs), stem cells from apical papilla and non-OSCs including bone marrow MSCs (BMMSCs), foreskin fibroblasts and dermal fibroblasts using non-neurosphere-mediated or neurosphere-mediated methods to guide them toward neuronal lineages. Cells were subjected to RT-qPCR, immunocytofluorescence to detect the expression of neurogenic genes or electrophysiological analysis at final stage of maturation. We found that induced DPSCs and GMSCs overall appeared to be more neurogenic compared to other cells either morphologically or levels of neurogenic gene expression. Nonetheless, of all the neural induction methods employed, only one neurosphere-mediated method yielded electrophysiological properties of functional neurons. Under this method, cells expressed increased neural stem cell markers, nestin and SOX1, in the first phase of differentiation. Neuronal-like cells expressed βIII-tubulin, CNPase, GFAP, MAP-2, NFM, pan-Nav, GAD67, Nav1.6, NF1, NSE, PSD95, and synapsin after the second phase of differentiation to maturity. Electrophysiological experiments revealed that 8.3% of DPSC-derived neuronal cells and 21.2% of GMSC-derived neuronal cells displayed action potential, although no spontaneous excitatory/inhibitory postsynaptic action potential was observed. We conclude that DPSCs and GMSCs have the potential to become neuronal cells in vitro, therefore, these cells may be used as a source for neural regeneration.

Quantitative Association of Anatomical and Functional Classes of Olfactory Bulb Neurons.

Juxtaglomerular cells (JGCs) of the olfactory bulb (OB) glomerular layer (GL) play a fundamental role in olfactory information processing. Their variability in morphology, physiology, and connectivity suggests distinct functions. The quantitative understanding of population-wise morphological and physiological properties and a comprehensive classification based on quantitative parameters, however, is still lacking, impeding the analysis of microcircuits. Here, we provide multivariate clustering of 95 in vitro sampled cells from the GL of the mouse (male or female C57BL/6) OB and perform detailed morphological and physiological characterization for the seven computed JGC types. Using a classifier based on a subselection of parameters, we identified the neuron types in paired recordings to characterize their functional connectivity. We found that 4 of the 7 clusters comply with prevailing concepts of GL cell types, whereas the other 3 represent own distinct entities. We have labeled these entities horizontal superficial tufted cell (hSTC), vertical superficial tufted cell, and microglomerular cell (MGC): The hSTC is a tufted cell with a lateral dendrite that much like mitral cells and tufted cells receives excitatory inputs from the external tufted cell but likewise serves as an excitatory element for glomerular interneurons. The vertical superficial tufted cell, on the other hand, represents a tufted cell type with vertically projecting basal dendrites. We further define the MGC, characterized by a small dendritic tree and plateau action potentials. In addition to olfactory nerve-driven and external tufted cell driven interneurons, these MGCs represent a third functionally distinct type, the hSTC-driven interneurons. The presented correlative analysis helps to bridge the gap between branching patterns and cellular functional properties, permitting the integration of results from in vivo recordings, advanced morphological tools, and connectomics.SIGNIFICANCE STATEMENT The variance of neuron properties is a feature across mammalian cerebral circuits, contributing to signal processing and adding computational robustness to the networks. It is particularly noticeable in the glomerular layer of the olfactory bulb, the first site of olfactory information processing. We provide the first unbiased population-wise multivariate analysis to correlate morphological and physiological parameters of juxtaglomerular cells. We identify seven cell types, including four previously described neuron types, and identify further three distinct classes. The presented correlative analysis of morphological and physiological parameters gives an opportunity to predict morphological classes from physiological measurements or the functional properties of neurons from morphology and opens the way to integrate results from in vivo recordings, advanced morphological tools, and connectomics.

Layer- and Cell Type-Specific Modulation of Excitatory Neuronal Activity in the Neocortex.

