Neurobiology Laboratory - Professor Max R. Bennett
(numbered references refer to those in the accompanying list of publications)
Research in the Neurobiology Laboratory has been concerned with elucidating the mechanisms governing the formation and function of synapses. This has involvedinvestigations using the most suitable preparations from the peripheral nervoussystem, particularly the autonomic and somatic neuromuscular junctions.
The mechanism of transmission at autonomic neuromuscularjunctions and synapses
NANC (nonadrenergic and noncholinergic) nerve terminals on smooth musclecells were discovered in the Laboratory in 1963(5). In the subsequent thirty yearsthis has produced a significant change in our understanding of the peripheralnervous system, with ATP, nitric oxide and neuropeptides now identified astransmitters. This work subsequently led in 1964 to the description of how theclassical transmitters noradrenaline and acetylcholine together with the NANCtransmitters act on receptors to produce permeability changes in smooth muscle ofthe kind expected from biophysical studies of synapses elsewhere, a result notthen expected for the autonomic neuroeffector junction (3). The specificity of thisjunction was then elucidated at the biophysical level by showing how smoothmuscle cells integrate direct synaptic inputs to their receptors together with thoseradiating indirectly into the cell through the syncytial couplings from the action onreceptors belonging to other smooth muscle cells, a concept involving syncytialintegration of transmission (141). This produced the standard model of theautonomic neuroeffector junction, described in detail in a Physiological SocietyMonograph 'Autonomic Neuromuscular Transmission' (15).
Studies of the mechanism of transmitter secretion at sympathetic neuroeffectorjunctions have been taken to a new degree of resolution by the introduction in theLaboratory of techniques for visualizing single live varicosities for the purposesof recording from these with loose-patch microelectrodes placed over thevaricosities (114, 120). This work has shown that there is a non-uniformprobability for secretion at individual varicosities along single terminals; each ofthese varicosities, however, shows the same calcium dependence for transmitterrelease independent of the probability for secretion on arrival of a nerve impulse.There is considerable variability in the size of the transmitter packets releasedfrom varicosities (142) which may arise because of the simultaneous release ofmore than one vesicle at a release site (144). These observations on singlevaricosities have recently been extended to single visualized boutons on pelvicganglion cells (168). Loose-patch electrode recordings from these show that theyrelease transmitter packets that saturate the postsynaptic receptor patch whichcontains only about 100 receptors; in this case it is not known how the transmitteris packaged. These observations on single boutons are the first to be made in vivoanywhere in the nervous system. The intraterminal calcium dependence of theincrease in efficacy of transmitter release by a test impulse following at differentintervals after a conditioning impulse at nerve terminals on ganglion cells hasbeen determined by introducing calcium indicators specifically into the terminals(148). This work has shown for the first time at any nerve terminal that there is alinear releationship between the intraterminal calcium concentration and theincreased efficacy of transmission associated with facilitation, augmentation andposttetanic potentiation.
The formation of somatic motor-nerve terminals and theirprobability for secretion
Research in the Laboratory established that reinnervating motor axons uniquelyform synaptic terminals if they make contact with denervated synaptic sites onstriated muscle, indicating unequivocally for the first time that this site containsinformation that triggers terminal formation (33). Subsequent research showed thatthis site is not present on developing muscle but is induced to form on the muscleby the first contacting motor axon (24). Once formed, the site becomes transientlyhyperinnervated by many axons whose later elimination gives rise to atopographical map between the motor neurone pool in the spinal cord and themuscle (102). These observations have given rise to the concept ofsynapse-formation molecules that contain information concerning the specificity ofthe synaptic connection and its topographical layout in the set of target cells, nowcalled the dual constraint hypothesis.
Somatic motor- nerve terminal are not homogeneous, some release sites or activezones in the terminal possess relatively high probabilities for secretion of aquantum of transmitter whilst others have virtually zero probability for release onarrival of the nerve impulse (87); there is no evidence for multiple packets ofrelease from an active zone at these terminals (163). These very low probabilityactive zones are mixed in amongst the high probability zones (103). The levels ofprobability are subject to hormonal control and are particularly plastic duringdevelopment, when all the active zones of a terminal may be turned off; this occursprior to the physical removal of a terminal during the competition of terminals fora synaptic site that accompanies the period of hyperinnervation (97). Theseobservations have led to the concept that there are endogenous factors concernedwith setting the distribution of secretion probabilities over a terminals releasesites (105;152).
The survival of central neurones
The Laboratory first showed that specific classes of neurones in the centralnervous system are provided with growth factors necessary for survival from cellsin their immediate environment (96). This involved introducing a method foridentifying specific classes of central neurones such as retinal ganglion cells, aftertheir dissociation into tissue culture, for the purposes of using them to bioassay forgrowth factors derived from other cells. He showed that rat retinal ganglion cellsdie in large numbers during normal development, although they can be saved fromdegeneration by growth factors provided by glial cells surrounding their cellbodies,then subsequently by glial cells surrounding their axons and finally by theirtarget cells. These observations gave the first quantitative study of neurone celldeath in the brain together with evidence that this could be prevented by factorsprovided by cells surrounding these neurones.
New concepts developed from research in theNeurobiology Laboratory.
1.Syncytial integration of junctional transmission.
The mechanism whereby electrically coupled smooth muscle cells integratedirect synaptic inputs on their receptors at autonomic neuromuscularjunctions with those radiating indirectly into the cells through syncytialcouplings.
2.NANC junctional transmission.
The first definitive evidence that transmission at autonomic neuromuscularjunctions may involve transmitters other than noradrenaline or acetylcholine.
3.Non-uniform properties of different varicosities at junctions.
The concept that transmission at the different varicosities of an autonomicneuromuscular junction involves different probabilities for the release ofpackets of transmitter.
4.Synapse formation molecules at motor-nerve terminals.
The idea that molecules reside at mature synaptic sites on striated musclecells that may trigger nerve terminal formation there; these molecules arecreated at the sites by motor- nerve terminals during development.
5.Plasticity of the probability for secretion at motor-nerveterminals.
The discovery that different release sites within a single motor-nerveterminal possess different probabilities for secretion. These probabilitiesmay be significantly reduced during development with subsequent regressionof the nerve terminal. They are also subject to modulation by seasonalchanges in amphibia.
6.Sequential selection of neurone survival.
The idea that central neurones compete for growth factors provided bydifferent cells in their environment in a sequential way during development.The only neurones that mature are those that survive all these selectiveprocedures.