NARCOTICS RESEARCH LABORATORY

Nickolas A Lavidis


RESEARCH REPORT

Personnel in 1996 and 1997

Dr Nickolas A Lavidis      Senior Research Officer (NHMRC)/Senior Lecturer           1978-96
Dr Kerry A Nichol          Research Officer (0.2) NHMRC                              1996-
Darren Warren              PhD student                                               1992-
David Kim                  PhD student                                               1995-
Justin Morgan              PhD student                                               1996-
Irene Chin                 PhD student                                               1996-
Elizabeth Fletcher         BSc(Dent)(Hons)                                           1996
Marco D'Arbe               BSc(Hons)                                                 1997
Those with M Bennett Supervisor, NA Lavidis Assoc Supervisor:
Greg T Macleod             PhD student                                               1995-
Yee Q Ling                 PhD student                                               1995-

This Laboratory is concerned with identifying the changes in effectiveness in nerve to nerve communication during the development of tolerance and dependence to drugs such as morphine, heroin and clonidine (a drug which has been used to lower blood pressure and, as a replacement for methadone to reduce the severity of acute heroin withdrawal).

RESEARCH in 1996

Neurotrophic factor(s) released from vasa deferentia during chronic morphine treatment

EK Fletcher, KA Nichol, NA Lavidis

Acute administration of morphine to the mouse vas deferens producess a decrease in communication between sympathetic varicosities and smooth muscle. Chronic morphine treatment (CMT) induces an increase in the efficacy of transmitter release from sympathetic varicosities resulting in a decrease in the effectiveness of morphine to inhibit transmitter release. It has recently been shown that synaptic activity can alter the production of neurotrophic factors. Neurotrophic factors have been shown to increase the evoked synaptic currents and the frequency of spontaneous transmitter release possibly by increasing the phosphorylation of synapsin I and/or phosphorylation of voltage dependent calcium channels. The possibility that the increase in efficacy of neurotransmission at sympathetic varicosities induced by chronic morphine treatment (CMT), may parallel an increase in the levels of neurotrophic factor(s) released by smooth muscle cells of the mouse vas deferens was examined. CMT mice received morphine, 10 to 100 mg/kg, 3 times/day, for 7 to 9 days, were anaesthetised with halothane and killed by cervical fracture. The left vas deferens from each animal was electrophysiologically studied using small diameter electrodes (10 to 15 µm) to record excitatory junctional currents (EJCs) from known numbers of sympathetic varicosities. The right vas deferens was transversely cut into small pieces of 0.5 mm and placed on poly-l-ornithine coated 35 mm tissue culture dishes containing serum free defined media. Each plate contained a whole dorsal root ganglion (DRG) obtained from 9 to 11 day chick embryos. After 27 hours incubation the cultures were fixed with 2.5% glutaraldehyde in phosphate buffer and videotaped. Dorsal root ganglions (DRGs) cultured in the presence of CMT vasa deferentia exhibited 2 to 5 times greater neurite outgrowth than control vasa deferentia (n = 12). Most of the increase was reduced by anti-NGF. The prescence of neurotrophin-3, neurotrophin-4 and brain derived neurotrophic factor was also investigated. Thus extent of neurite outgrowth correlates with probability of transmitter release.

The role of N- and P- type calcium channels in regulating transmitter release from sympathetic varicosities of the mouse vas deferens

DK Kim, NA Lavidis

Evoked transmitter release from the sympathetic varicosities of the mouse vas deferens is highly dependent on extracellular calcium ([Ca2+]o). Most spontaneous transmitter release is also dependent on [Ca2+]o with about 20% of the spontaneous release persisting in zero [Ca2+]o. Of the 5 known voltage dependent calcium channels known only N- and P- type calcium channels have been shown to affect transmitter release at the sympathetic varicosities of the guinea-pig vas deferens. In this study the effect of various calcium channel blockers on spontaneous and evoked transmitter release has been investigated in order to identify the types of voltage-dependent calcium channels found on the presynaptic membrane of the sympathetic varicosities of the mouse vas deferens.

Sympathetic varicosities were visualised using DiCO2-fluorescence before placing an extracellular electrode over small groups of varicosities to record both evoked (EJCs) and spontaneous excitatory junctional currents (SEJCs). N- type calcium channels were blocked with w-conotoxin (w-CTX) GVIA while P- type calcium channels were blocked w-CTX MVIIA and L- type calcium channels with nifedipine. w-CTX MVIIA (16 nM) decreased evoked average EJC amplitude by 19.6 ± 10.6% (n = 5) and in one preparation there was no effect. w-CTX GVIA (40 nM) decreased evoked average EJC amplitude by 79 ± 8% (n = 6). Nifedipine (10 µM) did not effect evoked EJC amplitude (n = 3). SEJC frequency was not affected by any of the calcium channel blockers used.

Evoked transmitter release was found to be dependent on calcium entry predominantly via N-type calcium channels and partly P-type calcium channels. Spontaneous transmitter release, although greatly reduced in [Ca2+]o, was not affected by N-, P- or L- type calcium channel blockers.

