NEUROBIOLOGY LABORATORY

Maxwell R. Bennett

  

  

RESEARCH

PERSONNEL in 1994 and 1995

Dr Maxwell R. Bennett Professor (in-charge) University 1969-
Dr Leonard G. Motin Postdoctoral Fellow ARC 1994-
Dr Lynne J. Cottee Research Officer (0.5) NHMRC 1993-
Lorraine Kerr Research Officer (0.5) NHMRC 1994-
Les Farnell Research Officer (0.5) ARC 1994-
*Yon-Qi Lin Research Officer (PhD 0.8, from '95) ARC 1990-
*Gregory T. MacLeod Research Assistant (0.6 in '94; casual+PhD from '95) NHMRC 1991-
Michael I. Stewart Research Assistant (0.5) ARC 1995-
Rowena Henery PhD student (Supervisor: W. Gibson, Dept of Maths) NHMRC 1995-
Dr Kerry A. Nichol PhD student (RA 0.6 in '94; SRO 0.4 from '95) NHMRC 1988-
*Shanker Karunanithi PhD student (RA 0.6 in '94; 1.0 from '95) NHMRC 1989-
Matthew Larkum PhD student APA 1991-
*Darren A. Warren PhD student 1992-
Jennifer Kearns PhD student (Supervisor: W. Gibson, Dept of Maths) 1994-
*Maria Theodorou BMedSc(Hons) student 1994
Keith L. Brain BSc(Med)(Hons) student in '94; PhD from '95 APA 1994-
Melissa J. Bradley BSc(Med)(Hons) student 1994
Chandra Balachandran BSc(Med)(Hons) student 1994
Glen W. Bennett BSc(Med)(Hons) student 1994
*Svetlana Cherepanoff GradDipSc student (0.5) 1993-94
*David Kyunghwan Kim BMedSc(Hons) (co-supervisor in '95: N. Lavidis) 1995
Ayesha Hakim BMedSc(Hons) (co-supervisor: R. Dampney) 1994
Thomas Kim BMedSc(Hons) student 1995
Justin Morgan BMedSc(Hons) student 1995
Andrea Santoro BMedSc(Hons) student 1995
Marcha Tuyau BMedSc(Hons) student (co-supervisor: R. Dampney) 1995
Jennifer Cantrill Administrative Assistant (0.2) ARC 1982-
Zofia Dreher Senior Research Officer (0.5) ARC 1993-

* Indicates Max Bennett: Supervisor; Nickolas Lavidis: Associate Supervisor
Current effective full-time personnel (with all co-supervised taken as 0.5) = 12.8

  
Research in this Laboratory is concerned with understanding the determinants of secretion of neurotransmitters at nerve terminals and processes involved in the plasticity of these terminals.
  

PROJECTS in 1994

Mechanisms of transmitter release at sympathetic varicosities
Ultrastructural reconstruction of single sympathetic-nerve varicosities in mouse vas deferens has shown that these can come into closest apposition of between several hundred nanometres to forty nanometres of smooth muscle cells. Theoretical analysis of secretion of ATP from these shows that several hundred ATP molecules must be released in a quantum if the characteristics of the junctional current are to be simulated. Statistical analysis of the occurrence of pairs of junctional currents of like amplitude from varicosities does not support the conjecture that ATP secretion is always monoquantal.

Postganglionic sympathetic nerves to the vas deferens also contain nitric oxide synthase. The Laboratory has been able to show that release of endogenous nitric oxide increased the contractile activity of the vas deferens without apparently modulating the transmitter release.

Syncytial integration of autonomic transmission
Sympathetic neuromuscular transmission occurs to smooth muscle cells in an electrically coupled synctium. We have previously described how current flows in the syncytium following the secretion of a quantum of transmitter. This was based, however, on an approximate representation of a smooth muscle cell as a lumped resistance and capacitance. This year we have solved the problem for a syncytium in which the smooth muscle cells are represented by a distributed cable network. Quantal secretory mechanisms at preganglionic nerve terminals.

The changes in calcium concentration during facilitation, augmentation and post-tetanic potentiation have been determined for pre-ganglionic calyciform nerve terminals in avian ciliary ganglia. The different time courses of elevated transmitter release were accompanied by identical periods of elevated calcium. Quantal secretion from single boutons on pelvic ganglion cells shows that these are likely to be monoquantal. The existence of multiquantal secretion seems to be related to the occurrence of closely apposed boutons, as revealed by confocal microscopy.

