Address correspondence and reprint requests to Dr. I. M. Najm at S51 9500 Euclid Ave., Cleveland, OH 44195, U.S.A. E-mail: firstname.lastname@example.org
Summary: Purpose: A lack of selective α1-adrenergic receptor (α1-ARs) agonists and antagonists has made it difficult to clarify the precise function of these receptors in the CNS. We recently generated transgenic mice that overexpress either wild-type or a constitutively active mutant α1B-AR in tissues that normally express the receptor. Both wild-type and mutant mice showed an age-progressive neurodegeneration with locomotor impairment and probable stress-induced motor events, which can be partially reversed by α1-AR antagonists. We hypothesized that the wild-type and mutant mice may exhibit spontaneous epileptogenicity as compared with normal (nontransgenic) mice.
Methods: Normal, wild-type, and mutant mice were studied. Twenty mice (1 year old) underwent prolonged video-EEG monitoring over a 4-week period. Raw EEG data were blindly analyzed by visual inspection for the presence of interictal and ictal epileptic activities.
Results: During the acute postoperative period (≤3 days), both wild-type (26.1 ± 8.07 spikes/day) and mutant mice (116.87 ± 55.13) exhibited more frequent interictal spikes than did normal mice (2.17 ± 0.75; p value, <0.05), but all three groups showed EEG and clinical seizures. During the later monitoring periods (>3 days), wild-type and mutant mice showed more frequent interictal spikes (15.44 ± 4.07; p < 0.01; and 6.05 ± 2.46; p < 0.05, respectively) as compared with normal mice (0.41 ± 0.41), but only mutant mice had spontaneous clinical seizures (means ± SEM).
Conclusions: The selective overexpression of the α1B-AR is associated with increased in vivo spontaneous interictal epileptogenicity and EEG/behavioral seizures. These results suggest a possible role (direct or indirect) for the α1B-ARs in the development and expression of epileptogenicity.
The adrenergic receptors (ARs) are currently classified into three main subclasses; α1-, α2-, and β-receptors, each with three or more subtypes. These receptors modulate the sympathetic nervous system via the peripheral and central effects of epinephrine and norepinephrine. α1-AR subtypes (α1A, α1B, and α1D) belong to a family of seven transmembrane-spanning domain receptors (G protein–coupled receptors; GPCRs) that are predominantly coupled to the G protein, Gq/11, and to calcium mobilization (1). The distribution of α1-AR subtypes in the brain has been determined by either ligand binding on tissue and/or autoradiography or hybridization of messenger RNAs (mRNAs) (2). α1-ARs are highly abundant in the cerebral cortex and hippocampus of the mouse and human.
The α1-ARs are the least understood of the central ARs because of the lack of selective agonists and antagonists for these receptors. The activation of α1-ARs can have a facilitatory effect on neuronal transmission in the neocortex (3). In the somatosensory areas of the cortex, α1-AR activation has been found to increase the excitation seen after administration of glutamate or acetylcholine (4). α1-ARs also cause excitatory responses in subcortical areas such as the medial and lateral geniculate nuclei, the reticular thalamic nucleus, dorsal raphe, and spinal motor neurons. It appears that this activation is largely due to a decrease in resting potassium conductance and not to an increase in calcium conductance (4). α1-ARs also may affect many brain functions through nonneuronal mechanisms because they also are localized in glial cells (5).
Previous studies on the role of the α1-AR system in the initiation or propagation of seizures are scant. One study has reported that strychnine-induced convulsive behavior in mice is potentiated by noradrenergic stimulation, possibly through a α1-AR action (6). Involvement of these receptors in seizure phenotypes also may be through modulation of other neurotransmitter receptors such as N-methyl-d-aspartate (NMDA) (7) or γ-aminobutyric acid (GABA) receptors (8).
