Biomedical subjects

M S Cynader

Publications and source records attributed to M S Cynader.

At least 37 records · Page 2Linked to original sources

PubMed1997

Visualization of muscarinic cholinergic receptors on chick cardiomyocytes and their involvement in phosphatidylcholine hydrolysis.

The purpose of this study was to visualize muscarinic receptors and their distribution on cardiomyocytes and to examine the effects of muscarinic cholinergic receptor (mACh-R) stimulation with carbachol on phosphatidylcholine hydrolysis. Cardiomyocytes were prepared as primary culture from 7-day-old chick embryo hearts. Cardiomyocytes, grown on cover slips, were labelled with BODIPY PZ, a fluorescent analog of the muscarinic receptor antagonist pirenzepine, and examined with a laser scanning confocal microscope, mACh-R clusters were visualized and were fairly homogeneous in size with diameters ranging from 0.5 to 1.0 micron. The number of receptor clusters per cell was 83.5 +/- 6.8 (mean +/- SEM) and clusters were found at the periphery of the cell. Cardiomyocytes, grown as a monolayer in dishes, were treated with the 10(-4) M carbachol, a mACh-R agonist, and the effects on phosphatidylcholine hydrolysis were ascertained in cells preincubated with [methyl-3H]choline for 18 h. Cells were washed, lysed, and subjected to thin-layer chromatography to separate [3H]choline in various metabolites of phosphatidylcholine. Carbachol significantly (p < 0.05) increased intracellular free choline and decreased cellular phospholipid consistent with phosphatidylcholine hydrolysis. Carbachol increased the amount of [3H]choline that effluxed out of the cardiomyocyte into the medium. Carbachol-induced choline efflux was not prevented by pretreatment with n-butanol, a phospholipase D inhibitor, suggesting that other lipases such as phospholipase C are the major enzyme involved in phosphatidylcholine hydrolysis. Pertussis toxin prevented carbachol-induced choline efflux and the changes in intracellular free choline and phospholipid. An action of carbachol through G proteins was supported by the ability of pertussis toxin to antagonize the carbachol-induced reduction in cAMP generation from isoproterenol. In summary, mACh-Rs, visualized in living cardiomyocytes, were peripheral to the nucleus. Phosphatidylcholine hydrolysis induced by mACh-R stimulation may be a signal transduction pathway for mACh-R in the cardiomyocyte, operating through inhibitory G proteins sensitive to pertussis toxin.

1-Butanol↗

PubMed1997

Glaucomatous optic nerve cupping as an optic neuropathy.

Intraocular pressure (IOP), which causes the lamina cribrosa to bulge backward, produces a pressure gradient along the axoplasm of exiting optic nerve axons, and challenges the circulation, interacts with presently unknown physiologic or anatomic factors to harm the optic nerve and causes loss of vision. Present treatment of glaucoma is limited to efforts to lower IOP. Future treatments may be directed at other contributing anatomic or physiologic abnormalities that permit IOP to be harmful, or at some step in the pathway from insult to mortal injury of the axon or cell body.

Animals↗

PubMed1996

Nerve growth factor induced modification of presynaptic elements in adult visual cortex in vivo.

Nerve growth factor (NGF) has been shown to play important roles in neuronal survival, growth and differentiation. Recently, we have found that intracortical infusion of NGF into adult cat visual cortex can recreate ocular dominance plasticity, suggesting that NGF is also involved in activity-dependent modification of synaptic connectivity in the adult brain. To further explore the mechanisms of NGF-induced plasticity in adult visual cortex, we studied two presynaptic markers: GAP-43 and synaptophysin. Immunocytochemical staining showed that NGF-treatment of adult visual cortex selectively increased the level of the phosphorylated form of GAP-43, while the total level of GAP-43 was not changed. These results demonstrate that NGF-treatment stimulates phosphorylation processes of GAP-43 in vivo. In addition, NGF-treatment of adult visual cortex increased the level of synaptophysin immunoreactivity. Since the phosphorylated form of GAP-43 is known to be enriched in the membrane skeleton of growth cones and of developing synapses, and the phosphorylation of GAP-43 has been linked with events that underlie synaptic plasticity, and since synaptophysin is a major component of presynaptic vesicles, our results suggest that NGF-treatment of adult visual cortex modulates presynaptic terminals, possibly by inducing axonal sprouting and formation of new synapses, and that these changes may play a role in the NGF-induced functional plasticity.

