Chapter
6 "Withdrawal substance" in cerebrospinal fluid of morphine-abstinent
rats
Abstract - The behavioral, electrophysiological (visual evoked potentials, VEP) and in vitro effects of cerebrospinal fluid (CSF) taken from the donor rat have been investigated in the recipient rat and guinea-pig isolated ileum. The CSF of spontaneous morphine-abstinent donor rat precipitated in morphine-dependent recipient rat an opioid withdrawal syndrome, which was characterized by a decrease in the VEP peak latency N3 and amplitudes N2P3 and P3-N3. The CSF-induced withdrawal syndrome was behaviorally less severe and electrophysiologically less prominent, but qualitatively - identical to the naloxone-induced abstinence. However, in contrast to naloxone, the CSF from spontaneous morphine-abstinent rat did not contract the morphinedependent isolated guinea-pig ileum. Chromatographic analysis of CSF samples from naive, morphine-dependent or morphine-abstinent rats reveal distinct fractions, containing an active component present only in CSF of morphine-abstinent rats. The estimated relative molecular mass of this active component was around 50 kDa and the short retention time on the reversedphase column suggests the high hydrophobicity. The results indicate that spontaneous morphine-abstinent donors synthesize and release certain quantity of putative "withdrawal substance" in the CSF, which is without naloxone-like properties. This further suggests, that the CSF- and naloxone-precipitated withdrawal in the morphine-dependent recipients are mediated by activation of different neuronal mechanisms. Part of these data are published in Regulatory Peptides 1: 5227-S228, 1994.
It
was shown that cerebrospinal fluid (CSF) from morphine-abstinent donor rats,
precipitates an opiate withdrawal syndrome in morphine-dependent recipient rats
(Malin et al., 1987). These authors found an increased level of octapeptide
F-8-F-NH2 (Phe-Leu-PheGln-Pro-Gln-Arg-Phe-NH Z)-like immunoreactivity in CSF of
morphine-dependent rats (Malin et al., 1990a). The octapeptide F-8-F-NH2
precipitates withdrawal syndrome in morphine-dependent rats, but not in the
naive ones (Malin et al., 1990b). However, the role of this peptide in the CSF
during morphine dependence and withdrawal remains unclear.
In order to test this hypothesis, we examined the electrophysiological effects of CSF withdrawn from abstinent rats, by monitoring the peak latencies and amplitudes of visual evoked potentials (VEP) during CSF-precipitated withdrawal syndrome in rats. The effect of CSF from spontaneous morphine-abstinent rats was also studied in naive rats, morphine-dependent rats and morphine-dependent isolated guinea-pig ileum. In addition, we analyzed CSF samples from morphine-abstinent, morphine-dependent and naive rats by high-performance liquid chromatography (reversed-phase and gel-filtration techniques).
Materials and methods Animals
Male Wistar rats (TNO, Zeist),
weighing 200-300 g were housed in groups and had a free access to food and
water. The room was maintained on 12 h light-dark cycle (lights on 8.00 h), with
constant temperature (21° C) and humidity (55%).
Procedures Surgical procedure
All animals were anaesthetized with
pentobarbital (60 mg/kg, i.p.). The donor rats were implanted with a chronic
cannula, placed into the cisterna magna in order to withdraw CSF from conscious
animals (Bouman and Van Wimersma Greidanus, 1979). In recipient rats, receiving
CSF from donor rats, a cannula was placed into the lateral ventricle (Paakkari,
1980). Rats, involved in the VEP experiments were all recipient rats and, in
addition to the lateral ventricle cannula, they were implanted with stainless
steel screw electrodes over the right and left visual cortex (7 mm posterior to
the bregma and 3 mm lateral to the midline). A reference electrode was placed in
the frontal sinus. The electrodes were soldered to a miniature socket and
attached to the skull with dental acrylate. In the recovery period (7 days), all
operated animals were housed individually with food and water ad libitum. At the
end of the experiments, the placement of lateral ventricle cannula was confirmed
by injection of methylene blue.
Morphine dependence and abstinence
Morphine dependence was induced by
treating animals with morphine for 8 days (twice daily, 9h and 17h). A starting
dose of morphine was 10 mg/kg/injection, which increased daily to 20, 20, 40,
reaching a final dose of 80 mg/kg/injection on the 5t h day. Rats treated with
distilled water (1.0 mi/kg) for 8 days (twice daily, 9h and 17h), formed a group
of naive animals. Morphine-dependent rats were considered as spontaneous
morphine-abstinent, 6 h following the last morphine injection.
The control experiments were
performed in naive and morphine-dependent rats on the 7th day of drug treatment.
