iPSCs in Neurodegenerative Disorders: A Unique Platform for Clinical Research and Personalized Medicine.

In the past, several animal disease models were developed to study the molecular mechanism of neurological diseases and discover new therapies, but the lack of equivalent animal models has minimized the success rate. A number of critical issues remain unresolved, such as high costs for developing animal models, ethical issues, and lack of resemblance with human disease. Due to poor initial screening and assessment of the molecules, more than 90% of drugs fail during the final step of the human clinical trial. To overcome these limitations, a new approach has been developed based on induced pluripotent stem cells (iPSCs). The discovery of iPSCs has provided a new roadmap for clinical translation research and regeneration therapy. In this article, we discuss the potential role of patient-derived iPSCs in neurological diseases and their contribution to scientific and clinical research for developing disease models and for developing a roadmap for future medicine. The contribution of humaniPSCs in the most common neurodegenerative diseases (e.g., Parkinson's disease and Alzheimer's disease, diabetic neuropathy, stroke, and spinal cord injury) were examined and ranked as per their published literature on PUBMED. We have observed that Parkinson's disease scored highest, followed by Alzheimer's disease. Furthermore, we also explored recent advancements in the field of personalized medicine, such as the patient-on-a-chip concept, where iPSCs can be grown on 3D matrices inside microfluidic devices to create an in vitro disease model for personalized medicine.

NMDA Receptors in Astrocytes.

Astrocytes support glutamatergic neurotransmission in the central nervous system through multiple mechanisms which include: (i) glutamate clearance and control over glutamate spillover due to operation of glutamate transporters; (ii) supply of obligatory glutamate precursor glutamine via operation of glutamate-glutamine shuttle; (iii) supply of L-serine, the indispensable precursor of positive NMDA receptors neuromodulator D-serine and (iv) through overall homoeostatic control of the synaptic cleft. Astroglial cells express an extended complement of ionotropic and metabotropic glutamate receptors, which mediate glutamatergic input to astrocytes. In particular a sub-population of astrocytes in the cortex and in the spinal cord express specific type of NMDA receptors assembled from two GluN1, one GluN2C or D and one GluN3 subunits. This composition underlies low Mg2+ sensitivity thus making astroglial NMDA receptors operational at resting membrane potential. These NMDA receptors generate ionic signals in astrocytes and are linked to several astroglial homoeostatic molecular cascades.

Jan Evangelista Purkyne (1787-1869) and his instruments for microscopic research in the field of neuroscience.

The findings obtained by the famous nineteenth-century Czech scientist Jan Evangelista Purkyne (1787-1869) in the field of microscopic structure of animal and human tissues, including the brain, spinal cord, and nerves, have already been described in depth in a number of older and newer publications. The present article contains an overview of the instruments and tools that Purkyne and his assistants used for microscopic research of tissue histology. Some of these instruments were developed either by Purkyne alone, such as the microtomic compressor, or together with his assistant Adolph Oschatz, such as the microtome. A brief overview of the development of the cutting engines suggests that the first microtome, a prototype of modern sliding microtomes, was designed and constructed under the supervision of Purkyne at the Institute of Physiology in Wroclaw. Purkyne and his assistants, thus, not only obtained important findings of animal and human nervous and other tissues but also substantially contributed to the development of instruments and tools for their study, a fact often forgotten today.

Vincenc Alexandr Bohdálek (1801-1883): Czech anatomist and neuroscientist of the nineteenth century.

Vincenc Alexandr Bohdálek (Vincenz Alexander Bochdalek) was a well-known anatomist and pathologist in the nineteenth century. Today, however, his name is all but forgotten. Bohdálek described a number of anatomical structures; some of them became eponyms. Unfortunately, his findings concerning the innervation of the eye, upper jaw, hard palate, auditory system, and meninges are little known today. This current overview is based on available archival sources and provides an insight into his results in the field of nervous system research, which account for almost half his work. Bohdálek can clearly be considered a pioneer in the field we now call functional anatomy, as he tried to find a physiological explanation for the anatomical and pathological findings he observed. The work and results of this truly outstanding neuroscientist of his time are thus again available to current and future generations of neuroscientists and neuroanatomists.