From an anatomical point of view the neocortex is subdivided into up to six layers depending on the cortical area. This subdivision has been described already by Meynert and Brodmann in the late 19/early 20. century and is mainly based on cytoarchitectonic features such as the size and location of the pyramidal cell bodies. Hence, cortical lamination is originally an anatomical concept based on the distribution of excitatory neuron. However, it has become apparent in recent years that apart from the layer-specific differences in morphological features, many functional properties of neurons are also dependent on cortical layer or cell type. Such functional differences include changes in neuronal excitability and synaptic activity by neuromodulatory transmitters. Many of these neuromodulators are released from axonal afferents from subcortical brain regions while others are released intrinsically. In this review we aim to describe layer- and cell-type specific differences in the effects of neuromodulator receptors in excitatory neurons in layers 2–6 of different cortical areas. We will focus on the neuromodulator systems using adenosine, acetylcholine, dopamine, and orexin/hypocretin as examples because these neuromodulator systems show important differences in receptor type and distribution, mode of release and functional mechanisms and effects. We try to summarize how layer- and cell type-specific neuromodulation may affect synaptic signaling in cortical microcircuits.

Reproducible and efficient generation of functionally active neurons from human hiPSCs for preclinical disease modeling

The use of human induced pluripotent stem cell (hiPSC)-derived neuronal cultures to study the mechanisms of neurological disorders is often limited by low efficiency and high variability in differentiation of functional neurons. Here we compare the functional properties of neurons in cultures prepared with two hiPSC differentiation protocols, both plated on astroglial feeder layers. Using a protocol with an expandable intermediate stage, only a small percentage of cells with neuronal morphology were excitable by 21–23days in culture. In contrast, a direct differentiation strategy of the same hiPSC line produced cultures in which the majority of neurons fired action potentials as early as 4–5days. By 35–38days over 80% of the neurons fired repetitively and many fired spontaneously. Spontaneous post-synaptic currents were observed in ~40% of the neurons at 4–5days and in ~80% by 21–23days. The majority (75%) received both glutamatergic and GABAergic spontaneous postsynaptic currents. The rate and degree of maturation of excitability and synaptic activity was similar between multiple independent platings from a single hiPSC line, and between two different control hiPSC lines. Cultures of rapidly functional neurons will facilitate identification of cellular mechanisms underlying genetically defined neurological disorders and development of novel therapeutics.

CD40 is a major regulator of dendrite growth from developing excitatory and inhibitory neurons.

Dendrite size and morphology are key determinants of the functional properties of neurons and neural circuits. Here we show that CD40, a member of the TNF receptor superfamily, is a major regulator of dendrite growth and elaboration in the developing brain. The dendrites of hippocampal excitatory neurons were markedly stunted in Cd40-/- mice, whereas those of striatal inhibitory neurons were much more exuberant. These striking and opposite phenotypic changes were also observed in excitatory and inhibitory neurons cultured from Cd40-/- mice and were rescued by soluble CD40. The changes in excitatory and inhibitory neurons cultured from Cd40-/- mice were mimicked in neurons of Cd40+/+ mice by treatment with soluble CD40L and were dependent on PKC-β and PKC-γ, respectively. These results suggest that CD40-activated CD40L reverse signalling has striking and opposite effects on the growth and elaboration of dendrites among major classes of brain neurons by PKC-dependent mechanisms.

Direct conversion of human fibroblasts to functional excitatory cortical neurons integrating into human neural networks

BackgroundHuman fibroblasts can be directly converted to several subtypes of neurons, but cortical projection neurons have not been generated.MethodsHere we screened for transcription factor combinations that could potentially convert human fibroblasts to functional excitatory cortical neurons. The induced cortical (iCtx) cells were analyzed for cortical neuronal identity using immunocytochemistry, single-cell quantitative polymerase chain reaction (qPCR), electrophysiology, and their ability to integrate into human neural networks in vitro and ex vivo using electrophysiology and rabies virus tracing.ResultsWe show that a combination of three transcription factors, BRN2, MYT1L, and FEZF2, have the ability to directly convert human fibroblasts to functional excitatory cortical neurons. The conversion efficiency was increased to about 16% by treatment with small molecules and microRNAs. The iCtx cells exhibited electrophysiological properties of functional neurons, had pyramidal-like cell morphology, and expressed key cortical projection neuronal markers. Single-cell analysis of iCtx cells revealed a complex gene expression profile, a subpopulation of them displaying a molecular signature closely resembling that of human fetal primary cortical neurons. The iCtx cells received synaptic inputs from co-cultured human fetal primary cortical neurons, contained spines, and expressed the postsynaptic excitatory scaffold protein PSD95. When transplanted ex vivo to organotypic cultures of adult human cerebral cortex, the iCtx cells exhibited morphological and electrophysiological properties of mature neurons, integrated structurally into the cortical tissue, and received synaptic inputs from adult human neurons.ConclusionsOur findings indicate that functional excitatory cortical neurons, generated here for the first time by direct conversion of human somatic cells, have the capacity for synaptic integration into adult human cortex.

