Biomedical subjects

F van Huizen

Publications and source records attributed to F van Huizen.

14 recordsLinked to original sources

PubMed1994

Muscarinic receptor characteristics and regulation in rat cerebral cortex: changes during development, aging and the oestrous cycle.

The effects of postnatal development, aging and the oestrous cycle on muscarinic acetylcholine receptor (mAChR) properties were examined in in vitro living slices of rat neocortex. Using the hydrophilic antagonist ([3H]NMS) to label cell surface mAChRs, an increase in both Bmax and Kd was found during the first postnatal weeks. These values peaked at between 20-40 days postnatally and then declined to adult levels. After 3 months of age, a steady decline in receptor number started: it was 10.1% lower at 10 months and 38.7% lower at 17 months of age. In contrast, Kd values increased, being 31.7 and 20% higher respectively at these ages. Carbachol-induced (4 h at 37 degrees C) down-regulation of receptor number was approximately 22.2% in newborn and 26.1% in adult (3-month-old) rats, but only 16.3% at 20-40 days of age. The degree of carbachol-induced down-regulation of mAChR was not affected in the older animals. Veratridine, which increases neural activity, also induced a significant reduction in [3H]NMS binding sites of 11.4% in rats aged 0-20 days and 22.4% in 3-month-old rats, but at 20-40 and 40-60 days of age no significant down-regulation of receptor number was observed. Furthermore, down-regulation was absent in the 10-month-old rats as well. Since a great variation in Bmax and Kd values was seen in 3-month-old females but not in male rats, we investigated mAChR characteristics during the oestrous cycle of female rats. In pro-oestrus, mACh receptor number was increased and affinity decreased in comparison with di-oestrus.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1993

Agonist-induced down-regulation of human 5-HT1A and 5-HT2 receptors in Swiss 3T3 cells.

We have used single cell clones of Swiss 3T3 cells transfected with genes for the human 5-HT1A or 5-HT2 receptor to study down-regulation and desensitization. After pre-incubation of the cells with serotonin agonists, a time-dependent decrease in [3H]8-hydroxy-2-(di-n-propylamino) tetralin or [3H]ketanserin binding was observed. The pertussis toxin sensitive, 5-HT mediated inhibition of forskolin-stimulated cAMP accumulation in 5-HT1A receptor transfected cells was diminished by 68% after a 2 h pre-incubation of the cells with 10 microM 5-HT. The pertussis toxin insensitive, 5-HT mediated PI turnover in 5-HT2 receptor transfected cells was decreased by 65% after pre-treatment. While this decrease was paralleled by a decreased potency of 5-HT to stimulate PI turnover, in 5-HT1A cells the potency of 5-HT to inhibit cAMP formation was comparable to control values. The down-regulation and desensitization of the 5-HT2 receptor can be explained by phosphorylation via activated PKC. In contrast, the attenuation of the 5-HT1A receptor-coupled inhibition of cAMP accumulation has to occur by an alternative, as yet unknown, molecular mechanism.

3T3 Cells↗

PubMed1993

Muscarinic receptor regulation and 2nd messenger responses in rat neocortex cultures.

Primary cultures of dissociated cerebral cortex cells were used to characterize the muscarinic acetylcholinergic receptors (mAChR) present and to study receptor down-regulation and receptor mediated 2nd messenger responses induced by muscarinic agonists. Binding of the hydrophilic antagonist [3H]N-methyl scopolamine ([3H]NMS) to the cultured cells was saturated after one hour at 4 degrees C with a Kd of 93 pM and a Bmax of 958 fmol/mg protein. Competition binding studies with several antagonists and agonists indicated that the mAChR present in the culture were of a mixed M1/M3 subtype. The number of muscarinic receptors at the cell surface decreased by 60% after one hour pre-incubation of the cultures with 10 microM carbachol or oxotremorine. After down-regulation with carbachol affinity for pirenzepine was decreased, while low affinity sites for 4-DAMP were lost, indicating that especially M1 subtypes are sensitive to this type of regulation. Carbachol and oxotremorine-M induced a 2-3 fold increase in phosphatidyl inositide (PI) turnover, which was blocked with high affinity by both pirenzepine and 4-DAMP. Down-regulation of the mAChR and stimulation of PI-turnover by agonists with different potency and intrinsic activity appeared highly correlated. These data suggest that activation of the PI second-messenger system is involved in the desensitization and down-regulation of the muscarinic acetylcholine receptor.

