Retzius Neurons Research Articles

Novel model of cortical–meningeal organoid co-culture system improves human cortical brain organoid cytoarchitecture

Human cortical organoids (hCOs), derived from human induced pluripotent stem cells (iPSCs), provide a platform to interrogate mechanisms of human brain development and diseases in complex three- dimensional tissues. However, current hCO development methods lack important non-neural tissues, such as the surrounding meningeal layer, that have been shown to be essential for normal corticogenesis and brain development. Here, we first generated hCOs from a single rosette to create more homogenous organoids with consistent size around 250 µm by day 5. We then took advantage of a 3D co-culture system to encapsulate brain organoids with a thin layer of meningeal cells from the very early stages of cortical development. Immunostaining analysis was performed to display different cortical layer markers during different stages of development. Real-time monitoring of organoid development using IncuCyte displayed enhanced morphology and increased growth rate over time. We found that meningeal-encapsulated organoids illustrated better laminar organization by exhibiting higher expression of REELIN by Cajal–Retzius neurons. Presence of meningeal cells resulted in a greater expansion of TBR2 intermediate progenitor cells (IPCs), the deep cortical layer (CTIP2) and upper cortical layer (BRN2). Finally, meningeal-encapsulated organoids enhanced outer radial glial and astrocyte formation illustrated by stronger expression of HOPX and GFAP markers, respectively. This study presents a novel 3D co-culture platform to more closely mimic the in vivo cortical brain structure and enable us to better investigating mechanisms underlying the neurodevelopmental disorders during embryonic development.

Early Regional Patterning in the Human Prefrontal Cortex Revealed by Laminar Dynamics of Deep Projection Neuron Markers.

Early regional patterning and laminar position of cortical projection neurons is determined by activation and deactivation of transcriptional factors (TFs) and RNA binding proteins (RBPs) that regulate spatiotemporal framework of neurogenetic processes (proliferation, migration, aggregation, postmigratory differentiation, molecular identity acquisition, axonal growth, dendritic development, and synaptogenesis) within transient cellular compartments. Deep-layer projection neurons (DPN), subplate (SPN), and Cajal-Retzius neurons (CRN) are early-born cells involved in the establishment of basic laminar and regional cortical architecture; nonetheless, laminar dynamics of their molecular transcriptional markers remain underexplored. Here we aimed to analyze laminar dynamics of DPN markers, i.e., transcription factors TBR1, CTIP2, TLE4, SOX5, and RBP CELF1 on histological serial sections of the human frontal cortex between 7.5-15 postconceptional weeks (PCW) in reference to transient proliferative, migratory, and postmigratory compartments. The subtle signs of regional patterning were seen during the late preplate phase in the pattern of sublaminar organization of TBR1+/Reelin+ CRN and TBR1+ pioneering SPN. During the cortical plate (CP)-formation phase, TBR1+ neurons became radially aligned, forming continuity from a well-developed subventricular zone to CP showing clear lateral to medial regional gradients. The most prominent regional patterning was seen during the subplate formation phase (around 13 PCW) when a unique feature of the orbitobasal frontal cortex displays a "double plate" pattern. In other portions of the frontal cortex (lateral, dorsal, medial) deep portion of CP becomes loose and composed of TBR1+, CTIP2+, TLE4+, and CELF1+ neurons of layer six and later-born SPN, which later become constituents of the expanded SP (around 15 PCW). Overall, TFs and RBPs mark characteristic regional laminar dynamics of DPN, SPN, and CRN subpopulations during remarkably early fetal phases of the highly ordered association cortex development.

The Thermodynamically Expensive Contribution of Three Calcium Sources to Somatic Release of Serotonin.

The soma, dendrites and axon of neurons may display calcium-dependent release of transmitters and peptides. Such release is named extrasynaptic for occurring in absence of synaptic structures. This review describes the cooperative actions of three calcium sources on somatic exocytosis. Emphasis is given to the somatic release of serotonin by the classical leech Retzius neuron, which has allowed detailed studies on the fine steps from excitation to exocytosis. Trains of action potentials induce transmembrane calcium entry through L-type channels. For action potential frequencies above 5 Hz, summation of calcium transients on individual action potentials activates the second calcium source: ryanodine receptors produce calcium-induced calcium release. The resulting calcium tsunami activates mitochondrial ATP synthesis to fuel transport of vesicles to the plasma membrane. Serotonin that is released maintains a large-scale exocytosis by activating the third calcium source: serotonin autoreceptors coupled to phospholipase C promote IP3 production. Activated IP3 receptors in peripheral endoplasmic reticulum release calcium that promotes vesicle fusion. The Swiss-clock workings of the machinery for somatic exocytosis has a striking disadvantage. The essential calcium-releasing endoplasmic reticulum near the plasma membrane hinders the vesicle transport, drastically reducing the thermodynamic efficiency of the ATP expenses and elevating the energy cost of release.