Discovering the structure of nerve tissue: Part 3: From Jan Evangelista Purkyne to Ludwig Mauthner.

The previous works of Purkyne, Valentin, and Remak showed that the central and peripheral nervous systems contained not only nerve fibers but also cellular elements. The use of microscopes and new fixation techniques enabled them to accurately obtain data on the structure of nerve tissue and consequently in many European universities microscopes started to become widely used in histological and morphological studies. The present review summarizes important discoveries concerning the structure of neural tissue, mostly from vertebrates, during the period from 1838 to 1865. This review describes the discoveries of famous as well as less well-known scholars of the time, who contributed significantly to current understandings about the structure of neural tissue. The period is characterized by the first descriptions of different types of nerve cells and the first attempts of a cytoarchitectonic description of the spinal cord and brain. During the same time, the concept of a neuroglial tissue was introduced, first as a tissue for "gluing" nerve fibers, cells, and blood capillaries into one unit, but later some glial cells were described for the first time. Questions arose as to whether or not cells in ganglia and the central nervous system had the same morphological and functional properties, and whether nerve fibers and cell bodies were interconnected. Microscopic techniques started to be used for the examination of physiological as well as pathological nerve tissues. The overall state of knowledge was just a step away from the emergence of the concept of neurons and glial cells.

Sodium-calcium exchanger and R-type Ca(2+) channels mediate spontaneous [Ca(2+)]i oscillations in magnocellular neurones of the rat supraoptic nucleus.

Isolated supraoptic neurones generate spontaneous [Ca(2+)]i oscillations in isolated conditions. Here we report in depth analysis of the contribution of plasmalemmal ion channels (Ca(2+), Na(+)), Na(+)/Ca(2+) exchanger (NCX), intracellular Ca(2+) release channels (InsP3Rs and RyRs), Ca(2+) storage organelles, plasma membrane Ca(2+) pump and intracellular signal transduction cascades into spontaneous Ca(2+) activity. While removal of extracellular Ca(2+) or incubation with non-specific voltage-gated Ca(2+) channel (VGCC) blocker Cd(2+) suppressed the oscillations, neither Ni(2+) nor TTA-P2, the T-type VGCC blockers, had an effect. Inhibitors of VGCC nicardipine, ω-conotoxin GVIA, ω-conotoxin MVIIC, ω-agatoxin IVA (for L-, N-, P and P/Q-type channels, respectively) did not affect [Ca(2+)]i oscillations. In contrast, a specific R-type VGCC blocker SNX-482 attenuated [Ca(2+)]i oscillations. Incubation with TTX had no effect, whereas removal of the extracellular Na(+) or application of an inhibitor of the reverse operation mode of Na(+)/Ca(2+) exchanger KB-R7943 blocked the oscillations. The mitochondrial uncoupler CCCP irreversibly blocked spontaneous [Ca(2+)]i activity. Exposure of neurones to Ca(2+) mobilisers (thapsigargin, cyclopiazonic acid, caffeine and ryanodine); 4-aminopyridine (A-type K(+) current blocker); phospholipase C and adenylyl cyclase pathways blockers U-73122, Rp-cAMP, SQ-22536 and H-89 had no effect. Oscillations were blocked by GABA, but not by glutamate, apamin or dynorphin. In conclusion, spontaneous oscillations in magnocellular neurones are mediated by a concerted action of R-type Ca(2+) channels and the NCX fluctuating between forward and reverse modes.

Physiology of spontaneous [Ca(2+)]i oscillations in the isolated vasopressin and oxytocin neurones of the rat supraoptic nucleus.

