Extract
Note: Please read the complete
full text with Figures and Tables at
Introduction
Benzodiazepines (BDZ) have been widely used as anxiolytics, sedatives, hypnotics and anticonvulsants. BDZ
produce therapeutic effects through the major fast
inhibitory neurotransmitter receptors,
GABAA receptors, in the central nervous system (CNS). BDZ positively modulate
GABAA receptors by increasing their affinity for GABA and
thus reduce the excitability of the post-synaptic neurons.
However, long-term treatment with BDZ often leads to
drug tolerance, which means the therapeutic efficacy of the
drug decreases after a period of continuous administration.
Another consequence of chronic administration of BDZ is
the dependence manifested by the aggravation of primary
symptoms and/or appearance of abstinence syndrome, which
includes anxiety, discomfort, diarrhea, loss of bodyweight
and salivating when the drug is withdrawn abruptly.
Tolerance and dependence are frequently encountered in the
clinical use of BDZ as an antiepileptic medicine. The usefulness
of BDZ is therefore limited, although most have primarily
satisfactory antiepileptic efficacies.
Many studies have focused on the mechanisms of
tolerance and dependence. Changes of binding capacity, gene
expression and metabolism in neural receptors, including
GABAA receptors[1], glutamatergic
receptors[2] and peripheral BDZ
receptors[3], were studied in the presence of
tolerance and/or dependence. Other factors, such as nitric
oxide[4], calcium-channel
blockers[5], protein
kinases[6] and bicarbonate
radicals[7] were also studied. However, the exact
mechanism underlying the tolerance and dependence is still
unknown.
Neuropeptide Y (NPY) is a polypeptide of 36 amino acid
residues widely distributed in the nervous system. This
neuropeptide regulates various functions such as food
uptake, blood pressure, circadian rhythm and anxiety. It
induces the central and peripheral activities through at least
six receptor subtypes called
Y1-Y6 that belong to the
G-protein coupled receptor
superfamily[8].
After a period of BDZ treatment, the glutamatergic
system was modified, which was thought to be one of the
reasons underlying tolerance to BDZ. For example, an increase
of 206% in in vitro glutamate release was found in the
hippocampus of animals chronically treated with
lorazepam[2]. Based on the facts that the NPYergic system inhibited the
release of glutamate from presynaptic
membranes[9] and that NPY was reduced in the CNS in alcohol
dependence[10], the changes of NPYergic system in rat models with BDZ
tolerance and dependence attracted our attention.
In this study, rat models of anticonvulsant tolerance and
dependence to flurazepam (FZP, a BDZ) were established
and the changes of NPY and its receptors
(Y1, Y2, and Y5) in
the hippocampus of these rat models were investigated.
Materials and methods
The establishment of rat models and the experiments on
rats in this study were approved by the Ethics Committee of
Animal Experiments of Peking University First Hospital.
Rat model of FZP tolerance The rat model of FZP
tolerance used was produced by following the method
developed by Rosenberg[11]. Male Sprague-Dawley rats (initial
weight 180-200 g) were housed in a climate-controlled room
with free access to standard rat food. FZP dissolved in a
0.02% saccharin solution was given as drinking water for 1
week (FZP-tolerant group, n=10). The concentration of FZP
was adjusted to provide a daily dose of up to 100 mg/kg for
the first 3 d and 150 mg/kg for the next 4 d. All rats consumed
an average concentration of more than 100 mg/kg FZP daily.
Control rats (n=10) were handled identically, but received
0.02% saccharin solution without the drug. After one week
of treatment, rats were given saccharin solution as drinking
water. Withdrawal signs such as overt ataxia, anxiety and
hyperactivity were not found with this
treatment[12].