Abnormally Increased Secretion in Olfactory Neuronal Precursors from a Case of Schizophrenia Is Modulated by Melatonin: A Pilot Study.

The alterations that underlie the pathophysiology of schizophrenia (SCZ) include the dysregulation of structural and functional properties of neurons. Among these, the secretion of neurotransmitters and hormones, which plays a key role for neuronal communication and development, is altered. Neuronal precursors from the human olfactory epithelium have been recently characterized as a reliable model for studying the etiopathogenesis of neuropsychiatric diseases. Our previous work has shown that melatonin enhances the development of morphological and functional features of cloned olfactory neuronal precursors (ONPs) from a healthy subject. In this work we found that primary cultures of ONPs obtained from a schizophrenic patient display an increased potassium-evoked secretion, when compared with ONPs from an age- and gender-matched healthy control subject (HCS). Secretion was evaluated by FM1-43 fluorescence cumulative changes in response to depolarization. Interestingly, a 12 h-melatonin treatment modulated the abnormally increased secretion in SCZ ONPs and brought it to levels similar to those found in the HCS ONPs. Our results suggest that the actin cytoskeleton might be a target for melatonin effects, since it induces the thickening of actin microfilament bundles. Further research will address the mechanisms by which melatonin modulates neurochemical secretion from ONPs.

The organization of frequency and binaural cues in the gerbil inferior colliculus.

The inferior colliculus (IC) is the common target of separate pathways that transmit different types of auditory information. Beyond tonotopy, little is known about the organization of response properties within the 3-dimensional layout of the auditory midbrain in most species. Through study of interaural time difference (ITD) processing, the functional properties of neurons can be readily characterized and related to specific pathways. To characterize the representation of ITDs relative to the frequency and hodological organization of the IC, the properties of neurons were recorded and the sites recovered histologically. Subdivisions of the IC were identified based on cytochrome oxidase (CO) histochemistry. The results were plotted within a framework formed by an MRI atlas of the gerbil brain. The central nucleus was composed of two parts, and lateral and dorsal cortical areas were identified. The lateral part of the central nucleus had the highest CO activity in the IC and a high proportion of neurons sensitive to ITDs. The medial portion had lower CO activity and fewer ITD-sensitive neurons. A common tonotopy with a dorsolateral to ventromedial gradient of low to high frequencies spanned the two regions. The distribution of physiological responses was in close agreement with known patterns of ascending inputs. An understanding of the 3-dimensional organization of the IC is needed to specify how the single tonotopic representation in the IC central nucleus leads to the multiple tonotopic representations in core areas of the auditory cortex.

Directly Converted Human Fibroblasts Mature to Neurons and Show Long-Term Survival in Adult Rodent Hippocampus.

Direct conversion of human somatic cells to induced neurons (iNs), using lineage-specific transcription factors has opened new opportunities for cell therapy in a number of neurological diseases, including epilepsy. In most severe cases of epilepsy, seizures often originate in the hippocampus, where populations of inhibitory interneurons degenerate. Thus, iNs could be of potential use to replace these lost interneurons. It is not known, however, if iNs survive and maintain functional neuronal properties for prolonged time periods in in vivo. We transplanted human fibroblast-derived iNs into the adult rat hippocampus and observed a progressive morphological differentiation, with more developed dendritic arborisation at six months as compared to one month. This was accompanied by mature electrophysiological properties and fast high amplitude action potentials at six months after transplantation. This proof-of-principle study suggests that human iNs can be developed as a candidate source for cell replacement therapy in temporal lobe epilepsy.