Animals↗

PubMed1996

Increased beta-amyloid precursor protein mRNA in the rat cerebral cortex and hippocampus after chronic systemic atropine treatment.

Rats were treated with once-daily subcutaneous injections of atropine or normal saline for 10 days. Cryostat sections of fresh-frozen brain were subjected to quantitative muscarinic receptor ([3H]quinuclidinylbenzilate (QNB)) binding autoradiography, and quantitative in-situ hybridization autoradiography for beta-amyloid precursor protein (beta-APP) mRNA using an oligonucleotide probe recognizing all major isoforms. QNB binding in the atropine-treated group was increased 6-7% in the areas measured (dentate gyrus, CA1, and cerebral cortex), confirming that the treatment was effective in inducing muscarinic receptor upregulation. Hybridization signal for beta-APP mRNA was increased 15-20% in the atropine-treated group in the same regions. As chronic atropine treatment models the muscarinic effects of cholinergic denervation, these results suggest that age-related cholinergic neuron loss may result in upregulation of beta-APP.

Amyloid beta-Protein Precursor↗

PubMed1996

Synergistic interactions between noradrenaline and glutamate in cytosolic calcium influx in cultured visual cortical neurons.

In primary neuronal cultures derived from the visual cortex of embryonic day 16-18 rats, intracellular free calcium concentration, [Ca2+]i, was increased by bath application of glutamate in a dose-dependent manner. Noradrenaline applied alone had relatively small effects. However, when glutamate concentrations eliciting modest increases in [Ca2+]i were applied together with 1 microM noradrenaline, the increase in [Ca2+]i could be enhanced by a factor of up to eight. The synergistic effect was seen in 147 neurons out of a total of 215 cells observed in 54 experiments. The observed enhancement was much more obvious at low doses of glutamate than with higher doses, augmenting all submaximal calcium responses to similar asymptotic levels. 2-Amino-5-phosphonovalerate (APV), the NMDA receptor antagonist, completely blocked the adrenergic enhancing effect (29/29 cells in 8 experiments). Among the antagonists specific to alpha 1, alpha 2 and beta subtypes of adrenoceptors, the beta antagonist propranolol most completely blocked the enhancing effect (13/14 cells in 4 experiments, reducing the effect by an amplitude of 90%). The involvement of the beta receptor pathway was further supported by the ability of a cAMP analog to mimic the enhancing effect of noradrenaline. On the other hand, an alpha 1 blocker showed no effect and an alpha 2 blocker showed only a relatively small effect. These results suggest that receptors for noradrenaline and glutamate colocalize on postsynaptic cortical cells and that adrenergic modulation of glutamate induced calcium influx most likely operate through the beta receptor pathway. It is further postulated that cortical ocular dominance plasticity may be at least partially implemented via a calcium dependent cascade.

2-Amino-5-phosphonovalerate↗

PubMed1996

Differential expression of neurofilament protein in the visual system of the vervet monkey.

It has been previously reported that the monoclonal antibody SMI-32 reveals a characteristic pattern of immunostaining which may be used to delineate various cortical modules in the monkey visual system. We wished to examine staining patterns with this antibody at both the lateral geniculate nucleus (LGN) and cortical levels with regard to magno- and parvocellular processing schemes in the vervet monkey. Using standard immunohistochemical procedures, we have found that the M-layers of the LGN were intensely stained in comparison to P-layers and that there were regional variations in staining within the visual cortex that reflected this input. The transition between areas V1 and V2 was especially prominent due to differences in the laminar staining profiles. Another striking result was found within the superior temporal sulcus where heavy SMI-32 immunostaining confined to the floor of the sulcus coincided with a similar zone of intense myelin staining. We have also found a number of other areas within the intraparietal and lateral sulci that show foci of heavy SMI-32 staining. As with Cat-301 immunostaining, the regional variabilities that are observed with SMI-32 in the visual cortex reflect molecular distinctions that may provide further criteria for functional segmentation.

Animals↗

PubMed1995

Synaptic depression in visual cortex tissue slices: an in vitro model for cortical neuron adaptation.

