George B. Koelle

A NEW GENERAL CONCEPT OF THE NEUROHUMORAL FUNCTIONS OF ACETYLCHOLINE AND ACETYLCHOLINESTERASE

THERE is surely no other compound for which a neurohumoral function has been so well established as it has for acetylcholine (ACh). The early work of Loewi, Dale, Feldberg, and their collaborators which first indicated the occurrence of cholinergic transmission at various sites has been extensively confirmed and amplified by subsequent investigators. The sequence of events which is now generally accepted as taking place during the passage of an impulse across a cholinergic synapse, such as those of the autonomic ganglia, is as follows (Grundfest, 1957) : With the arrival of the nerve action potential (NAP) at the terminals of the preganglionic axon, the transmitter, ACh, is liberated from an intra-axonal storage site; it diffuses across the narrow (at most, a few hundred A) synaptic cleft, and combines with receptor groups on the ganglion cell membrane, causing the development of a localised non-propagated depolarisation, known as the postsynaptic potential (PSP) ; the latter initiates electrogenically a NAP, which is propagated along the postganglionic fibre ; the polarised state of the postsynaptic membrane is restored with the rapid destruction of the synaptic transmitter by the enzyme, acetylcholinesterase (AChE). The work in our laboratory over the past several years has been concerned chiefly with the correlation of histochemical studies of the cytological localization of neuronal AChE with pharmacological investigations of the effects of anticholinesterase (anti-ChE) agents, with the general aim of elucidating the physiological functions of AChE and ACh. Our findings and those of several other investigators have not been fully consistent with the description of the steps involved in cholinergic transmission given in the foregoing brief account. In order to explain these discrepancies, a working hypothesis was proposed (Koelle, 1961), according to which the ACh liberated by the NAP acts initially at the same presynaptic terminals to bring about the liberation of additional quanta of ACh, and it is the secondarily released, increased amount of ACh which acts at the postsynaptic site to effect transmission. In many types of non-cholinergic neurons, it is equally likely that a similar mechanism is involved, in which the initial liberation of ACh promotes the release of another neurohumoral transmitter from the same nerve endings. Finally, it was suggested that at peripheral sensory receptors the specific stimulus may activate the release of ACh from either the accesssory cells or the * Based on a Special University of London lecture given at King’s College, London, on February 23, 1961.

Localization of Acetylcholinesterase in the Neurohypophysis and its Functional Implications.

Summary Neurohypophyses of 7 cats were stained for AChE activity by AThCh method. In the infundibular stalk, fibers showed light staining, whereas fibers, their vesicular swellings, and terminations showed moderate staining in the infundibular process of the posterior lobe. No significant AChE activity was noted in the anterior or intermediate lobe. Thus, in conjunction with findings of an earlier study (7), neurons of the hypothalamico-neurohypophyseal tract appear to contain light to moderate concentrations of AChE throughout their full lengths. These findings are consistent with previous proposals (7,8) that liberation of endocrine secretions of the neurohypophysis is controlled by liberation of ACh from the same terminals.

Acetylcholinesterase Regeneration in Peripheral Nerve after Irreversible Inactivation

The return of acetylcholinesterase activity was studied in several cholinergic structures in the cat after irreversible inactivation by diisopropylfluoro-phosphate. It was found that enzymic activity returned uniformly along the hypoglossal and cervical sympathetic nerve trunks. No evidence for somatoaxonal convection of enzyme was obtained.

Effects of inactivation of butyrylcholinesterase on steady state and regenerating levels of ganglionic acetylcholinesterase.

