Professor Max Bennett - Research Contributions
On the biophysics of synapse formation and function (Numbers cited refer to papers listed under Publications)
Professor Max Bennett's research has involved, for over 40 years, development of quantitative analyses of the function of excitable cells at synapses utilizing electrical engineering models. This research has been along three main lines of enquiry into synaptic function, namely the formation of synapses by nerve terminals, their secretion of transmitters and the interaction of these transmitters with receptors.
1. The function of transmitter receptors
The concept of transmitters released onto receptors was pioneered by Elliot (for a history, see 230) using the sympathetic nerves to smooth muscle preparation. Bennett showed in the early 1960's, following Elliott's work, that smooth muscles possess receptors for transmitters other than those discovered 50 years earlier for acetylcholine and noradrenaline. These new receptors were termed then non - adrenergic non - cholinergic (acronym, NANC; see 186). About 30 different NANC receptor types have been identified in the subsequent years, some of which are channel-receptors arranged in small patches on the muscle membranes. The currents that flow at these patches on binding transmitter have been quantitatively accounted for in terms of the kinetics of interaction between transmitter and the patch (127, 154,242). Bennett's research on activation of the these receptors, using electrical circuit theory, showed that the currents which flow are not restricted to a single muscle cell but rather pass into other muscle cells throughout what is effectively an electrical syncytium of cells (3, 13, 15,20). Such currents, if of appropriate spatial and temporal extent, can initiate propagating action potentials that were shown to be due to an influx of calcium ions - the first calcium action potential discovered (10; for a history see 213). This work provided an understanding, amongst other things, of why nerve trunks are restricted to the adventitial surface of blood vessels: the receptors activated there generate currents that propagate into the media of the vessel and in most cases can control smooth muscle cells even as far as the intimal surface.
In 1963 Bennett discovered a NANC transmitter which acts on G protein - receptors. This transmitter was later identified as A TP. Transmitters that act on G protein - receptors can diffuse for long distances in the smooth muscle syncytium, for example in the media of blood vessels, before being metabolized. This allows for the activation of multiple receptor patches throughout the syncytium and has been quantitatively described by Bennett and his colleagues (266), as has been the desensitization, internalization and sequestration of the G protein - receptor -transmitter complex on the cells (255, 256). The subsequent action of the contractile machinery by calcium released in the cells through G-protein - the model has also quantitatively accounted for coupled receptor activation in the syncytium.
The above research completed for the first time a model of a muscle cell from activation of receptors to contraction. It has been able to predict and consolidate a large array of experimental data. A virtual muscle cell, activated by receptors, is now available for analyzing a large range of quantitative data and to plan future experiments.
2. The secretion of transmitter at nerve terminals.
Katz (see 234, 263) pioneered the mechanism of transmitter release, who first showed that transmitter is released in packages from terminals at the somatic neuromuscular junction on skeletal muscle. Bennett and his colleagues subsequently showed that such nerve terminals could be considered as electrical cables with strategic release sites for transmitter secretion occurring at regular intervals along their length (216, 221). Electrical analysis of the signals generated by these packages of transmitter showed that they are of uniform size (175). These release sites have different probabilities for transmitter secretion as can be shown by measuring the electrical fields around each of the release sites on transmitter secretion (233).
The key question arises as to what determines the different probabilities of release at the different sites. Modeling shows that the possibility of an autoinhibitory mechanism whereby a package released at one site on the cable inhibits release at another site is not adequate to explain the non - uniform probability (111, 168). Modeling of the kinetics of calcium movements that trigger the release of transmitter packages at a release site shows that the probability of release depends simply on the number of transmitter - package - associated secretory proteins (termed a synaptosecretosome (189)) that each synapse possesses (231, 232). A quantitative model of the facilitated release of such packages at different release sites along the terminal cable by a test impulse following a conditioning impulse at different intervals then gives a quantitative description of all experimental observations on transmitter release at short intervals (266).