Synaptic depression was assessed from intracellular recordings in cortical tissue slices. Evoked postsynaptic potentials exhibited synaptic depression with an exponential or double exponential decrease (time constants: < 1-30 s) in amplitude during repetitive afferent stimulation by short trains of suprathreshold stimuli. Depressed synaptic responses with an exponential time course (time constants: 10 s-8 min) during presentation of similar short trains of stimuli every 5 or 10 s. Cortical cells recorded extracellularly in cat visual cortex show similar time constants of response decrement during adaptation to moving stripes. Postsynaptic voltage- or ion-regulated conductances and chloride conductances do not appear to be involved in synaptic depression. Input resistance changes and effects of injection of chloride indicate a lack of GABAA receptor-mediated effects. Hyperpolarizing or depolarizing neurons, and pairing polarization with afferent stimulation, also did not affect synaptic depression. This distinguishes these processes from long-term depression and long-term potentiation. Our results suggest that the most likely mechanisms of synaptic depression and adaptation in cortical cells are presynaptic decrease in transmitter release and/or receptor desensitization. Short-term postsynaptic changes may also occur after synaptic depression.

Adaptation, Physiological↗

PubMed1994

Nerve growth factor-induced ocular dominance plasticity in adult cat visual cortex.

Activity-dependent modifiability of cortical ocular dominance occurs only during early postnatal life, within the so-called "critical period," but not thereafter in adult visual cortex. To examine the role of neurotrophins in the activity- and age-dependent stimulation-induced modifiability of visual cortex, we tested whether intracortical infusion of nerve growth factor could induce ocular dominance plasticity in adult visual cortex. Nerve growth factor was continuously infused, by means of osmotic minipumps, into striate cortex of adult cats for 2 weeks. At the time of minipump implantation, one eyelid of the experimental animals was sutured closed. After 3 weeks of monocular deprivation, the ocular dominance distribution of neurons in the striate cortex was assessed using single unit recording. We found that monocular deprivation imposed on adult animals in conjunction with nerve growth factor infusion causes an ocular dominance shift toward the deprived eye. Although the underlying mechanisms remain uncertain, the results indicate that nerve growth factor can enhance activity-dependent synaptic modification and remodeling in adult visual cortex.

Age Factors↗

PubMed1994

A study of tachykinin-immunoreactivity in the cat visual cortex.

The localization of tachykinin-immunoreactivity in the cat visual cortex (area 17) was investigated using immunohistochemical methods. Strong laminar specificity was observed, with immunoreactivity highest in layer V, followed by layers I, VI, II and III, and the lowest density in layer IV. Most of the immunoreactive product was localized in neuronal processes. A few immunopositive cell bodies were also present. The immunopositive neurons were non-pyramidal, multipolar, or bipolar in shape, and mostly found in layer V. There were particularly dense immunopositive fibers and varicosities around somata in layer V. These may represent tachykinin-containing presynaptic terminals (boutons). The results provide anatomical evidence that tachykinins may primarily affect layer V neurons in the cat visual cortex.

Animals↗

PubMed1994

Identification of cDNA clones expressed selectively during the critical period for visual cortex development by subtractive hybridization.

We have used the method of subtractive hybridization to isolate cDNA clones of mRNAs expressed in abundance in the visual cortex of 30-day-old kittens but absent or in lower abundance in the adult cat visual cortex. Of 12,000 colonies screened, 200 clones which hybridized to the subtracted probe were isolated and characterized. Northern blots confirmed the specificity of the vast majority of the isolated clones. 120 of the 200 clones were sequenced and the EMBL and GenBank (release 76) database were searched for known identities using FASTA and BLAST programs. Twenty-seven of these sequenced clones were identifiable. The identities showed that these sequences code for proteins involved in a variety of cellular processes. These include cell-cell interaction (TAPA-1, contactin, tachykinin receptor, phospholipase A2), cellular remodeling (C1q beta isoform, heat shock protein), neurofilament assembly (alpha tubulin and alpha internexin), neurotransmitter release (VAMP-2, amphiphysin, carboxypeptidase E, scg 10 and proton channel), energy metabolism (mitochondrial hinge protein, ADP/ATP transporter, cytochrome oxidase subunits), RNA processing (helix destabilizing protein, ribonucleoprotein) and protein synthesis (eIF-4A initiation factor, ribosomal protein S27). The results show that gene expression in the kitten visual cortex differs rather little from that of the adult visual cortex since over 98% of the sequences appear common. The relatively rare kitten-specific sequences are likely to form the basis for the critical period plasticity in this system.

