Prazosin increases immobility episodes in taiep ra

Neuroscience Letters 412 (2007) 159–162
Prazosin increases immobility episodes in taiep rats without changes
in the properties of ␣1 receptors
Ma.-del-Carmen Cort´es a , Jos´e A. Arias-Monta˜no b , Jos´e-R. Eguibar a,∗
Instituto de Fisiolog´ıa and Secretar´ıa General, Benem´erita Universidad Aut´onoma de Puebla, Apdo. Postal 5-66, C.P. 72430 Puebla, Pue., M´exico
b Departamento de Fisiolog´ıa, Biof´ısica y Neurociencias, CINVESTAV, M´
exico, D.F. M´exico
Received 26 September 2006; received in revised form 31 October 2006; accepted 31 October 2006
The taiep rat is a myelin mutant in which immobility episodes (IEs) can be induced in adult males by gripping. EEG recordings during grippinginduced IEs show a rapid eye movement (REM) sleep-like pattern, similar to that reported for narcolepsy-cataplexy suggesting that IEs represent
a disorder of REM-sleep. An ␣2 adrenoceptor agonist increases gripping-induced IEs, whereas ␣2 antagonists decrease these. We have studied the
effect of prazosin on IEs and the levels of ␣1 adrenoceptors were evaluated in cerebro-cortical homogenates of taiep and control rats. Systemic
administration of prazosin results in a significant increase in both the frequency and duration of gripping-induced IEs. Our results show that
cerebro-cortical tissue is not an adequate candidate for the expression of cataplexy-like symptoms, but prazosin, an ␣1 antagonist, is a potent
inducer of gripping-induced immobility episodes in taiep rats.
© 2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: Narcolepsy; Cataplexy; Noradrenaline; Myelin mutant; REM sleep; Monoamines
Taiep rats carry a hereditary autosomal, recessive myelin disorder that leads to the development of a progressive neurological
syndrome, which is characterized by tremor, ataxia, immobility episodes (IEs), epilepsy, and finally paralysis of the
hindlimbs [8]. The rats were named after the acronym (taiep)
of the symptoms and morphologically show hypomyelination
followed by progressive demyelination of the CNS axons [11].
Ultrastructurally, oligodendrocytes reveal an accumulation of
microtubules leading to alterations in transport mechanisms
At age 8–12 months, both spontaneous and gripping-induced
IEs are observed in taiep rats, with males being more susceptible
[4]. Electroencephalographic (EEG) recordings obtained during
IEs resemble those characteristic of rapid eye movement (REM)
sleep [4] suggesting that IEs represent a disorder of REM sleep
generation similar to narcolepsy-cataplexy in canines [17].
Adrenergic transmission appears to be involved in grippinginduced IEs in taiep rats because systemic administration of ␣2
agonists significantly increased their frequency and duration,
whereas ␣2 antagonists decrease these [6]. Prazosin, a well-
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E-mail address: [email protected] (J.-R. Eguibar).
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known antihypertensive agent acting as selective antagonist at
␣1 adrenoceptors, is also a potent inducer of cataplexy episodes
in dogs and humans [1,7,14,15]. In this work, we tested the
effect of systemic prazosin on gripping-induced IEs in taiep rats
and searched for possible changes in the expression of brain
␣1 adrenoceptors by analyzing the binding of [3 H]-prazosin to
cerebro-cortical homogenates.
Taiep rats were supplied by our animal house facilities. Animals were under a 12:12 h light:dark cycle (lights on at 07:00 h),
at 21 ± 2 ◦ C and 30–45% relative humidity, with free access
to water and food. Rats were tested at 08:00 h, at the peak in
susceptibility to gripping-induced IEs [4].
Tests were done in acrylic cages. The IEs were induced by
sequentially gripping the base of the rat’s tail and 5 min later
gripping around the thorax for 10 s. If an IE is not induced, the
animal is put into the observation box [4]. The duration of the
IE and the latency to the first IE were recorded.