Tolerance to FZP was evaluated 12 h after
discontinuation of FZP intake. Because of the very rapid
biotransformation of FZP and its metabolites in rats, and the
corresponding plasma half-life
(T1/2) of less than 2 h, the residual drug
should not interfere with the anticonvulsant test at this
time[13]. FZP dissolved in distilled water (100 mg/mL) was
diluted to 20 mg/ml with normal saline for injection. This
solution was injected intraperitoneally to deliver a dose of
20 mg/kg. After 30 min, 20 mg/mL pentylenetetrazol (PTZ,
freshly prepared in normal saline) was infused at a
constant rate of 0.5 mL/min into a tail vein, and clonus of the
front legs was monitored to determine the threshold of
PTZ-induced seizures. The onset time of clonus was recorded,
and the PTZ threshold (mg/kg) was then derived. Rats in the
control group were tested along with those in the
FZP-tolerant group using the same procedure. FZP tolerance was
determined by the decrease of PTZ threshold as compared
with that of the control group. After the PTZ threshold test,
the brain was removed and stored at -70 ºC immediately.
Rat model of FZP dependence We integrated the
methods reported by Rosenberg[11] and Izzo
et al[14] to establish a rat model of FZP dependence. The animals used, drug
dosage, method of drug intake and PTZ threshold test were
from the method reported by
Rosenberg[11], but the days of drug administration and the dependence test were from the
method reported by Izzo et
al[14]. To obtain a more stable blood concentration, male Sprague-Dawley rats were given
FZP dissolved in 0.02% saccharin solution as drinking water
instead of an oral gavage of diazepam three times
daily[14]. For rats in the FZP-dependent group
(n=10), FZP was given at increasing doses for 14 d: d 1-3, 100
mg·kg-1·d-1; d 4-7, 150
mg·kg-1·d-1; d 8-10, 200
mg·kg-1·d-1; and d 11-14, 250
mg·kg-1·d-1. Control rats
(n=10) were handled identically, but received 0.02% saccharin solution without the drug
as drinking water. The emergence of dependence was
determined by the susceptibility to PTZ-induced seizures 96 h
after termination of the 14-day FZP administration. PTZ (20
mg/mL) was infused at a constant rate of 0.5 mL/min into a
tail vein and the infusion was discontinued at the first sign
of tonic-clonic seizures. The PTZ threshold (mg/kg) was
derived from the amount of PTZ used to induce tonic-clonic
seizures. Rats in the control group were treated identically.
FZP dependence was determined by the decrease of the PTZ
threshold as compared with that of the control group. After
the PTZ threshold test, the brain was removed and stored at
-70 ºC immediately.
PTZ-induced seizures themselves may also cause changes in the brain. To minimize these interferences, we
killed rats immediately after the PTZ threshold test and
compared the changes in the hippocampus with those from the
respective control rats.
Reagents FZP was purchased from DaZhong
Pharmaceutical (Shanghai, China).
Rats were from the Animal Department of Peking
University Health Sciences Center. PTZ and anti-NPY antibody
were from Sigma-Aldrich (Saint Louis, MO, USA).
RNase-free DNase I was obtained from Promega (Madison, WI, USA),
Trizol reagent and M-MuLV reverse transcriptase were from
Gibco (Rockville, MD, USA). The sequences of
oligonucleotide primers for the amplification of preproNPY (386 bp),
Y1 (426 bp), Y2 (393 bp), and
Y5 (391 bp) cDNA are: NPY-F, 5¡¯- TATCCCTGCTCGTGTGTTTG-3¡¯; NPY-R, 5¡¯-
AACGACAA-CAAGGGAAATGG-3¡¯; Y1-F, 5¡¯-
ACTCTCACAGGCTGTC-TTAC-3¡¯; Y1-R, 5¡¯- ATAGTCTCGTAGTCGTCGTC-3¡¯;
Y2-F, 5¡¯-AGCCTTTCCACCCTGCTAAT-3¡¯;
Y2-R, 5¡¯-GTGAATGGCA-TCCAACCTCT-3¡¯;
Y5-F, 5¡¯-CACCTAGCCGTTCCAGAA-AA-3¡¯;
Y5-R, 5¡¯- GGGCTCTCAAGTCTGCTTTG-3¡¯.