The magnocellular vasopressin (AVP) and oxytocin (OT) neurones exhibit specific electrophysiological behaviour, synthesise AVP and OT peptides and secrete them into the neurohypophysial system in response to various physiological stimulations. The activity of these neurones is regulated by the very same peptides released either somato-dendritically or when applied to supraoptic nucleus (SON) preparations in vitro. The AVP and OT, secreted somato-dendritically (i.e. in the SON proper) act through specific autoreceptors, induce distinct Ca(2+) signals and regulate cellular events. Here, we demonstrate that about 70% of freshly isolated individual SON neurones from the adult non-transgenic or transgenic rats bearing AVP (AVP-eGFP) or OT (OT-mRFP1) markers, produce distinct spontaneous [Ca(2+)]i oscillations. In the neurones identified (through specific fluorescence), about 80% of AVP neurones and about 60% of OT neurones exhibited these oscillations. Exposure to AVP triggered [Ca(2+)]i oscillations in silent AVP neurones, or modified the oscillatory pattern in spontaneously active cells. Hyper- and hypo-osmotic stimuli (325 or 275 mOsmol/l) respectively intensified or inhibited spontaneous [Ca(2+)]i dynamics. In rats dehydrated for 3 or 5days almost 90% of neurones displayed spontaneous [Ca(2+)]i oscillations. More than 80% of OT-mRFP1 neurones from 3 to 6-day-lactating rats were oscillatory vs. about 44% (OT-mRFP1 neurones) in virgins. Together, these results unveil for the first time that both AVP and OT neurones maintain, via Ca(2+) signals, their remarkable intrinsic in vivo physiological properties in an isolated condition.

The dissertation on pain by Jan Krtitel Bohác published in 1746.

It is reported that continuous attention to the topic of pain in the Czech lands started only in the sixties of the twentieth century. Newly discovered archival documents show, however, that the subject of pain was studied at the Prague Medical Faculty more than 200 years before. In 1746 one of the medical students, Jan Krtitel Bohác (John Baptist Bohadsch) defended his dissertation on pain, titled "De Doloribus in Genere." Unlike other dissertations of the time, Bohác's treatise was not a mere transcription of the teaching texts. A detailed examination of his dissertation shows that it contained a thesis for a disputation, an act that took place regularly at that time during university study. Only 22 years old, Bohác showed in his dissertation a perfect knowledge of the contemporary literature, providing a more accurate classification of the causes and effects of pain. He also disagreed with statements and conclusions of authorities at the beginning of the eighteenth century and even offered his own opinion. The present work summarizes the concept of the nervous system and pain in the middle of the eighteenth century and compares them with the translation of selected parts of Bohác's formerly unknown dissertation.

Discovering the structure of nerve tissue: part 1: from Marcello Malpighi to Christian Berres.

The invention of the microscope at the beginning of the seventeenth century was a pivotal event for subsequent studies of the microscopic structure of nerve tissue. The present article, using translations of the original texts, presents a recollection of the discoveries made during the second half of the seventeenth century up to the beginning of the nineteenth century by prominent scholars as well as those nearly forgotten today. The findings in the field of neuroanatomy are collected together into a coherent form and in chronological order, showing the progress of the discoveries from a historical perspective. The early scientists discovered, and then repeatedly confirmed, that nerve tissue was remarkably similar over a wide range of animal forms. While they offered little detail, and much of what was described was flawed because of various technical restraints of the time, what they did report was very similar from animal to animal. Their studies, however, in parallel with the improvement of microscopic techniques as well as the processing and fixation of animal tissues, helped to create fertile ground for a number of important neurohistological discoveries in the first half of the nineteenth century.

Discovering the Structure of Nerve Tissue: Part 2: Gabriel Valentin, Robert Remak, and Jan Evangelista Purkyne.

During the 1830s, the use of improved microscopic techniques together with new histological methods, including tissue fixation, allowed more precise data to be obtained concerning the structure of nerve tissue of animals as well as humans. The present article, based on the translations of original texts never before published, brings together for the first time the discoveries of famous scholars Gustav Valentin, Robert Remak, and Jan Evangelista Purkyne, who made their significant discoveries in the field of neuroscience almost simultaneously and shows how their findings affected each other. In addition, this article also contains digitally remastered and reconstructed figures published in the original works of Valentin, Remak, and Purkyne and they are displayed for the first time in high quality. Although the fundamental discoveries of these famous scholars did not imply the discovery of nerve cells as we know them today, they were certainly a very important basis for further research of many other eminent scholars during the second half of the nineteenth century.

