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Keywords:

  • Epilepsy;
  • α1b-Adrenergic receptor;
  • Noradrenaline;
  • Neurodegeneration;
  • Sensitization;
  • Methylenedioxymethamphetamine

Summary

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Purpose: The role of α1b-adrenergic receptor (α1b-AR) in relation with neuronal degeneration, drug addiction, and seizure susceptibility has recently emerged. In particular, mice that overexpress α1b-AR undergo spontaneous epileptic seizures and progressive neuronal loss in a variety of brain areas. Therefore, one should expect that the blockade of α1b-AR leads to anticonvulsant and neuroprotective effects. However, the lack of α1b-AR antagonists does not allow testing of this hypothesis.

Methods: The development of α1b-AR knockout (KO) mice led us to measure seizure susceptibility and neurodegeneration following systemic excitotoxins in these mice.

Results: We found that α1b-AR KO mice are markedly resistant to kainate- and pilocarpine-induced seizures. Moreover, when marked seizure duration and severity are obtained by doubling the dose of chemoconvulsants in α1b-AR KO, neuronal degeneration never occurs.

Conclusions: These data indicate that α1b-AR per se plays a fundamental role in the mechanisms responsible for seizure onset, severity, and duration, whereas the brain damage observed in α1b-AR–overexpressing mice is likely to be a secondary phenomenon. In fact, the absence of α1b-AR confers resistance to neurotoxicity induced by seizures/chemoconvulsants. These data, although confirming a pivotal role of α1b-AR in modulating seizure threshold and neuronal death, offer a novel target, which may be used to develop novel anticonvulsants and neuroprotective agents.

The role played by α1-adrenergic receptors (α1-ARs) in vivo, when challenged using α1-agonists or antagonists often led to conflicting results. This becomes even more confused when considering the effects of α1-agonists and antagonists in epileptic seizures (Weinshenker & Szot, 2002; Giorgi et al., 2004).

In recent years, the genetic modeling of a mouse strain that overexpresses α1b-ARs led to spontaneous seizures and widespread degeneration (Zuscik et al., 2000). Overactivity of α1b-ARs is supposed to trigger neuronal death, which depends upon allosteric interaction between α1b-AR and N-methyl-d-aspartate (NMDA)–glutamate receptors, according to Paladini et al. (2001). Spontaneous seizures observed in this strain of mice may be the consequence of enhanced recruitment of glutamate NMDA receptors induced by α1b-AR overexpression (Paladini et al., 2001).

However, when considering the presence of a diffuse neuronal loss, which is also present early in limbic regions of these mice, it is debatable whether seizure activity occurs as a primary effect of the overexpression of α1b-ARs or is instead the consequence of an extensive neuronal damage.

To solve this question in the present study we used the opposite approach. In fact, we used mice lacking α1b-AR (knockout, KO). These mice were challenged with epileptogenic compounds: If overexpression of α1b-AR is the primary trigger for spontaneous seizures we expect that α1b-AR KO mice are resistant to seizures, compared with the wild-type (WT). Conversely, if spontaneous seizures observed in α1b-AR–overexpressing mice are the consequence of the brain damage, α1b-AR KO mice should not be refractory to seizures. Again, if overexpression of α1b-ARs is detrimental for neuronal survival independently of seizure-triggering effects, we expect an increased resistance to excitotoxicity in α1b-AR KO mice.

We recently demonstrated that mice previously exposed to amphetamines develop seizures as part of a latent brain hyperexcitability caused by limbic sensitization (Giorgi et al., 2005); since α1b-AR KO mice are resistant to sensitization induced by amphetamines (Drouin et al., 2002), we also evaluated whether the absence of α1b-ARs suppresses amphetamine-induced seizure susceptibility.