Animals↗

PubMed1989

Characterization of muscarinic acetylcholine receptors in rat cerebral cortex slices with concomitant morphological and physiological assessment of tissue viability.

We have begun studies on regulatory mechanisms of muscarinic acetylcholine receptors (mAChRs) in slices of rat cerebral cortex. This paper, the first of two, deals with the viability of the cells in the slices (a prerequisite for studying receptor regulation) and provides a characterization of binding sites for [3H]N-methyl scopolamine ([3H]NMS) and [3H]quinuclidinyl benzylate ([3H]QNB) in this preparation. Trypan blue exclusion tests in 400-microns-thick cortical slices showed a number of dead cells in a 100 microns zone from each cut edge, for a total of about 15-30% of all cells in the slice. In agreement with previous reports, electron microscopy revealed healthy tissue in the middle of the slice, but after incubation for several hours, swollen cells and dendrites were seen without cytoplasmic organelles. Axon terminals, however, were still seen to synapse upon these processes. Electrophysiological single unit recordings showed spontaneous action potentials in the slices. For receptor binding experiments, slices were incubated with either [3H]NMS, a hydrophilic mAChR ligand which does not penetrate the cell membrane, or the lipophilic ligand [3H]QNB which readily enters cells. For both ligands, equilibrium binding was reached after 8 h at 4 degrees C, and after 3 h at 30 degrees C. Binding of both ligands could be displaced by unlabelled atropine sulphate, NMS or QNB. Saturation binding curves yielded a Bmax of 2187 fmol/mg protein for [3H]QNB (reflecting all mAChRs) and 1335 fmol/mg protein for [3H]NMS (only mAChRs on the cell surface) at 30 degrees C. Kd values were 8.2 and 5.2 nM for [3H]QNB and [3H]NMS, respectively. These values are high compared with values obtained from homogenates, frozen sections or dissociated cells, and presumably reflect the use of intact, living tissue. These data are probably a better reflection of the actual, in vivo mAChR number and affinity than those obtained from dead tissue. This slice preparation suggests itself as a simple but effective method with which to study the regulation of mAChRs in living brain tissue.

Animals↗

PubMed1989

A role for potassium channels in the regulation of cortical muscarinic acetylcholine receptors in an in vitro slice preparation.

The rules underlying muscarinic acetylcholine receptor (mAChR) regulation in an in vitro cortical slice preparation of adult rats were examined following various alterations of bioelectric activity and following agonist stimulation. Muscarinic ACh antagonists [3H]N-methyl scopolamine ([3H]NMS) or [3H]quinuclidinyl benzylate ([3H]QNB) were used to label cell surface vs total (i.e. surface and internal) receptors, respectively. Depolarization of neural membranes for 4 h at 22-37 degrees C using veratridine or high external potassium (K+) led to a temperature-dependent down-regulation of surface mAChR of 26.2% and 11.3%. Total mAChRs decreased by 37.6% and 8.1%. Addition of picrotoxin and glutamic acid also led to decreases in mAChRs. Increases in inward chloride ion current induced by gamma-aminobutyric acid (GABA) or gold chloride had no significant effect on mAChRs. Blockade of calcium channels and synaptic transmission by magnesium or cobalt and postsynaptic calcium channels with nifedipine showed a significant effect on mAChRs only in the latter case. In contrast, agonist stimulation using carbachol led to a large down-regulation for both [3H]NMS and [3H]QNB (26.1%, 35.9%). ACh decreased [3H]QNB binding by 33.9%, but had little effect on [3H]NMS binding (6.3%). For [3H]QNB binding sites the effects of carbachol appeared to summate with those of veratridine. Down-regulation of [3H]NMS labelled mAChRs by carbachol and veratridine had an estimated half-time of 30 min and 2 h, respectively. Neither the effects of veratridine nor carbachol could be antagonized by tetrodotoxin (TTX), showing that the effects were not due to an increase in sodium ion currents. However, a common thread linking the various agents which induce mAChR down-regulation appears to involve changes in potassium (K+) current. Potassium channel blockers tetraethylammonium chloride (TEA), 4-aminopyridine (4-AP) and apamin had little independent effect on mAChR number, but prevented veratridine-induced down-regulation, presumably through a blockade of K+- and Ca2+-dependent K+-channels. Only TEA and 4-AP diminished carbachol-induced down-regulation suggesting that this effect involves only the non Ca2+-dependent K+-channels. It thus appears that mAChR regulation in the rat cerebral cortex is linked to changes in active K+-channel currents: activation of the K+-channel by depolarization-induced changes in K+ current or by agonist stimulation leading to changes in the selective K+ currents stimulate mAChR down-regulation; blockage of the K+-channels prevents this down-regulation.

