Norepinephrine-Deficient Mice Have Increased Susceptibility to Seizure
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The Journal of Neuroscience, December 15, 1999, 19(24):10985–10992 Norepinephrine-Deficient Mice Have Increased Susceptibility to Seizure-Inducing Stimuli Patricia Szot,1,6 David Weinshenker,2,5 Sylvia S. White,1 Carol A. Robbins,3 Nicole C. Rust,2,5 Philip A. Schwartzkroin,3,4 and Richard D. Palmiter2,5 Departments of 1Psychiatry and Behavioral Science, 2Biochemistry, 3Neurological Surgery, and 4Physiology/Biophysics and 5Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195, and 6GRECC, Puget Sound Health Care System, Seattle, Washington 98108 Several lines of evidence suggest that norepinephrine (NE) can acid. c-Fos mRNA expression in the cortex, hippocampus (CA1 modulate seizure activity. However, the experimental methods and CA3), and amygdala was increased in Dbh 2/2 mice in used in the past cannot exclude the possible role of other association with flurothyl-induced seizures. Enhanced seizure neurotransmitters coreleased with NE from noradrenergic ter- susceptibility to flurothyl and increased seizure-induced c-fos minals. We have assessed the seizure susceptibility of geneti- mRNA expression were reversed by pretreatment with L-threo- cally engineered mice that lack NE. Seizure susceptibility was 3,4-dihydroxyphenylserine, which partially restores the NE con- determined in the dopamine b-hydroxylase null mutant (Dbh tent in Dbh 2/2 mice. These genetically engineered mice 2/2) mouse using four different convulsant stimuli: 2,2,2- confirm unambiguously the potent effects of the noradrenergic trifluroethyl ether (flurothyl), pentylenetetrazol (PTZ), kainic acid, system in modulating epileptogenicity and illustrate the unique and high-decibel sound. Dbh 2/2 mice demonstrated en- opportunity offered by Dbh 2/2 mice for elucidating the path- hanced susceptibility (i.e., lower threshold) compared with lit- ways through which NE can regulate seizure activity. termate heterozygous (Dbh 1/2) controls to flurothyl, PTZ, kainic acid, and audiogenic seizures and enhanced sensitivity Key words: dopamine b-hydroxylase; c-fos mRNA; norepi- (i.e., seizure severity and mortality) to flurothyl, PTZ, and kainic nephrine; flurothyl; epilepsy; seizure; kainic acid Chen et al. (1954) first suggested that the noradrenergic system Although there is significant evidence that the NE system is modifies seizure activity. Since then, four major observations anticonvulsant, there are several considerations that temper one’s have supported an anticonvulsant role for norepinephrine (NE): confidence in the hypothesis that NE, itself, reduces seizure (1) selective lesioning of noradrenergic neurons (with sensitivity. For example, although the lesioning studies (i.e., 6-hydroxydopamine or DSP-4) increases seizure susceptibility to chemical destruction of noradrenergic terminals) reduce the a variety of convulsant stimuli (Arnold et al., 1973; Jerlicz et al., amount of NE release, this manipulation also reduces the release 1978; Mason and Corcoran, 1979; Snead, 1987; Trottier et al., of other transmitters coreleased with NE. The neuropeptides 1988; Sullivan and Osorio, 1991; Mishra et al., 1994); (2) direct galanin and neuropeptide Y (NPY) and the neurotransmitter stimulation of the locus coeruleus (LC, the major concentration adenosine (i.e., ATP) are released at noradrenergic terminals and of noradrenergic cell bodies in the CNS) and the subsequent have been shown to exert anticonvulsant effects against several release of NE reduce CNS sensitivity to convulsant stimuli (Libet convulsant stimuli (Murray et al., 1985; Mazarati et al., 1992, et al., 1977; Turski et al., 1989); (3) genetically epilepsy-prone rats 1998; Dichter, 1994; Erickson et al., 1996; Baraban et al., 1997). A (GEPRs), a widely used animal model of epilepsy, have deficient similar argument can be made for the anticonvulsant effect of presynaptic NE content, NE turnover, tyrosine hydroxylase lev- direct LC stimulation, which results in the release not only of NE b els, dopamine -hydroxylase (DBH) levels, and NE uptake (Jobe but also of these cotransmitters. The enhanced seizure sensitivity et al., 1984; Dailey and Jobe, 1986; Browning et al., 1989; Laut- of the GEPRs may not be caused solely by their abnormal nor- erborn and Ribak, 1989; Dailey et al., 1991); and (4) adrenergic adrenergic system, because these animals also have abnormalities a a agonists acting at the -2 adrenoreceptor ( 2-AR) have anticon- in their central serotonergic, GABAergic, and excitatory amino vulsant action (Papanicolaou et al., 1982; Baran et al., 1985; acid systems (Faingold et al., 1986; Dailey et al., 1992; Meyerhoff Loscher and Czuczwar, 1987; Fletcher and Forster, 1988; Jackson et al., 1992); moreover, other animal models