Extrasynaptic Communication.

Streams of action potentials or long depolarizations evoke a massive exocytosis of transmitters and peptides from the surface of dendrites, axons and cell bodies of different neuron types. Such mode of exocytosis is known as extrasynaptic for occurring without utilization of synaptic structures. Most transmitters and all peptides can be released extrasynaptically. Neurons may discharge their contents with relative independence from the axon, soma and dendrites. Extrasynaptic exocytosis takes fractions of a second in varicosities or minutes in the soma or dendrites, but its effects last from seconds to hours. Unlike synaptic exocytosis, which is well localized, extrasynaptic exocytosis is diffuse and affects neuronal circuits, glia and blood vessels. Molecules that are liberated may reach extrasynaptic receptors microns away. The coupling between excitation and exocytosis follows a multistep mechanism, different from that at synapses, but similar to that for the release of hormones. The steps from excitation to exocytosis have been studied step by step for the vital transmitter serotonin in leech Retzius neurons. The events leading to serotonin exocytosis occur similarly for the release of other transmitters and peptides in central and peripheral neurons. Extrasynaptic exocytosis occurs commonly onto glial cells, which react by releasing the same or other transmitters. In the last section, we discuss how illumination of the retina evokes extrasynaptic release of dopamine and ATP. Dopamine contributes to light-adaptation; ATP activates glia, which mediates an increase in blood flow and oxygenation. A proper understanding of the workings of the nervous system requires the understanding of extrasynaptic communication.

The electrophysiological effects of cadmium on Retzius nerve cells of the leech Haemopis sanguisuga

Cadmium is considered one of the most toxic heavy metals which can cause cytotoxicity in multiple organs including the brain. Despite many studies over the past decades, the cellular and molecular mechanisms underlying its neurotoxicity remain unclear. The present study was designed to examine the acute effects of cadmium chloride (CdCl2) on the electrical activity of the Retzius nerve cells of leech Haemopis sanguisuga using electrophysiological techniques. CdCl2, in concentrations of 10–100 μM, produced a dose- and time-dependent depolarization of Retzius neurons, paralleled by an increase in firing frequency and action potential duration. To examine potential mechanisms, we studied the effects of cadmium on the outward potassium current upon depolarization using a point microelectrode voltage-clamp technique. Reduction of the fast, and partial inhibition of the slow outward current were observed after adding 50 and 100 μM CdCl2 in the external fluid. The present results support the view that the effect of cadmium on the outward potassium channel may be a potential contributing mechanism for cadmium-induced neurotoxic damage. The proposed mechanism of cadmium action on the electrical properties of leech Retzius neurons might have broader significance concerning not only the leeches but vertebrate brains as well.

Specific Localization of an Auto-inhibition Mechanism at Presynaptic Terminals of Identified Serotonergic Neurons

Auto-regulation mechanisms in serotonergic neurons regulate their electrical activity and secretion. Since these neurons release serotonin from different structural compartments – including presynaptic terminals, soma, axons and dendrites – through different mechanisms, autoregulation mechanisms are also likely to be different at each compartment. Here we show that a chloride-mediated auto-inhibitory mechanism is exclusively localized at presynaptic terminals, but not at extrasynaptic release sites, in serotonergic Retzius neurons of the leech. An auto-inhibition response was observed immediately after intracellular stimulation with an electrode placed in the soma, in neurons that were isolated and cultured retaining an axonal stump, where presynaptic terminals are formed near the soma, but not in somata isolated without axon, where no synaptic terminals are formed, nor in neurons in the nerve ganglion, where terminals are electrotonically distant from the soma. Furthermore, no auto-inhibition response was detected in either condition during the longer time course of somatic secretion. This shows that the auto-inhibition effects are unique to nerve terminals. We further determined that serotonin released from peri-synaptic dense-core vesicles contributes to auto-inhibition in the terminals, since blockade of L-type calcium channels, which are required to stimulate extrasynaptic but not synaptic release, decreased the amplitude of the auto-inhibition response. Our results show that the auto-regulation mechanism at presynaptic terminals is unique and different from that described in the soma of these neurons, further highlighting the differences in the mechanisms regulating serotonin release from different neuronal compartments, which expand the possibilities of a single neuron to perform multiple functions in the nervous system.

