Only the most significant papers have been listed; these do not include biophysical modelling papers; for a complete set see Neurobiology and Biophysics of Synapses: Click Here
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Discovery I
Discovery II
Discovery III
Discovery IV
DISCOVERY I:
That there exists transmitter substances in the autonomic nervous system that are neither noradrenaline nor acetylcholine (for which I introduced the acronym NANC synapses), one of which is Adenosine Triphosphate (ATP; later termed Purinergic transmitters [1]).
BENNETT, M.R. (1965) A Study of Transmission from Autonomic Nerves to Smooth Muscle. MSc Thesis. University of Melbourne
BENNETT, M.R., BURNSTOCK, G. & HOLMAN, M.E. (1966). Transmission from intramural inhibitory nerves to the smooth muscle of the guinea-pig taenia coli. J. Physiol. (Lond.) 182: 541-558.
NANC (Non-Adrenergic, Non-Cholinergic) transmission. Individual inhibitory junction potentials (IJPs) in a taenia coli muscle, recorded with an intracellular electrode, before (a) and after (b) bathing the muscle with cholinergic (atropine) and noradrenergic (guanethidine) blocking drugs. No change was observed in the characteristics of the IJP due to single stimulations, indicating that the nerves were not releasing acetylcholine or noradrenaline. This was the first identification of NANC transmission (from Fig. 12 in chapter 2 of MSc thesis of Bennett (1965); also see Bennett et al., 1966)
LEMON, G., BROCKHAUSEN, J., LI, G.H., GIBSON, W.G. & BENNETT, M.R. (2004) Calcium
Mobilization and spontaneous transient outward current characteristics upon agonist activation of P2Y2 receptors in smooth muscle. Biophys. J. 88: 1507-1523
P2Y2-GFP in A7r5 cells under different experimental conditions. (A) Confocal images of A7r5 cells showing the clustering of P2Y2-GFP in the membrane 24 h after transection (a), and then the gradual loss of many of the clusters after exposure of the cells to 10 mM UTP for 1 min (b), 2 min (c) and 5 min (d); higher magnification inserts in a and d are taken from the solid square regions and show dense clusters of P2Y2-GFP in a that are largely lost in d. Note that in addition to the loss of many of the clusters, there is a gradual rounding up of cells in the presence of the agonist. (B) Confocal images of P2Y2-GFP as in A, but this time in the presence of monensin (5 mM); note no loss of P2Y2- GFP fluorescence, but the cells still round up. (C) Confocal images of P2Y2-GFP as in A, but this time in the presence of suramin (10 mM); note again no loss of P2Y2-GFP as in A, but this time in the presence of suramin (10 mM); note again no loss of P2Y2-GFP fluorescence, and in this case the cells do not round up. Calibration bar is 10 mm. (from Fig. 4 in Lemon Brockhausen, Li, Gibson, Bennett, 2004).
[1] For 50 years an incorrect account of these discoveries was widely disseminated. The correct account, given by the person that did the experiments, is clarified in the following correspondence:Bennett, M.R. 2013 The discovery of a new class of synaptic transmitters in smooth muscle 50 years ago and amelioration of coronary artery thrombosis. Acta Physiol (Oxf) 207(2):236-243
Burnstock, G.. 2013. Letter to the Editor. Acta Physiol (Oxf) 208, 138.
The discovery of non-adrenergic, non-cholinergic transmission: reply to the criticisms of G.Burnstock (2013) (GB) and Abbracchio et al. (2013) (A et al.) of M. Bennett’s (MB) account of this history in Acta Physiol (2013) Feb; 207
(2):236–43. Acta Physiol 2013, 292-293
HANSEN,M.A., BALCAR,V.J., BARDEN,J.A., BENNETT,M.R. (1998) The distribution of single
P2X1-receptor clusters on smooth muscle cells in relation to nerve varicosities in the rat urinary bladder. J.Neurocytol. 27(7): 529-539.
Confocal images of the distribution of P2x1 receptor clusters with respect to single varicosities labelled with AbSV2 on smooth muscle cells in a region of the detrusor smooth muscle. In this double-label experiment, AbSV2 immunoreactivity is shown on the left (A), rendered in green, with AbP2x1 immunoreactivity on the right (B), rendered in red. A color merge of the two images is presented in a stereo view (C, D). In (A), the arrowhead points to an SV2-labelled varicosity not co- localized with a P2x1 receptor-cluster representing about 4.5% of such clusters. The arrowheads in (B) point to large P2x1 receptor clusters not co-localized with SV2 and associated with varicosities; the arrow points to an example of a small P2x1 receptor cluster and associated with varicosities. The arrowhead in (D) points to an example of a group of large P2x1 receptor clusters co-localized with varicosities. Calibration: 19 um. Images (E), (F) and (G) show individual varicosities with SV2 staining green and P2x1 staining red. (E) shows a varicosity that appears to be almost surrounded by P2x1 labelling. (F) and (G) show receptors that are confined to an ellipse that almost, but not quite, represents the projection of the varicosity onto the XY plane. Scale bars: 0.5 um. (H) is a low magnification view demonstrating the overall labelling observed in a cryosection of rat bladder detrusor. Calibration: 50 um. The boxed area in (H) is magnified in (I) and (J), which demonstrate the co-localization of SV2 and P2X2 labelling of large varicosities on a nerve. (I) shows SV2 immunoreactivity in green and (J) shows P2X2 immunoreactivity in red. Scale 5 um. (from Hansen et al., 1998).