of epilepsy have a et al., 1991). higher than normal central NE content (Noebels, 1986; Hara et al., 1993). Finally, the a2-AR pharmacological studies are difficult Received July 7, 1999; revised Sept. 28, 1999; accepted Sept. 30, 1999. to interpret because the effect of clonidine (a2-AR agonist) on This work was supported by the National Alliance for Research on Schizophrenia and Depression (P.S.), the Department of Veterans Affairs (P.S.), National Institutes seizure-induced activity can be biphasic, nonexistent, or even of Health Grant NS-18895 (P.A.S.), and the Howard Hughes Medical Institute proconvulsant (King and Burnham, 1982; Tacke and Kolonen, (D.W. and N.C.R). We thank Sumitomo Pharmaceuticals for their generous dona- a tion of DOPS. 1984; Lapin and Ryzor, 1990). Such multiple responses to 2-AR P.S. and D.W. contributed equally to this work. agonists may be caused by the localization of the affected a2-AR. Correspondence should be addressed to Dr. Patricia Szot, Geriatric Research, Activation of presynaptic a2-AR autoreceptors would reduce Education, and Clinical Center (182B), Veterans Affairs Medical Center, 1660 South Columbian Way, Seattle, WA 98108. E-mail: [email protected]. transmitter released at NE terminals (L’Heureux et al., 1986), Copyright © 1999 Society for Neuroscience 0270-6474/99/1910985-08$05.00/0 whereas activation of postsynaptic a2-ARs would mimic the effect 10986 J. Neurosci., December 15, 1999, 19(24):10985–10992 Szot et al. • Seizure Susceptibility in Dbh Knock-Out Mice of released NE. Because it has not been determined whether the 1/2 and Dbh 2/2 mice was determined by exposing animals to a 115 dB anticonvulsant effect of a2-AR agonists is mediated via pre- or sound for 60 sec with an SR Pilot (San Diego Instruments, San Diego, CA). After the sound was started, the mouse was closely monitored for postsynaptic receptors, it remains unclear whether increased NE occurrence of a seizure. If no seizure occurred, the sound was terminated release is anti- or proconvulsant. after 60 sec. If a seizure was observed during the 60 sec period, the sound Taken together, these studies suggest that changes in norad- was immediately terminated, and the animal was removed. Mice were renergic functions (terminal NE content or release) can modulate scored as exhibiting or not exhibiting a seizure. seizure activity, but they do not resolve the issue of whether NE c-Fos mRNA expression after flurothyl-induced seizures. The mice that survived flurothyl-induced generalized seizures were killed by cervical is, itself, anticonvulsant. It is this issue that we have addressed dislocation 1 hr after the seizure [Dbh 1/2 (n 5 8) and Dbh 2/2 (n 5 with the DBH null mutant (Dbh 2/2) mouse. These animals 6) without DOPS; Dbh 1/2 (n 5 8) and Dbh 2/2 (n 5 9) with DOPS]. selectively lack NE and epinephrine (dopamine content tends to To determine basal c-fos mRNA expression Dbh 1/2 (n 5 6) and Dbh 2 2 5 be elevated) because DBH is required for the conversion of / (n 6) mice (same age as the flurothyl-tested mice) were also killed. Brains were collected from each animal and immediately frozen on dry dopamine to NE (Thomas et al., 1998). ice. Twenty micrometer coronal sections containing neocortex and hip- pocampus were cut on a cryostat and mounted onto Fisher Superfrost MATERIALS AND METHODS slides (Fisher Scientific, Houston, TX). Slides were stored at 270°C until assayed. Animals. Mice were derived from a hybrid line (129/Sv/Ev and C57BL/ 2 2 1 2 Tissue preparation and labeling of the c-fos oligonucleotide was per- 6J). Dbh / and heterozygote (Dbh / ) mice were bred as described formed as described previously (Szot et al., 1997). The c-fos oligonucle- previously (Thomas et al., 1995). Mice were maintained on a 12 hr otide probe was a 51-base probe complementary to nucleotides 270–319 light/dark cycle in a specific pathogen-free facility at the University of of the c-fos mRNA (Curran et al., 1987). The oligonucleotide probe was Washington (Seattle, WA). Food and water were available ad libitum, 39-end-labeled with [ 33P]dATP (New England Nuclear, Boston, MA) and animals were maintained according to the guidelines outlined in the using terminal deoxyribonucleotidyl transferase (Life Technologies, NIH Guide for Care and Use of Laboratory Animals. All animal proce- Gaithersburg, MD) and then purified on NEN-Sorb columns (New dures were approved by the University of Washington Animal Care 2 2 England Nuclear). The c-fos hybridization buffer for the flurothyl- Committee. Genotype was deduced from phenotype (Dbh / mice induced seizure assay contained 0.3 3 10 6 cpm/50 ml. The c-fos hybrid- exhibit delayed growth during adolescence and ptosis), and a subset of ization buffer for the basal assay contained 0.4 3 10 6 cpm/50 ml. mice was confirmed by PCR (Thomas et al., 1995).