The effects of single and repeated injections of tetramonoisopropyl pyrophosphortetramide (iso‐OMPA), a selective inactivator of butyrylcholinesterase (BuChE), were studied on the ganglionic and muscular levels of BuChE and acetylcholinesterase (AChE) in cats during the steady state and following the irreversible inactivation of both enzymes by isopropylmethylphosphonofluoridate (sarin). Single intravenous injections of iso‐OMPA, 3.0 or 6.0 μmol/kg, produced nearly total inactivation of BuChE with no immediate effect on the AChE of the superior cervical (SCG), stellate (StG), and ciliary (CG) ganglia and inferior oblique (10) muscle; regeneration of BuChE occurred at approximately the same rate in the three ganglia, and at 4–6 days the AChE levels were significantly elevated. When single doses of iso‐OMPA were given 1 h following sarin, 2.0 μmol/kg, intravenously, there was a slight increase in the rate of AChE regeneration during the ensuing 2 days. With the repeated injection of iso‐OMPA, 3.0 μmol/kg every 48 h, there was a consistent but not statistically significant reduction in AChE regeneration at 4, 6, 12, and 18 days following sarin in all 3 ganglia. Similar treatment with iso‐OMPA alone produced significant increases in ganglionic AChE at all these periods excepting the longest. The daily injection of iso‐OMPA for 6 days, which maintained ganglionic BuChE at approx 2% of the control values, produced significant reductions in AChE regeneration, but again significant increases in ganglionic AChE levels in cats that did not receive sarin. The IO muscle did not exhibit these effects. A working hypothesis is proposed, that BuChE is a precursor of ganglionic AChE, and that the level of BuChE participates in the regulation of AChE synthesis by inhibition of a preceding rate‐limiting step.

Selective, near-total, irreversible inactivation of peripheral pseudocholin-esterase and acetylcholinesterase in cats In vivo

Abstract Tetramonoisopropyl pyrophosphortetramide (Iso-OMPA) was confirmed to be a highly selective inactivator of cat pseudocholinesterase (BuChE) over wide ranges of concentration and temperature in vitro ; intravenous doses of 3.0 or 6.0 μmoles (approximately 1.0 or 2.0 mg)/kg produced nearly total inactivation of peripheral BuChE, with no detectable inactivation of acetylcholinesterase (AChE). Selective inactivation of AChE in vivo was obtained as follows: after a dose of mephentermine, 2.0 to 3.0 mg/kg, i.v., to sustain blood pressure, cats received an intravenous dose of 10-(α-diethylaminopropionyl) phenothiazine HCl (Astra 1397) up to 200 μmoles (73 mg)/kg, which produces selective reversible inhibition of BuChE; they were then given 1.0 μmole (0.332 mg) 2-diethyoxyphosphinylthioethyldimethylamine acid oxalate (217 AO)/kg, which is sufficient when given alone to produce essentially total, irreversible inactivation of AChE and BuChE. Under these conditions, approximately one-third of the autonomic ganglionic BuChE, but none of the AChE, was protected and restored to activity. Quantitative results, obtained by a spectrophotometric method, were confirmed histochemically.

Acetylcholinesterase: Method for Demonstration in Amacrine Cells of Rabbit Retina

The activity of acetylcholinesterase in the inner plexiform layer of the rabbit retina was not affected detectably by prior section of the optic nerve. After the animals were treated with diisopropyl phosphorofluoridate, acetylcholinesterase reappeared in the somata of the amacrine cells and in certain cells of the ganglion cell layer before it reappeared in the inner plexiform fibers. This confirms the normal presence of acetylcholinesterase at the former site. The possible role of acetylcholine in intraretinal transmission is considered.

EFFECTS OF PROTECTION OF BUTYRYLCHOLINESTERASE ON REGENERATION OF GANGLIONIC ACETYLCHOLINESTERASE

The regeneration of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) was followed in the superior cervical (SCG), stellate (StG), and ciliary (CG) ganglia and inferior oblique (IO) muscle of cats for the 3‐day period following their inactivation by isopropylmethylphosphonofluoridate (sarin; 2.0 μmol/kg intravenously), with and without preservation of over half the butyrylcholinesterase activity by the prior intravenous infusion of 10‐(α‐diethylaminopropionyl) phenothiazine HCl (Astra 1397; 100 or 200 μmol/kg). Rates of regeneration of AChE in the three ganglia exhibited the same sequence as their relative proportions of AChE‐containing cholinergic ganglion cells (SCG < StG < CG); no such difference was found in the rates of butyrylcholinesterase regeneration. The mean AChE levels in all three ganglia were higher at 48 h in the cats that had received Astra 1397 (100 μmol/kg) prior to sarin. This finding is interpreted as evidence that BuChE may function as a precursor in the synthesis of AChE.

