Download Effects of galanin on wide-dynamic range neuron activity

Regulatory Peptides 95 (2000) 19–23
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Effects of galanin on wide-dynamic range neuron activity in the spinal
dorsal horn of rats with sciatic nerve ligation
a
a
b
Shi-Lian Xu , Yan-Ping Zhang , Thomas Lundeberg , Long-Chuan Yu
a
a,b ,
*
Department of Physiology, College of Life Sciences, and National Laboratory of Biomembranes and Membrane Biotechnology, Peking University,
Beijing 100871, PR China
b
Department of Physiology and Pharmacology, and Department of Medical Rehabilitation, Karolinska Institutet, 171 77 Stockholm, Sweden
Received 20 October 1999; received in revised form 12 May 2000; accepted 24 May 2000
Abstract
Galanin is a 29-amino acid peptide with a suggested role in nociception. The effect of galanin on wide-dynamic range neuron discharge
frequency in rats with nerve ligation, used as a model of neurogenic pain, was investigated by extracellular recording methods. Seven to
14 days after sciatic nerve ligation, 0.1, 0.5 or 1 nmol of galanin was administered directly on the dorsal surface of the L3–L5 spinal cord
of rats with sciatic nerve ligation. It was found that galanin inhibited the activity of wide-dynamic range neurons dose-dependently, an
effect was more pronounced in sciatic nerve ligated rats than intact rats. Furthermore, when 1 nmol of galantide, the galanin antagonist,
was administered on the dorsal surface of the L3–L5 spinal cord, the wide-dynamic range neuron discharge frequency increased
significantly. The results suggest that galanin plays an important role in the modulation of presumed nociception in mononeuropathy.
 2000 Elsevier Science B.V. All rights reserved.
Keywords: Galanin receptor; Extracellular recording; Neuron discharge frequency; Mononeuropathy; Nociception
1. Introduction
Galanin, a 29-amino acid peptide, is widely distributed
in the central and the peripheral nervous system [1].
Galanin-like immunoreactivity was densely distributed in
the superficial layers of the spinal dorsal horn and present
in capsaicin-sensitive primary sensory neurons, suggesting
that this neuropeptide is involved in the transmission or
modulation of nociceptive information at the spinal cord
level [2,3]. Galanin inhibited the nociceptive flexor reflex
[4], and the inhibitory effect of galanin on the flexor reflex
was enhanced after sciatic nerve injury [5]. It has been
suggested that galanin may serve as an endogenous antinociceptive analgesic [6]. Yu and collaborators reported
that intrathecal administration of galanin produced dose*Corresponding author. Tel.: 1 86-10-6275-1867; fax: 1 86-10-62751526.
E-mail address: [email protected] (L.-C. Yu).
dependent increases in hindpaw withdrawal latency to both
noxious heat and mechanical stimulation in rats with
mononeuropathy [7]. Recent studies in our laboratory
demonstrated that wide-dynamic range (WDR) neuron
discharge frequency decreased significantly after administration of galanin in intact rats (unpublished data). The aim
of the present study was to elucidate the effect of galanin
on WDR neuron activity in rats with sciatic nerve ligation.
2. Materials and methods
2.1. Animals and surgery
Experiments were performed on 12 adult male Sprague–
Dawley rats weighing 200–250 g (The experimental
Animal Center of Beijing Medical University, Beijing, PR
China). The rats were housed one per cage with free access
to food and water. All experiments were conducted
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S.-L. Xu et al. / Regulatory Peptides 95 (2000) 19 – 23
according to the guidelines of the animal ethical committee
of Karolinska Institutet and every effort was made to
minimize animal suffering.
The rat was anesthetized with pentobarbital sodium
injected intraperitoneally (50 mg / kg). The left sciatic
nerve was exposed just below the division of the semitendinosis branch. Four loose ligations (4.0 chromic gut) were
made around the nerve with a 1.0–1.5 mm interval
between each of them. The ligation was carefully manipulated so that the nerve was barely constricted. The muscle
and skin layers were closed with sutures and the animals
allowed to recover.
