Controlled release of dopamine from a polymeric brain implant: in vitro characterization

A biocompatible polymeric matrix system for the long-term controlled release of dopamine has been developed. Solid particles of this bioactive agent were encapsulated in ethylene-vinyl acetate copolymer (EVAc). Following immersion in an aqueous buffer solution, the release rate of dopamine from the polymer matrix was found to depend on the initial concentration of dopamine in the polymer. After coating the matrix devices with an additional impermeable layer of EVAc, constant rates of release were obtained by creating a cavity in this impermeable layer. The observed experiments are consistent with a diffusion-limited model of dopamine release; all the in vitro experimental results were therefore correlated by the effective diffusion coefficient of dopamine through the porous polymer network. These results are discussed in terms of potential design modifications to achieve desired release characteristics for a variety of neuroactive substances, including neurotransmitters or their precursors.     

Controlled release of dopamine from a polymeric brain implant: in vivo characterization

Intracerebral microdialysis was used to evaluate the long-term in vivo release of dopamine from ethylene-vinyl acetate (EVAc)-dopamine copolymer matrix discs for up to 65 days following striatal implantation. Dopamine release occurred through a single cavity present on one side of the disc, which was otherwise fully coated with an additional, impermeable layer of EVAc. At 20 days following implantation of the device, extracellular concentrations of dopamine within the striatum reached micromolar levels, over 200-fold greater than control values. Release of dopamine was shown to be stable and maintained for the 2-month duration of the experiment. Histological examination confirmed the biocompatible nature of the implant. There are potential applications of this technology to the treatment of Parkinson's disease and other neurological and psychiatric disorders.     

Controlled release of nerve growth factor from a polymeric implant

Recent studies suggest that neurotrophic factors applied directly to brain tissue may enhance regeneration in the central nervous system. Biocompatible polymeric implants providing a controlled release of nerve growth factor (NGF) for over one month were developed. The released nerve growth factor stimulated neurite sprouting in cultured PC12 cells. While a model polymer with demonstrated biocompatibility was used for the present study, the methods can be extended to other polymer systems. Controlled release implants may be useful in the treatment of Alzheimer's disease.     

In vitro differentiation of neuron-like cells

The pluripotent embryonal carcinoma, PCC4uva, differentiates into multiple cell types under standard culture conditions. Neuron-like cells first appear at 8 days of culture, and by Day 15 they represent approximately 3% of the cell population. We promoted neural differentiation by treatment with retinoic acid and dibutyryl cyclic adenosine monophosphate. Under these optimized culture conditions, neuron-like cells appear on the third day of culture, and by the sixth day, neuron-like cells represent 40% of the total cell population. After 10 days, the neuron-like cells represent approximately 60–80% of total cells. At this time the cells form large clusters of cell bodies that are interconnected by neurite fascicles. The cells express the neuron-specific proteins; Map2, Tau, neurofilaments, and the type III β-tubulin isotype. We have recently used this developing system to investigate the effects of laminin substrates on neural differentiation.     

In vitro expression of tyrosine hydroxylase by a subpopulation of rat melanotrophs is down-regulated by dopamine

Some rat melanotrophs express in vivo tyrosine hydroxylase mRNA. In this report we show, by Western-blotting, that cultures of adult rat melanotrophs, but not adenohypophyseal cells, express tyrosine hydroxylase. Immunocytochemical analyses confirmed the existence of a subpopulation of melanotrophs expressing tyrosine hydroxylase. Bromocryptine (2.5 × 10⁻⁷ M), a D2 dopamine agonist, down-regulated melanotroph tyrosine hydroxylase expression in a time-dependent manner; initial effect was detected at 15 h and maximum at 3 days treatment (reduction to about 40% of control values). Down-regulation at 3 days was dose-dependent (ED₅₀ around 2 × 10⁻⁹ M). This decrease was reversed by sulpiride, a D2 dopamine antagonist. The cell number was slightly increased by bromocryptine treatment. These data suggest tyrosine hydroxylase expression in melanotrophs being under tonic inhibitory control by dopamine innervation in vivo.     