Jirí Procháska (1749-1820): Part 2: "De structura nervorum"--studies on a structure of the nervous system.

The treatise "De structura nervorum" by Jirí Procháska was published in 1779 and is remarkable not only for its anatomical and histological findings but also for its historical introduction, which contains a detailed bibliographical review of the contemporary knowledge of the structure of the nervous tissue. Unfortunately, the treatise has never been translated from the Latin language, but it deserves further analysis as a historical document about the level of neuroscience research conducted by a famous Czech scholar. The present article includes a historical overview of the contemporary knowledge of the structure of the nervous tissue up to the late eighteenth century from the perspective of today, a translation of selected chapters from Prochaska's treatise (a historical introduction about the medieval knowledge of the structure of the nervous tissue and an interpretation of his neurohistological observations), and an analysis of Jirí Prochaska's results in light of current knowledge.

Jirí Procháska (1749-1820): part 1: a significant Czech anatomist, physiologist and neuroscientist of the eighteenth century.

Jirí (George) Procháska (1749-1820), a Czech anatomist, physiologist, and neuroscientist of the eighteenth century, ranks among the major figures of Czech and European cultural history. The works of Jirí Procháska, due to historical circumstances, were published mostly in Latin and only some in German. However, given that only one treatise was partially translated into English, the results of his extensive research activities are currently unavailable to the international scientific community. The achievements of Jirí Procháska undoubtedly belong to the major intellectual heritage of European science and certainly deserve attention as such, although his research reflected the time in which he lived and therefore has been reevaluated by later researchers. Undoubtedly, it is our duty not only to remember the work and legacy of Jirí Procháska, which significantly influenced the development of our knowledge, but also to try to critically assess his contribution in terms of today. This article surveys the important biographical events of Jirí Procháska's life, taking into account the significance of his research.

Cell death/proliferation and alterations in glial morphology contribute to changes in diffusivity in the rat hippocampus after hypoxia-ischemia.

To understand the structural alterations that underlie early and late changes in hippocampal diffusivity after hypoxia/ischemia (H/I), the changes in apparent diffusion coefficient of water (ADC(W)) were studied in 8-week-old rats after H/I using diffusion-weighted magnetic resonance imaging (DW-MRI). In the hippocampal CA1 region, ADC(W) analyses were performed during 6 months of reperfusion and compared with alterations in cell number/cell-type composition, glial morphology, and extracellular space (ECS) diffusion parameters obtained by the real-time iontophoretic method. In the early phases of reperfusion (1 to 3 days) neuronal cell death, glial proliferation, and developing gliosis were accompanied by an ADC(W) decrease and tortuosity increase. Interestingly, ECS volume fraction was decreased only first day after H/I. In the late phases of reperfusion (starting 1 month after H/I), when the CA1 region consisted mainly of microglia, astrocytes, and NG2-glia with markedly altered morphology, ADC(W), ECS volume fraction and tortuosity were increased. Three-dimensional confocal morphometry revealed enlarged astrocytes and shrunken NG2-glia, and in both the contribution of cell soma/processes to total cell volume was markedly increased/decreased. In summary, the ADC(W) increase in the CA1 region underlain by altered cellular composition and glial morphology suggests that considerable changes in extracellular signal transmission might occur in the late phases of reperfusion after H/I.

Impact of global cerebral ischemia on K+ channel expression and membrane properties of glial cells in the rat hippocampus.

