NARCOTICS RESEARCH LABORATORY

Nickolas A Lavidis

RESEARCH REPORT


PERSONNEL in 1995 and 1996

Dr Nickolas A Lavidis RD Wright Fellow (NHMRC) University 1978- Senior Lecturer

Dr Kerry A Nichol Research Officer (0.4) NHMRC 1983-

David Kim PhD student (from 96) 1995-

Justin Morgan PhD student (shared 1996- s'vision: R Einstein, Dept Pharmacol)

Irene Chin BMedSc(Hons) student 1996-

Elizabeth Fletcher BSc(Dent)(Hons) student 1996-

(Those with M Bennett: Supervisor; N Lavidis: Associate Supervisor in 95)

Darren Warren PhD student 1992-95

Greg T Macleod PhD student 1995-

Yon-Qi Lin PhD student 1995-

Current effective full-time personnel (with fractional counting of co-supervised) = 5.8


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).

PROJECTS in 1995

Transmitter release from sympathetic varicosities of the mouse vas deferens following chronic clonidine treatment

J Morgan, I Chin, NA Lavidis

(collaborator: R Einstein, Dept of Pharmacology)

In the mouse vas deferens, the a2-adrenoceptor agonist, clonidine, and opioid receptor agonist, morphine, decrease the amplitude of the excitatory junctional potential (EJP) without affecting nerve terminal impulse propagation. Increasing extracellular calcium concentration decreases the effect of these drugs. The acute inhibitory effects of morphine on transmitter release are reversed following chronic morphine treatment. In 1995 the Lab reported an increase in transmitter release efficacy of the sympathetic varicosities of the mouse vas deferens following chronic clonidine treatment (1 to 5 mg/kg, twice/day for 7-8 days). Small diameter electrodes (10-15 µm) were placed over varicosities visualized using DiOC 2(5)-fluorescence to record the nerve terminal impulse, evoked and spontaneous transmitter release. Only sympathetic varicosities with probabilities of transmitter release greater than 0.01 were chosen for this study. In these varicosities the decrease in transmitter release induced by clonidine (1 µM) in control preparations (bathed in 2 mM Ca2+ was not observed in clonidine-treated preparations. When the clonidine was acutely withdrawn from preparations and yohimbine (1 µM) added, transmitter release was increased to more than 5 times the average level of transmitter release recorded compared to control preparations (n7). Thus, the adaptive mechanism(s) occurring in the sympathetic varicosities of the mouse vas deferens to the sustained presence of clonidine produce an increase in the probability of transmitter release and are probably similar to the adaptive changes in transmitter release evoked by chronic morphine treatment.

Changes in the levels of intravaricosity free calcium during acute morphine withdrawal

DK Kim, NA Lavidis

In mouse vasa deferentia chronically treated with morphine (CMT), the ability of morphine to inhibit transmitter release decreases as the effectiveness of sympathetic varicosities to release transmitter increases. This adaptation of increasing the efficacy of transmission is clearly evident when the morphine is withdrawn acutely. The adaptive mechanism(s) responsible for the increased efficacy of transmitter release during morphine treatment have not been clearly identified. Spontaneous transmitter release is thought to be dependent on both intracellular and extracellular Ca2+ concentrations. Spontaneous excitatory junctional current (SEJC) frequency was used as a probe to monitor possible changes in concentrations of free Ca2+ within varicosities during acute withdrawal from chronic morphine treatment. Increasing the extracellular calcium concentration from 1 to 4 mM increased the frequency of 2.1 ± 0.4 SE (n17). When extracellular calcium was reduced to zero, SEJC frequency decreased to 20% when compared to SEJC frequency in the presence of 2 mM Ca2+. The amplitude distribution of SEJCs was not altered by changing extracellular Ca2+. In control vasa deferentia, application of 1 µM morphine in the tissue bathing solution increased the frequency of SEJCs in 4 out of 6 experiments but this was not significant at the P<0.05 level, even though evoked transmitter release was reduced by 81 ± 9 SE % (n = 6). In vasa deferentia treated chronically with morphine, removal of morphine from the bathing solution and addition of naloxone (1 µM) produced a 4-11 fold increase in the frequency of SEJCs without any alteration in the amplitude distribution of SEJCs. In 4 out of 6 experiments SEJCs were released in short bursts which occurred regularly every 1.3 to 2.7 min, with each burst lasting approx 0.1-0.3 min. This increase in SEJC frequency was not abolished by removal of extracellular calcium from the bathing solution, even though evoked transmitter release was abolished. Thus, morphine increases the frequency of SEJCs and at the same time increases the probability of evoked transmitter secretion from sympathetic varicosities of the mouse vas deferens. These effects are probably mediated by an increase in the concentration of free calcium ions within varicosities.

Facilitation in transmitter release during nerve stimulation with trains of impulses from chronically morphine treated sympathetic varicosities.