Methods

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Animals

The generation of α1b-AR KO mice (kindly provided by S. Cotecchia, Université de Lausanne, Switzerland) has been described previously (Cavalli et al., 1997). Briefly, 129/C57 BL is the common genetic background for KO mice and their WT controls, which derive from the same germline of microinjected clones. For each strain, mice from different litters were randomly intercrossed to obtain WT and KO progenies and never intercrossed with other strains or mated with those from the same litters; therefore, their genetic background should not be too different from that of the animals described in the previous study. Mice were kept under environmentally controlled conditions with food and water ad libitum. Experiments were carried out following the National Institutes of Health Guidelines for Animal Care and Use.

Part I: Evaluation of the role of α1b-AR in epilepsy

Testing seizure susceptibility with epileptogenic stimuli

Both α1b-AR KO and WT mice were treated with epileptogenic compounds administered at different convulsive doses by i.p. injections. Kainic acid and pilocarpine were used. Kainic acid (Sigma Chemical Co., St Louis, MO, U.S.A.) was administered at the dose of 35 or 70 mg/kg dissolved in 200 μl of distilled water. Kainic acid induces in mice limbic seizures characterized by a rapid progression from facial movements and forelimb clonus to repetitive rearing and falling up to occasional “pop corn” behavior (Mckhann et al., 2003). Pilocarpine (Sigma) was injected at the dose of 200 and 400 mg/kg dissolved in 200 μl of phosphate-buffered saline; 30 min before pilocarpine treatment, mice were injected with 1 mg/kg body weight methyl-scopolamine (Sigma) to block peripheral cholinergic actions of pilocarpine (Turski et al., 1984).

Limbic seizures observed in these experiments were scored according to a modified Racine’s scale (Racine, 1972) as follows: score 1 = repetitive intense jaw clonus; score 2 = forelimb clonus; score 3 = brief (<5 s) rearing with forelimb clonus; score 4 = prolonged (>10 s) bilateral forelimb clonus; score 5 = clonus of the four limbs with rearing and loss of balance. Scores 1–3 were considered mild seizures, whereas 4–5 were considered severe seizures.

EEG electrode implantation and recordings

Mice were anesthetized with chloral-hydrate (400 mg/kg, i.p., Sigma Chemical Co.) and placed in a Kopf stereotaxic apparatus. For each mouse, two handmade steel epidural electrodes were inserted through a hole drilled in the skull. The flattened (and subsequently enlarged) end of the electrodes possessed a recording surface of 2.25 mm2. One electrode was placed above the left frontal cortex (recording), and one below the occipital bone (reference). The electrodes were kept in place by dental acrylic. Recording electrodes impedance was ∼3kΩ.

Starting at 48 h after electrodes implantation, electroencephalography (EEG) recordings were obtained from WT and KO mice before and after administration of epileptogenic agents by using a dedicated EEG apparatus (Vega Polygraph; E.S.A.O.T.E. Biomedica, Florence, Italy). EEG recording started at 10 min before the injection of chemoconvulsants.

Morphologic studies

The occurrence of neuronal damage following comparable seizure duration was verified by using staining with cresyl violet and Fluoro Jade B (gentle gift from Larry Schmued, NCTR, FDA Jefferson, AR, USA). Under chloral hydrate anaesthesia, mice were perfused transcardially with 4% paraformaldehyde. According to the original method of Schmued and Hopkins (2000), 20-μm slides were stained with Fluoro-Jade B and were examined using an epifluorescent microscope with blue-violet excitation light set at 450 nm. Fluoro-Jade B–stained cells emit a typical yellow color. Cresyl violet–stained slides were analyzed by light microscopy (Wetzlar Orthoplan; Leitz, Milan, Italy).

Part II: Evaluation of seizure susceptibility developed following sensitization to methylenedioxymethamphetamine

Schedule of methylenedioxymethamphetamine administration

Alpha1b-AR KO and WT mice were treated with repeated injections of methylenedioxymethamphetamine (MDMA) (Institute of Forensic Toxicology, University of Pisa, Italy). A sensitizing regimen was administered, and mice behavior was observed in the course of treatment (Itzhak et al., 2003). In pilot studies, we had identified a sensitizing regimen of MDMA in WT mice. A single dose of MDMA 2.5 mg/Kg was not able to modify motor behavior in mice; motility in the open field box and frequency of the rearing were comparable to mice treated with saline. Mice received daily i.p. injection of MDMA at a dose of 2.5 mg/kg for a total number of 25 injections. A group of control mice received at the same time treatment with saline.

