Noradrenaline effects on cultured rat sensory neurones exposed to morphine in utero or in vitro

Neuroscience Letters 193 (1995) 57-60

Noradrenaline

effects on cultured rat sensory neurones exposed to morphine in utero or in vitro ValCrie Villibea, Ian R. Neeringb,*

aPrince of Wales Medical Research Institute, High Street, Randwick, NSW 2031, Australia bSchool of Physiology and Pharmacology, University of New South Wales, P.O. Box I, Kensington, NSW 2033, Australia

Received 3 April 1995; revised version received 22 May 1995; accepted 25 May 1995

Abstract

Dorsal root ganglion (DRG) neurones cultured from newborn rats were chronically exposed to 1 PM morphine. Untreated cells and in vitro morphine treated cells were cultured for 1 or 2 weeks from control offspring while in utero morphine treated cells were cultured for 1 week from offspring of morphine dependent rats. Noradrenaline (1 PM) was applied by pressure ejection. Intracellular recordings measured the change in action potential duration (APD). Noradrenaline significantly decreased the APD of l-week-old untreated cells while the 2-week-old untreated cells and the groups of morphine treated cells showed no significant change of APD following the drug application. Thus, newborn rat DRG neurones demonstrated a different sensitivity to noradrenaline with the culture duration or with morphine exposure. Keywords:

Dorsal root ganglion neurones; Culture; Prenatal morphine exposure; Noradrenaline; Action potential duration; Intracellular

recordings

Noradrenaline has been reported to decrease the APD of cultured chick embryo dorsal root ganglion neurones in 75% [lo] or 92% [6] of the cells tested. As the effects of noradrenaline and opioids are known to be mediated by the same intracellular pathway [4,5,8,12,17], we investigated the possibility that responses to such non-opioid compounds might be reduced by chronic opioid presence. Studies on rat spinal cord and dorsal root ganglion cells have demonstrated such effects. Attali et al. [2] have shown that chronic exposure to K-agonists reduced the potency of a*-adrenergic and muscarinic agonists on adenylate cyclase activity. Moreover, opiate and adrenergic receptors are both involved in the manifestation of withdrawal [9]. suggesting that the effects of adrenergic agonists may alter with opioid dependence. Our investigation focused on the development of two models of chronic morphine exposure and their possible modulation on the DKG neuronal sensitivity to noradrenaline. We compared in vitro morphine treated cells and DRG cells cultured from offspring whose mothers were dependent upon morphine. The importance of culture * Corresponding author, Tel.: +61 2 385 2563; Fax: +61 2 385 1059.

duration was also examined as no information is available on this matter. This study set out to compare the effects of long-term neuronal morphine exposure both in utero and in vitro. Accordingly, a number of cell groups have been delineated: (a) cells from control offspring cultured and examined after l-2 weeks; these neurones were called ‘untreated cells’; (b) cells from offspring of morphine exposed mothers (6-13 days following osmotic pump implantation); these neurones were cultured for 1 week in medium containing 1 PM morphine before study; the total period of exposure of these cells to morphine was therefore 2-3 weeks; these neurones were called ‘in utero morphine treated cells’; (c) cells from control offspring maintained in morphine (1 FM) and examined after l2 weeks in culture; these cells were denoted as ‘in vitro morphine treated cells’. Female rats, either primiparous or with one preceding litter, were mated. After 10-15 days pregnancy, an osmotic minipump (2mL2, Alza Corp.) was implanted subcutaneously at the back of the neck. The surgery was performed using either intramuscular xylazine (10 mg/kg) + ketamine (50 mg/kg) or inhaled ether. Implanted pumps

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V. VilliPre, I.R. Neering I Neuroscience Letters 193 (1995) 57-60