Prazosin hydrochloride (25–800 ␮g/kg) and WB-4101
hydrochloride (1–1000 ␮g/kg) were purchased from Sigma–
Aldrich. The drugs were freshly dissolved in sterile water,
expressed as a free drug, and the dosage volume adjusted to
1 mL/kg weight of rat. Animals received an intraperitoneal
injection of sterile water as a control and then prazosin in an
increasing dose scheme every 48 h. Behavioral analysis was
M.-d.-C. Cort´es et al. / Neuroscience Letters 412 (2007) 159–162
made by two observers, one of them blind to the drug tested.
Analysis of data were done with a χ2 -test, followed by a Dunn’s
test with P < 0.05 considered statistically significant [20].
Before each gripping-induced IE, we measured the behavioral state of the rats as (0)-indicating somnolence, (1)-quiet rat,
(2)-low activity such as sniffing and horizontal head movements,
(3)-regular activity as grooming, scratching, and locomotion,
and (4)-high activity indicating displacement and other motor
activities such as grooming, scratching, or even jumping behavior [6]. Somnolence was characterized by closed eyes, no
movement, and regular respiration.
The binding of [3 H]-prazosin to the cerebral cortex
homogenates was determined in age-matched control and taiep
rats. The animals were killed by decapitation, the brains were
quickly removed, and the cerebral cortex was dissected out.
Tissues were individually placed into plastic tubes, frozen by
immersion in liquid nitrogen, and stored at −70 ◦ C. On the day
of the binding assay, tissues were individually homogenized in
20 volumes of 10 mM TRIS–HCl buffer, pH 7.4, containing
1 mM EGTA. The homogenates were centrifuged at 20,000×g
for 30 min and the pellets resuspended in 10 mM TRIS–HCl
buffer and centrifuged again. Pellets thus formed were resuspended (∼4 mg protein/mL) [10] in incubation buffer (50 mM
TRIS–HCl, 140 mM NaCl, 5 mM MgCl2 , pH 7.4).
The radioligand assays were done simultaneously and contained 1 mL buffer, 2 nM [3 H]-prazosin (77.2 Ci/mmol, New
England Nuclear), ∼400 ␮g protein, and increasing concentrations (10−10 M–10−6 M) of unlabelled prazosin. Equilibration
was for 45 min at 25 ◦ C and termination of the reaction was
by filtration through Whatman GF/B glass fiber paper presoaked in 0.3% polyethylenimine. Nonspecific binding was
determined as that insensitive to 10 ␮M phentolamine (Sigma)
and accounted for 25–30% of total binding. Radioactivity was
measured by scintillation counting. Inhibition curves were fitted by a nonlinear regression to a logistic (Hill) equation using
the program Prism 4.03 (GraphPad). Values for the dissociation constant (Kd ) were calculated according to the equation
Kd = IC50 −(concentration of [3 H]-prazosin). The maximum
binding (Bmax ) was estimated by correcting for fractional occupancy (α) by using the equation α = Ka [D]/{Ka [D] + 1}, where
Ka is the affinity constant (1/Kd ) and [D] is the concentration of
[3 H]-prazosin (2 nM).
All procedures described in this study were in accordance
with the “Guide for the Care and Use of Laboratory Animals” of the Mexican Council for Animal Care as approved
by the BUAP Animal Care Committee and are also in accordance with guide for the care and use of laboratory animals
The prazosin dose range was 25–800 ␮g/kg in increasing successive doses at 48 h intervals. No signs of behavioral alteration
or discomfort were observed with this dose scheme.
Fig. 1A shows that whereas prazosin doses between 25 and
200 ␮g/kg had no effect on gripping-induced IEs, the two highest
doses tested (400 and 800 ␮g/kg) did induce a statistically significant increase in the analyzed behavior (χ2 -test = 29.9, d.f. = 6,
P < 0.001, followed by Dunn’s test, P < 0.05) with a maximum
effect of 216 ± 22% of control (8.2 ± 0.6 versus 3.8 ± 0.7 IEs,
mean ± S.E.M., n = 7).
