Evidence for the exclusive expression of functional homomeric α7 nAChRs in hypothalamic histaminergic tuberomammillary neurons in rats.

Hypothalamic histaminergic tuberomammillary (TM) neurons in rats express high densities of nicotinic acetylcholine receptors (nAChRs) whose Ca(2+) permeability, kinetic and pharmacological properties are similar to those of heterologous homomeric α7 nAChRs. However, native α7 nAChR subunits can co-assemble with ß or α5 nAChR subunits to form functional heteromeric α7-containing α7ß or α7α5 nAChRs with kinetics and pharmacology similar to those of α7 homomers. Therefore, although TM nAChRs have been used as an ex vivo model of functional α7 homomers, the molecular makeup of TM nAChRs has not been determined and the expression of functional α7-containing heteromers in TM neurons has not been excluded. To determine the profile of TM nAChR subunit transcripts, we have conducted single-cell qRT-PCR experiments using acutely dissociated TM neurons in rats. TM neurons were found to express transcripts of only principal α3, α6 and α7 nAChR subunits. Transcripts of other known mammalian neuronal subunits (α2, α4-5, α9-10, ß2-4) were not detected. In the absence of ß and α5 subunits, the expression of functional α7-containing heteromers in TM neurons is highly unlikely because principal α3, α6 and α7 nAChR subunits alone are not known to form functional heteromeric nAChRs. These results support the exclusive expression of native functional α7 homomers in rat TM neurons and introduce these neurons as a unique reliable source of native functional homomeric α7 nAChRs suitable for ex vivo and in vitro pharmacological assays in developing selective α7 nAChR agents.

Differences 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.

DBA mice are models of sudden unexpected death in epilepsy (SUDEP) that exhibit audiogenic generalized convulsive seizures (GCS), ending in death due to respiratory arrest (RA). Serotonin (5-HT) normally enhances respiration in response to elevated CO(2) levels, which occur during GCS in patients. Fluoxetine, a selective serotonin reuptake inhibitor (SSRI), blocks GCS-induced SUDEP in both DBA/2 and DBA/1 mice. This study examined the effects of a 5-HT(2B/2C) agonist (m-chlorophenylpiperazine, mCPP) to test the generality of serotonergic effects on DBA mice. In DBA/2 mice mCPP pre-treatment [5 or 10 (but not 2) mg/kg, i.p.] significantly reduced RA incidence without blocking seizure susceptibility. However, in DBA/1 mice mCPP in doses up to 40mg/kg was ineffective in blocking seizure-induced RA, and 60mg/kg was toxic. The cause of this strain difference was perplexing. Previous studies showed that brainstem 5-HT receptor protein expression was abnormal in DBA/2 mice. Therefore, expression of 5-HT receptor proteins in the medial-caudal brainstem of DBA/1 mice was evaluated using Western blots. In DBA1/mice 5-HT(2C) and 5-HT(3B) receptor expression levels were significantly reduced, as seen previously in DBA/2 mice. However, 5-HT(2B) receptor expression was also reduced in DBA/1 mice, contrasting with the 5-HT(2B) receptor elevation seen in DBA/2 mice. This difference may explain the differential effects of the 5-HT(2B/2C) agonist in these SUDEP models. mCPP blocked RA in DBA/2 mice and concomitantly reduced tonic seizures, which also occurs. Fluoxetine is the only agent tested that blocks RA selectively in these SUDEP models, which may be clinically relevant.

Abnormal serotonin receptor expression in DBA/2 mice associated with susceptibility to sudden death due to respiratory arrest.

Previous studies indicate that DBA/2 mice may be a useful model of human sudden unexpected death in epilepsy (SUDEP), since these mice exhibit generalized convulsive seizures followed by respiratory arrest (RA). Respiratory deficits, following generalized convulsive seizures, are observed prior to SUDEP in patients. RA that occurs in DBA/2 mice following sound-induced seizures can be prevented by treatments that activate serotonin (5-HT) receptors. 5-HT receptor subtypes in brainstem respiratory centers are important in regulating normal respiration. The present study compared the expression of 5-HT subtype receptor proteins in excised brainstem tissue, containing the rostral ventral medulla respiratory region in DBA/2 mice vs. seizure-resistant C57BL/6J mice, using Western blot analysis. The results indicate that expression of specific 5-HT(2C), 5-HT(3), and 5-HT(4) receptor proteins in the brainstem tissue of DBA/2 mice is significantly diminished, while expression of the 5-HT(2B) receptors is significantly enhanced as compared to C57BL/6J mice. No difference in expression of 5-HT transporter protein is seen. These findings suggest that the DBA/2 mice are susceptible to RA, in part, because of the altered expression of 5-HT receptors. Preliminary studies indicate that 5-HT(2C) receptors may be particularly important, since a 5-HT(2C) agonist is very effective in blocking RA in DBA/2 mice.

Adenosine receptor antagonists and behavioral activation in NF-kappaB p50 subunit knockout mice.

AIMS: Our previous work revealed that mice lacking the p50 subunit of transcription factor nuclear factor kappa B (NF-kappaB) (p50 KO mice) and genetically intact F2 mice have similar locomotion under basal conditions, yet p50 KO mice show greater locomotor activation after caffeine ingestion. In this report, we test whether KO mice display altered caffeine pharmacokinetics or increased caffeine-induced DA turnover relative to F2 mice, and evaluate the impact of intraperitoneal administration of specific adenosine and DA receptor antagonists on locomotor activity. MAIN METHODS: Concentrations of DA and caffeine were measured using high performance liquid chromatography. DA turnover was measured after treatment of mice with an inhibitor of tyrosine hydroxylase. Locomotor activity was measured using telemetry. KEY FINDINGS: The data reveal that 1) caffeine concentrations in blood and brain are similar in KO and F2 mice after oral or intraperitoneal administration; 2) KO mice show greater DA turnover under basal conditions, but turnover is similar in both strains after caffeine administration; 3) the specific A2AAR antagonist SCH 58261 induces greater locomotion in KO versus F2 mice; and 4) the activating effect of SCH 58261 in KO mice is prevented by prior treatment with the D2R antagonist raclopride. SIGNIFICANCE: These findings support the conclusions that 1) A2AAR has a major impact on behavioral activation of p50 KO mice, and 2) D2R mediated neurotransmission is important to this effect.

Migration of cochlear lateral wall cells.

The role of apoptosis and proliferation in maintenance of cochlear lateral wall cells was examined. The methods employed for detection of apoptosis were the Hoechst fluorescence stain and TUNEL (TdT-mediated dUTP-biotin nick-end-labeling) assay, and proliferations were 5-bromo-2'-deoxyuridine (BrdU) incorporation and presence of the proliferating cell nuclear antigen. The incidence of apoptosis in the strial marginal cell was 50% greater (32.9+/-3.7%) than strial intermediate and basal cells but similar to spiral ligament cells. Although division of marginal strial cells was rarely detected, a significant number of proliferating cells in the remaining stria vascularis and spiral ligament were observed. These data implied that replacement of marginal cells arose elsewhere and could be followed by a BrdU-deoxythymidine pulse-chase study. At 2 h post injection, nuclear BrdU in marginal cells was not detected; however, by 24 h post injection, 20-25% of marginal cell nuclei were BrdU-positive. These observations are consistent with the hypothesis that marginal cells were replaced by underlying cells. Cell migration appears to be an important mechanism for preserving the function and structure of the stria vascularis.