Unconjugated bilirubin modulates neuronal signaling only in wild-type mice, but not after ablation of the R-type/Cav 2.3 voltage-gated calcium channel

Abstract

Introduction: The relationship between blood metabolites and hemoglobin degradation products (BMHDPs) formed in the cerebrospinal fluid and the development of vasospasm and delayed cerebral ischemia (DCI) after aneurysmal subarachnoid hemorrhage (aSAH) has been the focus of several previous studies, but their molecular and cellular targets remain to be elucidated.

Methods: Because BMHDP-induced changes in Ca_v 2.3 channel function are thought to contribute to DCI after aSAH, we studied their modulation by unconjugated bilirubin (UCB) in an organotypical neuronal network from wild-type (WT) and Ca_v 2.3-deficient animals (KO). Murine retinae were isolated from WT and KO and superfused with nutrient solution. Electroretinograms were recorded before, during, and after superfusion with UCB. Transretinal signaling was analyzed as b-wave, implicit time, and area under the curve (AUC).

Results: Superfusion of UCB significantly attenuated the b-wave amplitude in the isolated retina from wild-type mice by 14.9% (P < 0.05), followed by gradual partial recovery (P = 0.09). Correspondingly, AUC decreased significantly with superfusion of UCB (P < 0.05). During washout, the b-wave amplitude returned to baseline (P = 0.2839). The effects of UCB were absent in Ca_v 2.3-deficient mice, lacking the expression of Ca_v 2.3 as proofed on the biochemical level.

Conclusions: Ex vivo neuronal recording in the murine retina is able to detect transient impairment of transretinal signaling by UCB in WT, but not in KO. This new model may be useful to further clarify the role of calcium channels in neuronal signal alteration in the presence of BHMDPs.

Keywords: bilirubin; blood degradation products; ex vivo; isolated vertebrate retina; murine ERG.

Figure 1

Expression analysis of Ca_v2.3 after SDS‐polyacrylamide gel electrophoresis and Western blot. Microsomal membranes were isolated from the retina and the neocortex of Ca_v2.3 null mutants and wild‐type littermates.12 Lane 1: 49 μg protein from Ca_v2.3‐deficient retina (−|−). Lane 3: 34 μg protein from Ca_v2.3‐competent retinae (+|+). Microsomal membranes from murine neocortices of Ca_v2.3‐deficient mice were used as negative (−|−) and from Ca_v2.3‐competent mice as positive controls (+|+), respectively. Lane 5: 32 μg protein from Cav2.3‐competent neocortical membranes. Lane 6 and 8, 72 μg of neocortical microsomal protein from Ca_v2.3‐competent or Ca_v2.3‐deficient mice, respectively. In lanes 2, 4, and 7, no protein was loaded. Peptide‐directed antibodies directed against a common epitope in all Ca_v2.3 splice variants (anti‐a1E‐com) were used as the primary antibody during Western blot analysis.9 Note that the predicted size of the full length Cav2.3 protein (262 kDa) is marked by the arrowheads

Figure 2

Schematic representation of an electroretinogram (ERG) recorded from the isolated and superfused murine retina and the parameters quantified for analysis. Latency, implicit times of the b‐wave; FWHM, full width at half maximum of the b‐wave; t _(50%), time (ms) after the flash required for the b‐wave amplitude to decay to 50% of its maximal value; AUC₁ and AUC₂, area under the curve (AUC) of the b‐wave

Figure 3

ERG recordings from superfused murine retinae. (A) B‐wave amplitude in response to repetitive light stimulation every 3 min in wild‐type mice (n = 16). UCB was only added after reaching an equilibrium of b‐wave amplitude with albumin (pre‐UCB, black circles). Superfusion with UCB 5 μmol/L lasted 45 min and was followed by a washout of the retina by albumin only. UCB significantly decreased the b‐wave amplitude after approximately 18‐24 min (P < 0.05, corrected with Holm‐Bonferroni, dark gray circles), with partial recovery toward the end of UCB superfusion (P = 0.09, corrected, gray circles). After washout, the b‐wave amplitude recovered completely (n = 16; wild type, light gray circles). (B) UCB effects were not observed in Ca_v2.3‐deficient mice (n = 14). After washout, the b‐wave amplitude tended to decrease (P = 0.09, corrected)

Figure 4

ERG responses of the isolated and superfused murine retina. Data analysis of the selected ERGs shown in Figure 3 before and after superfusion with unconjugated bilirubin (UCB) in the presence of albumin 5 μmol/L/10 μmol/L. (A) Effect of UCB on the transretinal signaling in wild‐type mice (n = 16). The b‐wave amplitude decreased significantly after 18‐24 min superfusion with UCB by 14.9% (UCB₁, P < 0.05, corrected with Holm‐Bonferroni) and spontaneously increased again after 39‐45 min (UCB₂, P = 0.09, corrected). After washout with albumin 10 μmol/L only, the b‐wave amplitude returned to baseline. (B) Corresponding ERG traces from the normalized data presented in panel A. Superfusion with UCB (dark gray curve) decreased the (b)‐wave after 18‐24 min, followed by partial recovery (light gray curve) after 39‐45 min. (C) Effect of UCB on the transretinal signaling in Ca_v2.3‐deficient mice (n = 14). The b‐wave amplitude was not affected by UCB (P = 0.8077). After washout, the b‐wave tended to decrease by 11.6% (P = 0.09, corrected), independent from UCB (Figure 2C). (D) Corresponding ERG traces for normalized data are presented in panel C. After superfusion with UCB, the b‐wave amplitude was unaffected (UCB₁, dark gray curve vs UCB₂, light gray curve). The duration of light stimulus was always 500 ms, and it is indicated by a white bar beyond the traces