Investigation of Changes in Membrane Potential and Rhythmic Activity of the Retzius Neuron upon Stimulation of the Sensory P-Neuron

It was found that in "natural neural networks", ganglia of the nervous system of a leech, the frequency of rhythmic excitation (a series of nerve impulses, RE) of one neuron is modulated upon activation of other neurons. Changes in the electrophysiological characteristics of the leech Retzius cell in response to electrical stimulation of one of the sensory neurons (P-cells) were revealed. Registration of changes in the membrane potential of neurons, as well as electrical stimulation of the P-cell was carried out using microelectrodes introduced into the cells. It was found that during electrical stimulation, P-cells increase the frequency of spontaneous RE of Retzius cells, the membrane potential of P-cells increases, but Retzius cells do not change. With an increase in the duration of stimulation, the RE frequency increases in both the P-cell and the Retzius cell. It has been found that RE Retzius cells, upon stimulation of P-cells, arise against the background of RE of the Retzius cell. Thus, during RE of sensory neurons and synaptic transmission to the Retzius cell, RE frequency modulation occurs. According to the authors, changes in the frequency of spontaneous RE of the Retzius neuron in the "natural neural network" are associated not only with a change in the RE frequency during excitation along nerve fibers from skin receptors, but also with the transformation of RE both between cells of one ganglion and between cells in different ganglia leech nerve chain

Purinergic modulation of neuronal gap junction circuits in the CNS of the leech.

Gap junctions (GJs) are widely distributed in brains across the animal kingdom. To visualize the GJ- coupled networks of two major mechanosensory neurons in the ganglia of medicinal leeches, we injected these cells with the GJ-permeable tracer Neurobiotin. When diffusion time was limited to only 30min, tracer coupling was highly variable for both cells, suggesting a possible modulation of GJ permeability. In invertebrates the innexins (homologs of vertebrate pannexins) form the GJs. Because extracellular adenosine triphosphate (ATP) modulates pannexin and leech innexin hemichannel permeability and is released by leech glial cells following injury, we tested the effects of bath application of ATP after the injection of Neurobiotin and observed a significant increase in the number of neurons tracer coupled to the sensory neurons. This effect required the elevation of intracellular Ca2+ and could be produced by bath application of caffeine. Conversely, scavenging endogenous extracellular ATP with the ATPase apyrase decreased the number of coupled cells. ATP also increased electrical conductance and tracer permeability between the bilateral Retzius neurons. This modulatory effect of ATP on GJ coupling was blocked by siRNA knockdown of a P1-like adenosine receptor. Finally, exposure of leech ganglia to extracellular ATP induced a characteristic low frequency (<0.3Hz) rhythmic bursting activity that was roughly synchronous among multiple neurons, a behavior that was significantly attenuated by the GJ blocker octanol. These findings highlight the mediation by ATP of a robust physiological mechanism for modifying neuronal circuits by rapidly recruiting neurons into active networks and entraining synchronized bursting activity.

Взаимодействие между нервной и секреторной функциями в осуществлении интегративной деятельности нейросекреторной клетки Ретциуса пиявки

Based on the literature review and our own experimental data, it is shown how the two most important functions of the Retzius neuron (RN) of the medical leech — secretory and electrical — are related to each other. During low-frequency activation, RN behaves like a typical nerve cell, releasing serotonin from the presynaptic endings. During high-frequency stimulation, RN demonstrates properties a secretory cell, releasing serotonin by somatic exocytosis. The mechanism of autoinhibition (secretory function), revealed during highfrequency synaptic stimulation of RN, allows to prevent hyperactivation of its impulse activity (nervous function), which, in turn, through autoregulation inhibits hyperactivation of somatic exocytosis (secretory function). These data demonstrate that RN is a unique neurosecretory cell with polyfunctional activity.

Changes in the Frequency of Rhythmic Excitation of Retzius Cells during Thermal Stimulation of Leech Skin.