In vitro induction of apoptosis of developing brain cells by 5-azacytidine

We had found that 5-azacytidine (5Az), a cytidine analogue, could produce apoptosis of fetal developing neuronal cells on the day after injection of the agent into dams by the i.p. route at 11 days of gestation. To make a further understanding of the phenomenon by comparing the results between the in-vivo and in-vitro system, this study was carried out. Entire cephalic parts of the fetuses were collected aseptically at days 11 of gestation and a mixed culture, consisting of neuronal and mesenchymal cells, was established after one week of incubation. The silver-staining revealed pyknotic nuclei and loss of dendrites of neuronal cells in the lower (5 μg/ml) dose of the 5 Az-added group. SEM showed shrinkage of the cell body and blebbing formation on the cell surface. TEM evoked margination, segmentation and complete condensation of the nuclear chromatin. Scattered positive signals identical to the apoptotic cells and aggregated fragmented DNA were detected by the TUNEL method. Treatment of higher doses of 5Az (50 and 500 μg/ml), however, induced necrosis of both neuronal and mesenchymal cells, light- and electron-microscopically. On the contrary, the control group (0 μg/ml) showed normal development of neuronal cells and very few positive signals of physiological apoptosis. It was concluded that 5Az could induce apoptosis of developing neuronal cells at lower doses, but necrosis of a wider cell population at higher doses. Involvement of hypomethylation, an important biochemical function of 5Az, in apoptosis was also speculated.     

In vitro measurements of cholinergic activity in brain regions of hibernating ground squirrels

High affinity choline uptake (HACU) and choline acetyltransferase (ChAT) activity were measured in synaptosomal P2 fractions from four brain regions in a mammalian hibernator, the golden-mantled ground squirrel. The 14CO2 evolution from [6-14C]glucose was also measured. Comparisons were made across the euthermic (not hibernating) and hibernation state in synaptosomes from cortex (CTX), preoptic area and hypothalamus (POA/HYP), olfactory apparatus (OA), and hippocampus (HPC). HACU was significantly increased in the CTX, from hibernating ground squirrels compared to euthermic animals. ChAT activity was significantly increased in the CTX and OA from hibernating animals. No change in either cholinergic marker was evident for the POA/HYP and HPC. The evolution of 14CO2 from [6-14C]glucose was generally, though not significantly, higher for synaptosomes from euthermic animals compared to hibernating animals. The results are discussed with reference to the involvement of cholinergic mechanisms in the control of hibernation.     

In vitro and in situ formation of neuron-glia junctions

The formation of neuron-glia junctions during neurogenesis has been considered as an error of the neuronal specificity mechanism or as a consequence of the artificial conditions when observed in central nervous system tissue culture. We report the demonstration of the regular existence of synaptic-like junctions in tissue cultures prepared from different regions of chick central nervous system as well as the occurrence of such intercellular contacts in the cerebellum of adult and normal frogs. The formation of neuron-glia junctions in tissue culture during normal neurogenesis and their presence in adult frogs seem to indicate that those types of neuron-glia relationships are not a simple biologic error. Rather, we propose that they may represent a primitive, but normal neuron-glia interaction.     

