Research ReportDifferences in serotonin receptor expression in the brainstem may explain the differential ability of a serotonin agonist to block seizure-induced sudden death in DBA/2 vs. DBA/1 mice
Highlights
► A 5-HT2B/2C agonist (mCPP) blocks seizure-induced death in DBA/2 mice. ► mCPP is not effective in blocking seizure-induced death in DBA/1 mice. ► Decreased brainstem 5-HT2B receptor protein occurred in DBA/1 mice. ► 5-HT2B receptor expression in DBA/2 mice was elevated in previous studies. ► This difference in 5-HT2B expression may explain the difference in mCPP effects.
Introduction
Two genetically-related models of sudden unexpected death in epilepsy (SUDEP) in DBA mice have been proposed (Faingold et al., 2010a, Tupal and Faingold, 2006, Venit et al., 2004). SUDEP is an important clinical problem, estimated to be the cause of death in a significant percentage of patients with epilepsy (Asadi-Pooya and Sperling, 2009, Hughes, 2009, Sperling, 2001). The overall risk of sudden death is more than 20 times higher than that in the general population (Shorvon and Tomson, 2011). Several different causes for SUDEP have been proposed, including seizure-related respiratory depression, cardiac arrhythmia, cerebral depression, and autonomic dysfunction (Bateman et al., 2008, Hughes, 2009, Richerson and Buchanan, 2011, Shorvon and Tomson, 2011).
The establishment of useful animal models is important for the development of preventative therapies of SUDEP. Evidence for DBA/2 mice as a useful model for SUDEP due to respiratory depression has been well-documented, and effective approaches to prevention of seizure-induced sudden death in this model have also been demonstrated (Tupal and Faingold, 2006, Venit et al., 2004). The only effective pharmacological preventative therapy for sudden death in the DBA mice models that has been shown, thus far, is the administration of fluoxetine, a selective serotonin reuptake inhibitor (SSRI) (Faingold et al., 2011, Tupal and Faingold, 2006). However, receptors for serotonin (5-hydroxytryptamine, 5-HT), exist in many subtypes (Terry et al., 2008). The increase in 5-HT availability induced by SSRIs would likely activate all of these 5-HT receptor subtypes, potentially leading to a number of unwanted adverse effects, as seen with the clinical use of SSRIs (Rief et al., 2009). Therefore, 5-HT receptor subtype-selective agents currently under development have the potential to possess greater selectivity by activating the specific 5-HT receptors that are involved in the susceptibility of the DBA mice to SUDEP (Uteshev et al., 2010).
In these DBA mouse models, the generalized convulsive seizures (GCS) are induced by acoustic stimuli, and the cause of seizure-induced sudden death is respiratory arrest (RA), leading to subsequent cardiac failure (Faingold et al., 2010a, Faingold et al., 2010b, Tupal and Faingold, 2006), which is consistent with the findings in many cases of human SUDEP, although the clinical definition of SUDEP does not require evidence of a seizure (Bateman et al., 2010b, Blum, 2009, Tomson et al., 2008). Enhancing 5-HT availability in the DBA mouse SUDEP models is postulated to prevent seizure-induced RA based on the role that 5-HT plays in normal respiration and arousal mechanisms. 5-HT normally acts on the brainstem respiratory network to enhance respiration in the medullary respiratory centers and mediate arousal via the ascending arousal network in response to elevated levels of CO2 (Benarroch, 2007, Buchanan and Richerson, 2010, Budzinska, 2009, Feldman et al., 2003, Hodges and Richerson, 2010, Richter et al., 1999). CO2 levels are known to rise in epileptic patients in association with generalized convulsive seizures (GCS) related to the accompanying respiratory depression (Bateman et al., 2008, Bateman et al., 2010a, Bateman et al., 2010b).
The expression of 5-HT2C, 5-HT3B, and 5-HT4 subtype receptor proteins in the brainstem respiratory region of DBA/2 mice is significantly diminished, while expression of the 5-HT2B receptors is significantly enhanced as compared to control C57BL/6J mice (Uteshev et al., 2010). These data suggest that a selective 5-HT agonist might be effective in blocking seizure-induced sudden death in DBA/2 mice, and the present study examined this hypothesis by administering a 5-HT2B/2C agonist (m-chlorophenylpiperazine, mCPP) to evaluate the ability of this agent to prevent seizure-induced RA in DBA/2 mice.
The DBA/2 model of SUDEP is subject to an important limitation, because consistent susceptibility to seizure-induced RA only lasts for approximately one week (Tupal and Faingold, 2006). However, a closely-related model of SUDEP, DBA/1 mice, which are also subject to sudden death due to respiratory arrest that follows their seizures, exhibit susceptibility to SUDEP that lasts for several months (Faingold et al., 2010a). Seizure-induced sudden death in DBA/1 mice can also be blocked by prior administration of the SSRI, fluoxetine, although the dose required is considerably higher than the effective dose in DBA/2 mice (Faingold et al., 2011).
The question arises if the expression of 5-HT subtype receptor proteins in the respiratory region of DBA/1 mice is abnormal and whether it parallels the expression profile seen in DBA/2 mice, and this issue was also examined in the present study. Finally, a comparison was made of the actions of a 5-HT2B/2C agonist (mCPP) in the DBA/1 vs. DBA/2 mice to evaluate whether the ability of this agent to affect seizure-related parameters differs quantitatively or qualitatively between the two SUDEP models.
Section snippets
Seizure testing
The effects of mCPP (2–10 mg/kg, i.p) on seizure behaviors and seizure-induced RA were evaluated in the acute SUDEP model in DBA/2 mice (Fig. 1).
Neither saline nor the 2 mg/kg dose of mCPP administered 30 min prior to behavioral testing produced any significant change in the incidence of seizure behaviors or seizure-induced RA in DBA/2 mice (Fig. 1). By contrast, significant reductions in the incidence of seizure-induced RA were seen with the higher doses of mCPP. Thus, the 5 and 10 mg/kg of mCPP
Discussion
The results of the present study indicate that mCPP, a 5-HT2B/2C agonist, was effective in blocking seizure-induced RA without blocking seizures in DBA/2 mice, but mCPP was totally ineffective and even toxic in DBA/1 mice. DBA/2 mice are an acutely susceptible model of SUDEP that exhibits a short-term (7 day) (Tupal and Faingold, 2006) susceptibility to seizure-induced RA, while DBA/1 mice exhibit an extended susceptibility (up to 8 weeks) (Faingold et al., 2010a). The block of seizure-induced RA
Animals
Male DBA/2 (N = 44) and DBA/1 mice (N = 33) obtained from Harlan Laboratories were screened for susceptibility to acoustically-induced audiogenic seizures, leading to RA, on post-natal day 21 (DBA/2) or 21–35 (DBA/1), as previously described (Faingold et al., 2010a, Faingold et al., 2010b, Tupal and Faingold, 2006). The seizure-related behaviors were recorded on video for subsequent observational evaluation. Only those DBA mice that exhibited tonic hind limb extension, leading to RA, were used
Acknowledgments
We wish to thank Trish Ellis for her manuscript assistance; and the Citizens United for Research in Epilepsy (CURE) Grant award for funding this research on SUDEP and CIR-CNS.
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