Fig. 1. Effect of prazosin on the frequency and duration of gripping-induced immobility episodes in taiep rats. Values are expressed as means ± S.E.M. A) At 400
and 800 ␮g/kg prazosin induces a significant increase in the frequency of gripping-induced IEs (* P < 0.05, χ2 -test = 29.9, d.f. = 6, P < 0.001, followed by Dunn’s test
P < 0.05). B) The same prazosin doses produced a significant increase in the mean IE duration (* P < 0.05, χ2 -test = 36.8, d.f. = 6, P < 0.001, followed by Dunn’s test).
M.-d.-C. Cort´es et al. / Neuroscience Letters 412 (2007) 159–162
Table 1
[3 H]-prazosin binding to cerebro-cortical homogenates of control and taiep rats
Hill coefficient
(nH )
Kd (nM)
Bmax (fmol/mg
0.76 ± 0.04
0.80 ± 0.11
0.26 ± 0.05
0.19 ± 0.04NS
90 ± 9
90 ± 9NS
Values for Hill coefficient, dissociation constant (Kd ), and the maximum binding
(Bmax ) are mean ± S.E.M. from the number of determinations indicated, NS , Not
significantly different, P > 0.05 Student’s t-test.
The administration of prazosin also caused a dose-related
increase in the duration of IEs (Fig. 1B). For the IE frequency, statistical significance was detected only for the 400 and
800 ␮g/kg doses. For the 800 ␮g/kg dose, the mean IE duration
increased to 35.0 ± 1.5 s from 20.5 ± 1.0 s in the control session (χ2 -test = 36.8, d.f. = 6, P < 0.001, followed by Dunn’s test
P < 0.05, see Fig. 1B).
A significant decrease of latency to the first IE was also
observed with the 400 and 800 ␮g/kg doses (χ2 -test = 15.5,
d.f. = 6, P < 0.01, followed by Dunn’s test P < 0.05, Fig. 1C).
Prazosin also produced somnolence in a dose-related manner (χ2 -test = 30.7, d.f. = 6, P < 0.001, followed by Dunn’s test
P < 0.05, Fig. 1D).
Systemic administration of the selective ␣1A antagonist
WB-4101 (1–1000 ␮g/kg) did not modify the frequency of
gripping-induced IEs (χ2 -test = 11.3, d.f. = 7, P = 0.1, followed
by Dunn’s test P < 0.05; not illustrated), although somnolence
was detected (χ2 -test = 27.6, d.f. = 7, P < 0.001, followed by
Dunn’s test P < 0.05, not illustrated).
The concentration of [3 H]-prazosin present in the binding
assays (2 nM) yielded fractional occupancies of 88–91% on the
basis of the Kd estimates (Table 1). For none of the curves was the
fit to a two-site model significantly better that the fit to a one-site
model, indicating that [3 H]-prazosin bound to a single population of ␣1 adrenoceptors in accordance with the similarity of the
Hill coefficients (Table 1). Neither receptor levels (Bmax ) nor the
affinity for the radioligand (Kd estimates) were significantly different between taiep and age-matched control rats (see Table 1).
The antihypertensive drug prazosin [24] is contraindicated
in human narcoleptics because of severe exacerbation of cataplexy episodes [1]. In canine narcoleptics, Mignot et al. [14,15]
have also shown a potent exacerbation of cataplexy by prazosin,
which also blocked the improvement of cataplexy induced by
dextroamphetamine, protriptyline, and methoxamine. Further,
prazosin markedly increased the number of cataplexic attacks
and the elapsed time in both the food-elicited cataplexy test
(FECT) and the play-elicited cataplexy test (PECT) [15].
EEG recordings in taiep rats during the occurrence of IEs suggest that the latter represent a disorder of REM-sleep generation,
similar to narcolepsy-cataplexy in canines. We show herein that
prazosin enhances gripping-induced IEs, when administered at
doses of 400 and 800 ␮g/kg, indicating that in taiep rats ␣1
adrenoceptors are involved in the generation of IEs. The relatively low ability of prazosin to enter the brain may account for
the lack of effect of doses of 200 ␮g/kg and below [22].