contained morphine hydrochloride (Macfarlan Smith Ltd, Edinburgh) at a concentration such that they released the drug at the rate of 20 mg/kg per day to induce morphine dependence. Physical dependence was tested on a nonpregnant female rat which had received 10 mg/kg per day of morphine for 8 days: 10 mg/kg intraperitoneal naloxone precipitated withdrawal symptoms (body shakes, teeth chattering, ear bleaching, lacrimation and diarrhoea in less than 30 min). After birth, blood samples were taken from maternal rats by intracardiac puncture under ether anaesthesia. Blood samples from l-day-old rats were collected following decapitation. Analysis of morphine content of plasma samples was by gas-chromatography/mass-spectrometry following the method of Jones et al. [ 111. In general, higher free morphine concentrations were found in maternal plasma than in l-day-old offspring: respectively, 0.291 ng/@ and 0.114 ng/@ [18]. This finding is in agreement with that of Sanner and Woods [ 161. The level of morphine chosen for in vitro exposure was 0.3 pg/ml (= 1 PM) which corresponds to the average free morphine concentration found in maternal plasma. Dorsal root ganglion neurones were cultured from lday-old rats. Ganglia were removed under sterile conditions and pooled in Ca 2+-free, phosphate buffered saline solution (Dulbecco) containing 5 mg/ml glucose. Collagenase was added (final concentration, 0.01%) and the cell suspension was incubated at 37°C for 30 min. Trypsin was then added to achieve 0.1% concentration and the cells were incubated for a further 15 min at the same temperature. Cells were then dispersed by mechanical trituration using graded Pasteur pipette tips. Following washing and centrifugation of cells, they were suspended in Dulbecco’s modified Eagle’s medium (DMEM) and plated on coverslips in 16 mm wells. The coverslips were coated with rat tail collagen and poly-D-lysine (0.1 mg/ml) according to the method of Bornstein [3]. The culture medium consisted of DMEM supplemented with rat serum (7.5%), glutamine (55Opg/ml), vitamins, glucose (23 mM) and 50 III/ml penicillin/streptomycin (Flow Laboratories). Nerve growth factor (NGF-2.5S, Collaborative Research) was added to achieve a final concentration of 20 @ml. In some cultures (see below), morphine was also added to the medium (1 ,uM). After 2 days in culture, cytosine arabinoside (50 PM, final concentration) was added to avoid proliferation of non-neuronal cells. Culture plates were incubated at 37°C with 5% CO2 and 90% humidity, The DRG neurones were well adhered with processes after 5 days and, under the above conditions, remained viable in excess of 3 weeks. The culture medium was changed twice each week. Experiments were conducted on the stage of an inverted microscope (Nikon Diaphot) at room temperature. Coverslips with attached DRG neurones were transferred into a specially constructed chamber [14] on the microscope stage and continuously perfused with a balanced

salt solution (BSS) containing 130 mM NaCl, 3.5 mM KCl, 5 mM CaC12, 1 mM MgC12, 5 mM HEPES, 5 mM Dglucose, 24 n-&l sucrose, 10 mM tetraethylammonium chloride (TEA) (pH 7.4 adjusted with 1 M NaOH). TEA was used to increase the duration of the calcium component of the action potential in DRG neurones [ 1,191. Morphine (1 PM) was added to this medium when experiments were performed on morphine treated cells. Intracellular recordings were made from randomly chosen DRG neurones using microelectrodes (tip diameter 0.3 pm; Clark Instruments) filled with 0.3 M KC1 with a resistance of 60-80 MQ. Recordings were made using an Axoclamp 2A (Axon Instruments) set in bridge mode. Cells were stimulated at 0.1 Hz. Action potentials were recorded and stored on tape for subsequent analysis. Action potential duration was measured using a computer program which calculates the time between the peak of the AP and 50% of the total AP depolarisation. The action of noradrenaline (1 PM) on the electrical activity of DRG neurones was examined. This drug was applied 5 s before application of electrical stimuli (duration 2 ms; amplitude 2 nA) to evoke action potentials (AP). Application was done by pressure ejection using a ‘puffer’ pipette situated close to the cell (<150pm). Stock solutions of noradrenaline ((-)arterenol bitartrate, Sigma) were dissolved in distilled water containing 5% ascorbic acid. Appropriate aliquots were frozen to be used in individual experiments. Noradrenaline stock was dissolved in recording medium con-

A

I

5OmV

Fig. 1. Effects of noradrenaline (1pM) on the APD of l-week-old untreated neurones. (A) Noradrenaline decreased the APD of an 8 dayold untreated cell. (1) Control AP, (2) after noradrenaline application. Intermediate AP taken during the 3 min recovery from noradrenaline. (B) Noradrenaline increased the APD of an I-day-old untreated cell. (1) Control AP, (2) after noradrenaline application, (3) 1 min and (4) 5 min after drug application ceased. This effect was seen in only 2 of 22 cells.

V. Villihre, I.R. Neering I Neuroscience Letters 193 (1995) 57-60

taining 1% ascorbic acid before application. Pressure ejection of this solution had no effect on resting DRG electrical parameters and APD. Analysis of covariance was performed on the raw values of action potential duration before and after application of drugs. The effects of culture age and morphine exposure were tested by using orthogonal or nonorthogonal sets of contrasts. Data were grouped and presented as means * standard error of the mean (SEM). Noradrenaline was only applied for 5 s. It appears that this duration was sufficient to observe a significant and reversible response to noradrenaline. We chose one concentration of noradrenaline to compare its effects on DRG neurones chronically exposed to morphine in two different models. Noradrenaline is known to have depressant effects on mouse spinal cord ganglia [5] and on embryonic chick DRG neurones [6] at a concentration of O.l1 PM. Noradrenaline (1 @I) mostly shortened the APD of untreated neurones (F’c 0.05) (see Table 1). For this group, the mean va1ue.s of APD decreased from 35.5 + 3.8 ms to 26.1 f 3.5 ms.. An example of such a response is shown in Fig. 1A; the action potential shortened by 36 ms (69% of control duration) and regained its original duration 3 min after noradrenaline application. In only two cells of this group, was the APD increased; the effect was smaller and the recovery longer (see Fig. 1B). Noradrenaline did not alter tbe APD of in vitro or in utero morphine treated cells (P > 0.05) as much as it did in the l-week-old untreated group; the mean values of APD were relatively unchanged after noradrenaline (see Table 1).