Thermal stimulation of various parts of the skin in Hirudo medicinalis increases the frequency of spontaneous rhythmic excitation of Retzius neurons in leech ganglia. It was shown that the frequency of spontaneous rhythmic excitation of Retzius cells in the segmental ganglion increases only in response to thermal stimulation and returns to initial values upon cooling. This effect was also detected in neurons that are not directly connected by nerve fibers with the particular skin area. Changes in the frequency of spontaneous rhythmic excitation of Retzius cells in the segmental ganglion were observed during thermal stimulation of not only leech body, but also of the head and caudal suckers. These changes in spontaneous rhythmic excitation of Retzius cells in the segmental ganglion during thermal stimulation were observed in Hirudo medicinalis, but not in Macrobdella decora.

Two distinct electrophysiological mechanisms underlie extensive depolarization elicited by 2,4 diaminobutyric acid in leech Retzius neurons

Recent studies suggest that 2,4-DABA, a neurotoxic excitatory amino acid present in virtually all environments, but predominantly in aquatic ecosystems may be a risk factor for development of neurodegenerative diseases in animals and humans. Despite its neurotoxicity and potential environmental importance, mechanisms underlying the excitatory and putative excitotoxic action of 2,4-DABA in neurons are still unexplored. We previously reported on extensive two-stage membrane depolarization and functional disturbances in leech Retzius neurons induced by 2,4-DABA. Current study presents the first detailed look into the electrophysiological processes leading to this depolarization. Intracellular recordings were performed on Retzius neurons of the leech Haemopis sanguisuga using glass microelectrodes and input membrane resistance (IMR) was measured by injecting hyperpolarizing current pulses through these electrodes. Results show that the excitatory effect 2,4-DABA elicits on neurons’ membrane potential is dependent on sodium ions. Depolarizing effect of 5·10−3 mol/L 2,4-DABA in sodium-free solution was significantly diminished by 91% reducing it to 3.26 ± 0.62 mV and its two-stage nature was abrogated. In addition to being sodium-dependent, the depolarization of membrane potential induced by this amino acid is coupled with an increase of membrane permeability, as 2,4-DABA decreases IMR by 8.27 ± 1.47 MΩ (67.60%). Since present results highlight the role of sodium ions, we investigated the role of two putative sodium-dependent mechanisms in 2,4-DABA-induced excitatory effect – activation of ionotropic glutamate receptors and the electrogenic transporter for neutral amino acids. Excitatory effect of 5·10−3 mol/L 2,4-DABA was partially blocked by 10-5 mol/L 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) a non-NMDA receptor antagonist as the first stage of membrane depolarization was significantly reduced by 2.59 ± 0.98 mV (40%), whilst second stage remained unaltered. Moreover, involvement of the sodium-dependent transport system for neutral amino acids was investigated by equimolar co-application of 5·10−3 mol/L 2,4-DABA and L-alanine, a competitive inhibitor of this transporter. Although L-alanine exhibited no effect on the first stage of membrane depolarization elicited by 2,4-DABA, it substantially reduced the second stage (the overall membrane depolarization) from 39.63 ± 2.22 mV to 16.28 ± 2.58 mV, by 58.92%. We therefore propose that the electrophysiological effect of 2,4-DABA on Retzius neurons is mediated by two distinct mechanisms, i.e. by activation of ionotropic glutamate receptor that initiates the first stage of membrane depolarization followed by the stimulation of an electrogenic sodium-dependent neutral amino acid transporter, leading to additional influx of positive charge into the cell and the second stage of depolarization.

Thermodynamic Efficiency of Somatic Exocytosis of Serotonin.