Measurement of preproNPY and NPY receptor cDNA in
hippocampus by competitive RT-PCR Animals were
decapitated immediately after the PTZ threshold test. The
hippocampus was quickly removed and stored at -70 ºC until use.
Brain tissue was homogenized in Trizol reagent in a
pre-cooled mortar following manufacturer¡¯s
protocol[15]. Total RNA samples were treated with RNase-free DNase I to elimi
nate genomic DNA contamination before reverse
transcrip-tion. Total RNA of 3 µg was reversely transcribed into cDNA
using oligo d (T)15 as the primer and M-MuLV reverse
transcriptase.
The internal competitive standards for the measurement
of preproNPY, Y1, Y2, and
Y5 cDNA were made using PCR. These DNA fragments had the same sequences as their
respective PCR products, except that approximately 80 bp at
the downstream site of the forward primers were deleted.
Quantitative PCR was performed in a 0.2 mL tube containing
brain cDNA 1 µL, internal competitive standard 1 µL, 5
µmol/L each primer mixture 1µL, 2.5 mmol/L each dNTP mixture 1
µL, 1.5 mmol/L MgCl2, 10×PCR buffer 2.5 µL, and 1.25 unit
Taq DNA polymerase, in a total volume of 25 µL. PCR was run at
94 ºC 30 s, 62 ºC 30 s, and 72 ºC 40 s (93 ºC 1 min, 65 ºC 2 min
and 72 ºC 2 min for the amplification of
Y1 cDNA) for 35 cycles. PCR products were separated in a 1.5% agarose gel
and stained using ethidium bromide. The ratio of the optical
density of the two DNA bands under UV light was measured
by a gel image analysis system. A standard curve of the ratio
was drawn using the same conditions as described above,
except that brain cDNA was changed to a serial dilution of
the purified PCR product. The relative amount of the cDNA
in a sample was then obtained from the standard curve.
Immunohistochemistry of NPY in hippocampus
After anesthesia, rats were perfused through the left ventricle with
0.9% sodium chloride followed by 4% paraformaldehyde in
phosphate buffer solution for approximately 30 min. The
removed brain was sequentially soaked in 4%
paraformaldehyde in phosphate buffer solution for 24 h and 30% sucrose
at 4 ºC for 72 h. Frozen sections of 8-mm thickness in a
coronal plane were used for immunohistochemistry. One
brain section from the treated rat and one from the
respective control rat were placed on one slide and processed
together. After treatment with 0.3%
H2O2 to block the endogenous peroxidase, sections were incubated with 1:50
polyclonal anti-NPY antibody overnight at 4
ºC. The secondary antibody was biotinylated anti-rabbit IgG antibody
(Vector, Burlingame, CA, USA). Signals were visualized by
horseradish peroxidase conjugated avidin and
diamino-benzidine. The relative density of NPY-immunoreactive
material in neurons in the pyramidal cell layer of CA1,
CA3 region and granular cell layer of the dentate gyrus region was
measured in a defined area by a densitometer.
Statistical analysis Data were expressed as mean±SD.
Paired t-tests were used to evaluate the significance of
intergroup differences. P<0.05 was considered as significant.
Results
Rat models of FZP tolerance and dependence
FZP-tolerant model The rat model of FZP tolerance was
evaluated by studying the FZP anticonvulsant efficacy after
discontinuing the 7-d FZP treatment for 12 h when the brain
FZP should have been metabolized[13]. Anticonvulsive
efficacy of a single dose of 20 mg/kg FZP was measured by
studying the threshold of PTZ-induced seizures after 30 min.
In comparison with the control group, the average PTZ
threshold decreased by more than 3 times in the
FZP-tolerant group, indicating the successful establishment of an
FZP-tolerant model in rats (Table 1).