Extra-cellular recordings were performed at 7–10 days
after sciatic nerve ligation. Animals were anesthetized with
intraperitoneal pentobarbital sodium (50 mg / kg; maintained with intermittent dose of 10 mg / kg / h) and a
cannula was inserted into the trachea. The dorsal L3–L5
region of the spinal cord was exposed by laminectomy.
The vertebral column was stabilized by vertebral and hip
clamps. The spinal cord between L3 and L5 was placed on
a curved metal saddle, gently lifted 0.5 mm from the
vertebral canal, and then covered with thermal 0.9% saline
(378C). The animals were immobilized with intraperitoneal
gallamine triethiodide (100 mg / kg / h) and received artificial respiration (frequency: 90; tidal volume: 4–5 ml). In
order to maintain the body temperature of the rat within
physiological levels, a heating plate was placed under the
rat so that rectal temperature remained between 35 and
36.58C. At the end of the experiments, the rats were killed
with an overdose of pentobarbital sodium.
2.2. Recording and stimulation
Extracellular recording of lumbar dorsal horn units was
performed with a glass microelectrode filled with 3 M
NaCl (3–8 MV) [8–11] and advanced by a manual
hydraulic microdriver. Recording sites ranged 70–1100
mm below the dorsal surface of L3–L5 levels. The evoked
action potentials were displayed on an oscilloscope after
being passed through magnifiers. The output of the magnifier was also entered into a computer which was programmed to construct histograms. Stimulation was through
fine stainless needle electrodes inserted through the skin of
the ipsilateral hindpaw (4 V, 0.33 Hz, 3 ms wide pulses, 20
stimuli / trial). The electrical stimulation was delivered, and
the microelectrode was advanced to find neurons which
responded to both lightly brushing and pinching of the
skin, and to a greater degree to pinch than the others, those
neurons were confirmed to be WDR neurons [8–11]. Then
electrical stimulation was applied again, and the evoked
responses of individual neurons were recorded.
Stable extracellular recordings were obtained from the
dorsal horn neurons in L3 to L5 region of the spinal dorsal
horn. The number of the neuron discharges was recorded
and used to plot the frequency histograms. Each histogram
has 128 dots, and each dot’s duration was 11 ms, making
the duration of each histogram about 1.5 s. After 20
histogram pictures were recorded, the sum of the number
of discharges was calculated.
2.3. Experimental protocol
Once a WDR neuron was determined, electrical stimuli
were applied again and the evoked discharges were
recorded. Trigger inputs were used to make the stimuli and
recording synchronous. Each neuron was recorded for 30
min after the chemical applied to the dorsal surface of the
spinal cord. The neuron discharge frequency was recorded
at 2, 5, 10, 15, 20, 25 and 30 min after the administration.
Each time for recording lasted for 30 s. After recording for
30 min, the effect of chemicals was washed away. At least
a 30 min rest period was allowed for the next neuron
discharge recording.
2.4. Chemicals
Solutions for administration were prepared with sterilized saline, each with a volume of 10 ml of (1) 0.1, 0.5 or
1 nmol of galanin (rat-galanin, Sigma Chemical Company,
St. Louis, MO), (2) 1 nmol of galantide [Galanin (1–13)–
Substrate P (5–11) amide, Bachem, Feinchemikalien AG,
Switzerland], (3) 10 ml of 0.9% saline as a control.
2.5. Statistical analysis
The discharge frequency of WDR neuron was recorded
and used to plot the frequency histograms. Each histogram
was 1.5 s. After having recorded 20 histograms, they were
piled up and the sum of the discharge calculated. The
discharge frequency was presented as mean6standard
error of the mean (S.E.M.). The discharge frequencies
recorded during subsequent experiments were expressed as
percentage changes of the basal level of each neuron’s
discharge frequency. The difference between groups was
determined by two-way analysis of variance (ANOVA).