Through somatic exocytosis neurons liberate immense amounts of transmitter molecules that modulate the functioning of the nervous system. A stream of action potentials triggers an ATP-dependent transport of transmitter-containing vesicles to the plasma membrane, that ends with a large-scale exocytosis. It is commonly assumed that biological processes use metabolic energy with a high thermodynamic efficiency, meaning that most energy generates work with minor dissipation. However, the intricate ultrastructure underlying the pathway for the vesicle flow necessary for somatic exocytosis challenges this possibility. To study this problem here we first applied thermodynamic theory to quantify the efficiency of somatic exocytosis of the vital transmitter serotonin. Then we correlated the efficiency to the ultrastructure of the transport pathway of the vesicles. Exocytosis was evoked in cultured Retzius neurons of the leech by trains of 10 impulses delivered at 20 Hz. The kinetics of exocytosis was quantified from the gradual fluorescence increase of FM1-43 dye as it became incorporated into vesicles that underwent their exo-endocytosis cycle. By fitting a model of the vesicle transport carried by motor forces to the kinetics of exocytosis, we calculated the thermodynamic efficiency of the ATP expenses per vesicle, as the power of the transport divided by total energy ideally produced by the hydrolysis of ATP during the process. The efficiency was remarkably low (0.1–6.4%) and the values formed a W-shape distribution with the transport distances of the vesicles. Electron micrographs and fluorescent staining of the actin cortex indicated that the slopes of the W chart could be explained by the interaction of vesicles with the actin cortex and the calcium-releasing endoplasmic reticulum. We showed that the application of thermodynamic theory permitted to predict aspects of the intracellular structure. Our results suggest that the distribution of subcellular structures that are essential for somatic exocytosis abates the thermodynamic efficiency of the transport by hampering vesicle mobilization. It is remarkable that the modulation of the nervous system occurs at the expenses of an efficient use of metabolic energy.

Effects of Nimodipine, Calcium-Free Medium and Colchicine on Electrogenesis of Neurosecretory Retzius Cells in the Leech Hirudo medicinalis

Retzius neuron (RN) in the medicinal leech is known to function as a typical neurosecretory cell. This study addresses the relationship between its two functions, neural and paracrine. It was shown that synaptic activation of RN at 710-Hz frequencies causes the neurophysiological process of habituation. Experiments conducted in calcium-free solution and in that containing nimodipine and colchicine (which block somatic exocytosis of serotonin under these experimental conditions) demonstrate alterations in the electrophysiological characteristics of RN: the rate of spontaneous impulse activity (IA), action potential (AP) amplitude, and synaptic stimulation threshold. Under these conditions, RN generates AP to every stimulus even at frequencies of 7 to 10 Hz. Thus, while somatic exocytosis of serotonin is blocked, habituation does not develop. It is suggested that habituation of RN to high-frequency synaptic stimulation is mediated by the concurrent effects of two factors — stimulatory (via synaptic activation) and inhibitory (via autoinhibition).

Extensive depolarization and lack of recovery of leech Retzius neurons caused by 2,4 diaminobutyric acid

In this paper we present, for the first time, a detailed account of electrophysiological effects of 2,4-diaminobutyric acid (2,4-DABA). 2,4-DABA is a neurotoxic non-protein amino acid produced by Cyanobacteria with a possible link to neurodegenerative disorders in animals and humans. Intracellular recordings were performed on Retzius nerve cells of the leech Haemopis sanguisuga using glass microelectrodes filled with 3 mol/L KCl. Our results show that 2,4-DABA is an excitatory amino acid, causing membrane depolarization in a concentration-dependent manner. The most prominent depolarizations of 39.63±2.22 mV and 47.05±4.33 mV, induced by 5×10−3 and 10−2 mol/L 2,4-DABA respectively, are several times larger than maximal depolarizations induced by either Glutamate, Aspartate, β-N-methylamino-alanine (BMAA) or β-N-oxalylamino-alanine (BOAA) on our model. These 2,4-DABA induced depolarizations evolve through two distinct stages, which is a novel phenomenon in electrical cell activity upon application of an excitatory amino acid, at least on our model. Involvement of two separate mechanisms, suggested by the two stage phenomenon, is discussed in the paper. We also provide evidence that 2,4-DABA induces irreversible functional disturbances in neurons in a concentration-dependent manner, since only half of the cells recovered normal electrical activity after application of 5×10−3 mol/L 2,4-DABA, and none recovered after application of 10−2 mol/L 2,4-DABA. Effects of both L-2,4-DABA and DL-2,4-DABA were tested and are not significantly different.

Effects of Divalent Cations on Outward Potassium Currents in Leech Retzius Nerve Cells

AbstractThe present study examines the effects of divalent metals, cadmium (Cd2+) and manganese (Mn2+), on the outward potassium currents of Retzius cells in the hirudinid leeches Haemopis sanguisuga using conventional two-microelectrode voltageclamp techniques. The outward potassium current is activated by depolarization and plays an important role in determining both the neuronal excitability and action potential duration. A strong inhibition of the fast current and a clear reduction in the late currents of the outward current with 1 mM Cd2+were obtained, which indicated that both components are sensitive to this metal. Complete blockage of the fast and partial reduction of the slow outward currents was observed after adding 1 mM Mn2+to the extracellular fluid. These data show that the outward K+current in leech Retzius nerve cells comprises at least two components: a voltage-dependent K+current and a Ca2+- activated K+current. These observations also indicate that Cd2+is more eff ective than Mn2+in blocking ion fl ow through these channels and that suppressing Ca2+-activated K+outward currents can prolong the action potential in nerve cells.