The effect of chronic clonidine treatment on transmitter release from sympathetic varicosities of the mouse vas deferens

JJ Morgan, R Einstein and NA Lavidis

a2-adrenoceptor agonist (clonidine) and opiate agonist (morphine) decrease evoked transmitter release from sympathetic varicosities of the mouse vas deferens. The decrease in release is achieved without any noticeable change in the shape of the nerve terminal impulse (NTI). The inhibitory effect of morphine on transmitter release is reduced during chronic morphine treatment (CMT) of animals. Furthermore acute morphine withdrawal from CMT induces an enhancement of transmitter release. In this study we examined the effect of chronically clonidine treating animals on transmitter release efficacy.

Mice were treated twice per day with clonidine (1 - 5 mg/kg) for between 3 and 9 days. Vasa deferentia were isolated and treated with DiOC2 (0.1 µM) and fluoresced to visualize the sympathetic varicosities. Microelectrodes with tip diameters of 10 to 20 µm were placed over 3 to 5 varicosities to record the NTIs, spontaneous excitatory junctional currents (SEJCs) and excitatory junctional currents (EJCs). In most (11/14) control (untreated) preparations bath applied clonidine (1 µM) completely abolished EJCs but did not affect SEJC frequency. For the remaining 3 preparations the EJCs were greatly reduced. In chronically clonidine treated (CCT) preparations bath applied clonidine (1 µM) did not abolish EJCs when the preparations were from animals CCT for more than 7 days. In CCT preparations acute withdrawal of clonidine from the bathing solution induced a significant (P < 0.05) increase in EJCs and SEJC frequency.

Chronic chloride treatment was found to increase efficacy of neurotransmission. This observation and previously reported chronic morphine effects on neurotransmission from the same preparation indicate that chronic administration of agents which have presynaptic inhibitory effects on neurotransmitter release triggers an increase in the efficacy of neurotransmission.

RESEARCH PROPOSED for 1997

At the begining of 1997 the Laboratory moved to The University of Queensland where work will continue into the physical change responsible for the increase in probability of transmitter release from sympathetic varicosities of animals which had been chronically morphine or clonidine treated, the importance of a verity of calcium stores in producing the acute and chronic effects of morphine and clonidine, and the role of neurotrophic factors in increasing efficacy of neurotransmission in chronically morphine or clonidine treated animals.

COLLABORATIONS

Mechanism of neurotransmission in sympathetic varicosities of the mouse vas deferens and the release sites of the skeletal neuromuscular junction: Prof Max R Bennett (1984-present)

Action of opiates and clonidine on neurotransmission: Dr TC Cunnane, Univ of Oxford (1993-present).

Effect of stretching and integrins in transmitter release: Prof A Grinnell, Los Angeles (1995-present).

FACILITIES

Room 140 of the Anderson Stuart Building. Nick Lavidis's office was room 265. Electrophysiological facilities include an imaging unit and a tissue culture facility.

THESES PASSED in 1996

GradDipSc

Cherepanoff S (1996) A statistical analysis of spontaneous miniature endplate currents recorded at the toad (Bufo marinus) neuromuscular junction.

BMedSc(Hons)

Chin I (1996) Stress induced changes in transmitter release from sympathetic varicosities of the mouse vas deferens (Result: 1st Class).

SCHOLARSHIPS

Australian Postgraduate Award: K David Kim (1996-)

University of Sydney Medical Foundation: Irene Chin (1997-)

Dental Association: Elizabeth Fletcher (1996-)

OTHER RESEARCH ACTIVITIES in 1996

Refereeing

NHMRC project grants (3), ARC small grants (1).

Seminars

Dept of Pharmacology, Univ of Oxford, UK.

Dept of Physiology & Pharmacology, Univ of Queensland.

Theses marked

PhD: 3

News media

Addiction to stress (Aug-Oct):

Newspapers: The University of Sydney News, Daily Telegraph, Weekend Australian.

Magazines: Kosmos, Today's Life Science.

Radio: 2BL Sydney, ABC Brisbane, 5AAA Adelaide, 2GB Sydney, ABC Melbourne, 5AN Adelaide, ABC Wollongong.

5-Year Research Publications


FUNDING in 1996

NHMRC          Mechanism of transmitter               Bennett MR*          1994
               secretion at sympathetic               Lavidis NA           1995               
               nerve varicosities.                                         1996          $67,673
                                                                           1997          $78,299

NHMRC          Probability of quantal secretion       Bennett MR*          1996          $51,059
               at neuromuscular synapses.             Lavidis NA           1997          $68,164

NHMRC          Effect of chronic morphine             Lavidis NA          1996           $62,762
               treatment on sympathetic                                   1997           $65,760
               varicosities innervating the                         
               mouse vas deferens.

Ramaciotti     Effect of opiates on cytosolic         Lavidis NA          1997           $15,000
               calcium of sympathetic
               varicosities.

ARC Small      The effect of clonidine on             Lavidis NA          1997           $15,000
Grant          transmitter release.

*Also appears in report for MR Bennett

Total for 1996:  $181,494
Total for 1997:   $95,760


TEACHING REPORT

COURSES TAUGHT

TAFE (Ultimo): Associate Diploma of Podiatry

Lectures: 54 on general pharmacology

Setting exams: 12 h

Marking exams: 20 h


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