Nitric oxide control of autonomic synaptic transmission
We have shown that nitric oxide released in ciliary ganglia modulates transmitter release and can give rise to long-term potentiation. The existence of cAMP and PKC systems in the ganglia has been established, together with the fact that their non-hydrolysable analogues promote transmitter release. Nitric oxide has now been shown to lead to phosphorlyation of a protein in the embryonic ganglion, probably through cAMP, and that these embryonic ganglia also show enhanced transmitter release when exposed to nitric oxide.
Preganglionic sympathetic neurones contain nitric oxide synthase
In collaboration with Dr Dampney we have shown that endogenous nitric oxide released at these neurones has a powerful effect on renal nerve activity and hence on blood pressure.
Mechanisms of transmitter release at neuromuscular junctions
An experimental and theoretical study has been completed on quantal secretion at small numbers of release sites on visualized motor-nerve terminal branches, which shows that the stochastics of secretion can be modelled by a three component process: one involves the opening of voltage-dependent calcium channels and the diffusion of transmitter to the vesicle-associated proteins; second is the binding of calcium ions to a strategic protein (possibly synaptotagmin) and its conformational change; finally, there is the exocytosis of the synaptic vesicle. No autoinhibitory effects were detected in the release process itself. The theoretical conditions that must be fulfilled for the detection of subquantal release have been determined, but as yet no physiological evidence for this has been obtained. The theory of how spontaneous quantal secretion occurs following the spontaneous opening of a voltage-dependent calcium channel is complete; this is now being extended to the evoked quantal secretion following an impulse.
Computational role of hippocampal synapses
Having completed our theory of how mossy fibre synapses on CA3 pyramidal cells and their recurrent collaterals on NMDA receptors can be used to recall associative memories, we have sought to give an account of how the recurrent synapses can have their probability for secretion permanently up-regulated without a change in transcription in the neurone. In the experimental work, we have concentrated on our discovery that ATP antagonists modulate glutamate transmission in the CA3 and CA1 regions of hippocampus and that both ATP and non-hydrolysable ATP analogues produce currents in these neurones. ATP can generate long-term potentiation in CA3 and CA1 pyramidal neurones.
Plasticity of central neurones
Retinal ganglion cell survival following lesions to the optic nerve can be enhanced by supplying the vitreous with a proteoglycan isolated from the superior colliculus. We have recently shown that this proteoglycan controls the levels of calcium in the ganglion cells and that this may be the principal method by which it controls survival.
  

RESEARCH PLANNED for 1995

Mechanisms of transmitter release at sympathetic varicosities
We are at present determining theoretically how the secretion of noradrenaline from a varicosity modulates the secretion of ATP from other varicosities in a smooth muscle.

In addition an attempt is being made to identify single varicosities with the confocal microscope, as well as the distribution of P2-purinoceptors at these varicosities, after their quantal secretion probability has been determined. The vesicle-associated proteins concerned with the quantal release process are also being identified. In addition, quantal release at varicosities on blood vessels in the vas deferens is being determined, together with the interaction between quantal release probabilities at adjacent varicosities on the same terminal branch. The possibility that the slow evoked synaptic currents that occur at varicosities are due to a transmitter other than ATP is also under consideration.

Syncytial integration of autonomic transmission
Present work is now attempting to introduce a stochastic distribution of gap junctions into our model of the smooth muscle syncytium. There is also an effort to give a more realistic representation of current flow around an external recording electrode placed on a varicosity. The experimental work is concerned with determining the flow of current when several varicosities are recorded from, together with the differential of their synaptic potentials, in the mouse versus the guinea-pig vas deferens. The changes in these synaptic currents under conditions in which the electrical gap-junction coupling is broken is also being investigated.
Quantal secretory mechanisms at preganglionic nerve terminals
Our present effort is to identify the calcium-sequestering events responsible for the different time courses of increased synaptic efficacy following a train of preganglionic impulses, as well as to identify how they are modified, together with quantal secretion, by co-transmitters released from the terminal. The distribution of different vesicle-associated proteins in the terminal are also being investigated, along with the effects of antibodies to these on transmission. In addition an attempt is being made to use the loose-patch clamp technique on these preganglionic terminals to determine the channel currents that give rise to synaptic currents at boutons.

