Serotonin induces or inhibits neuritic regeneration of leech CNS neurons depending on neuronal identity

Recovery of motor function after central nervous system (CNS) injury is dependent on the regeneration capacity of the nervous system, which is a multifactorial process influenced, among other things, by the role of neuromodulators such as serotonin. The neurotransmitter serotonin can promote neuronal regeneration but there are also reports of it causing restriction, so it is important to clarify these divergent findings in order to understand the direct scope and side effects of potential pharmacological treatments. We evaluated the effect of serotonin on the extent of neuritic outgrowth and morphology of three different neuronal types in the leech Haementeria officinalis during their regeneration in vitro: Retzius interneurons (Rz), annulus erector (AE) motoneurons, and anterolateral number 1 (AL1) CNS neurons. Neurons were isolated and cultured in L15 medium, with or without serotonin. Growth parameters were registered and quantified, and observed differences were analyzed. The addition of serotonin was found to induce AL1 neurons to increase their average growth dramatically by 8.3-fold (P=0.02; n=5), and to have no clear effect on AE motoneurons (P=0.44; n=5). For Rz interneurons, which normally do not regenerate their neurites, the addition of concanavaline-A causes substantial growth, which serotonin was found to inhibit on average by 98% (P=0.02; n=5). The number of primary neurites and their branches were also affected. These results reveal that depending on the neuronal type, serotonin can promote, inhibit, or have no effect on neuronal regeneration. This suggests that after CNS injury, non-specific pharmacological treatments affecting serotonin may have different effects on different neuronal populations.

Implications and limitations of cellular reprogramming for psychiatric drug development

Human-induced pluripotent stem cells (hiPSCs) derived from somatic cells of patients have opened possibilities for in vitro modeling of the physiology of neural (and other) cells in psychiatric disease states. Issues in early stages of technology development include (1) establishing a library of cells from adequately phenotyped patients, (2) streamlining laborious, costly hiPSC derivation and characterization, (3) assessing whether mutations or other alterations introduced by reprogramming confound interpretation, (4) developing efficient differentiation strategies to relevant cell types, (5) identifying discernible cellular phenotypes meaningful for cyclic, stress induced or relapsing-remitting diseases, (6) converting phenotypes to screening assays suitable for genome-wide mechanistic studies or large collection compound testing and (7) controlling for variability in relation to disease specificity amidst low sample numbers. Coordination of material for reprogramming from patients well-characterized clinically, genetically and with neuroimaging are beginning, and initial studies have begun to identify cellular phenotypes. Finally, several psychiatric drugs have been found to alter reprogramming efficiency in vitro, suggesting further complexity in applying hiPSCs to psychiatric diseases or that some drugs influence neural differentiation moreso than generally recognized. Despite these challenges, studies utilizing hiPSCs may eventually serve to fill essential niches in the translational pipeline for the discovery of new therapeutics.

Differential Expressions of Synaptogenic Markers between Primary Cultured Cortical and Hippocampal Neurons

Primary dissociated neuronal cultures are widely used research tools to investigate of pathological mechanisms and to treat various central and peripheral nervous system problems including trauma and degenerative neuronal diseases. We introduced a protocol that utilizes hippocampal and cortical neurons from embryonic day 17 or 18 mice. We applied appropriate markers (GAP-43 and synaptophysin) to investigate whether neurite outgrowth and synaptogenesis can be distinguished at a particular period of time. GAP-43 was found along the neural processes in a typical granular pattern, and its expression increased proportionally as neurites lengthened during the early in vitro period. Unlike GAP-43, granular immunoreactive patterns of synaptophysin along the neurites were clearly found from day 2 in vitro with relatively high immunoreactive levels. Expression of synaptic markers from cortical neurons reached peak level earlier than that of hippocampal neurons, although neurite outgrowths of hippocampal neurons were faster than those of cortical neurons. The amount of peak synaptic markers expressed was also higher in cortical neurons than that in hippocampal neurons. These results strongly suggest the usefulness of primary cultured neurons from mice embryos for synaptic function and plasticity studies, because of their clear and typical patterns of morphology that establish synapses. Results from this study also suggest the proper amount of time in vitro according to neuronal types (cortical or hippocampal) when utilized in experiments related with synaptogenesis or synaptic activities.