4-Aminopyridine↗

PubMed1989

Phorbol 12,13-dibutyrate regulates muscarinic receptors in rat cerebral cortical slices by activating protein kinase C.

Stimulation of muscarinic acetylcholine receptors (mAChR) elicits phosphatidylinositol turnover, which yields inositol phosphates (InsP) and diacylglycerol (DG) the latter activating protein kinase C (PKC). Activating PKC with phorbol esters inhibits mAChR agonist-stimulated phosphoinositide hydrolysis and InsP production. A possible mechanism of this inhibition may be down-regulation of mAChR by PKC. In the present work, rat cortical slices were preincubated with phorbol 12,13-dibutyrate (PDBu) followed by binding assays for [3H]quinuclidinyl benzilate [( 3H]QNB), [N-methyl-3H]scopolamine [( 3H]NMS) or [3H]pirenzepine [( 3H]PZ). Our data demonstrate that activation of PKC by phorbol esters causes a rapid down-regulation of muscarinic cholinergic receptors. This down-regulation is also rapidly reversible. Receptors on the cell surface appear to be more sensitive to the effect of PKC than do internal ones. This down-regulation occurs by a decrease in the number of receptors, rather than by changes in receptor affinity. The results suggest that PKC may exert negative feedback on its own activation by down-regulating the receptors that normally elicit phosphatidylinositol turnover.

Animals↗

PubMed1989

Differential effects of quinolinic acid lesions on muscarinic acetylcholine receptors in cat visual cortex during postnatal development.

Quinolinic acid (QA) lesions of neurons in cat visual cortex were combined with conventional in vitro autoradiographic methods in order to define the cellular locus of the muscarinic acetylcholine receptor (mAChR). Animals of various postnatal ages had QA unilaterally injected into the visual cortex. Four to fourteen days later they were sacrificed and processed for electron microscopy (EM) or in vitro autoradiography. QA lesions at the various postnatal ages were found to eliminate intrinsic cortical neurons and their processes while leaving intact glia, fibers of passage and axon terminals from outside the lesion zone. Autoradiograms of visual cortex labelled with [3H]QNB (which labels M1 and M2 subtypes) showed an age-dependent loss of binding sites, with the greatest decreases occurring after 65 days postnatal. Examined separately, only the M1 mAChRs labelled with [3H]pirenzepine exhibited these age-dependent alterations. The results indicate a differential distribution of the M1 mAChRs during postnatal development. The loss of receptors late in postnatal life following QA suggests a dominantly neuronal locus; the relatively small loss early in postnatal life suggests a locus on other cellular elements.

Aging↗

PubMed1988

Cellular and subcellular localisation of muscarinic acetylcholine receptors during postnatal development of cat visual cortex using immunocytochemical procedures.