Astrocytes and NG2 glia respond to CNS injury by the formation of a glial scar. Since the changes in K(+) currents in astrocytes and NG2 glia that accompany glial scar formation might influence tissue outcome by altering K(+) ion homeostasis, we aimed to characterize the changes in K(+) currents in hippocampal astrocytes and NG2 glia during an extended time window of reperfusion after ischemic injury. Global cerebral ischemia was induced in adult rats by bilateral, 15-min common carotid artery occlusion combined with low-pressure oxygen ventilation. Using the patch-clamp technique, we investigated the membrane properties of hippocampal astrocytes and NG2 glia in situ 2 hours, 6 hours, 1 day, 3 days, 7 days or 5 weeks after ischemia. Astrocytes in the CA1 region of the hippocampus progressively depolarized starting 3 days after ischemia, which coincided with decreased Kir4.1 protein expression in the gliotic tissue. Other K(+) channels described previously in astrocytes, such as Kir2.1, Kir5.1 and TREK1, did not show any changes in their protein content in the hippocampus after ischemia; however, their expression switched from neurons to reactive astrocytes, as visualized by immunohistochemistry. NG2 glia displayed increased input resistance, decreased membrane capacitance, increased delayed outwardly rectifying and A-type K(+) currents and decreased inward K(+) currents 3 days after ischemia, accompanied by their proliferation. Our results show that the membrane properties of astrocytes after ischemia undergo complex alterations, which might profoundly influence the maintenance of K(+) homeostasis in the damaged tissue, while NG2 glia display membrane currents typical of proliferating cells.

REVIEW: Oxytocin: Crossing the bridge between basic science and pharmacotherapy.

Is oxytocin the hormone of happiness? Probably not. However, this small nine amino acid peptide is involved in a wide variety of physiological and pathological functions such as sexual activity, penile erection, ejaculation, pregnancy, uterus contraction, milk ejection, maternal behavior, osteoporosis, diabetes, cancer, social bonding, and stress, which makes oxytocin and its receptor potential candidates as targets for drug therapy. In this review, we address the issues of drug design and specificity and focus our discussion on recent findings on oxytocin and its heterotrimeric G protein-coupled receptor OTR. In this regard, we will highlight the following topics: (i) the role of oxytocin in behavior and affectivity, (ii) the relationship between oxytocin and stress with emphasis on the hypothalamo-pituitary-adrenal axis, (iii) the involvement of oxytocin in pain regulation and nociception, (iv) the specific action mechanisms of oxytocin on intracellular Ca²(+) in the hypothalamo neurohypophysial system (HNS) cell bodies, (v) newly generated transgenic rats tagged by a visible fluorescent protein to study the physiology of vasopressin and oxytocin, and (vi) the action of the neurohypophysial hormone outside the central nervous system, including the myometrium, heart and peripheral nervous system. As a short nine amino acid peptide, closely related to its partner peptide vasopressin, oxytocin appears to be ideal for the design of agonists and antagonists of its receptor. In addition, not only the hormone itself and its binding to OTR, but also its synthesis, storage and release can be endogenously and exogenously regulated to counteract pathophysiological states. Understanding the fundamental physiopharmacology of the effects of oxytocin is an important and necessary approach for developing a potential pharmacotherapy.

Neural stem/progenitor cells derived from the embryonic dorsal telencephalon of D6/GFP mice differentiate primarily into neurons after transplantation into a cortical lesion.

D6 is a promoter/enhancer of the mDach1 gene that is involved in the development of the neocortex and hippocampus. It is expressed by proliferating neural stem/progenitor cells (NSPCs) of the cortex at early stages of neurogenesis. The differentiation potential of NSPCs isolated from embryonic day 12 mouse embryos, in which the expression of green fluorescent protein (GFP) is driven by the D6 promoter/enhancer, has been studied in vitro and after transplantation into the intact adult rat brain as well as into the site of a photochemical lesion. The electrophysiological properties of D6/GFP-derived cells were studied using the whole-cell patch-clamp technique, and immunohistochemical analyses were carried out. D6/GFP-derived neurospheres expressed markers of radial glia and gave rise predominantly to immature neurons and GFAP-positive cells during in vitro differentiation. One week after transplantation into the intact brain or into the site of a photochemical lesion, transplanted cells expressed only neuronal markers. D6/GFP-derived neurons were characterised by the expression of tetrodotoxin-sensitive Na(+)-currents and K (A)- and K (DR) currents sensitive to 4-aminopyridine. They were able to fire repetitive action potentials and responded to the application of GABA. Our results indicate that after transplantation into the site of a photochemical lesion, D6/GFP-derived NSPCs survive and differentiate into neurons, and their membrane properties are comparable to those transplanted into the non-injured cortex. Therefore, region-specific D6/GFP-derived NSPCs represent a promising tool for studying neurogenesis and cell replacement in a damaged cellular environment.