DK Kim, NA Lavidis

The morphine induced decrease in transmitter release is not observed if mice are chronically treated with morphine for 7-9 days. In preparations treated chronically with morphine, acute morphine withdrawal induces an increase in the activity of sympathetic varicosities. In 1995 the possibility was examined that chronic treatment with morphine is altering the rate of Ca2+ sequestration within the varicosities by determining the time course of decay of facilitation. The level of facilitation was determined for very active varicosities and for varicosities which had vastly different levels of activity. There was an inverse relationship between the level of facilitation in the test impulse and average EJC amplitude of the conditioning nerve impulse. In control preparation, morphine decreased the average EJC amplitude and increased facilitation. In acute morphine withdrawal (AW) preparations, there was an increase in the average EJC amplitude and corresponding decrease in facilitation. Inhibiting autoinhibition with yohimbine (1 µM) did not significantly affect the average EJC amplitude but significantly increased facilitation. In control preparations, facilitation decayed exponentially with a time constant of 6 s. In AW preparations, the facilitation time constant of decay was 2.5 s. These results indicate that recruitment of sympathetic varicosities having a low probability of releasing transmitter is responsible for most of the facilitation observed during stimulation with trains of impulses. The time course of decay in facilitation was decreased from 6 to 2.5 s, suggesting an enhancement of Ca2+ sequestering within the sympathetic varicosities during CMT.

Possible role of neurotrophic factor(s) released from vasa deferentia during chronic opiate treatment

E Fletcher, K Nichol, NA Lavidis

The morphine and clonidine induced decrease in transmitter release is reduced following chronic morphine or clonidine treatment. This decrease in efficacy of these presynaptically acting inhibitors of transmitter release is a consequence of an increase in the probability of transmitter release by sympathetic varicosities. In 1995 the Lab examined the possibility that the increase in efficacy of sympathetic varicosities in releasing transmitter during chronic presynaptic inhibition may involve an increase in the production of neurotrophic factor(s) by smooth muscle cells of the mouse vas deferens. Mice were chronically treated with morphine (10-100 mg/kg, 3 times/day, for 7-9 days), then anaesthetized with halothane and killed by cervical fracture. The left vas deferens from each animal was electrophysiologically studied using small diameter electrodes (10-15 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 obtained from 9-11day-old chick embryos. After 27 h incubation, the cultures were fixed with 2.5% glutaraldehyde in phosphate buffer and videotaped. Ganglia cultured in the presence of vasa deferentia that had been treated chronically with morphine exhibited 2-5 times greater neurite outgrowth than control vasa deferentia (n >5). A comparison was also made between the extent of neurite outgrowth from dorsal root ganglia and the level of activity of sympathetic varicosities. The results indicated a positive correlation between neurite outgrowth from dorsal root ganglia and level of activity of the sympathetic varicosities of the mouse vas deferens.


RESEARCH PLANNED for 1996

In 1996 the Laboratory will investigate the physical change responsible for the increase in probability of transmitter release from sympathetic varicosities of animals which had been treated chronically with morphine or clonidine. The Lab will examine the importance of a verity of calcium stores in producing the acute and chronic effects of morphine and clonidine. Studies will also attempt to determine which neurotrophic factor is responsible for the increase in neurite outgrowth from chick dorsal root ganglia when co-cultured with vasa deferentia from chronically morphine treated animals.

COLLABORATIONS

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

Sensory innervation of smooth muscle cells:Dr Dirk Van Helden, Univ of Newcastle (1994-present).

Action of opiates on intracellular calcium ion concentration:Dr Thomas C Cunnane, Univ of Oxford (1993-present).

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

FACILITIES

The Laboratory occupies rooms 140 and 265 of the Anderson Stuart building. Dr Lavidis's office is room 265. Electrophysiological facilities include 1 unit, an imaging unit and a tissue culture facility.

THESIS PASSED in 1995

BMedSc(Hons)

Kim KD (1995) Effect of chronic morphine treatment on the frequency of spontaneous transmitter release and the time constant of facilitation. (Result: 1st Class; Awarded the Harding Burns prize).

SCHOLARSHIPS

Australian Postgraduate Award: KD Kim (1996-).

Harding Burns Prize: KD Kim (1996).

Colin Cormic Scholarship: E Fletcher (1996-97).

OTHER RESEARCH ACTIVITIES in 1995

Refereeing

Grant applications: for NHMRC (4), ARC small grants (1).

Seminars

Dept of Pharmacy, Univ of Sydney (May).

Dept of Physiology & Pharmacology, Univ of Queensland (Feb).

5-Year Research Publications


FUNDING in 1995 and 1996

NHMRC The effects of opiates on the Lavidis NA 1992

probability of quantal secretion 1993

at sympathetic varicosities 1994

(*Includes RD Wright Fellowship) 1995 *$66,345

NHMRC Mechanism of transmitter Bennett MR 1994

secretion at sympathetic Lavidis NA 1995 $68,755

varicosities 1996 $71,100

NHMRC Probability of quantal secretion Bennett MR 1994

at neuromuscular synapses Lavidis NA 1995 $51,815

1996 $51,200

NHMRC Effect of chronic morphine Lavidis NA 1995 $15,974

treatment on mouse vas deferens 1996 *$62,762

(*1996 amount includes RD Wright Fellowship) 1997

Ramaciotti The development of tolerance to Lavidis NA 1995 $20,000

opiates by sympathetic nerve

endings

Total for 1995: $222,889

Total for 1996: $185,062


TEACHING REPORT

COURSES TAUGHT

Medical Science 3: Advanced Neurosciences

Lectures: 8, on neuropharmacology.

Practicals: 2 students, 48 h.

Pharmacology to TAFE, Ultimo

Lectures: 108 on general pharmacology.

Setting exams: 12 h.

Marking exams: 20 h.

TEACHING HOURS in 1995

AdvN Ext Teach Total

Lectures 8 108 116

Practical consultations 48 ­ 48

Setting exams 1 12 13

Marking exams 5 20 25

Total formal contact teaching time in Department of Physiology = 56 h

Total formal contact teaching time at TAFE in Ultimo = 108 h


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