The behavioral signs similar to withdrawal symptoms occurred
The withdrawal signs were scored
according to the weighting factors described by Neal and Sparber (1986). In
short, the signs observed during a mild withdrawal syndrome were assigned with 1
(chewing, diarrhoea, grooming, rearing, irritability on touch). The weighting
factor 2 was given to the withdrawal signs, teeth-chattering, wet-dog shakes,
penile licking, ptosis and jumping. The sign rhinorrhoea, observed during severe
withdrawal was assigned a 3.
Visual Evoked Potentials (VEP)
A postoperative recovery period of 7
days was followed by habituation of the recipient to the recording procedure.
The method used for recording of VEP is described in the previous study of
Dzoljic et al., (1994). Briefly, the animal was placed in a test chamber and
after connecting the electrodes, a flash stimulus was induced at 1 per 7 s for
10 min. The habituation period lasted 3 days. This approach was selected, since
it has been shown that under these conditions VEP discharges stabilize after
several days (Bigler, 1975). The flash light was generated by a Grass S44
stimulator in a frequency of 0.14 Hz. Brain responses were amplified with a
Grass model 79 B, connected to an analog-digital convertor (Lab Master,
Scientific solutions Inc., Ohio, USA), triggered by the Grass S44 stimulator
after every flash light. A computer connected to the analog-digital convertor
performed the averaging of 25 VEP over a 800 ms epoch after every flash light
and printed the results. Stimulation was performed only in animals with open
eyes. The VEP parameters (peak latency and amplitude) were recorded in a total
of six sessions, namely 5, 15 and 30 min before drug administration (self-control)
and in the same time intervals after drug administration.
In vitro experiments
Male guinea-pigs (n=5, 600-900 g) were killed by a blow on the head. A 40 cm long segment of the small intestine was rapidly removed and placed in Krebs solution (room temperature). The terminal section of the guinea-pig ileum was used after discarding the portion of 10 cm closest to the ileo-caecal junction (Munro, 1953). The ileum was cut in eight 3-cm long segments. These segments were gently and thoroughly washed free of faecal matter by flushing Krebs solution through the lumen. Each streap of ileum was set up in a 8 ml organ bath containing Krebs solution and bubbled with 95% O z and 5% CO 2. Every 15 min the bath was perfused with fresh warm Krebs solution. The temperature and pH of the Krebs solution were maintained at 37°C and 7.4, respectively. The ileum was fixed at a resting tension of 1 g and allowed to equilibrate for 30 min. No drug was added in this time period. The spontaneous activity of the ileum was recorded isometrically. In order to induce morphine dependence, the ileum was exposed to morphine (1 uM) for 2 h (Cruz et al., 1991). The pieces of ileum not treated with morphine were considered as naive ileum. Exposure of ileum to CSF for 5 min was followed (after washing) by naloxone (0.1 uM) Naloxone remained in the bath also for 5 min. The CSF was made artificially or withdrawn from the donor rats (naive, morphine-dependent or morphine-abstinent) on the 8th day of drug treatment.
The contraction of the ileum was defined as the peak tension observed within 1 min after drug administration. In order to check the contractility of smooth muscle, each ileum was exposed to methacholine (0.1 uM) at the end of experiment. Only experiments with morphine-dependent ileum responding to methacholine and naloxone were taken as valid.
High-Performance Liquid
Chromatography (HPLC)
Pooled samples of CSF (approximately
240-300 ul total volume) taken from naive, morphine-dependent or
morphine-abstinent rats were analyzed using the SMART micropurification system (Pharmacia
Biotech., Uppsala, Sweden). The system was operated as described in previous
reports (Nyberg et al., 1991; Renlund et al., 1993). Briefly, a reversed-phase
column pRPC C2/C18 (2.1 x 10 mm) and a gel-filtration column Superdex 75 (3.2 x
300 mm) were used in this study. The CSF samples were filtered through a
nonsterile 45 um filter (Ultrafree-MC, Millipore, Bedford, MA, USA) and injected
into the system. The reversed-phase column was eluted with a 30 min linear
gradient from 0-60% acetonitrile, supplemented with 0.1% trifluoroacetic acid.
The flowrate was maintained at 50 pl/min and one-min fractions were collected.
The size separations (100 pl sample injected) were conducted using 20 mM
Tris-HCI buffer of pH 7.4 as the eluent. The collected material was stored at
-80° C until assayed.
Drugs
Morphine hydrochloride (OPG, Utrecht)
and naloxone hydrochloride (Research Biochemical Incorporation, England) were
dissolved in distilled water. The composition of solutions (expressed in mM) was
as follows: Krebs buffer - NaCl 118; KCl 4.7; CaC1 2 2.5; NaHCO3 25; KH 2PO 4
1.2; MgSO 4 1.2; glucose 5.55; Artificial cerebrospinal fluid - NaCl 138; KCl
3.3; CaC 2 2.2; MgC1 2 1.15; NaHCO3 2.1; NaH 2PO 4 0.6; urea 2.16; glucose 3.38.