3. The formation of synapses
In 1900 Langley showed that cutting the nerves to a ganglion results in growth of the nerves back into the ganglion in which they reconstitutes the previous pattern of innervation (for a history, see 212). In order to investigate the mechanism by which this occurs Bennett designed experiments on the reinnervation of skeletal muscle fibres where the site of synapse formation is in the middle of the fibres. On reinnervating a muscle the nerves only formed terminals at the original synaptic sites in the middle of the fibres (21). Even nerves from foreign muscles only formed synapses at these sites (22, 23), leading to the conclusion that synapse formation molecules must exist at the site which triggers nerves to form synapses there (34). A typographical distribution of nerves exists in a muscle that mirrors their rostro - caudal origin from the spinal cord (84). This is also reconstituted on reinnervation of a muscle indicating that synapse formation molecules possess typographical information.
A model based on the idea that the synaptic site possesses synapse formation molecules, which must be recognized, by appropriate molecules on the nerve terminals for a synapse to form has been developed (108). This quantitatively predicts all of the observations mentioned above as well as those carried out in many different laboratories. The theory has become known as the dual-constraint hypothesis for synapse formation (212). It has recently been extended to account for the role of growth factors as well as of synapse formation molecules in synapse formation (250).
Max Bennett has applied electrical engineering and applied mathematical principles to provide three major classes of quantitative models in order to describe the interaction between excitable cells. The first set of models has elucidated 'how current flows between smooth muscle cells on activation of transmitter receptors, leading to the initiation of impulses as well as how calcium signals in these cells leads to contraction. The second set shows how the probability of transmitter secretion from nerve terminals on skeletal muscle cells is determined by the spatial distribution of calcium channels and the influx of these ions. The final set of models provides a quantitative description of the minimal mechanisms that are involved in the formation of nerve terminals at synapses.
1. Discovery that nerve terminals exist in the peripheral nervous system that release neither noradrenaline nor acetylcholine (NANC synaptic transmission).
2. Identification of the first calcium action potential in either the vertebrate or invertebrate nervous systems (calcium action potential in smooth muscle cells).
3. Elucidation of the fact that synaptic transmission in smooth muscle occurs in an electrical syncytium (syncytial integration of synaptic transmission).
4.Determination of the fact that mature striated muscle cells possess molecules at particular sites on their surface which guide the formation of synapses by motor-nerve terminals there (synapse formation molecules).
5. Discovery that synapse formation molecules are induced on developing striated muscle cells by the ingrowing motor-nerve terminals.
6. First quantitative description of neuronal cell death in the developing mammalian central nervous system (retinal ganglion cell death).
7. Discovery that central mammalian neurons (retinal ganglion cells and motoneurons) can survive in vitro if supplied by factors derived from the cells on which they normally form synapses as well as from glial cells.
8. Determination that Schwann cells are at the leading edge of motor-nerve terminals as they grow into muscle to form synapses during development.
9. Discovery that each active zone of a single motor-nerve terminal possesses a distinct probability for secretion of the same size packet of transmitter
10. Discovery that individual varicosities of a single autonomic nerve terminal have different probabilities for secretion and different autoreceptors.
11. First description of calcium influxes at single synapses in an intact organ (varicosities of autonomic nerve terminals) following an impulse and that these f1uxes can occur spontaneously.
12. Discovery that ionotropic receptors at autonomic nerve terminals are organized in I mm diameter junctional clusters beneath individual varicosities as well as in 0.4 mm diameter clusters elsewhere over the muscle cells (localisation of P2x purinergic receptors).
13. Discovery that P2x receptor junctional clusters are formed under varicosities from the small clusters during development, and that their subunit composition is under hormonal control.
14. Discovery that terminal Schwann cells at mature and intact synapses are in a continual state of growing new processes that lead the formation of new functional synapses over periods of minutes.
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