3. Results
3.1. Effects of galanin on the WDR neuron discharge
frequency in rats with sciatic nerve ligation
Thirty-four WDR neurons were recorded before and 2,
5, 10, 15, 20, 25 and 30 min after application of galanin or
saline on the dorsal surface of the spinal cord. Compared
with the saline treated group (n 5 9), the WDR neuron
discharge frequency decreased significantly after administration of 0.1 nmol (n 5 7; F 5 17.61, P , 0.001), 0.5
nmol (n 5 8; F 5 39.04, P , 0.001) or 1 nmol of galanin
(n 5 10; F 5 263.97, P , 0.001), as shown in Fig. 1.
S.-L. Xu et al. / Regulatory Peptides 95 (2000) 19 – 23
Fig. 1. Effect of administration 0.1, 0.5 and 1 nmol of galanin to the
dorsal surface of the L3–L5 spinal cord on the discharge frequency of
WDR neurons in rats with sciatic nerve ligation. –s–: 1 ml of 0.9%
saline as control group; –h–: galanin 0.1 nmol; –d–: galanin 0.5 nmol;
–j–: galanin 1 nmol. Data are presented as mean 1 S.E.M. The
statistical difference between groups was evaluated by two-way analysis
of variance (ANOVA), ***P , 0.001 compared with the control group.
3.2. Effects of galantide on the WDR neuron discharge
frequency in rats with sciatic nerve ligation
The discharges of 16 WDR neurons were recorded in
rats with sciatic nerve ligation. After administration of 1
nmol of galantide, the discharge frequency of WDR
neurons (n 5 7) increased significantly (F 5 44.49, P ,
0.001) compared with the saline group (n 5 9). The results
are shown in Fig. 2.
3.3. The effect of galanin on the WDR neuron discharge
frequency in ligation rats compared with intact rats
21
Fig. 3. Effects of galanin on the discharge frequency of WDR neurons in
intact and rats with sciatic nerve ligation. Intact rats: –s–: galanin 0.5
nmol; –d–: galanin 1 nmol. Ligation rats: –h–: galanin 0.5 nmol; –j–:
galanin 1 nmol. Data are presented as mean 1 S.E.M. The statistical
difference between groups was evaluated by two-way analysis of variance
(ANOVA), ***P , 0.001 compared with the control group.
quency decreased significantly after administration of 1
nmol of galanin (n 5 9; F 5 52.03, P , 0.001) in intact
rats, but 0.5 nmol of galanin had no marked effect (F 5
0.05, P 5 0.83).
In sciatic nerve ligated rats, the discharge frequencies of
27 WDR neurons were recorded. After administration of
0.5 or 1 nmol of galanin the WDR neuron discharge
frequency decreased significantly (see above). Comparing
the effects of galanin on the WDR neuron activity in intact
rats and in rats with mononeuropathy, the effects of 0.5
nmol of galanin was more pronounced in mononeuropathic
rats as compared to intact rats (F 5 14.29, P , 0.001), as
shown in Fig. 3.
In intact rats, the discharge frequencies of 23 WDR
neurons were recorded. The WDR neuron discharge fre4. Discussion
Fig. 2. Effect of administration of 1 nmol of galantide on the discharge
frequency of WDR neurons in rats with sciatic nerve ligation. –s–: 1 ml
of 0.9% saline as control group; –d–: galantide 1 nmol. Data are
presented as mean 1 S.E.M. The statistical difference between groups was
evaluated by two-way analysis of variance (ANOVA), ***P , 0.001
compared with the control group.
The results of the present study demonstrated that
galanin inhibited the activity of wide-dynamic range
neurons in a dose-dependent fashion. The effect of galanin
was more pronounced in sciatic nerve ligated rats than
intact rats. Administration of the galanin antagonist galantide resulted in a significant increase in the wide-dynamic
range neuron discharge frequency.