Because the lack of specific α1-AR subtype agonists and antagonists has limited the detailed study of their function in the CNS, we attempted to clarify the precise function(s) of a single receptor by creating a transgenic mouse that systemically overexpresses either wild-type or constitutively active mutant forms of the receptor. Endogenous tissue expression of the α1B-AR is achieved by using the mouse α1B-AR promoter. We recently reported that these transgenic mice showed a neurodegeneration with a parkinsonian-like locomotor impairment and behavioral motor spells that were age progressive (9). The fact that these behavioral phenotypes could be partially rescued with the α1-AR antagonist terazosin indicated that α1-ARs signaling participated in the pathologic mechanism of this model. In this study, we characterize the in vivo electroencephalographic (EEG) correlates of the motor spells by using prolonged video-EEG monitoring.
Details about the transgenic construction and genotyping were previously described (9). In brief, the promoter sequence from the mouse α1B-AR gene was isolated from a mouse genomic library (129SVJ female liver; Stratagene, La Jolla, CA, U.S.A.) (10). Two separate transgenes were constructed by using two α1B-AR cDNA types (W, wild-type; T, triple mutant). The triple mutant encodes three different point mutations in the coding region of the α1B-AR that synergistically renders the receptor highly constitutively active (11). The Cleveland Clinic Foundation Transgenic Core Facility injected ∼200 copies of each transgene into the pronuclei of one-cell B6/CBA mouse embryos, which were surgically implanted into “pseudopregnant” female mice. Founder mice were identified, and subsequent generations were genotyped by Southern blot analysis of genomic DNA extracted from tail biopsies.
At 1 year of age, normal controls (NT, Nontransgenic), W, and T mice were anesthetized with sodium pentobarbital (60 mg/kg, i.p.), and placed into a stereotaxic apparatus (Kopf Instruments, Tujunga, CA, U.S.A.). Epidural electrodes (stainless steel screws) were inserted bilaterally in the skull over the frontal lobe (LF, left frontal cortex; RF, right frontal cortex), and parietal lobe (LP, left parietal cortex; RP, right parietal cortex), with the parameters of 2.0 mm anterior and 2.0 mm posterior, respectively, and 2.0 mm lateral from bregma. Each mouse was kept in a separate cage, with 12-h dark/light cycles, with free access to food and water.
Prolonged EEG monitoring
Continuous EEG recording with time-lapse video monitoring was simultaneously carried out over a 4-week period on each mouse included in the study. The electrodes were connected to a plastic plug connector (SMP-04V-BC; J.S.T. Corporation, Waukegan, IL, U.S.A.), which was tightly fixed to the skull with orthodontic resin (The Hygenic Corporation, Akron, OH, U.S.A.) and attached to a pendulous electroslip ring (MRS 35-06P; MT GIKEN, Tokyo, Japan). This swivel device enabled the acquisition of real-time noise-free EEGs, even when the mice were freely moving and during the spontaneous seizures. EEG data were digitally and acquired and stored (four channels per mouse) by using a Vangard system (Vangard, Cleveland, OH, U.S.A.). EEG data were acquired at a sampling rate of 100 Hz. Filter settings were as follows: low-frequency filter of 1 Hz and high-frequency filter of 70 Hz. Behavioral data were simultaneously filmed with time-lapse video recording for further analyses. The video-EEG monitoring was performed every other day for the week after electrode implantation, and 1 day (24 h) per week for the next 3 weeks (days 14, 21, and 28). The correlations between EEG events and overt motor events were assessed by using a split-screen video-monitoring system.
EEG recordings were analyzed for the presence of interictal and ictal activities by using bipolar differential montages. Interictal spikes were defined as paroxysmal electrical sharp activity for a duration of 20–150 ms, with amplitude ≥3 times the baseline EEG activity (Fig. 1A and B). Because the main purpose of the study is to document the presence of spontaneous epileptogenicity in T and W, and their brains are relatively small for our screw electrodes to analyze the generator source of epileptiform discharges, we did not try to map out the location of various epileptic activities. The rates of occurrence of all interictal spikes were calculated and expressed as spikes/day.