FZP-dependent model In contrast to the evaluation of
FZP tolerance, FZP dependence was estimated by studying
the PTZ threshold after discontinuing the 14-d FZP
treatment for 96 h when maximum withdrawal signs were expected
to occur[14]. As shown in Table 1, the average PTZ threshold
in the FZP-dependent model was lower than that in its
control group by more than 2 times, indicating that the
FZP-dependent model is acceptable for further study (Table 1).
Changes of preproNPY cDNA in hippocampus of
FZP-tolerant and -dependent rats In the hippocampus, preproNPY
cDNA was dramatically reduced both in FZP-tolerant and
-dependent groups, as compared with that of the respective
control groups (Table 2, Figure 1).
NPY-immunoreactive cells were found in many brain
regions, with the hippocampus showing the most
remarkable changes in FZP tolerant and dependent rats. In the
hippocampus of control animals, NPY-immunoreactive cells
were mostly located in CA1, CA3, and dentate gyrus regions.
NPY-immunoreactive cells were large multipolar or fusiform
neurons, and NPY positive material was found in their
cytoplasm and processes (Figure 2A, 2B). In the hippocampus
from FZP-tolerant and FZP-dependent rats,
NPY-immunoreactive material in the cytoplasm and processes was
significantly reduced as compared with that in the same regions of
the respective control rats (Figure 2C, 2D, Table 3).
Changes of NPY receptor cDNA in hippocampus of
FZP-tolerant and -dependent rats
Y1 and Y5 cDNA were decreased in the tolerant group as compared with those of the
respective control group. However, no quantitative changes
of these two cDNA were found in the FZP-dependent group.
In contrast, Y2 cDNA was increased significantly in the
FZP-tolerant group, but was decreased in the FZP-dependent
group, as compared with that of the respective controls
(Table 2, Figure 1).
Correlations between the threshold of PTZ-induced
seizures and the levels of preproNPY,
Y1, Y2, and Y5 cDNA in
hippocampus of FZP-tolerant and -dependent rats
The correlations were examined to show whether changes of NPY
and its receptors in the hippocampus were related to the
pathogenesis of FZP tolerance and dependence. The PTZ
threshold was positively correlated with the level of
preproNPY cDNA in the hippocampus both in FZP-tolerant
and -dependent groups (Figure 3A, 3B). In other words, the
lower the expression of NPY in the hippocampus, the higher
degree of tolerance and dependence the rat showed. The
PTZ threshold was negatively correlated with the level of
hippocampal Y2 cDNA in the FZP-tolerant group and was
positively correlated with the level of hippocampal
Y2 cDNA in the FZP-dependent group (Figure 3C, 3D). No correlation
between the PTZ threshold and the changes of
Y1 and Y5 cDNA were found (data not shown).
Discussion
Pharmacologically, BDZ produce their sedative, anxiolytic
and antiepileptic activities through a modulation action on
GABAA receptors. However, tolerance to BDZ and
dependence on BDZ are the two major adverse reactions in clinical
practice. Neuronal hyperexcitability may be the common
basis for the tolerance and dependence, although they may
have different manifestations.
Hippocampus is implicated in the generation and
modulation of seizure activities, and plays a central role in
controlling excitability of the CNS[16]. In the present study, the
hippocampus was separated to investigate the changes of
NPY and its receptors (Y1,
Y2, and Y5) in rat models with
anticonvulsant tolerance and dependence to FZP.
We found that preproNPY cDNA in the hippocampus
was significantly decreased in both the FZP-tolerant and
FZP-dependent groups. Immunohistochemistry also showed
the reduction of NPY-immunoreactive material in the
neuronal cytoplasm and neurofibers of CA1, CA3, and dentate
gyrus regions. Y1 and Y5 receptor cDNA were decreased in
the tolerant group but were not changed in the dependent
group. Y2 receptor cDNA was increased in the tolerant group
but was decreased in the dependent group.