N3DFix: an Algorithm for Automatic Removal of Swelling Artifacts in Neuronal Reconstructions.

It is well established that not only electrophysiology but also morphology plays an important role in shaping the functional properties of neurons. In order to properly quantify morphological features it is first necessary to translate observational histological data into 3-dimensional geometric reconstructions of the neuronal structures. This reconstruction process, independently of being manual or (semi-)automatic, requires several preparation steps (e.g. histological processing) before data acquisition using specialized software. Unfortunately these processing steps likely produce artifacts which are then carried to the reconstruction, such as tissue shrinkage and formation of swellings. If not accounted for and corrected, these artifacts can change significantly the results from morphometric analysis and computer simulations. Here we present N3DFix, an open-source software which uses a correction algorithm to automatically find and fix swelling artifacts in neuronal reconstructions. N3DFix works as a post-processing tool and therefore can be used in either manual or (semi-)automatic reconstructions. The algorithm's internal parameters have been defined using a "ground truth" dataset produced by a neuroanatomist, involving two complementary manual reconstruction procedures: in the first, neuronal topology was faithfully reconstructed, including all swelling artifacts; in the second procedure a meticulous correction of the artifacts was manually performed directly during neuronal tracing. The internal parameters of N3DFix were set to minimize the differences between manual amendments and the algorithm's corrections. It is shown that the performance of N3DFix is comparable to careful manual correction of the swelling artifacts. To promote easy access and wide adoption, N3DFix is available in NEURON, Vaa3D and Py3DN.

In vitro comparative analysis of human dental stem cells from a single donor and its neuronal differentiation potential evaluated by electrophysiology

AimsThe aim of this study was to find out a mesenchymal stem cells (MSCs) source from human dental tissues of the same donor (follicle, papilla and pulp), which exhibits higher neurogenic differentiation potential in vitro. Main methodsMSCs were isolated from dental tissues (follicle, papilla and pulp) by digestion method. All MSCs were analyzed for pluripotent makers by western blot, cell surface markers by flow cytometry, adipo- and osteocytes markers by RT-qPCR. The neuronal differentiated MSCs were characterized for neuronal specific markers by RT-qPCR and immunofluorescence. Functional neuronal properties were analyzed by electrophysiology and synaptic markers expression. Key findingsAll MSCs expressed pluripotent markers (Oct4, Sox2 and Nanog) and were found positive for mesenymal markers (CD44, CD90, CD105) while negative for hematopoietic markers (CD34 and CD45). Furthermore, MSCs were successfully differentiated into adipocytes, osteocytes and trans-differentiated into neuronal cells. Among them, dental pulp derived MSCs exhibits higher neurogenic differentiation potential, in term of expression of neuronal specific markers at both gene and protein level, and having higher Na+ and K+ current with the expression of synaptic markers. SignificanceThe three types of dental MSCs from a single donor broadly possessed similar cellular properties and can differentiate into neuronal cells; however, pulp derived MSCs showed higher neurogenic potential than the follicle and papilla, suggesting their use in future stem cells therapy for the treatment of neurodegenerative disorders.

Baicalein reverts L-valine-induced persistent sodium current up-modulation in primary cortical neurons

L-valine is a branched-chain amino acid (BCAA) largely used as dietary integrator by athletes and involved in some inherited rare diseases such as maple syrup urine disease. This pathology is caused by an altered BCAA metabolism with the accumulation of toxic keto acids in tissues and body fluids with consequent severe neurological symptoms. In animal models of BCAA accumulation, increased oxidative stress levels and lipid peroxidation have been reported. The aim of this study was to analyze both whether high BCAA concentrations in neurons induce reactive oxygen species (ROS) production and whether, by performing electrophysiological recordings, the neuronal functional properties are modified. Our results demonstrate that in primary cortical cultures, a high dose of valine increases ROS production and provokes neuronal hyperexcitability because the action potential frequencies and the persistent sodium current amplitudes increase significantly compared to non-treated neurons. Since Baicalein, a flavone obtained from the Scutellaria root, has been shown to act as a strong antioxidant with neuroprotective effects, we evaluated its possible antioxidant activity in primary cortical neurons chronically exposed to L-valine. The preincubation of cortical neurons with Baicalein prevents the ROS production and is able to revert both the neuronal hyperexcitability and the increase of the persistent sodium current, indicating a direct correlation between the ROS production and the altered physiological parameters. In conclusion, our data show that the electrophysiological alterations of cortical neurons elicited by high valine concentration are due to the increase in ROS production, suggesting much caution in the intake of BCAA dietary integrators.