BRAIN,K.L. & BENNETT,M.R. (1997) Calcium in sympathetic varicosities of mouse vas deferens during facilitation, augmentation and autoinhibition. J. Physiol. (Lond.) 502 (3): 521-536.
Calcium concentration in varicosities following a short tetanus. Images of Oregon-BAPTA intensity in three varicosities of a single nerve terminal at 10 different times prior to, during and after stimulation at 5 Hz for 5 impulses. The impulse commenced at zero time, with the number giving the time in seconds. Every second image collected is shown. The calcium concentration increases during the train and then declines, following the end of the train, towards its resting concentration. Calibration bar is 2 um. In the scale at the right, the upper part corresponds to the greatest measured fluorescence intensity and thus the highest calcium concentration. The scale is linear with respect to the fluorescent intensity (From Brain and Bennett, 1997).
DISCOVERY II:
Of the formation of synapses by growth cones and their subsequent pruning during development (first made at the neuromuscular synapse with PhD student Alan Pettigrew) and of naturally occurring death of CNS neurons at early developmental stages (first made with regard to retinal ganglion cells with honours student Rebecca Potts).
BENNETT, M.R. & PETTIGREW, A. G. (1974). The formation of synapses in striated muscle during development. J. Physiol. (Lond.) 241: 515-545.
Distribution of nerves in the rat hemidiaphragm at 15 days gestation. A, the primary (phrenic) nerve (p) enters near the centre of the hemidiaphragm and divides into two secondary nerve branches (s) which traverse the myotubes (my) at right angles and extend to the pools of myoblasts (mb) at the ends of the hemidiaphragm. Smaller tertiary nerve bundles (t) leave the secondary nerve trunks and run in general parallel to the mytubes and obliquely into the pools of myoblasts. The tertiary nerve branches extend almost the entire length of those myotubes which are adjacent to the pool of myoblasts. Calibration, 100 um. b, tertiary nerve bundles at the end of the secondary nerve trunk radiating into the pool of myoblasts (mb) at the end of the hemidiaphragm. Calibration, 50 um. c, single axons leaving the tertiary nerve branches at a position 1 mm from the point of nerve entry into the hemidiaphragm and growing out between and along the myotubes (my) in this region. The tertiary nerve bundles arise from the secondary trunk (s) at the bottom of the plate. Some axons have been retouched since they pass out of the plane of focus of the three photomicrographs use to make up the plate. Calibration, 30 um. (From Bennett and Pettigrew, 1974).
Frequency histograms of the number of nerve terminals per muscle cell of the rat hemidiaphragm, determined by electrophysiological means, during the last 6 days gestation and the first 4 weeks post-natal. Bennett and Pettigrew (1974)
DREHER, B., POTTS, R.A. & BENNETT, M.R. (1983). Evidence that the early post-natal reduction in the number of rat retinal ganglion cells is due to a wave of ganglion cell death. Neurosci. Lett. 36: 255-260.
Estimated number of cells containing HRP reaction products in retinal wholemounts of rats of different ages. The ages indicated on the abscissa refer the ages of the animals at the time of the perfusion. The block dots indicate the estimated number of labelled cells in the animals in which HRP had been injected 15-20 hrs before the perfusion. The triangles indicate the estimated numbers of labelled cells in the retinae of animals in which HRP was injected 12 hrs before birth. Finally, the open circles indicate the estimated numbers of labelled cells in the retinae contralateral to the ablated (within 12 hrs of birth) superior colliculus. HRP was injected in these animals 15-24 hrs before the perfusion. Inset: micrographs of the HRP-labeled cells in the whole mounted retina of 8-day old rat. HRP was injected on day one (5 hrs after birth of the animal). The picture was taken under a 100 x oil immersion objective. The photographed area is located about 1 mm from the periphery of the retina. Calibration bar = 10 um. Note that a number of cells located in the ganglion cell layer do not contain HRP reaction products. (from Dreher, Potts and Bennett, 1983).
MACLEOD, G., DICKENS, P. & BENNETT, M. (2001) Formation and Function of Synapses with Respect to Schwann Cells at the Motor Nerve Terminal Branches on Mature Amphibian (Bufo marinus) Muscle. J Neurosci. 21(7), 2380-2392.