BMP4 acts as a dorsal telencephalic morphogen in a mouse embryonic stem cell culture system.

ABSTRACTThe concept of a morphogen – a molecule that specifies two or more cell fates in a concentration-dependent manner – is paradigmatic in developmental biology. Much remains unknown, however, about the existence of morphogens in the developing vertebrate central nervous system (CNS), including the mouse dorsal telencephalic midline (DTM). Bone morphogenetic proteins (BMPs) are candidate DTM morphogens, and our previous work demonstrated BMP4 sufficiency to induce one DTM cell fate – that of choroid plexus epithelial cells (CPECs) – in a mouse embryonic stem cell (mESC) culture system. Here we used BMP4 in a modified mESC culture system to derive a second DTM fate, the cortical hem (CH). CH and CPEC markers were induced by BMP4 in a concentration-dependent manner consistent with in vivo development. BMP4 concentrations that led to CH fate also promoted markers for Cajal–Retzius neurons, which are known CH derivatives. Interestingly, single BMP4 administrations also sufficed for appropriate temporal regulation of CH, CPEC, and cortical genes, with initially broad and overlapping dose-response profiles that sharpened over time. BMP4 concentrations that yielded CH- or CPEC-enriched populations also had different steady-state levels of phospho-SMAD1/5/8, suggesting that differences in BMP signaling intensity underlie DTM fate choice. Surprisingly, inactivation of the cortical selector gene Lhx2 did not affect DTM expression levels, dose-response profiles, or timing in response to BMP4, although neural progenitor genes were downregulated. These data indicate that BMP4 can act as a classic morphogen to orchestrate both spatial and temporal aspects of DTM fate acquisition, and can do so in the absence of Lhx2.

On the Basis of Synaptic Integration Constancy during Growth of a Neuronal Circuit

We studied how a neuronal circuit composed of two neuron types connected by chemical and electrical synapses maintains constant its integrative capacities as neurons grow. For this we combined electrophysiological experiments with mathematical modeling in pairs of electrically-coupled Retzius neurons from postnatal to adult leeches. The electrically-coupled dendrites of both Retzius neurons receive a common chemical input, which produces excitatory postsynaptic potentials (EPSPs) with varying amplitudes. Each EPSP spreads to the soma, but also crosses the electrical synapse to arrive at the soma of the coupled neuron. The leak of synaptic current across the electrical synapse reduces the amplitude of the EPSPs in proportion to the coupling ratio. In addition, summation of EPSPs generated in both neurons generates the baseline action potentials of these serotonergic neurons. To study how integration is adjusted as neurons grow, we first studied the characteristics of the chemical and electrical connections onto the coupled dendrites of neuron pairs with soma diameters ranging from 21 to 75 μm. Then by feeding a mathematical model with the neuronal voltage responses to pseudorandom noise currents we obtained the values of the coupling ratio, the membrane resistance of the soma (rm) and dendrites (rdend), the space constant (λ) and the characteristic dendritic length (L = l/λ). We found that the EPSPs recorded from the somata were similar regardless on the neuron size. However, the amplitude of the EPSPs and the firing frequency of the neurons were inversely proportional to the coupling ratio of the neuron pair, which also was independent from the neuronal size. This data indicated that the integrative constancy relied on the passive membrane properties. We show that the growth of Retzius neurons was compensated by increasing the membrane resistance of the dendrites and therefore the λ value. By solely increasing the dendrite resistance this circuit maintains constant its integrative capacities as its neurons grow.