Nitric oxide leads to the phosphorylation of a protein in ciliary ganglia, and our present attempt is to identify this protein. One interesting possibility is that an ATP-dependent calcium-activated potassium channel which we have characterized may be the protein. Nitric oxide also promoted transmission at nerve terminals on preganglionic nerve terminals in the spinal cord.

Our present investigation is to examine the mechanism whereby nitric oxide exerts this effect at the spinal cord level, using our knowledge of the cellular mechanisms elucidated on ciliary ganglia to guide this research.

The existence of nitric oxide synthase in both preganglionic and postganglionic neurones has prompted a new research programme, in which we are investigating the role of nitric oxide in cell death of these neurones and the role of calcium in this process.

Mechanisms of transmitter release at neuromuscular junctions
The possibility that autoinhibitory processes occur over large but not small distances between release sites is now being investigated for individual motor-nerve terminal branches.

We are also at present developing an analysis of the physico-chemical events that are responsible for the exocytotic event at the motor-terminal release sites.

Computational role of hippocampal synapses
Our present theoretical work aims to give a more biologically plausible account of transmission at recurrent synapses on CA3 pyramidal neurones, including details of the exocytotic mechanism and how this changes during the laying down of memories. In particular, we are developing a continuous model of the recurrent network operation in which biologically plausible recurrent synapses are embedded. This will allow for the most detailed model of how memories are formed. Our present experimental investigations centre on the role of ATP in potentiation of transmission in the CA3 region of the hippocampus. In particular, the effect of ATP and glutamate on AMPA receptors in isolated patches from pyramidal neurones is being determined, together with possible modulation of the calcium levels in these neurones by ATP.
Plasticity of central neurones
Present work aims at determining the source of calcium that mediates the neurone survival enhancing capacity of proteoglycans and, if extraneuronal, what the membrane mechanism is that is mediating the action of the proteoglycan.
  

COLLABORATIONS

Quantal secretory mechanisms in the hippocampus:
Prof. Steve Redman, Division of Neuroscience, John Curtin School of Medical Research, ANU (1991-present).

Nitric oxide secretion at terminals:
Prof. Peter Dunkley and Dr John Rostas, School of Medicine, Univ. of Newcastle (1993-present).

Distribution of purinergic receptors in the brain:
Dr Vladimir Balcar, Univ. of Sydney (1994-present).

Models of the secretory process at nerve terminals, especially in the hippocampus:
Prof. John Robinson and Dr Bill Gibson, School of Mathematics & Statistics (1994-present).

Phosphorylation processes in nerve terminals:
Prof. Peter Dunkley and Dr John Rostas, School of Medicine, Univ. of Newcastle (1994-present).
  

FACILITIES

The Laboratory is located in rooms 125, 131, 133, 134, 137, 139 and 145A of the Anderson Stuart Building. Prof. Bennett's office is room 138. Electrophysiology facilities include 8 separate units, each consisting of computers, patch-clamp and voltage-clamp amplifiers, microscopes and computer-driven stimulators, as well as digital imaging. Room 125 is a fully-equipped biochemistry laboratory that includes tissue culture facilities, confocal Leitz microscope, FPLC protein purification system and Coulter Cell Sorter.
  

THESES PASSED in 1994

PhD

Karunanithi S (1994) The role of ATP in somatic and sympathetic neuromuscular transmission.

Nichol KA (1994) The regulation of retinal ganglion cell numbers during development.
  

SCHOLARSHIPS

Australian Postgraduate Awards

Larkum M, 1991-present.

Brain K (1994).
  

OTHER RESEARCH ACTIVITIES in 1994

Australian science administration

Australian Academy of Science

Member, Science Policy Committee (1984-present.

Member, Boden Conference Committee (1986-present).

Member, NSW Science Policy Committee (1991-present).

Australian National University

Member, Scientific Advisory Committee, National Research School of Biological Sciences (1991-present).

Reviewer, National University Strategic Development Fund.

Australian Medical Research and Development Corporation (AMRAD)

Member, Scientific Advisory Committee (1992-present).