In vivo and in vitro development of serotonergic neurons

The monoamines are one of the earliest developing neurotransmitter systems in the mammalian brain. The first part of this paper describes the normal ontogeny of the serotonergic (5-HT) system in the rat brain as studied using long survival 3H-thymidine autoradiography (time of neuronal genesis, time of origin) and the Falck-Hillarp histofluorescence method, electron microscopy, and immunocytochemistry (anti-5-HT). Due to their early ontogeny relative to other brain regions, 5-HT neurons (as well as monoamine neurons in general) have been suggested to exert some type of "trophic" influence on brain development. Results of pharmacological experiments designed to inhibit 5-HT synthesis in the embryonic rat brain by maternal treatment with p-chlorophenylalanine (pCPA) at a time when this monoamine might exert such an influence are discussed with regard to effects on the time course of neuronal genesis (time of origin) of 5-HT neurons and their target cells. These results, which prompted us to propose that 5-HT might act as a "differentiation signal" for certain of its target cells, are now discussed in light of our more recent immunocytochemical-autoradiographic studies (anti-5-HT, 3H-thymidine) which morphologically demonstrate close associations between developing 5-HT neurons and proliferating neuroepithelial cells in the embryonic brain. Postnatal studies using this immunocytochemical-autoradiographic method also provide evidence for interactions of 5-HT axons with proliferating glioblasts in the developing cerebellum and with immature granule cells and their precursors in the hippocampus. These findings, in conjunction with the results of our pCPA experiments, further enhance the possibility that 5-HT neurons could exert an epigenetic influence on the development of less differentiated cells with which they come into contact. Finally, preliminary studies using dissociated cell cultures containing 5-HT neurons suggest that interactions between 5-HT neurons and glial elements may be important for the differentiation of these neurons in vitro. Whether 5-HT neurons in turn influence the development of glial or neuronal cells in these cultures remains to be determined. These studies are evaluated with regard to a possible pre-transmission role for 5-HT during key phases of neuronal and glial genesis.     

In vitro characterization of a murine oligodendrocyte precursor cell line (BO-1) following spontaneous immortalization

The understanding of oligodendrocyte differentiation is crucial for designing therapies of demyelinating diseases. Oligodendrocyte precursor cells are of particular interest in this context, because of their remyelinating potential. Permanent cell lines, which are a versatile tool for studying oligodendrocyte physiology, have been so far mainly established from the rat CNS. In the present study, we describe a novel murine oligodendrocyte precursor cell line (BO-1) established by spontaneous immortalization using light microscopy, immunocytochemical phenotyping and genetic analysis. BO-1 cells displayed a bi- to multipolar morphology and expressed early oligodendrocytic lineage markers, such as A2B5 and NG-2. Expression of pre-oligodendrocyte (O4, CNPase) and mature oligodendrocyte markers (e.g. myelin basic protein) was found in about 30% and 1.5% of the cells, respectively. Addition of serum, known to promote type-2 astrocyte differentiation, significantly increased the number of GFAP-positive cells, while thyroid hormones, (T3/T4) known to foster oligodendrocyte differentiation, did not substantially alter the antigenic and gene expression of myelin markers. This deficiency might be related to the high intrinsic proliferation rate of BO-1 cells that was unaltered upon removal of mitogenic factors. Expression of O4 and CNPase in BO-1 cells could be significantly increased by co-culture with primary astrocytes suggesting that the differentiating potential of BO-1 cells was influenced by environmental factors and may have to be fully explored in future studies. In summary, the novel murine BO-1 cell line shares several characteristics with oligodendrocyte precursor cells but displays a restricted differentiation into mature oligodendrocytes.     

In vitro screening for anticonvulsant-induced teratogenesis: Drug alteration of cell adhesivity

Anticonvulsant-induced alteration in C6 glioma cell adhesivity has been evaluated in two independent in vitro assay systems. A centrifugal shear assay was employed to determine drug-induced change in cell-substratum adhesivity. Valproate and clonazepam were found to significantly increase cell-substratum adhesivity when cells were cultured at concentrations which were within twice their therapeutic plasma level. A second assay evaluated change in affinity for concanavalin A lectin coated surfaces to determine change in cell surface glycoconjugate expression. Valproate and clonazepam and, to a lesser extent, diazepam significantly decreased drug-exposed C6 glioma cell affinity for concanavalin A lectin coated surfaces. Valproate and clonazepam had approximate IC50 values of 0.75 mM and 75 μM, respectively. These findings are compared and discussed in relation to those obtained with an anti-proliferative assay which has been suggested to predict teratogen potential.     