Aging↗

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↗

PubMed1994

Muscarinic receptor M1 and M2 subtypes in the human eye: QNB, pirenzipine, oxotremorine, and AFDX-116 in vitro autoradiography.

Muscarinic cholinergic agents are used to lower intraocular pressure in the medical management of glaucoma and subtypes of muscarinic receptors have now been recognised in many tissues including the eye. To localise muscarinic receptors and their M1 and M2 subtypes in the human eye, in vitro ligand binding and autoradiographic techniques with densitometric quantitation on postmortem eye sections were used. As ligands, [3H] quinuclydinyl benzylate (QNB) (non-subtype specific muscarinic antagonist), [3H]pirenzipine (M1 antagonist), [3H]oxotremorine (M2 muscarinic agonist), [3H]AFDX-116(11[(2[diethylaminomethyl]1-piperidinyl)acetyl]5 , 11dihydro-6H-pyrido [2,3b][1,4]benzodiazepine-6-one) (M2 antagonist) were studied. Specific binding sites for QNB, pirenzipine, and AFDX-116 were localised in the entire ciliary muscle, the iris, and ciliary epithelium. [3H]oxotremorine localised only in the longitudinal portion of the ciliary muscle, and additionally, was not localised in the iris or ciliary epithelium. These results suggest that oxotremorine, by binding selectively to receptors on the longitudinal ciliary muscle and inducing its contraction, may modulate outflow facility independently from accommodation and miosis.

Adult↗

PubMed1994

Localization of M3 muscarinic receptor subtype and mRNA in the human eye.

Cholinergic agents used to lower intraocular pressure in the medical management of glaucoma act on muscarinic receptors in the eye. Recently, subtypes of muscarinic receptors have been recognized in many tissues including the eye. To localize the M3 muscarinic receptor subtype and m3 messenger RNA (mRNA) in the human eye, we used in vitro ligand binding and in situ hybridization techniques on post-mortem sections. We used an M3 antagonist, [3H]-4-DAMP ([3H]-4-diphenylacetoxy-N-methylpiperidine methiodide) to identify M3-binding sites and a [35S]-labelled oligonucleotide probe to detect and localize m3 mRNA. M3 and m3 mRNA were both localized in the ciliary muscle, ciliary epithelium, iris, corneal epithelium and anterior lens epithelium. The m3 transcript was also detected in the trabecular meshwork and corneal endothelium.

Acetylcholine↗

PubMed1994

Activity-dependent expression and distribution of M1 muscarinic ACh receptors in visual cortex neuronal cultures.

The fluorescently labeled muscarinic M1 receptor-selective antagonist BODIPY FL pirenzepine has been employed to study the activity-dependent distribution and expression of muscarinic M1 ACh receptors (M1AChRs) in cultured neurons derived from rat visual cortex. Displacement experiments showed that like pirenzepine, binding of BODIPY FL pirenzepine was specific to M1 receptors and its K(i) was similar to that of unlabeled pirenzepine. Using confocal laser scanning microscopy, M1 receptors were predominantly localized to cell bodies early in development in the culture environment. After 2 weeks in culture, the receptors showed labeling not only in cell bodies but also in neuritic processes, especially on the initial segments of the processes. Chronic membrane depolarization with 40 mM potassium chloride caused a dramatic increase in M1 receptor expression on these neurons. Conversely, blockade of neuronal activity with 0.1 microM TTX decreased expression of the receptors. Receptor expression increased after cells were treated chronically with 50 nM pirenzepine, whereas it decreased after exposure to 10 microM carbachol. The results demonstrate for the first time the exact location of muscarinic receptors in living cultured neurons and also the activity-dependent expression of M1 receptors on these neurons. Both chronic membrane depolarization and antagonist application upregulate receptor expression, whereas blocking bioelectrical activity or chronic agonist application downregulates expression.

Animals↗

PubMed1993

An interdigitated columnar mosaic of cytochrome oxidase, zinc, and neurotransmitter-related molecules in cat and monkey visual cortex.