Functional Deficiencies in Fragile X Neurons Derived from Human Embryonic Stem Cells.

Fragile X syndrome (FXS), the most common form of inherited mental retardation, is caused by silencing of the FMR1 gene. In this study, we describe for the first time the properties of neurons developed from human embryonic stem cells (hESCs) that carry the FMR1 mutation and are grown in culture for extended periods. These neurons are retarded compared with controls in several morphological and functional properties. In vitro neural differentiation of FX hESCs can thus serve as a most relevant system for the analysis of molecular mechanisms underlying the impaired neuronal functions in FXS.

Increasing Spontaneous Retinal Activity before Eye Opening Accelerates the Development of Geniculate Receptive Fields.

Visually evoked activity is necessary for the normal development of the visual system. However, little is known about the capacity for patterned spontaneous activity to drive the maturation of receptive fields before visual experience. Retinal waves provide instructive retinotopic information for the anatomical organization of the visual thalamus. To determine whether retinal waves also drive the maturation of functional responses, we increased the frequency of retinal waves pharmacologically in the ferret (Mustela putorius furo) during a period of retinogeniculate development before eye opening. The development of geniculate receptive fields after receiving these increased neural activities was measured using single-unit electrophysiology. We found that increased retinal waves accelerate the developmental reduction of geniculate receptive field sizes. This reduction is due to a decrease in receptive field center size rather than an increase in inhibitory surround strength. This work reveals an instructive role for patterned spontaneous activity in guiding the functional development of neural circuits.

Spatial processing: mapping function.

Studies reveal a gradient in the functional properties of neurons along the transverse axis of hippocampal CA3 and their likely contribution to pattern separation and pattern completion.

Cell-Type-Specific Activity in Prefrontal Cortex during Goal-Directed Behavior

The prefrontal cortex (PFC) plays a key role in controlling goal-directed behavior. Although a variety of task-related signals have been observed in the PFC, whether they are differentially encoded by various cell types remains unclear. Here we performed cellular-resolution microendoscopic Ca(2+) imaging from genetically defined cell types in the dorsomedial PFC of mice performing a PFC-dependent sensory discrimination task. We found that inhibitory interneurons of the same subtype were similar to each other, but different subtypes preferentially signaled different task-related events: somatostatin-positive neurons primarily signaled motor action (licking), vasoactive intestinal peptide-positive neurons responded strongly to action outcomes, whereas parvalbumin-positive neurons were less selective, responding to sensory cues, motor action, and trial outcomes. Compared to each interneuron subtype, pyramidal neurons showed much greater functional heterogeneity, and their responses varied across cortical layers. Such cell-type and laminar differences in neuronal functional properties may be crucial for local computation within the PFC microcircuit.

Distribution and effects of the muscarinic receptor subtypes in the primary visual cortex

Muscarinic cholinergic receptors modulate the activity and plasticity of the visual cortex. Muscarinic receptors are divided into five subtypes that are not homogeneously distributed throughout the cortical layers and cells types. This distribution results in complex action of the muscarinic receptors in the integration of visual stimuli. Selective activation of the different subtypes can either strengthen or weaken cortical connectivity (e.g., thalamocortical vs. corticocortical), i.e., it can influence the processing of certain stimuli over others. Moreover, muscarinic receptors differentially modulate some functional properties of neurons during experience-dependent activity and cognitive processes and they contribute to the fine-tuning of visual processing. These functions are involved in the mechanisms of attention, maturation and learning in the visual cortex. This minireview describes the anatomo-functional aspects of muscarinic modulation of the primary visual cortex’s (V1) microcircuitry.