Assessment of behavioral changes during MDMA exposure

Animals were observed in an open field box (80 × 80 cm). In each session, the number of open field squares crossed and the number of rearing episodes in each time interval (2 min) was counted for 30 min before and after MDMA injection.

Kainic acid after MDMA exposure

One month after the last exposure to MDMA/saline, α1b KO and WT mice were treated with kainic acid, administered i.p. at the dose of 10 mg/kg dissolved in 200 μl.

Statistical analysis

Counts of locomotor and rearing activity in open field were expressed as the means ± SEM and compared using analysis of variance (ANOVA) with Sheffé’s post hoc analysis (p < 0.05). Seizure incidence (occurrence of both episodic and prolonged seizures) and seizure severity were compared using the Mann-Whitney U test for nonparametric grouped data (p < 0.05).

Results

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Part I

Seizure activity

None of the α1b KO mice displayed seizures following kainic acid 35 mg/kg and pilocarpine 200 mg/kg. In contrast, kainic acid induced seizures in all WT, whereas pilocarpine induced seizures in 50% of WT (Fig. 1A).When doubling the doses, we observed moderate seizures also in KO mice (Fig. 1A).

image

Figure 1.   Seizure activity in wild-type (WT) and α1b-AR knockout (KO) mice. (A) Mice were injected with kainic acid (KA) and pilocarpine (PIL). KA (35 mg/kg) induces limbic seizures in all WT mice, whereas none of the KO mice exhibited seizures. Similarly, PIL (200 mg/kg) induces seizures in 50% of WT mice, but no seizures in KO. By doubling the doses of KA and PIL, WT had severe seizures, whereas some KO had moderate seizures. (B) Representative electroencephalography (EEG) traces of seizures induced by KA. KA (35 mg/kg) did not produce modification on the EEG trace of KO mice, whereas it induced seizures in WT. The EEG pattern corresponds to low-grade seizure as shown by brief runs of sharp waves, whereas high grade corresponds to prolonged sharp waves, spikes, and spike and waves. (C) Previous exposure to methylenedioxymethamphetamine (MDMA 2.5 mg/kg × 25 injections) induces seizure susceptibility in WT but not in KO mice.

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EEG recordings

In keeping with convulsive episodes, kainic acid 35 mg/kg and pilocarpine 200 mg/kg did not produce changes in EEG activity in KO mice. The EEG pattern corresponding to a mild seizure showed brief runs of sharp waves, whereas for severe seizures we observed prolonged sharp waves, spikes, and spike and waves. These EEG patterns were independent of the strain of mice and type of chemoconvulsant. Representative EEGs are shown in Fig. 1B.

Part II: Behavioral sensitization

Behavioral sensitization induced by repeated administration of MDMA

Repeated injections of MDMA (2.5 mg/kg) led to a progressive increase in locomotor activity (both open field and rearing) in WT animals. In contrast, no change in α1b KO locomotion was observed (Fig. 2).

image

Figure 2.   Knockout (KO) mice are resistant to behavioral sensitization induced by methylenedioxymethamphetamine (MDMA). Repeated injections of MDMA (2.5 mg/kg × 25 injections) led to progressive increase in locomotor activity measures as (A) open field and (B) and rearing in WT mice, which was related to the number of injections up to a plateau. In contrast, in KO, locomotion remained constant following repeated MDMA injections.

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Seizure activity after MDMA sensitization

As expected, a pronounced susceptibility for developing limbic seizures after a subthreshold dose of kainic acid was observed in WT mice. All of these mice developed kainic acid–induced serial limbic seizures starting shortly after the end of kainic acid injection (12 ± 3 min). In contrast, previous exposure to MDMA did not affect seizure susceptibility in KO mice (Fig. 1C).

