The extracellular matrix modulates the response of PC12 cells to nerve growth factor: cell aggregation versus neurite outgrowth on 3-dimensional laminin substrata.

PC12 cells attach well to plastic culture dishes coated with laminin, collagen, polylysine or a basement membrane extract (2-dimensional substrata) and, in the presence of NGF, extend short neurites within 1-2 days. However, on gels (3-dimensional substrata) reconstituted from a basement membrane extract (RBM), PC12 cells attach extending short processes transiently and within one day, form networks of small aggregates interconnected by process-bearing cells. By 3 days the network collapses into large aggregates that, in media supplemented with NGF, extended a halo of neurites resembling dorsal root ganglia in culture. Time-lapse video recordings indicate that cell motility on RBM gel is accompanied by extensive blebbing as well as extension of processes that attach to and pull together neighbouring cells. These cellular events may contribute to the disruption of the gel underneath aggregates that is apparent when cultures are stained with Coomasie Blue. Ultrastructural studies indicated that aggregates often have zonula adherens-type junctions where cell bodies and processes come in contact. PC12 cells seeded onto gels of laminin alone behave essentially the same as on RBM gels, whereas on collagen gels they behave as on 2-dimensional substrata and extended neurites rather than aggregate. The extent of aggregation increases with greater cell density and is enhanced significantly by NGF. Antisera to NGF reduce the NGF-enhancement of aggregation but do not block aggregation in the absence of NGF. Dibutyryl cAMP or epidermal growth factor, which stimulate process extension and cell division respectively, do not enhance aggregation. However, 3A3, a monoclonal antibody to a laminin/collagen receptor on PC12 cells and antibodies (Fab fragments) to the neural cell adhesion molecule both inhibit cell aggregation.

Histofluorescent labelling of catecholaminergic structures in rotifers (Aschelminthes). II. Males of Brachionus plicatilis and structures from sectioned females.

1. Catecholaminergic structures in the male Brachionus plicatilis were investigated, using aqueous dansylpropranolol as fluorescent label of neurotransmitter receptors. 2. All major organs of the male are innervated by catecholaminergic systems, that may also be involved in the regulation of copulatory behavior. 3. Cryostat-sectioned preparations of the female B. plicatilis were also investigated. They provided additional information to findings on whole animals reported in our previous paper (Keshmirian and Nogrady 1987 a).

Histofluorescent labelling of catecholaminergic structures in rotifers (Aschelminthes) in whole animals.

1. Catecholaminergic neuronal structures were investigated in the rotifers Brachionus plicatilis, Asplanchna priodonta and Asplanchna herricki, using three different aqueous histofluorescent methods. 2. The adrenergic receptors were labelled using the dansyl analog of propranolol, a beta-adrenergic blocker. Catecholamine neurotransmitters were visualized by derivatizing with glyoxylic acid and formaldehyde respectively. 3. Although all three methods lead to similar results, dansyl-propranolol gave the most rapid and strong fluorescence. 4. The results reveal a complex and highly developed catecholaminergic neuronal system in all adult organs and sensory structures. While developed embryos in the egg show strong fluorescence, immature eggs do not.

Rotifer neuropharmacology--III. Adrenergic drug effects on Brachionus plicatilis.

Norepinephrine (NE) induces three pharmacological effects in Brachionus plicatilis. As a result of excitation the rate of ciliary motion and swimming increases, and the animals flip their foot constantly at a rapid rate. This rapid foot flipping was used as a specific model to measure adrenergic effects in B. plicatilis. Phenylephrine induces the same effect at identical efficacy, while isoproterenol and salbutamol, two beta-agonists, show one-half and one-tenth NE efficacy. The beta blocker propranolol and the alpha blocker tolazoline both antagonize foot flipping induced by NE. However, propranolol shows antagonism because it causes foot paralysis by itself. Timolol, another beta blocker but without the membrane effect of propranolol, does not antagonize the alpha receptor mediated NE effect, nor does it cause foot paralysis. Propranolol, timolol and tolazoline also show agonist activity, inducing foot flipping. NE does not antagonize the foot paralysis induced by propranolol, only its anesthetic effect by delaying its onset. These results indicate that the foot flipping induced by NE is a receptor-mediated alpha adrenergic effect, while the foot paralysis is caused by membrane phenomena.

Rotifer neuropharmacology--I. Cholinergic drug effects on oviposition of Philodina acuticornis (Rotifera, Aschelminthes).

The presence of acetylcholine in the culture medium was found to result in egg retention in the bdelloid rotifer Philodina acuticornis in a dose-dependent fashion, with no other discernible physiological effect. When six to eight eggs accumulate, the animals distend, burst and die. No other cholinergic agonist has been shown to produce a similar effect. The antagonistic effect of six anticholinergic drugs and five acetylcholinesterase-inhibitor insecticides was investigated on egg retention. All compounds were found to inhibit this phenomenon to varying degrees, but on the basis of their EC50 neuromuscular blockers appear to be most active in inhibiting egg retention. We suggest, therefore, that egg retention is caused by a spasm or increased tone of the cloacal sphincter rather than a paralysis of muscles needed in the peristaltic expulsion of the egg. Five acetylcholine-esterase inhibitory insecticides also inhibit egg retention.

Rotifer neuropharmacology--II. Synergistic effect of acetylcholine on local anesthetic activity in Brachionus calyciflorus (Rotifera, Aschelminthes).

A number of compounds showing general anesthetic action in the rotifer Brachionus calyciflorus were investigated in the presence of acetylcholine. Non-ionizing anesthetics, including tricaine, showed no interaction with acetylcholine. However, highly ionized compounds like the local anesthetics procaine and lidocaine, the muscarinic blocker and local anesthetic atropine, and the beta-adrenergic blocker propranolol showed a synergistic effect with acetylcholine. ACh increased the general anesthetic effect of these compounds in a statistically highly significant dose-dependent fashion. To account for the mechanism of this unusual and novel effect it is proposed that these compounds interact with the anesthetic binding site of the rotifer cholinoceptor ionophore in the open state. It is also proposed that non-ionizing compounds have a general membrane effect only. In addition to anesthesia, atropine and propranolol cause foot paralysis in B. calyciflorus. This other novel effect is also enhanced by acetylcholine as well as decamethonium, a neuromuscular blocker.