Conference organization

Australian Neuroscience Colloquium, Newcastle (Oct).

Service to scientific societies

Foundation Chairperson, International Society for Autonomic Neuroscience (ISAN).

Member, Programme Committee of World Congress of Neuroscience (IBRO), Australian representative, Japan.

Convenor; responsible for Draft Constitution of The Sydney Institute for Biomedical Research.

University administration

Member, Academic Board.

Member, Faculty of Science Board of Post-graduate Studies.

Member, Faculty of Medicine Board of Post-graduate Studies.

Member, Standing Committee of Faculty of Medicine.

Co-ordinator for higher degree students enrolled through Dept of Physiology.

Refereeing

Journal articles:
for Neuroscience (2), Neuroscience Letters (6), Brain Research (2), Biophysical Journal (1).

Grant applications:
for NHMRC (6), NHMRC Fellowships (3), NHMRC Program Grants (2), ARC (6), ARC Fellowships (2), ANU (John Curtin School) Project Grants (12).
  

5-YEAR RESEARCH PUBLICATIONS

MOST RECENT TOTAL ANNUAL CITATIONS (for 1993): 195

  

FUNDING in 1994 and 1995

ARC Computational role of hippocampal
synapses
Bennett MR 1994 $110,000
Robinson J 1995 $112,573
Gibson W 1996

NHMRC Mechanisms of transmitter release
at neuromuscular synapses
Bennett MR 1994 $51,058
Lavidis N 1995 $51,815
1996

NHMRC Mechanisms of transmitter release
at sympathetic varicosities
Bennett MR 1994 $67,673
Lavidis N 1995 $68,755
1996

NHMRC Syncytial integration of
autonomic transmission
Bennett MR 1994 $35,240
Gibson W 1995 $35,804
Phillips W 1996

NHMRC Quantal secretory mechanisms
at preganglionic terminals
Bennett MR 1993
1994 $49,441
1995 $50,232

ARC
(Small)
Secretion and plasticity of
retinal ganglion cell terminals
Bennett MR 1993
1994 $24,500
1995 $24,500

NHF Nitric oxide control of synaptic
transmission
Bennett MR 1994 $45,000
Dunkley P 1995 $41,715
Rostas J

ARC
(Small)
The function of Hebbian
synapses
Bennett MR 1995 $21,030
Gibson WG 1996
Robinson J 1997

Plus confocal microscope equipment grants, shared with 3 other members of the Department
  
R.D. Wright Fellowship
NHMRC The effects of opiates on the
probability of quantal secretion
at sympathetic varicosities
(*Includes $10,000 maintenance)
Lavidis NA 1992
1993
1994 *$66,345

Total for 1994: Grants: $382,912; Fellowship to N. Lavidis: $66,345; Grand total: $449,257

Total for 1995: Grants: $406,424

  

  

TEACHING

COURSES TAUGHT

Medical Science: Human Life Sciences 2

Lectures: 18, on the brain and its functions.

Practical classes: 1, of 3 h, presented twice, on reflexes.

Tutorials: 3 on lecture material.

Examination: 6 short essay questions

Medical Science 3 (Advanced Neuroscience)

Lectures: 18, on the molecular basis of brain function.

Practicals: 16, each of 4 h, on electrophysiology of synaptic transmission.

Tutorials: 8, on scientific papers and 4 on neural network theory.

Examination: 5 short essay questions.

Medicine 3

Lectures: 1 Distinction lecture on the hippocampus.
  

TEACHING HOURS in 1994

HLS 2 MedSc 3 Med 3 Total
Lectures 18 18 1 37
Practical classes (no.) 6 (2) 64 (16) - 70
Tutorials (1 h each) 3 12 - 15

Total formal contact teaching time = 122 h

Time was also spent on lecture preparation, setting and marking of exams and essays.
  

  

ADMINISTRATION

(see also OTHER RESEARCH ACTIVITIES in 1994)

University administration

Member, Academic Board.

Member, Faculty of Science Board of Postgraduate Studies.

Member, Faculty of Medicine Board of Postgraduate Studies.

Member, Standing Committee, Faculty of Science.

Departmental administration

Co-ordinator for higher degree students

Course supervisor

Advanced Neuroscience 3 for BMedSc.