The volume of CSF administered i.c.v. was 80 ± 5 ul per recipient rat.
Statistical analysis
The data in relation to withdrawal
behaviour and electrophysiological study were statistically evaluated by using
the non-parametric Kruskal-Wallis one-way analysis of variance, followed by
Mann-Whitney U-test, with a level of P<0.05 being considered significant.
Results
Behaviour
Naive rats - recipients of CSF (Fig.
IA)
Administration of artificial CSF (80 ± 5 pl, i.c.v.) into naive animals (n=15) treated with vehicle for 7 days, did not alter the normal behaviour, characterized with occasional appearance of grooming, digging, scratching and rearing. On the following day (8' h day of the vehicle treatment) these animals, randomly divided into three groups, received the CSF
(80 ± 5 ul, i.c.v.) taken from the naive rats (n=5), morphine-dependent (n=4) or spontaneous morphine-abstinent rats (n=6). The behaviour of animals in all three groups receiving various samples of CSF remained unaltered.
Morphine-dependent rats - recipients
of CSF or treated with naloxone (Fig. IB) Administration of artificial CSF into
dependent animals (n=26, on the 7th day of morphine treatment) did not change
the behaviour. On the following day (8 th day of morphine treatment) these
animals, randomly divided into three groups, received CSF obtained from the
naive (n=6), morphine-dependent (n=5) or spontaneous morphine-abstinent (n=15)
donor rats. No behavioral changes were observed in the morphine-dependent rats,
receiving CSF from the naive or morphine-dependent donors. However,
morphinedependent recipients treated with CSF taken from spontaneous
morphine-abstinent rats exhibited a significant increase in the expression of
withdrawal syndrome. In order to compare the severity of CSF- and
naloxone-precipitated withdrawals, an additional group of dependent rats (n=5)
was treated with vehicle (1.0 ml/kg, i.p., on the 7th day of morphine treatment)
and naloxone (4.0 mg/kg, i.p. on the following day). It was found that
naloxone-precipitated withdrawal was significantly more severe than the
withdrawal induced by CSF.
Visual Evoked Potentials (VEP) Naive rats - recipients of CSF (Fig. 2) Artificial CSF or CSF taken from naive (n=5) or spontaneous morphine-abstinent donor rats (n=5) and administered into naive recipient rats (15 animals equally divided in three groups for each particular sample of CSF) did not change their peak latencies (Fig. 2A) or amplitude values (Fig. 2B).
Fig. 2. Effect of CSF (80 ± 5 VI, i.c.v.) on the peak latencies (A) and
amplitude values (B) of VEP in naive recipient rats. The artificial CSF (O) or
CSF withdrawn from the naive (•, n=5) or spontaneous morphine-abstinent (0,
n=5) donor rats were administered into three groups of naive recipient rats (5
animals for each different sample of CSF). The 0%-line, taken as self-control
indicates the average of peak latencies and amplitude values, measured in three
sessions (5, 15 and 30 min) before administration of CSF. Data are expressed as
% ± SEM of altered peak latencies and amplitude values compared to self-control.
Note that none of the CSF samples altered VEP parameters (latency and amplitude)
in native recipients rats.Morphine-dependent rats - recipients of CSF (Fig. 3) Artificial CSF or CSF taken from the
naive donors (n=5) were administered to morphinedependent rats (10 animals
equally divided into two groups for each particular sample of CSF). No
significant changes in the peak latencies (Fig. 3A) or amplitudes (Fig. 313)
were observed. However, CSF taken from spontaneous morphine-abstinent donor rats
(n=6) significantly decreased the peak latency N3 (Fig. 3A), and amplitude
values of N2-P3 and P3-N3 (Fig. 313) in morphine-dependent recipients (n=6). Morphine-dependent rats - treated with vehicle and naloxone (Fig. 4) Fig. 4. Effect of vehicle (distilled
water, 1.0 ml/kg, i.p.) and naloxone (4.0 mg/kg, i.p.) on the peak latencies (A)
and amplitude values (B) of visual evoked potentials (VEP) in morphine-dependent
rats (n=9). Vehicle (E0) or naloxone (0) were administered on the 7`" and
8t h day of morphine treatment, respectively. The 0%-line, taken as self-control
indicates the average of peak latencies and amplitude values, measured in three
sessions (5, 15 and 30 min) before administration of CSF. Data are expressed as
% ± SEM of altered peak latencies and amplitude values compared to selfcontrol.