There is considerable physiological evidence for the compartmentalization of mammalian visual cortex into functional columnar modules, representing features of visual information processing such as eye and orientation specificity. However, anatomical markers of visual cortical compartmentalization have been described only for primate visual cortex. In this report, we describe an interdigitated mosaic of four neuroactive molecules which demarcate two distinct columnar systems in the kitten visual cortex. Serotonin 1C receptors and synaptic zinc were found to demarcate columns within layer IV of kitten visual cortex, which were interdigitated with a second, patchy system characterized by increased levels of cytochrome oxidase and acetylcholinesterase. In primate visual cortex, as well as in the kitten, synaptic zinc was periodically distributed in a manner precisely complementary to cytochrome oxidase. These findings provide an anatomical framework on which unifying hypotheses of the functional organization of columnar systems in mammalian visual cortex can be built.

Acetylcholinesterase↗

PubMed1993

Immunocytochemical localization of enkephalin in the cat visual cortex.

The localization of enkephalin-immunoreactivity in the cat visual cortex (area 17) was analyzed by using immunohistochemical methods with a monoclonal antibody directed against enkephalin. The majority of the immunoreactive product was localized in neuronal processes. The density of immunopositive fibers was greatest in layer VI, with moderate staining in layers I, II, III and V, and the least dense staining in layer IV. Layer IVab neurons showed a striking concentration of immunopositive puncta around their cell bodies. Immunopositive neurons were scarcely present in the visual cortex. They were found in all cortical layers, but mostly in layer VI. The immunopositive neurons were non-pyramidal, mostly multipolar in shape and occasionally bipolar. The results provide anatomical evidence that enkephalin may have modulatory effects on visual cortical neurons.

Animals↗

PubMed1993

Spectral motion produces an auditory after-effect.

Distortions of perception following prolonged exposure to an unvarying sensory stimulus have been observed since at least the third century BC. The motion after-effect is a familiar experience in which, after a few minutes of viewing objects moving in a single direction, a stationary object appears to move in the opposite direction. Similar after-effects have been observed for many visual stimuli, including tilted lines, colours, stereoscopic depth, curvature, spatial frequency, contrast, rotation and motion in depth. In contrast to the rich variety of visual after-effects reported since the 1960s, reports of analogous auditory adaptation effects only appeared in the 1970s, but have continued since then. Some effects of sound source spatial movement perception after adaptation to a spatially moving sound source have been reported. Here we report an auditory perceptual after-effect analogous to the visual motion after-effect, which is caused by adaptation to auditory spectral (frequency) motion. After a few minutes of listening to a simple spectral pattern moving upwards or downwards in frequency space, the same pattern sounds as though it is drifting in the opposite direction when it is stationary. The effect shows binaural transfer, implying that it is generated at the level after binaural interaction. After-effects produced by the motion of spectral peaks are independent of those produced by spectral notches, suggesting separate processing channels for spectral peaks and notches.

Auditory Perception↗

PubMed1993

Immunocytochemical study of GABAA receptors in the cat visual cortex.

The laminar distribution and morphological structures associated with GABAA receptor immunoreactivity in the cat visual cortex were studied by using two different polyclonal antibodies directed either against the purified GABAA receptor protein (antibody "967") or against a specific domain of the beta 1-subunit of the GABAA receptor (antibody "Q"). Immunoblots of cat visual cortex tissue with these antibodies revealed that antibody "Q" recognizes only one subunit, namely the beta 1-subunit of the GABAA receptor, and that antibody "967" recognizes three subunits. Both antibodies produced very similar staining patterns, indicating that the beta 1-subunit may be an essential component of the GABAA receptor in the cat visual cortex. The typical staining pattern showed a clear membrane structure around neuronal somata. Using cell body shape criteria, immunopositive neurons included both pyramidal cells in cortical layers II, III, and V, and nonpyramidal cells in all cortical layers. Immunopositive neurons were uniformly distributed in layers II to VI, whereas the density of immunopositive cells in layer I was lower. Some immunopositive neurons were also found in the white matter underlying the visual cortex. In gray matter, immunopositive structures also included dendrites, especially the proximal dendrites, and axon initial segments of pyramidal neurons. The immunopositive processes usually ran vertically toward the pial surface. Some astrocytes were also immunostained. They were localized in layer I and in the white matter. The overall pattern of immunostaining was similar in areas 17, 18, and 19.

Amino Acid Sequence↗