A peripheral nerve ligation model in rats was set up with
many of the features which are seen in neuropathic pain in
patients [12,13]. Ligation of the sciatic nerve resulted in
oedema of the nerve and a decrease in perineural blood
supply eventually resulting in damage to the peripheral
axons [14,15]. The damage was accompanied by ‘hyperalgesia’ and / or ‘allodynia’ which developed over the
following 7–14 days [12,13].
Galanin, a possible endogenous analgesic peptide, was
found to be up-regulated in primary sensory neurons
following complete sciatic nerve injury [16]. Partial nerve
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S.-L. Xu et al. / Regulatory Peptides 95 (2000) 19 – 23
injury resulted in severer ‘neuropathic pain’ as compared
to the behavioural changes seen after complete nerve
injury [16]. Furthermore, ligation of the nerve resulted in a
more pronounced up-regulation of galanin as compared to
complete nerve injury [16]. After partial sciatic nerve
ligation, the number of galanin-immunoreactive neurons
were found to be significantly increased in the ipsilateral
dorsal root ganglia suggesting that galanin might serve as
an endogenous analgesic in ‘neuropathic pain’ [16].
In support of the role of galanin in neuropathic pain are
the findings that intrathecal administration of galanin
produced an inhibitory effect on the transmission of
presumed nociceptive information in the spinal cord [17].
Recently, Yu and collaborators reported that intrathecal
administration of 3 and 6 nmol of galanin produced
significant increases in hindpaw withdrawal latency to both
noxious heat and mechanical stimulation in rats with
sciatic nerve loose ligation [7]. Our recent study demonstrated that the discharge frequency of the WDR neuron
decreased significantly after the administration of galanin
in intact rats (unpublished data). The present study demonstrated that the discharge frequency of the WDR neurons
decreased significantly after the administration of galanin
in rats with mononeuropathy. Furthermore, we also demonstrate that 0.5 nmol of galanin results in a significant
inhibition of discharge frequency in ligated rats as compared with intact rats. Our results are supported by the
findings showing that galanin expression in primary sensory neurons was up-regulated, and that the inhibitory
action of galanin enhanced after sciatic nerve injury [18].
Galantide (galanin (1–13)–substance P (5–11) amide),
the antagonist of galanin, can block the inhibitory actions
of galanin [19]. Unlike in intact rats where galantide had
no effect on the discharge frequency of WDR neurons, in
the present study administration of galantide alone increased the discharge frequency of WDR neurons in rats
with nerve ligation. These findings are supported by recent
reports showing that a tight ligation of the L7 spinal nerve
lead to a variety of neuropathic symptoms 2 weeks after
surgery, and an increase in the galanin immunoreactivity in
laminae I and II [20]. Hokfelt and his collaborators
reported that galantide enhanced the nociceptive reflex in
spinalized rats, an effect being more pronounced in ligated
rats as compared to intact rats [18]. These results indicate
that there may be an up-regulation of galanin and galanin
receptor after periphery nerve injury. Similar situations
were observed in endogenous opioid systems. Lee et al.
reported that intravenous injection of morphine or
DAMGO significantly increased the struggle latency to
paw immersion in a hot water bath in rats with mononeuropathy induced by four loose ligatures around the
common sciatic nerve [21]. The effects of morphine and
DAMGO were more potent and more prolonged in nerve
ligated rats than in intact rats [21].
In summary, the present study demonstrated that galanin
inhibited the activity of wide-dynamic range neurons dose-
dependently, an effect more pronounced in sciatic nerve
ligated rats than in intact rats. Furthermore, when galantide, a galanin antagonist, was administered on the dorsal
surface of the L3–L5 spinal cord, the wide-dynamic range
neuron discharge frequency increased significantly indicating an up-regulation of galanin and its receptor system in
rats with sciatic nerve ligation. The results suggest that
galanin plays an important role in the modulation of
presumed nociception in mononeuropathy.
Acknowledgements
This study was supported by funds from the National
Natural Science Foundation of China (NSFC), the Natural
Science Research Foundation of Peking University, the
Karolinska Institutet Foundation and the Foundation for
Acupuncture and Alternative Treatment Methods.
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