A rapid method of freezing biopsied muscle: In vitro study development

This work compares the efficacy of varying the concentrations of cryoprotectants when freezing samples of rat muscles for later use in tissue culture. The best yields were obtained with DMSO associated with glycerol and sucrose; before plating, the best results indicated 45% of cells with respect to controls, and delayed (24–36 h) myotube maturation. Maturation was studied by analysing the molecular forms of acetylcholinesterase and the ability of myotubes to form synapses in the presence of neurons, in fresh and frozen muscle.     

Actions of feeding-relevant agents on hypothalamic glucose-responsive neurons In vitro

Effects of three feeding-relevant agents on the single-unit activity of the hypothalamic ventromedial nucleus (VMN) were investigated with tissue slice preparations. Changes in ambient glucose concentrations altered the activity of 39% of the 129 VMN neurons recorded. The predominant effect of glucose was facilitation. Norepinephrine (NE) affected all of the 29 glucose-responsive neurons tested, but only some (33/47) of the neurons unresponsive to glucose. Unlike that of glucose, the effects of NE on neuronal activity were distributed across excitatory, inhibitory, or biphasic responses. In vivo estrogen treatment did not affect neuronal responses to glucose, it modulated the type of neuronal responses to NE from glucose-responsive, but not glucose-unresponsive neurons. These results indicate that glucose, NE, and estrogen act on a common, glucose-responsive, population of VMN neurons but, as judged from their effects on neuronal activity, through different cellular mechanisms. Accordingly, glucose-responsive neurons may serve to integrate information mediated by different feeding-relevant agents.     

In vitro findings of alterations in intracellular calcium homeostasis in schizophrenia

The pathogenesis of schizophrenia involves several complex cellular mechanisms and is not well understood. Recent research has demonstrated an association between primary disturbances characteristic of the disease, including altered dopaminergic and glutamatergic neurotransmission, and impairments in neuronal calcium (Ca²⁺) homeostasis and signaling. Emerging Ca²⁺ hypothesis links and unifies various cellular processes involved in the pathogenesis of schizophrenia and suggests a central role of dysregulation of Ca²⁺ homeostasis in the etiology of the disease. This review explores the in vitro data on Ca²⁺ homeostasis and signaling in schizophrenia. Major limitation in this research is the lack of schizophrenia markers and validated disease models. As indicated in this review, one way to overcome these limitations may be analyses of Ca²⁺ signalosomes in peripheral cells from schizophrenia patients. Validation of animal models of schizophrenia may permit the application of advanced Ca²⁺ imaging techniques in living animals.     

Loss of H-corticosterone binding capacity in hippocampal slices maintained In vitro

Nuclear uptake of 3H-corticosterone was studied in hippocampal slices subjected to varying periods of preincubation before application of steroid. Uptake capacity declined rapidly toward nonspecific levels over the course of 3 hours from the time of cutting. Glucocorticoid receptors in the cytosol fraction of hippocampal slices also were found to decline, and Scatchard analysis indicated a loss of corticosterone binding sites rather than a change in binding affinity. These results are discussed in terms of the reported effects of corticosterone in vitro.     

Effects of metabolic alterations on dopamine release in an in vitro model of neostriatal ischaemia

Release of neurotransmitters, including dopamine (DA), plays a central role in neuronal death during cerebral ischaemia. We investigated the effects of changes in energy demand and supply on DA release in cerebral ischaemia in vitro. Rat striatal slices were superfused (400 ml/h) with an artificial cerebrospinal fluid at 34°C, unless otherwise stated. Ischaemia were mimicked by removal of O₂ and reduction in glucose concentration from 4 to 2 mM. DA release was monitored by voltammetry. The profile of ischaemia-induced DA release was temperature-dependent. Hypothermia (to 24°C) delayed, slowed, and reduced ischaemia-induced DA release relative to 34°C. Pretreatment of the slices for 3 h with creatine (25 mM) delayed and slowed ischaemia-induced DA release. Conversely, blockade of Na⁺/K⁺ ATPase with ouabain induced an anoxic depolarisation and rapid DA release similar to ischaemia. In summary, the onset of DA release in this model is controlled by the balance between energy supply and utilisation. Strategies that increase availability of energy substrates for the membrane sodium pump (i.e., pre-incubation with creatine) or decrease their utilisation (hypothermia) slow and delay DA release. Hypothermia may owe part of its neuroprotective effect to a delay and slowing of ischaemia-induced release of DA and/or other neurotransmitters.     