Table 1 Effects of 1 PM noradmnaline on the APD (ms) of l-day-old rat DRG neurones Time

n

APD (ms, mean rt SEM)

in culture Control

(wee@

Noradrenaline

Untreated

1

22

35.5 + 3.8

26.1 f 3.5*

Untreated

2

25

48.3 + 8.5

45.8 f 8.0

In vitro

1

15

36.0 i 6.5

35.4 f 5.8

2

15

44.2 f 8.1

46.1 f 8.4

1

30

29.7 i 4.1

30.6 k 4.2

morphine treated In vitro morphine treated In utero morphine treated n is the number of cells studied per group. Statistical comparisons were performed by using analysis of covariance: *P < 0.05

9 .i

120

1

100

e

80

z 2

60

a

59

40

0

20

40

60

80

WI

,120

140

160

control APD hs)

Fig. 2. Control APD values and values of APD after acute treatment with 1pM noradrenaline. Each point represents measurements from one rat DRG neurone. The median line represents no change of APD.

Fig. 2 shows the values of control APD and the values of APD after noradrenaline for each. neurone tested. It demonstrates that noradrenaline principally decreased the APD of untreated neurones. Only, a few morphine treated neurones were able to give a specific response to noradrenaline. The analysis of variance demonstrated that the APD values of the five groups of DRG neurones were not significantly different. When the control values of APD were compared, the P value was 0.256 and when the APD values after noradrenaline were compared, the P value was 0.181. Because no variability was found between groups, it was possible to compare the values of APD before and after noradrenaline for all groups. The analysis of covariante was performed and it demonstrated that noradrenaline had a significant effect on the APD of DRG neurones (P = 0.032). The comparison group by group was performed by using sets of contrasts. Noradrenaline was shown to have a significant effect only on l-week-old untreated cells compared to 2-week-old untreated cells (P c 0.05). The effects of noradrenaline on l-week-old untreated cells were also significantly different to those on in vitro morphine treated cells (P c 0.05) and in utero morphine treated cells (P c 0.05). The effects of noradrenaline on the two models of chronic morphine exposure were not significantly different (P > 0.05). Noradrenaline effects on 2-week-old untreated cells were also not significantly different from those on morphine treated cells (P > 0.05). Acute and chronic opioid effects have already been studied on DRG neurones. The originality of this study was to compare in vitro chronic morphine exposure with a combined in vitro/in utero exposure. This paper described the effects of noradrenaline at a chosen concentration on DRG neurones cultured for 1 or 2 weeks and exposed differently to morphine. We have expressed our results in terms of the change in action potential duration of the DRG neurones. These results demonstrated the electrical heterogeneity of DRG neurones. In embryonic chick DRG neurones, noradrenaline (50 PM) reduced the APD in 75% of the cells tested by an

60

V. VilliLre, I.R. Neering I Neuroscience Letters 193 (1995) 57-60

average of 43 f 4.7% [lo] . When our results were similarly expressed, the major effect of 1 PM noradrenaline was a 44 * 9% decrease of the APD on 55% of l-weekold untreated cells. It appears that fewer DRG neurones cultured from newborn rats are responsive to noradrenaline than neurones cultured from chick embryos. Dunlap and Fischbach [6] also reported that 92% embryonic chick DRG neurones were responsive to ,uM noradrenaline. Previous studies have shown that other cellular models (e.g. locus coeruleus neurones) responded differently to adrenergic drugs through antenatal and postnatal development [13]. The amplitude and duration of noradrenaline effects on our DRG neurones are similar to those reported in embryonic chick DRG neurones [6,7]. Noradrenaline decreases the APD by decreasing a calcium conductance [7] and this effect is mediated by G-proteins

HOI. The responses to noradrenaline observed on l-weekold untreated cells were affected by the culture duration and the chronic morphine exposure. This suggests that there is a change in modulatory non-opiate effects due to culture duration and morphine exposure. These modulatory effects seem to have developed during culture as no difference was found between the two models of morphine exposure and the longest culture duration. It is possible that they also develop during prenatal morphine exposure but, in this case, they would not be different to those developing during in vitro morphine exposure. For these reasons, it would be interesting to compare the concentration-response curves and ECso for noradrenaline in the different groups of neurones. In our study, morphine and culture duration might interfere with the development of catecholamine receptors. The culture was performed on newborn rats and postnatal life is a critical moment for the development of these receptors. They develop in early neonatal life and they are ready for binding 2 or 3 weeks after birth [ 151. In summary, newborn rat DRG neurones were less responsive to noradrenaline after in vitro or combined in vitro/in utero morphine exposure. The data presented showed clearly that the time in culture was critical even for only 1 week difference.

We thank National Health and Medical Research Council of Australia for their financial support and A/Prof Duncan for his help and the use of his biomedical massspectrometry unit. We wish to thank particularly Tim Charlton for his assistance with the GUMS analysis and Mr Park and Cho for their assistance with the statistical analysis.

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