In taiep rats, ␣2 adrenoceptor agonists increase the frequency
of gripping-induced IEs, whereas ␣2 antagonists decreased or
prevented such episodes [6]. Because ␣2 adrenoceptors function mainly as autoreceptors, either at nerve terminals or on
neuronal bodies [19,21]; the latter findings could be related to
a reduction in adrenergic transmission explaining why postsynaptic blockade of ␣1 adrenoceptors by prazosin results in an
enhancement of gripping-induced IEs in taiep rats. Indeed, Wu
et al. [23] reported that, in narcoleptic dogs, neurons of the locus
coeruleus cease discharging during periods of cataplexy.
Adrenoceptors of the ␣1B subtype have been proposed to
control canine cataplexy [16]. Our data show that in taiep rats
the prazosin action was not mimicked by the ␣1 antagonist
WB-4101, which shows 20- and 7-fold higher affinity for the
␣1A and ␣1D subtypes than for the ␣1B subtype [12], indicating
that in taiep rats prazosin enhances gripping-induced IEs by a
main action at ␣1B adrenoceptors. Similar results are reported in
canine narcolepsy in which ␣1B adrenoceptors are more involved
than other subtypes of adrenoceptors.
Mignot et al. [14,13] previously reported a significant change
in [3 H]-prazosin binding sites (32% increase) restricted to
the amigdala of homozygous narcoleptic Dobermans and that
such an increase was solely caused by ␣1B adrenoceptor upregulation. Changes in the expression of brain ␣1 adrenoceptors
could, therefore, underlie modifications in adrenergic transmission. However, our data showed that in the cerebral cortex, one of
the regions of the brain with the highest receptor densities [2]; ␣1
adrenoceptors were not significantly different between taiep and
control rats suggesting that the cortex is not a likely candidate
for the expression of cataplexy-like symptoms. Because in our
determinations no regional dissection was attempted, changes
in particular brain areas could have been missed and thus, a
detailed characterization is required to gain further insight into
the ␣1 adrenoceptor-mediated mechanisms involved in IEs.
The ␣1 antagonist prazosin induces a significant increase in
both the frequency and duration of gripping-induced IEs without significant differences in cerebro-cortical ␣1 adrenoceptor
In summary, our results show that postsynaptic ␣1 adrenoceptors participate in gripping-induced IEs in taiep rats. Although
the precise locus of action remains to be established, brain-stem
areas involved in the control of muscle tone and REM sleep
mechanisms [9] are potential candidates.
This research was supported by the CONACyT grant
43674-A, VIEP-BUAP grant 04/SAL/06-I to J.R. Eguibar and
PROMEP-BUAP-688 to MCC. We are grateful to A. Ugarte, J.
Garc´ıa, and J. Lazcano for maintenance of taiep rats and to Dr.
Ellis Glazier for editing the English-language text.
[1] M.S. Aldrich, A.E. Rogers, Exacerbation of human cataplexy by prazosin,
Sleep 12 (1989) 254–256.
[2] D. Arcos, A. Sierra, A. Nu˜nez, G. Flores, J. Aceves, J.A. Arias-Monta˜no,
Noradrenaline increases the firing of a population of subthalamic neurons
through the activation of ␣1 -adrenoceptors, Neuropharmacology 45 (2003)
M.-d.-C. Cort´es et al. / Neuroscience Letters 412 (2007) 159–162
[3] D. Clark, Guide for the Care and Use of Laboratory Animals, National
Academy of Sciences, 2002, pp. 148.
[4] M.C. Cort´es, B. Gavito, M.L. Ita, J. Valencia, J.R. Eguibar, Characterization
of the spontaneous and gripping-induced immobility episodes on taiep rats,
Synapse 58 (2005) 95–101.
[5] E. Couve, J.F. Cabello, J. Krsulovic, M. Roncagliolo, Binding of microtubules to transitional elements in oligodendrocytes of the myelin mutant
taiep rat, J. Neurosci. Res. 47 (1997) 573–581.