In vitro induction and in vivo expression of bcl-2 in the hNT neurons

Bcl-2 encodes membrane-associated proteins that suppress programmed cell death in cells of various origins. Compelling evidence suggests that bcl-2 is also involved in neuronal differentiation and axonal regeneration. The human Neuro-Teratocarcinoma (hNT) neurons constitute a terminally differentiated human neuronal cell line that is derived from the Ntera-2/clone D1 (NT2) precursors upon retinoic acid (RA) treatment. After transplantation into the central nervous system (CNS), the hNT neurons survive, engraft, maintain their neuronal identity, and extend long neurite outgrowth. We were particularly interested in the intracellular determinants that confer these post-transplant characteristics to the hNT neurons. Thus, we asked whether the hNT neurons express bcl-2 after transplantation into the rat striatum and if RA induction of the neuronal lineage is mediated by bcl-2. The grafted hNT neurons were first identified using three different antibodies that recognize human-specific epitopes, anti-hMit, anti-hNuc, and NuMA. After a 1-month post-transplant survival time, NuMA immunostaining revealed that 12% of the hNT neurons survived the transplantation. These neurons extended long neuritic processes within the striatum, as demonstrated using the human-specific antibody against the midsize neurofilament subunit HO14. Importantly, we found that 85% of the implanted hNT neurons expressed bcl-2 and that the in vitro induction of the neuronal lineage from the NT2 precursors with RA resulted in an upregulation of bcl-2 expression. Together, these data suggest that the differentiation of the hNT neurons to a neuronal lineage could be mediated at least partially by bcl-2.     

In vitro culture and labeling of neural cell aggregates followed by transplantation

Septal, cortical, or whole brain fetal (E15-17) cells were dissociated and cultured in serum-supplemented Dulbecco minimum essential medium under rotating culture conditions. Preincubation and exposure to cytosine arabinoside was utilized to produce "neuron-rich" cultures. Fluorescent latex microbeads were added to cultures at seeding time or early after aggregate formation. All cell types were found to incorporate the fluorescent beads, although apparently not to the same extent. Two- to five-day-old aggregates tended to attach and grow neurites after their transfer to poly-l-lysine- or Matrigel-coated dishes under stationary conditions. Early aggregates transplanted to the hippocampus of adult rats developed into identifiable grafts, with fluorescent-labeled cells. We conclude that "young" neural cell aggregates maintain their ability to undergo two basic phenomena for cellular interaction, i.e., attachment and neuritic growth. Floating aggregates may provide a convenient cellular condition whenever culturing of neural cells is to be used before grafting them into a host animal.     

Effects of ATP and AMP on hippocampal neurones of the rat In vitro

Effects of iontophoretic application of ATP and AMP were tested on glutamate-evoked extracellular spike activity recorded from single neurones in the CA1 pyramidal cell layer of the rat hippocampal slice in vitro. Glutamate, ATP and AMP were applied to the dendritic region of CA1. ATP depressed 11 of 25 units tested; AMP depressed 10 of 18 units tested. The remaining units were unaffected. Typically, the response to either purine developed slowly and persisted after the ejecting current was terminated, full recovery requiring 2-3 min. ATP and AMP were roughly equipotent. All units were tested with control current and none was affected. These results demonstrate a depressant effect of ATP and AMP on glutamate-evoked activity and therefore support earlier evidence of a postsynaptic depressant effect of purines in rat hippocampus.