Differential calcium signalling in neuronal-glial networks.

Calcium ions are the probably the most ancient, the most universal and omnipresent intracellular signalling molecules, which are involved in regulation of a host of cellular functional reactions. In the nervous system Ca2+ signalling is intimately involved in information transfer and integration within neural circuits. Local Ca2+ signals or Ca2+ microdomains control neurotransmitter release; more global Ca2+ signals regulate synaptic strength and accomplish postsynaptic processing. In the glial syncytium Ca2+ ions provide for glial "Ca2+ excitability", convey long-range signalling by means of propagating Ca2+ waves and control the release of gliotransmitters. Differential Ca2+ signals in various elements of neural circuits represent therefore molecular mechanisms of integration in the nervous system.

Three-dimensional confocal morphometry - a new approach for studying dynamic changes in cell morphology in brain slices.

Pathological states in the central nervous system lead to dramatic changes in the activity of neuroactive substances in the extracellular space, to changes in ionic homeostasis and often to cell swelling. To quantify changes in cell morphology over a certain period of time, we employed a new technique, three-dimensional confocal morphometry. In our experiments, performed on enhanced green fluorescent protein/glial fibrillary acidic protein astrocytes in brain slices in situ and thus preserving the extracellular microenvironment, confocal morphometry revealed that the application of hypotonic solution evoked two types of volume change. In one population of astrocytes, hypotonic stress evoked small cell volume changes followed by a regulatory volume decrease, while in the second population volume changes were significantly larger without subsequent volume regulation. Three-dimensional cell reconstruction revealed that even though the total astrocyte volume increased during hypotonic stress, the morphological changes in various cell compartments and processes were more complex than have been previously shown, including swelling, shrinking and structural rearrangement. Our data show that astrocytes in brain slices in situ during hypotonic stress display complex behaviour. One population of astrocytes is highly capable of cell volume regulation, while the second population is characterized by prominent cell swelling, accompanied by plastic changes in morphology. It is possible to speculate that these two astrocyte populations play different roles during physiological and pathological states.

High extracellular K(+) evokes changes in voltage-dependent K(+) and Na (+) currents and volume regulation in astrocytes.

[K(+)](e) increase accompanies many pathological states in the CNS and evokes changes in astrocyte morphology and glial fibrillary acidic protein expression, leading to astrogliosis. Changes in the electrophysiological properties and volume regulation of astrocytes during the early stages of astrocytic activation were studied using the patch-clamp technique in spinal cords from 10-day-old rats after incubation in 50 mM K(+). In complex astrocytes, incubation in high K(+) caused depolarization, an input resistance increase, a decrease in membrane capacitance, and an increase in the current densities (CDs) of voltage-dependent K(+) and Na(+) currents. In passive astrocytes, the reversal potential shifted to more positive values and CDs decreased. No changes were observed in astrocyte precursors. Under hypotonic stress, astrocytes in spinal cords pre-exposed to high K(+) revealed a decreased K(+) accumulation around the cell membrane after a depolarizing prepulse, suggesting altered volume regulation. 3D confocal morphometry and the direct visualization of astrocytes in enhanced green fluorescent protein/glial fibrillary acidic protein mice showed a smaller degree of cell swelling in spinal cords pre-exposed to high K(+) compared to controls. We conclude that exposure to high K(+), an early event leading to astrogliosis, caused not only morphological changes in astrocytes but also changes in their membrane properties and cell volume regulation.