[6] J.R. Eguibar, M.C. Cort´es, J. Valencia, J.A. Arias-Monta˜no, ␣2 Adrenoceptors are involved in the regulation of the gripping-induced
immobility episodes in taiep rats, Synapse 60 (2006) 362–370.
[7] C. Guilleminault, E. Mignot, M. Aldrich, M.A. Quera-Salva, M. Tiberge,
M. Partinen, Prazosin contraindicated in patients with narcolepsy, Lancet
332 (1988) 511.
[8] B. Holmgren, R. Urb´a-Holmgren, L. Riboni, E.C. Vega-Saenz de Miera,
Sprague–Dawley rat mutant with tremor, ataxia, tonic immobility episodes,
epilepsy and paralysis, Lab Anim. Sci. 39 (1989) 226–228.
[9] Y.Y. Lai, J.M. Siegel, Muscle tone suppression and stepping produced by
stimulation of midbrain and rostral pontine reticular formation, J. Neurosci.
10 (1990) 2727–2734.
[10] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randal, Protein measurement
with the Folin phenol reagent, J. Biol. Chem. 193 (1951) 265–275.
[11] K.F. Lunn, M.K. Clayton, I.D. Duncan, The temporal progression
of the myelination defect in the taiep rat, J. Neurocytol. 26 (1997)
[12] M.C. Michel, B. Kenny, D.A. Schwinn, Classification of ␣1 -adrenoceptors
subtypes, Naunyn Schmiedeberg’s Arch. Pharmacol. 352 (1995) 1–10.
[13] E. Mignot, S.S. Bowersox, J. Maddaluno, W. Dement, R. Ciaranello, Evidence for multiple [3 H]-prazosin binding sites in canine brain membranes,
Brain Res. 486 (1989) 56–66.
[14] E. Mignot, C. Guilleminault, S. Bowersox, A. Rappaport, W.C. Dement,
Effect of alpha 1-adrenoceptors blockade with prazosin in canine narcolepsy., Brain Res. 15 (1988) 184–188.
[15] E. Mignot, C. Guilleminault, S.S. Bowersox, A. Rappaport, W.C. Dement,
Role of central alpha-1 adrenoceptors in canine narcolepsy, J. Clin. Invest.
82 (1988) 885–894.
[16] S. Nishino, B. Fruhstorfer, J. Arrigoni, C. Guilleminaul, W.C. Dement, E.
Mignot, Further characterization of the alpha-1 receptor subtype involved
in the control of cataplexy in canine narcolepsy, J. Pharmacol. Exp. Ther.
264 (1993) 1079–1084.
[17] S. Nishino, E. Mignot, Pharmacological aspects of human and canine narcolepsy, Prog. Neurobiol. 52 (1997) 27–78.
[18] L.T. O’Connor, B.D. Gotees, E. Couve, J. Song, I.D. Duncan, Intracellular
distribution of myelin protein gene products is altered in oligodendrocytes
of the taiep rat, Mol. Cell. Neurosci. 16 (2000) 396–407.
[19] R.R. Ruffolo Jr., J.P. Hieble, ␣-Adrenoceptors, Pharmacol. Ther. 61 (1994)
[20] S. Siegel, Non Parametric Statistics for the Behavioral Sciences, McGrawBook Company, New York, 1970.
[21] K. Starke, Presynaptic autoreceptors in the third decade: focus on ␣2 adrenoceptors, J. Neurochem. 78 (2001) 685–693.
[22] E.A. Stone, H. Rosengarten, Y. Lin, D. Quatermain, Pharmacological
blockade of brain ␣1 -adrenoceptors as measured by ex vivo [3 H]prazosin
binding is correlated with behavioral immobility, Eur. J. Pharmacol. 420
(2001) 97–102.
[23] M.F. Wu, S.A. Gulyani, E. Yau, E. Mignot, B. Phan, J.M. Siegel, Locus
coeruleus neurons: cessation of activity during cataplexy, Neuroscience 91
(1999) 1389–1399.
[24] S. Yusuf, Preventing vascular events due to elevated blood pressure, Circulation 113 (2006) 2166–2168.