A monoclonal antibody against the muscarinic acetylcholine receptor was used to study the distribution of this receptor within the cat visual cortex at the light and electron microscopic level. Immunolabelling was found to be distributed mainly in cell bodies and dendrites in both young and adult cats. The laminar distribution, however, changed during development from cortical layers IV and to a lesser extent II in kittens of 28-42 days of age into one favouring the upper (L I-III) and lower layers (L V and VI) in kittens and cats more then 60 days of age. Electronmicroscopy revealed staining of membranes of cell bodies and dendrites and a great amount of intra-cellular staining of vesicles and internal membranes. Since the receptors are found on cell bodies and dendrites in both kittens and adults this indicates that different populations of cortical cells express these muscarinic receptors at different postnatal ages.

Aging↗

PubMed1987

Accelerated neural network formation in rat cerebral cortex cultures chronically disinhibited with picrotoxin.

Our aim was to determine if chronic blockade of GABAergic inhibitory synaptic activity, monitored electrophysiologically at the neuronal level, would affect synapse formation and ultrastructure in dissociated fetal rat cerebral cortex cultures. This was achieved by adding picrotoxin to the serum-free growth medium in a dose that induced continuous epileptiform discharges throughout the culture period. Light and electron microscopic analysis suggested an accelerated synaptic network formation in the experimental cultures during the first 2 weeks in vitro. The elimination of excess synapses (mainly on spines), which normally takes place during the fourth week in vitro, occurred 1 week earlier in the presence of picrotoxin. Finally, the experimental cultures showed smaller spine synapses throughout the entire culture period. Because these effects were opposite those induced by chronic tetrodotoxin-blockade of spontaneous bioelectric activity in a previous study, the underlying causal factor could be the respective intensification and suppression of neuronal activity in the two experiments. An appropriate balance between excitatory and inhibitory synaptic drive seems therefore to be important for normal maturation of neocortical circuitry.

Action Potentials↗

PubMed1987

Indications for a critical period for synapse elimination in developing rat cerebral cortex cultures.

It was observed in an earlier study that chronic tetrodotoxin (TTX) blockade of spontaneous bioelectric activity (SBA) in rat cerebral cortex cultures prevented the large-scale elimination of synapses which normally occurs during the fourth week in vitro. This prompted us to study whether the persisting high synapse density during long-term TTX-treatment would still return to the "normal' low level after restoration of SBA. Therefore, cultures grown in TTX-supplemented medium for 5 weeks were switched to control medium for an additional week prior to fixation. Electron microscopic analysis showed that the numerical synapse density remained at a high level, thus suggesting the presence of a critical period whereafter bioelectrically controlled elimination of redundant connections no longer occurs. In contrast, the mean size of synaptic structures depended only on the functional state of the tissue at the moment of fixation, being larger in TTX-silenced cultures than in bioelectrically active ones regardless of treatment during the first 5 weeks in vitro.

Animals↗

PubMed1985

An improved EPTA staining method for synapses in rat cerebral cortex cultures.

Ethanolic phosphotungstic acid (EPTA) staining has proved to be a useful tool in electron microscopic studies on synapse formation and maturation in the developing brain. However, the available staining procedures did not give satisfactory contrast between synaptic structures and background in reaggregated cerebral cortex tissue grown in vitro. Such contrast is a prerequisite for reliable quantitative studies. We therefore varied several steps of the staining procedure and have succeeded in obtaining a modification which gives the required contrast. A short rinsing time after EPTA staining turned out especially to be very crucial.

Animals↗

PubMed1985

Synaptogenesis in rat cerebral cortex cultures is affected during chronic blockade of spontaneous bioelectric activity by tetrodotoxin.

Reaggregated occipital cortex cells of 19-day-old fetal rats were grown in a serum-free, chemically defined medium, and chronically exposed to impulse-blocking levels of tetrodotoxin (TTX) in order to study the role of bioelectric activity in synaptogenesis. As judged by phase-contrast microscopy, no differences were noticed in the development of neuronal networks in the TTX-treated vs control cultures. In addition, when TTX was withdrawn from experimental cultures at any stage of development, bioelectric activity qualitatively comparable to that of the control cultures appeared within 1 min. However, quantitative stereological EM analysis revealed a significant retardation in synapse formation and ultrastructural maturation of synaptic junctions during the first 3 weeks. Around 23 days in vitro, the central zone of the reaggregates in control cultures started to degenerate, but not earlier then day 27 in TTX-treated cultures. During this time, the control, but not the experimental cultures showed (in intact tissue regions mainly situated at the outside of the aggregates) a large and selective loss of spine synapses. It is concluded that functional blockade not only retards the early growth and maturation of synaptic networks but also prevents the later occurring selective loss of spine synapses.