Significant changes of peak latencies and amplitude values versus self-control
are indicated by * (P<0.05). Note that naloxone induced a significant
decrease of peak latencies (P3 and N3) and amplitude values (N2-P3 and P3-N3) of
VEP in morphine-dependent rats. Fig. 5. Effect of CSF (80 ± 5 pl,
1>, 2T, 3T, 4T), naloxone (0.1 uM, 5T) and methacholine (0.1 uM, 6T) on naive
and morphine-dependent guinea-pig ileum in vitro. The CSF added into the bath
was made artificially (1>) or withdrawn from the naive (2T),
morphine-dependent (3T) or spontaneous morphine-abstinent (4T) donor rats. Note
that no any sample of CSF (1>, 2T, 3T, 4T) had an effect on muscle tonus,
while naloxone (5T) induced a contraction only in the morphinedependent ileum.
Methacholine (6T) induced a contraction in both, naive and morphine-dependent
ileum. High-Performance Liquid
Chromatography (HPLC) Discussion Behaviour Visual Evoked Potentials (VEP) Peak latencies: The CSF taken from
the spontaneous morphine-abstinent donor rat induced a decrease of peak latency
N3 in morphine-dependent recipient, which indicates a stimulation of central
neurotransmission. The CSF-induced decrease of peak latency was less prominent
compared to the effect of naloxone. This is a good reflection of similar
differences observed in respect to the severity between the withdrawal syndromes
induced by CSF and naloxone. The development of N3 component is a result of
massive discharge of lateral geniculate units (Bigler, 1975), while the
components P2, N2 and P3 represent a diffuse activity between thalamus, midbrain
and cortex (Creel et al., 1974). The peaks N3 and P3 reflect an arousal level in
the brain (Joseph et al., 1981). A decrease of peak latencies during naloxone-
or CSF-precipitated withdrawal suggest an increase of neuronal excitability in
the mentioned brain areas. VEP amplitude values: The CSF taken
from the spontaneous morphine abstinent donor rats and naloxone induced a
decrease of amplitude values of several peaks (N2-P3 and P3 N3) in the
morphine-dependent recipients. Decrease of amplitude values reflects a neuronal
depression, which is rather unexpected finding, since naloxone-precipitated
withdrawal was described as a state of psychomotor stimulation (Wise and Bozarth,
1987). The naloxone-induced decrease of VEP amplitudes contrasted also to the
suggested epilep togenic properties of naloxone, since this opioid receptor
antagonist induced an increase of photically evoked discharges in the naive
conscious rats (Shearer et al., 1984). However, the possibility that excitation
of some inhibitory neurons may lead to depression of other neurons in the visual
pathways of morphine-dependent rat, might explain these controver sies.
Furthermore, an anticonvulsant effect of naloxone has also been demonstrated (Carter-Snead
III and Bearden, 1982). In vitro experiments High-Performance Liquid Chromatography (HPLC) study The bioactive component partially isolated in this study, seems to be
different from the octapeptide F-8-F-NH2, due to the higher hydrophobicity and
much lower molecular mass of the latter (Kivipelto et al., 1989; Labrouche et
al., 1993). We can not, however, exclude at this moment, that such a component
might bound to a larger protein, affecting its chromatographic and spectral
properties. Attempts to determine this factor in CSF of morphine-abstinent rats
as well as the examination of bioactivity of these fractions are in progress. Concluding, this study shows that a "withdrawal substance", not yet
chemically defined is synthesized and released in CSF during the development of
spontaneous morphine abstinence. This substance is formed in sufficiently high
concentrations to induce a withdrawal in morphine-dependent recipient rats.
Regarding to the fact that total CSF volume in a 300 g rat is about 580 ul (Lai
et al., 1983) and that total CSF volume of rats is replaced completely within
10-25 min (Bouman and Van Wimersma Greidanus, 1979), the release of this
substance during withdrawal has to be very abundant. The CSF taken from the
spontaneous morphine-abstinent rats decreased the VEP peak latencies and
amplitude values, which is identical to the corresponding effects of naloxone in
the morphine-dependent rats. However, data from the in vitro study indicate that
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Administration of CSF samples taken from morphine-abstinent rats (donors) into
the lateral ventricle of morphine-dependent rats (recipients), precipitated a
withdrawal syndrome, which could not be observed in the naive recipient rats.
The CSF-induced withdrawal syndrome was less severe, but qualitatively identical
to the naloxoneprecipitated withdrawal syndrome. Artificial CSF or CSF from the
naive or morphinedependent animals did not induce withdrawal syndrome in no any
group of recipient animals. These results confirmed the earlier data (Malin et
al., 1987) related to the withdrawal induced by CSF from morphine-abstinent
rats.