To date, only one paper has been published on the
relationship between the NPYergic system and drug tolerance
and/or dependence[17]: contingent tolerance to carbamazepine
after repeatedly giving the drug may be associated with the
down-regulation of NPY. However, no data about the
changes of the NPYergic system following long-term
treatment of BDZ have been found in the published literature.
The level of preproNPY cDNA in the hippocampus
reversely relating to the degree of tolerance and dependence
may suggest that the changes of preproNPY cDNA are
involved in the pathogenesis of FZP tolerance and dependence.
NPY, one of the most abundant and widely distributed
neuropeptides in the central and peripheral nervous systems,
acts as an endogenous modulator to reduce seizure activities.
In the hippocampus, the inhibition of epileptiform discharges
after NPY treatment was attributed to the decrease of
glutamate released from the presynaptic nerve terminals
through blocking of the G-protein dependent calcium
channel[18]. Therefore, the decrease of cellular NPY we found
may result in neuronal hyperexcitability that is the
fundamental pathological state of BDZ tolerance and dependence.
Six subtypes of NPY receptors,
Y1-Y6, have been identified. Their pharmacological characteristics are very
similar, although the identity of amino acid sequences is as
low as 30% among these receptor
subtypes[19]. Of the six receptor subtypes,
Y1, Y2, and Y5 are the major functional
receptors and are expressed abundantly. In rodents, changes
in the Y1 receptor and its mRNA in the CNS can be induced
by various stimuli, suggesting the multiple functions of the
Y1 receptor[20]. The
Y2 receptor demonstrates a predominant role in modulation of seizure
activity[21]. The Y5 receptor
may also be involved in the modulation of anticonvulsant
activity in rodents[22,23]. Theoretically, drug tolerance and
dependence are caused by the imbalance of excitation over
inhibition in the brain, and NPY receptors would decrease in
association with the decrease of NPY. The different changes
to NPY receptors between the FZP-tolerant and
FZP-dependent group and the increase of
Y2 in FZP-tolerant rats in this study can not be satisfactorily explained because of the
insufficient and conflicting data regarding
Y1, Y2, and Y5
receptors in the regulation of neuronal excitability at the present
time. More experiments should be performed on the
distribution and ligand binding capacity of the three subtype
receptors in FZP tolerance and dependence.
Our preliminary results suggest that the decrease of NPY
in the hippocampus plays an important role in the
generation of anticonvulsant tolerance and dependence following
long-term FZP treatment. The NPYergic system may be a
new target for the comprehension of these adverse effects
of BDZ.
References
References
1 Brown MJ, Wood MD, Coldwell MC, Bristow DR.
Gamma-aminobutyric acid A receptor function is desensitised in rat
cultured cerebellar granule cells following chronic flunitrazepam
treatment. J Neurochem 1998; 71: 1232-40.
2 Bonavita C, Ferrero A, Cereseto M, Velardez M, Rubio M,
Wikinski S. Adaptive changes in the rat hippocampal gluta-
matergic neurotransmission are observed during long-term
treatment with lorazepam. Psychopharmacology (Berl) 2003; 166:
163-7.
3 Byrnes JJ, Miller LG, Perkins K, Greenblatt DJ, Shader RI. Chronic
benzodiazepine administration. XI. Concurrent administration
of PK11195 attenuates lorazepam discontinuation effects.
Neuropsychopharmacology 1993; 8: 267-73.
4 Gupta N, Bhargava VK, Pandhi P. Tolerance and withdrawal to
anticonvulsant action of clonazepam: role of nitric oxide.
Methods Find Exp Clin Pharmacol 2000; 22: 229-35.
5 Mizoguchi H, Yoshiike M, Suzuki T, Misawa M. Effects of
Ca2+ channel blockers on physical dependence on diazepam in mice.
Life Sci 1993; 53: PL365-70.