current concepts of synaptic structure and function

Most of our attention at this meeting will be focused on the neuromuscular junction or motor end plate (MEP) of skeletal muscle in our search for both the basis of its functional defect in myasthenia gravis and improved means for the treatment of this condition. Our present concept of the structure of the MEP originates chiefly from the brilliant light microscopic studies of Couteaux’ and Couteaux and Taxi.2 Their conclusions received general confirmation, as well as considerable extension and amplification in the early electron microscopic studies of Robertson3 and those of subsequent investigators, reviewed by Csillik.4 Perhaps the best appreciation of the complex architecture of the MEP can be derived from examination of the beautiful three-dimensional models reconstructed by Andersson-Cedergrens from electron micrographs of a great number of serial sections (FIGURE 1 ) . The MEP was described briefly in an earlier reviewB as follows: “As the motor nerve fiber approaches the muscle fiber, the former loses its myelin sheath, then terminates as a series of coils which, viewed from above has a pretzel-like appearance. The motor nerve terminal lies in a depression, known as the synaptic gutter, on the surface of the muscle fiber. There appears to be no organized cellular structure within the synaptic cleft, the space between the lower surface of the axoplasmic membrane and the modified sarcoplasmic membrane, or the subneural apparatus. The latter is folded into a complex series of invaginations which greatly increase its surface area; it is at the surface of this membrane that the receptor sites are presumably located. The distance across the synaptic cleft here is approximately 500 AU; its edges at the surface of the muscle fiber appear to be covered by teloglial cells, derivatives of the Schwann sheath cells, as is the upper surface of the axonal terminal. Within the axonal terminal are dense concentrations of both synaptic vesicles and mitochondria; the former represent packets or quanta of ACh (estimated at approximately 1,OOO molecules per vesicle), and the latter contain oxidative enzymes that probably provide energy for the preliminary steps in its synthesis.” Synapses elsewhere in the peripheral and central nervous system differ from the MEP in certain important respects. “The synaptic clefts elsewhere are narrower than that of the neuromuscular junction, the distance between the preand postsynaptic membranes ranging from 120 to 300 AU. Both membranes show significant thickening and increased electron density at their region of apposition. They are bridged by a series of thin, parallel intersynaptic filaments which apparently serve to anchor their spatial relationship. At many synapses, a subsynaptic web of h e filaments or canaliculi extends from the postsynaptic membrane into the underlying cytoplasm for variable distances; its function is not known. In the same region of axosomatic synapses, at several sites, dense accumulations of Nissl substance (the ribonucleic acid of the endoplasmic reticulum) have been noted; this observation has raised the interesting speculation of the possible involvement of Nissl substance in protein synthesis associated with the consolidation

COMPARISON OF THE GOLD-THIOCHOLINE AND GOLD-THIOLACETIC ACID METHODS FOR THE HISTOCHEMICAL LOCALIZATION OF ACETYLCHOLINESTERASE AND CHOLINESTERASES

The thiocholine (ThCh) and thiolacetic acid (ThAc) methods for the histochemical localization of acetylcholinesterase (AChE) and cholinesterases (ChEs) were modified to permit the substitution of aurous gold for copper and lead, respectively, as the capturing agent. The chief advantage of the modifications is that the precipitates formed (AuThCh-phosphate and AuS) have fine, colloidal dimensions and high electron density, thus improving their potential usefulness as electron microscopic methods. By means of selective inhibitors of AChE (10–5 M BW 284), of ChEs ( 10–7 M DFP or 3.10–8 M Nu-683), and of both enzymes (10–5 M DFP or eserine), it was shown that the gold thiocholine (AuThCh) method retains the high specificity of the original ThCh method, whereas the gold thiolacetic acid (AuThAc) method, like the lead ThAc procedure, is considerably less specific. However, the AuThAc method appears to permit finer localization than the AuTuCh technique.

A THIOCHOLINE-LEAD FERROCYANIDE METHOD FOR ACETYLCHOLINESTERASE

The principle employed in the thiocholine-copper ferrocyanide method of Karnovsky and Roots has been adopted for the development of a similar procedure in which lead, complexed with Tris-acetate buffer, is used as the trapping agent for the ferrocyanide ion, formed by the preferential reduction of ferricyanide by thiocholine released enzymatically from acetylthiocholine. The resulting colloidal, faintly yellowish white precipitate, Pb2Fe(CN)6, can be viewed directly by light or phase contrast microscopy, or more easily following its conversion to PbS. The method has been shown to produce extremely fine localization of acetylcholinesterase in the cat superior cervical and stellate ganglia and at the motor end plates of mouse intercostal muscle.