Animals↗

PubMed1984

Towards an improved serum-free, chemically defined medium for long-term culturing of cerebral cortex tissue.

The present study describes a series of experiments which have led to a substantially improved serum-free, chemically defined medium (CDM) for long-term culturing of reaggregated fetal rat cerebral cortex tissue. A reduction of the original medium concentrations of the hormones insuline, T3 and corticosterone, on the one hand, and an enrichment of the medium with the vitamins A, C and E, the unsaturated fatty acids linoleic and linolenic acid, and biotin, L-carnitine, D(+)-galactose, glutathione (reduced) and ethanolamine, on the other hand, formed the most important chemical adjustments of the medium. With the aid of this CDM (encoded R12), the light- and electron microscopic architecture of the tissue could be kept in a good condition (superior to that seen earlier in serum-supplemented medium) up to 23 days in vitro. From that time on, the neuronal network lying between the reaggregates degenerated for the largest part, while a portion of the large neurons (probably pyramidal cells) plus some of the neuronal network within the reaggregates degenerated too. This degeneration process continued during the following weeks, but the reaggregates nevertheless retained most of their mass, so that both small and large neuronal cell bodies (visible in transparent regions at the edge of the reaggregates) remained in good condition up to at least 103 DIV. Stout, thick nerve bundles interconnecting the reaggregates, also survived up to this point. Electron microscopic evaluation of such 'aged' reaggregates revealed degenerating as well as healthy regions. The latter had indeed retained healthy-looking pyramidal and non-pyramidal neurons, embedded within a dense neuropil which was often traversed by myelinated axons. The numerical synapse density in such selected, healthy tissue regions reached its maximum during the sixth week in vitro, followed by a rapid decrease and a stabilization at about half the peak values. The present culture system has opened the possibility for performing controlled quantitative studies on the relationship between structure and function of cerebral cortex tissues during development and aging, on its dependence on nutrients, hormones and drugs, and on special factors synthesized by the tissue and released into the nutrient medium.

Animals↗

PubMedDate not supplied

Binding of a biotinylated neurotrophic ACTH(4-9) analogue, Org 2766, to neurofilament-positive cells in primary or cell line cultures.

To study the putative binding sites of the neurotrophic peptide Org 2766, an analogue of ACTH(4-9) [H-Met(O2)-Glu-His-Phe-D-Lys-Phe-OH], biotinylated forms of the peptide were used. After fixation, cultures of rat spinal cord and dorsal root ganglia were incubated with 4-10 microM of biotinyl-Org 2766 (b-Org 2766). Binding of both N- and C-terminally biotinylated Org 2766 was seen to phase-bright, round cells with thin processes, but not to flat, orthogonal-shaped cells with tapering processes. The b-Org 2766 binding was displaceable by an excess of nonbiotinylated Org 2766. Light and electron microscopy showed that the biotinylated peptide binds to a cytoplasmatic component as well as to the cell membrane. Double-labeling experiments with b-Org 2766 and an antibody (RT-97) to a high molecular weight neurofilament protein in dorsal root ganglion cultures showed, using fluorescence and confocal scanning laser microscopy, that all b-Org 2766 binding cells were neurofilament positive. Biotinylated Org 2766 did also bind to the neuronally differentiated cells in cultures of the human neuroblastoma cell line SK-N-SH, but not to those differentiated into epithelial cells. The present data suggest that the neurotrophic peptide Org 2766 binds specifically to cell types with neuronal characteristics.

Adrenocorticotropic Hormone↗