6 Lilly SM, Zeng XJ, Tietz EI. Role of protein kinase A in
GABAA receptor dysfunction in CA1 pyramidal cells following chronic
benzodiazepine treatment. J Neurochem 2003; 85: 988-98.
7 Zeng XJ, Tietz EI. Role of bicarbonate ion in mediating
decreased synaptic conductance in benzodiazepine tolerant
hippocampal CA1 pyramidal neurons. Brain Res 2000; 868: 202-14.
8 Blomqvist AG, Herzog H. Y-receptor subtypes — how many
more? Trends Neurosci 1997; 20: 294-8.
9 Colmers WF, Lukowiak K, Pittman QJ. Neuropeptide Y action
in the rat hippocampal slice: site and mechanism of presynaptic
inhibition. J Neurosci 1988; 8: 3827-37.
10 Bison S, Crews F. Alcohol withdrawal increases neuropeptide Y
immunoreactivity in rat brain. Alcohol Clin Exp Res 2003; 27:
1173-83.
11 Rosenberg HC. Differential expression of benzodiazepine
anticonvulsant cross-tolerance according to time following flurazepam
or diazepam treatment. Pharmacol Biochem Behav 1995; 51:
363-8.
12 Rosenberg HC, Chiu TH. Tolerance during chronic
benzodiazepine treatment associated with decreased receptor binding. Eur
J Pharmacol 1981; 70: 453-60.
13 Lau CE, Falk JL, Dolan S, Tang M. Simultaneous determination
of flurazepam and five metabolites in serum by
high-performance liquid chromatography and its application to
pharmacokinetic studies in rats. J Chromatogr 1987; 423: 251-9.
14 Izzo E, Auta J, Impagnatiello F, Pesold C, Guidotti A, Costa E.
Glutamic acid decarboxylase and glutamate receptor changes
during tolerance and dependence to benzodiazepines. Proc Natl
Acad Sci USA 2001; 98: 3483-8.
15 Sammbrook J, Fritsch EF, Maniatis T. Molecular cloning, a
laboratory manual, 3rd Ed. Cold Spring Harbor (NY): Cold
Spring Harbor Laboratory Press; 2001. p 8.86-9.
16 Loscher W, Ebert U. The role of the piriform cortex in kindling.
Prog Neurobiol 1996; 50: 427-81.
17 Weiss SR, Clark M, Rosen JB, Smith MA, Post RM. Contingent
tolerance to the anticonvulsant effects of carbamazepine:
relationship to loss of endogenous adaptive mechanisms. Brain Res
Brain Res Rev 1995; 20: 305-25.
18 Qian J, Colmers WF, Saggau P. Inhibition of synaptic
transmission by neuropeptide Y in rat hippocampal area CA1:
modulation of presynaptic Ca2+ entry. J Neurosci 1997; 17: 8169-77.
19 Parker RM, Herzog H. Regional distribution of Y-receptor
subtype mRNAs in rat brain. Eur J Neurosci 1999; 11: 1431-48.
20 Gariboldi M, Conti M, Cavaleri D, Samanin R, Vezzani A. Anti
convulsant properties of BIBP3226, a non-peptide selective
antagonist at neuropeptide Y Y1 receptors. Eur J Neurosci 1998;
10: 757-9.
21 Kaga T, Fujimiya M, Inui A. Emerging functions of
neuropeptide Y Y(2) receptors in the brain. Peptides 2001; 22: 501-6.
22 Woldbye DP, Larsen PJ, Mikkelsen JD, Klemp K, Madsen TM,
Bolwig TG. Powerful inhibition of kainic acid seizures by
neuropeptide Y via Y5-like receptors. Nat Med 1997; 3: 761-4.
23 Wu YF, Li SB. Neuropeptide Y expression in mouse
hippocampus and its role in neuronal excitotoxicity. Acta Pharmacol Sin
2005; 26: 63-8.
|