Simple and Novel Three Dimensional Neuronal Cell Culture Using a Micro Mesh Scaffold

Conventional method of cell culture studies has been performed on two-dimensional substrates. Recently, three-dimensional (3D) cell culture platforms have been a subject of interest as cells in 3D has significant differences in cell differentiation and behavior. Here we report a novel approach of 3D cell culture using a nylon micro mesh (NMM) as a cell culture scaffold. NMM is commonly used in cell culture laboratory, which eliminates the requirement of special technicality for biological laboratories. Furthermore, it is made of a micro-meter thick nylon fibers, which was adequate to engineer in cellular scales. We demonstrate the feasibility of the NMM as a 3D scaffold using E18 rat hippocampal neurons. NMM could be coated with cell adhesive coatings (polylysine or polyelectrolyte) and neurons showed good viability. Cells were also encapsulated in an agarose hydrogel and cultured in 3D using NMM. In addition, the 3D pattern of NMM could be used as a guidance cue for neurite outgrowth. The flexible and elastic properties of NMMs made it easier to handle the scaffold and also readily applicable for large-scale tissue engineering applications.

Neuroprotection of estrogen against injury induced by ?-amyloid protein (25-35) in rat cortical neurons / 中国临床药理学与治疗学

AIM: To study the effect of estrogen on injury induced by ?-amyloid protein (A?) in primary cultures of rat cortical neurons. METHODS: The effect of 17?-E_2 on A?(25-35)-induced cell death in primary rat cortical neurons was observed by phase contrast light microscopy, Giemsa staining and determination of lactate dehydrohenase (LDH) release rate. RESULTS: A?(25-35) induced cell death in rat primary cortical neurons. Forty eight hours pretreatment with 17?-E_2 protected rat primary cortical neurons from A?(25-35)-induced injury. CONCLUSION: A? evokes toxicity in rat primary cortical neurons. Estrogen can protect the rat primary cortical neurons against injury induced by A? (25-35).

Synaptic Localization of NMDA Receptor and Shank Protein in Hippocampal Neuron in Vitro / 대한해부학회지

The existence of NMDA receptor and a new organizer protein, Shank, in the postsynaptic density was studied with the cultured hippocampal neurons using by double immunofluorescence method. The hippocampi from embryonic 18 days were dissected and hippocampal neurons were obtained from dissociated hippocampi with 0.25% trypsin and 0.1% DNase in PBS. The hippocampal neurons were plated with density 3,600/cm2 on the poly-L-lysine coated coverglass and cultured 37degrees C, 5% CO2 incubator for 5 weeks. The N2 supplemented MEM was used as a culture medium. Following results are obtained from experiments: 1. The 3~5 minor processes from the cell body of hippocampal neurons were observed at 20 hr in vitro. One of the minor processes was elongated and looked like an axon, and another minor processes showed dendritic branching pattern with slender in thickness. 2. The excitatory NMDA receptor colocalized with PSD-95 which is the postsynaptic density protein. The presynaptic protein, synapsin 1, was closely apposed with PSD-95. 3. Shank which is an organizer protein colocalize with NMDA receptor/PSD-95 complex in the postsynaptic density. Shank proteins may be concerned with the cluster formation of NMDA receptor/PSD-95 in the postsynaptic membrane.

IDENTIFICATION OF SEROTONIN-LIKE IMMUNOREACTIVE NEURONS IN NEURONAL CULTURES FROM E8 CHICK EMBRYO FOREBRAIN / 解剖学报

The expression of serotonin-like immunoreactivity in day 4 neuronal cultures from E8 chick embryo forebrains was investigated by PAP immunocytochemical method. It was found that 98% of the dissociated forebrain cells cultured on laminin substratum were neuron specific enolase immunoreactive positive; and 10.4% of these neurons were mainly multipolar or bipolar and serotonin immunoreactive positive. In order to verify whether the remaining 2% NSE negative forebrain cells were neuroglial cells, we had used anti-glial fibrillary acid protein antibody to show that only 2% of the cultured forebrain cells were composed of GFAP immunoreactive positive cells, i. e. neuroglial cells.

IMMUNOCYTOCHEMICAL STUDY OF GONADOTROPIN-RELEASING HORMONE (GnRH) NEURONS IN DISSOCIATED CULTURES OF NEWBORN RAT HYPOTHALAMUS / 解剖学报

Gonadotropin-Releasing Hormone (GnRH) immunoreactive neurons in dissociated cell culture from newborn rat hypothalamus were investigated on days 1,3,5,and 7 in vitro by means of the im-munocytochemical method. The results showed that GnRH was expressed in the first day of culture. GnRH neurons accounted for 12.1-14. 8% of the total neurons in culture,and they were mainly bipolar in type. There were growth cones on the end of GnRH processes. Various patterns of intercellular contacts between GnRH neurons and between GnRH and other neurons were also observed. These findings indicate that the cultured GnRH neurons exhibit the morphological and functional characteristics of the GnRH neurons in vivo,and serve as morphological evidence for pulsatile secretion of GnRH and its regulation.