Neurodegeneration

In WT mice, following pilocarpine (200 mg/kg) or kainic acid (35 mg/kg) there was a marked cell loss and neuronal degeneration as shown by cresyl violet and Fluoro Jade-B, respectively. These phenomena were most prominent in the hilus of the hippocampus and were also evident in the subregion CA1 and CA3. In contrast, no evidence of cell loss or degenerating neurons was observed in KO mice, even with doubling of the dose of each chemoconvulsant (Fig. 3). This means that neither an equivalent dose of excitotoxin nor an equivalent amount of seizure severity and duration produced comparable cell loss in α1b-AR KO mice.

image

Figure 3.   Knockout (KO) mice are resistant to chemoconvulsant-induced brain damage. For the same high-grade seizures, KO mice did not have the marked cell loss in the various subfields of the hippocampal formation.

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Discussion

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

These experiments demonstrate that α1b-AR plays a fundamental role in the mechanisms responsible for seizure generation, severity, and duration. Moreover, this receptor plays a crucial role in preventing neuron loss and neurodegeneration (Battaglia et al., 2003). Finally, the presence of α1b-AR is critical in the development of sensitization and long-lasting brain hyperexcitability following repeated administration of MDMA. This latter effect extends to the regulation of seizure threshold, which is lowered following repeated administration of low daily MDMA doses.

Therefore, the present data we collected in α1b-AR KO mice mirror those obtained in mice overexpressing α1b-AR. This allows us to conclude that α1b-AR is critical to promote cell death, seizure activity, and behavioral sensitization. These conclusions are justified even in the absence of specific pharmacologic ligands acting as agonists or antagonists at α1b-AR, since the appropriate use of genetically engineered mice (done by combining results obtained in mice overexpressing or knocked out for the same gene) provide convincing evidence for the role of such fascinating receptors.

Although the overexpression of α1b-AR produces spontaneous seizures (Zuscik et al., 2000; Kunieda et al., 2002), we found that the lack of α1b-AR confers a marked resistance to seizures induced by different chemoconvulsants. Again, although overexpression of α1b-AR produces spontaneous neuronal degeneration, which affects the hippocampal formation (Zuscik et al., 2000), we found that lack of α1b-AR suppresses cell loss and neuronal degeneration in the same brain area. In keeping with the suppression of amphetamine-induced sensitization observed in α1b-AR KO mice by Drouin et al. (2002), here we observed the lack of MDMA-induced sensitization in the same strain of mice. Again, such a lack of sensitization was bound to the absence of long-lasting limbic hyperexcitability, which is otherwise responsible for a lowered seizure threshold in MDMA pretreated mice (Giorgi et al., 2005).

Moreover, the data we obtained offer an answer to the potential linkage between seizure susceptibility and brain damage. In fact, the spontaneous occurrence of neurodegeneration and seizures in mice that overexpress α1b-AR left open the question of whether seizure susceptibility was primarily affected in these mice or it was instead the consequence of the brain damage. Our data indicate that the first hypothesis is correct; this was further confirmed by data obtained by doubling the dose of the excitotoxins. In fact, if overexpression of α1b-ARs was detrimental for neuronal survival independent of the triggering of seizures, we expect an increased resistance to excitotoxins in α1b-AR KO mice, as found in the present work. Incidentally, this is in line with the concept that toxicity induced by kainic acid in mice at the dose of 35 mg/kg is not the consequence of seizure activity but the result of its excitotoxic properties, as recently suggested by Benkovic et al. (2006). Therefore, the role of α1b-AR in seizures and neuronal degeneration is not causally related but it is rather the consequence of two independent mechanisms, which eventually may lead to synergistic detrimental effects.

Therefore, we might hypothesize that a novel antiepileptic therapy, based on the blockade of α1b-AR receptor, is expected to be both neuroprotective and anticonvulsant. Such a therapeutic approach should be particularly effective in protecting from seizure-induced brain damage.

Acknowledgment

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

Disclosure: The authors declare no conflicts of interest.

References

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References