Individual clusters of synaptic vesicles, indicated by FM1-43 blobs, can appear or disappear from the ends of terminal branches over hours. F1-43 blobs are shown along the distal portions of six terminal branches (A-F) after an initial staining with FM1-43 (left panel) and the same terminal branches are shown after restaining 16 hrs later (right panel). A-C show the appearance of new clusters of FM1-43 stained vesicles on these terminal branches (asterisk), whereas D-F show the disappearance of such clusters over the same period (asterisk). The scale bar shown in A is the same for all images. (from Macleod, Dickens and Bennett, 2001).
The intensity of FM1-43 staining along the entire length of terminal branches of motor- nerve terminals. A: simple terminal branch, about 40 um long, stained with FM2-43. P and D indicate the most proximal and most distal points of the branch, respectively. B: profile of the intensity of the FM1-43 staining along the branch at intervals of 0.13 um. C: average amount of FM1-43 fluorescence per blob in each one-tenth of the terminal length along the branch in A. D: average distance between the midpoint of the blobs in each one-tenth of the terminal length for the branch in A. E: complex of what is perhaps two parallel terminal branches, probably in the same synaptic gutter, stained with FM1-43. P and D are defined as in A. F-H: same as B-D, respectively; no allowance has been made for the fact that there may be two terminal branches present. Calibration in A is given by the abscissa in B and the abscissa in F gives the scale for E. (from MacLeod, Gan and Bennett, 1999).
DISCOVERY III:
That the loss of grey matter in particular parts of the brain following stress is due to the loss of synapses in these parts that leads to the regression of dendrites (this work required the guidance of a multidisciplinary research team).
KASSEM, M.S., LAGOPOULOS, J., STRAIT-GARDNER, T., PRICE, W.S., CHOHAN, T.W., ARNOLD, J.C., HATTON, S.N., & BENNETT, M.R. (2013) Stress-induced grey matter loss determined by MRI is primarily due to loss of dendrites and their synapses. Mol Neurobiol. 47(2):645- 61
Significant decreases occur in stressed animals in the cumulative length of apical dendrites of neurons, that is the sum of all different order dendrites in the Anterior Cingulate Cortex and CA1 hippocampus. a, photomicrograph of Golgi-stained pyramidal CA1 neurons of a non-stressed mouse. b, dendrites (at the same magnification as in (a) of a pyramidal CA1 neuron of a stressed mouse with shorter cumulative dendritic length (from Kassem et al., 2013).
Volume of grey matter taken up by different volumes of its cellular constituents and their processes. Percentages are determined from the review of existing literature as calculated in Table 5 in Kassem et al., 2013
In stressed animals, there is a linear relationship between the loss of Grey Matter Volume determined with high resolution Magnetic Resonance Imaging and the cumulative loss of dendritic volume such that the former can mostly account for the latter. (from Kassem et al., 2013).
DISCOVERY IV:
That the negative BOLD signals observed with fMRI s can be predicted using the Hyder-Rothman-Bennett theory relating synapse activity to energy utilization (critically requiring the expertise of my close colleague, Dr Bill Gibson)
BENNETT,M.R., FARNELL, G., & GIBSON, W. (2019) Quantitative relations between transient BOLD responses, cortical energetics, and impulse firing in different cortical regions. J Neurophysiol. 122(3):1226-1237
Plots of the blood oxygen level-dependent BOLD signal , ΔS/S0, as a function of time (t) for various neuronal inputs Δ<u>, calculated for comparison with the observations of Hanlon et al., (2016) on patients with schizophrenia (SP) compared with healthy control subjects (HC). The baseline firing rate is (<u>)0 = 1.2, and the Hyder-Rothman-Bennett (HRB)-Friston theory is used to calculate ΔS/So. In each case the neuronal input Δ<u>is shown as a dashed line of the same color as the corresponding BOLD signal (scale on the right axis): blue for healthy control subjects and red for schizophrenic patients. They compared with curves in Figure 2B of Hanlon et al., (2016): aDMN, A2 and PCUN. Note that excitatory impulse firing is not turned off, after -6s, to nearly the same extent in the schizophrenia patients compared with control subjects. aDMN, anterior node of the default mode network; A2, secondary auditory cortex; PCUN, precuneus. (from Bennett, Farnell and Gibson, 2019).
Flow chart showing the steps in the models considered: normalized cerebral blood flow (CBF) f = CBF/(CBF)0, normalized cerebral metabolic rate of oxygen (CMRO2) r = CMRO2/(CMRO2)0, normalized cerebral blood volume (CBV) v = CBV/(CBV)0, normalized deoxyhemoglobin content (dHb) q = _dHb/(dHb)0, E = oxygen extraction fraction (OEF), E0 =(OEF)0. The input to the Friston model is a scaled neuronal activity, z; the input to the Hyder-Rothman-Bennett (HRB) model is the baseline neuronal firing frequency (<μ>)0 together with the change in frequency due to activity Δ<μ>. The HRB model can then be interfaced to either the Davis model (path HRB-Davis) or the Friston-Buxton model (path HRB-Friston) to give an expression for the incremental blood oxygen level-dependent (BOLD) signal, ΔS/S0. (from Figure 1 in Bennett, Farnell & Gibson, 2019).