As shown in Fig. 1C and D, the following patterns were used to define EEG seizures: (a) paroxysmal fast low-amplitude spiking (>20 Hz) followed by subsequent buildup of rhythmic activity, or (b) buildup of rhythmic activity with a gradual increase in amplitude that leads to repetitive spiking and sudden suppression followed by rhythmic activity/repetitive spiking for a duration of >10 s. These patterns are similar to those described in rats and in humans (12). The rates of occurrence of ictal events were calculated and expressed as the number of seizures/24 h. Behaviorally the seizures typically start with arrest of activity followed by generalized tonic and clonic activity with rearing of the animal.
We used the Mann–Whitney test to analyze statistical differences of these data independently, along with their temporal course after the surgery.
Prolonged video-EEG recordings
The total duration of EEG monitoring for each mouse ranged from 72 to 168 h (mean, 121.2 h). There were no significant differences in the average monitoring periods between all three experimental groups; 116.0 ± 4.0 h for normal controls (NT), 115.2 ± 19.2 h for W, and 128 ± 11.3 h for T mice. Recorded seizures were behaviorally characterized by generalized motor activity involving both forelimbs and hindlimbs bilaterally. As the video recordings used a time-lapse video recording system, further detailed semiologic characterization of the epileptic seizures was not possible.
Interictal epileptiform discharges
As shown in Fig. 2, no interictal spikes were seen in controls (NT) after 14 days of electrode implantation. T and W mice showed a significantly higher number of spikes during both the early (≤3 days) and late (>3 days) monitoring periods as compared with normal controls (Table 1 and Fig. 2). During the late stages of monitoring, T mice continued to exhibit a tendency toward higher daily spike frequencies as compared with the W mice.
Table 1. Frequency of interictal and ictal activity in three mouse groups in 24 h
Early (≤3 days)
Late (>3 days)
Early (≤3 days)
Late (>3 days)
NT, nontransgenic (normal control mice); W, wild-type mice; T, triple-mutant.
a p < 0.01, bp < 0.05, compared with NT group (Mann–Whitney test).
Rare seizures occurred in the period (≤3 days) immediately after surgical implantation of recording electrodes in the control mice. However, a higher frequency of seizures was seen in both the T and W mice during the same immediate period (Fig. 3 and Table 1). Interestingly, although interictal spiking continued to occur in W and T mice, only the T mice continued to exhibit rare seizures during the later stages of monitoring (Table 1). Recorded seizures were not of generalized onset. Ictal events arose from various locations that included left frontoparietal (Fig. 1C), right frontoparietal (Fig. 1D), and bifrontal (not shown).
The current study shows that mice overexpressing the α1B-AR exhibit spontaneous interictal epileptic spikes and seizures. We previously showed that systemic α1B-ARs overactivity is associated with hindlimb dysfunction, beginning at age 3 months, and progressively worsens. Neurodegeneration in the cerebellum, spinal cord, and basal ganglia is histologically evident at age 8–10 months. Severity of the symptoms was associated with the different transgenic constructs, with the T being much more severe than W, consistent with the various degrees of constitutive activity. At 7 months, transgenic mice did manifest behavioral seizures after exposure to stressors such as intraperitoneal saline injection (9). Older transgenic mice (age 12 months) showed generalized motor events. The relationships between the development of seizures and neurodegeneration remain unclear. Although no recordings were made in younger animals, our previous observations and current results are suggestive of multiple factors (including age effect) that culminate in the expression of epilepsy in the transgenic mice.
In humans, insights to the genetics of epilepsy have emerged from studies of twins and linkage analyses of pedigrees with familial epilepsies (13–15). Because human and rodent nervous systems respond similarly to seizure-provoking stimuli (16,17), it is possible that biochemical and physiologic mechanisms of naturally occurring convulsive disorders also are similar in these species. Many forms of epilepsy in humans and diverse species have prominent genetic determinations (18). Because the genetic constitution of the mouse is better known and more easily manipulated than that of other mammalian species, the mouse may serve as an excellent animal model for genetic and biochemical studies of epilepsy (19–22).