The pathology of incipient polymicrogyria

ObjectiveTo characterise the early tissue changes of post encephaloclastic polymicrogyria in the human fetus. MethodsWe identified and reviewed the clinical histories and autopsy pathology of post ischemic fetal cerebral cortical injury at less than 30weeks gestational age (GA). The histology of local cortical abnormalities was examined with neuronal, glial, microglial and vascular immunohistochemical markers. ResultsWe identified eight cases ranging from 18 to 29weeks GA: 5 cases show full thickness cortical infarcts and 3 show periSylvian post-ischemic necrosis of the cerebral cortex. The maximal age is less than 10weeks after injury. There are abnormalities in gross fissuration as early as one month after injury. Disruption of the pia limitans was associated with a microglial and glial response and full thickness cortical injury. Macrophages were often seen accumulating deep to abnormal cortex. Hyperplasia of the subpial granular cell layer was universal in perilesional cortex. Cajal Retzius neuron hyperplasia, aggregation, and both superficial and deep displacement were noted. Where there was loss and dispersal of early cortical pyramidal neurons there was usually no pseudolaminar necrosis. Radial glia by 18weeks GA showed altered growth patterns and lateral branching. Altered migration of primitive elements was often prominent. Particularly prior to 20weeks GA subadjacent subplate neurons showed striking hypertrophy. ConclusionsThe array of histological changes encompasses all tissue elements of the affected brains, early in the evolution polymicrogyria. Although subpial alterations were ubiquitous, not all changes are referable to alterations in the pia limitans. The role of the necroinflammatory response in the genesis of abnormal cytoarchitecture deserves further study.

The neurotoxic effects of hydrogen peroxide and copper in Retzius nerve cells of the leech Haemopis sanguisuga.

ABSTRACTOxidative stress and the generation of reactive oxygen species (ROS) play an important role in cellular damage. Electrophysiological analyses have shown that membrane transport proteins are susceptible to ROS. In the present study, oxidative stress was induced in Retzius nerve cells of the leech Haemopis sanguisuga by bath application of 1 mM of hydrogen peroxide (H2O2) and 0.02 mM of copper (Cu) for 20 min. The H2O2/Cu(II) produced considerable changes in the electrical properties of the Retzius nerve cells. Intracellular recording of the resting membrane potential revealed that the neuronal membrane was depolarized in the presence of H2O2/Cu(II). We found that the amplitude of action potentials decreased, while the duration augmented in a progressive way along the drug exposure time. The combined application of H2O2 and Cu(II) caused an initial excitation followed by depression of the spontaneous electrical activity. Voltage-clamp recordings revealed a second effect of the oxidant, a powerful inhibition of the outward potassium channels responsible for the repolarization of action potentials. The neurotoxic effect of H2O2/Cu(II) on the spontaneous spike electrogenesis and outward K+ current of Retzius nerve cells was reduced in the presence of hydroxyl radical scavengers, dimethylthiourea and dimethyl sulfoxide, but not mannitol. This study provides evidence for the oxidative modification of outward potassium channels in Retzius nerve cells. The oxidative mechanism of the H2O2/Cu(II) system action on the electrical properties of Retzius neurons proposed in this study might have a wider significance, referring not only to leeches but also to mammalian neurons.

Effect of low potassium concentration on cadmium induced epileptiform activity of leech retzius neurons

Epilepsies have a large significance and require detailed investigation of cellular mechanisms that lead to this disorder. Environmental, especially industrial, toxins are having increasingly more prominent role in these investigations. The aim of our research was to investigate the significance of Cd2+ in generation of epileptiform electrical activity of neurons, and the role of Na+/K+ pump in mechanisms that lead to cessation of this activity. Experiments were performed on Retzius nerve cells of the leech Haemopis sanguisuga. Intracellularly placed microelectrodes were used to measure membrane potential changes upon administration of Cd2+ (100 µmol/l), and the same concentration of Cd2+ in low K+ (1 mmol/l) solution. In our experiments Cd2+ led to generation of rhythmic repetitive oscillatory activity. This activity closely resembles paroxysmal depolarizing shifts (PDS) which represent the cellular basis of epilepsy. Cd2+ induced epileptiform activity had the following characteristics: frequency of 3.9±0.8 PDS/minute, PDS duration of 4.0±0.3 s, and PDS amplitude of 8.1±0.7 mV. Cd2+ induces effects similar to those of Ni2+ and Co2+, but in 30 times smaller concentration. Application of Cd2+ in low K+ solution led to a significant reduction of PDS frequency (by 2.34±0.55 PDS/minute, p<0.05, Student's t-test), highly significant increase in PDS duration (by 2.84±0.23 s, p<0.01, Student's t-test) and highly significant reduction in PDS amplitude (by 1.91±0.33 mV, p=0.01, Student's t-test). Our results show that Cd2+ is a potent initiator of epileptiform activity, and that Na+/K+ pump significantly affects this activity and has a potentially important role in mechanisms that lead to its cessation.