EEG survey of mapped, single-locus mutations began in 1979 and has helped to define electrographic seizure types in these mutants (23). Major single-locus models are the tottering (tg/tg) mutant and the lethargic mouse (lh/lh). The tottering (tg/tg) mice exhibit spontaneous absence seizures accompanied by bilaterally synchronous spike–wave discharges (chromosome 8, autosomal recessive). Metabolic activity was increased bilaterally in selected brainstem structures (24). Conversely, multifactorial epilepsy models may be represented in the El (epilepsy-like) mice and AGSs (audiogenic seizures) mice (25,26). Although NMDA receptors may be involved in the genetic susceptibility to these seizures (27,28), it also was significantly influenced by a number of external and internal environmental factors (29,30).
Although most of these mutants emerged spontaneously and allowed the establishment of seizure-prone strains by selective breeding, transgenic techniques can create some mutations with two purposes: to disrupt or to overexpress the gene product. The most common genetic alteration generated by transgenic approaches is gene disruption, which can be introduced either randomly or intentionally. The first reported epilepsy syndrome to result from a targeted gene disruption was observed in a mutant mouse strain lacking the serotonin2C (5-HT2C) receptor (31). The mechanism for this syndrome is still unknown.
Manipulation of other biogenic amine receptors also has resulted in insights into epileptogenesis. Norepinephrine (NE) is unique among neurotransmitter monoamines in that it reduces the susceptibility of the CNS to epileptiform activity (32), which may be mediated by cyclic adenosine monophosphate (AMP) in the brain (33). Moreover, in vitro experiments on rat hippocampal tissue showed that NE has a biphasic effect on evoked potentials, with β-ARs mediating an increase and the α-ARs eliciting a decrease in the amplitude of the population spikes (34). NE also has a modulating effect on other neurotransmitter receptors such as NMDA (7) and GABA (8) receptors. Moreover, the α2A-AR gene was mapped to a region of chromosome 10q close to the one affected in a form of temporal lobe epilepsy (TLE) (35,36). Pralong et al. (37,38) showed that α2-ARs mediate hyperpolarization of neurons by activating a potassium conductance and strong inhibition of the glutamate-mediated synaptic transmission in the entorhinal cortex without affecting inhibitory postsynaptic potentials. α2-ARs have been reported to mediate the antiepileptic effect in amygdala and pyriform cortex for the kindling development (39). Activation of the α2-ARs may protect against seizures in the audiogenic seizures model (40). A point mutation in the α2A-adrenergic subtype in the mouse genome, which effectively results in an α2a-AR knockout phenotype because of very low expression, resulted in the suppression of epileptogenesis (41). However, it is proposed that the α2-Ars–induced blockade of tonic seizures is due to control of seizure propagation rather than seizure initiation (42,43).
The mechanism(s) by which α1B-ARs lead to the in vivo expression of spontaneous epileptogenicity remain(s) unknown. Because our transgenic model is opposite to expectations arising from direct actions of the α1B-AR, and there is no evidence that the α1-ARs are directly involved in triggering epilepsy, it is suggested that α1B-AR–induced epileptogenesis may be through their downstream interactions with other neurotransmitter receptors. This also is consistent with previous reports that suggest that the potential epileptogenic or antiepileptogenic effects of the ARs may be through their interactions with other neurotransmitter receptors such as NMDA and GABA (44–47). Indeed, work in progress suggests that the α1B-AR somehow upregulates certain subunits of the NMDA receptor while downregulating GABAA and does so in a time-frame consistent with seizure manifestation (manuscript in preparation). Our current results suggest a role of the α1B-ARs in the development of epileptogenicity.
Acknowledgment: This study was supported by the Research Program Committees of the Cleveland Clinic Foundation and the National Institutes of Health grants 1K08-NS02046 to I.N. and 1RO1-HL61438 to D.M.P.