Sulforhodamine 101 (SR101) has recently been identified as a cell type-selective fluorescent marker of astrocytes, both in vivo and in slice preparations. As a result, its use as an experimental tool has rapidly accelerated, based on SR101 as a phenotypic reporter. In the present study, we have found that SR101 is also a potent bioactive compound. In particular, we observed that SR101 induced LTP-IE, increases in synaptic efficacy, and spontaneous firing in hippocampal slices. Strikingly, the concentration of SR101 that caused sustained changes in neuronal excitability was 10-25-fold lower than that typically used to tag live astrocytes. Moreover, injection of SR101 into the adult rat hippocampus evoked epileptic seizures.
One of the key observations in this study was that SR101 increased intrinsic neuronal excitability (LTP-IE). The membrane potential threshold for evoking action potentials decreased slowly and reached its lowest level 120 −140 min after 10 min exposure to 1 μM SR101. The reduction of the AP threshold was remarkably consistent, and all cells studied exhibiting a significant decrease in the AP threshold. Neuronal excitability increased because the AP threshold fell from −48.6 ± 0.9 mV to −61.5 ± 2.5 mV. Thus, the threshold for generating AP was just 3 mV higher than the resting membrane potential (−64.7 ± 0.6 mV). In addition, SR101 also induced a long-lasting increase in eEPSCs that further increases synaptic inputs and spontaneous firing. SR101-induced LTP-IE is distinct from LTP-IE resulted from activity-dependent inhibition of afterhyperpolarization (AHP) (Saar et al., 1998
; Ireland and Abraham, 2002
; Melyan et al., 2002
; Sourdet et al., 2003
; Melyan et al., 2004
) and increase of input resistance (Campanac et al., 2008
). However, SR101 induced LTP-IE shared the similarity of NMDAR-dependency with correlated stimulation-induced LTP-IE (Xu et al., 2005
SR101 potentiated synaptic NMDAR currents evoked by stimulating Schaffer collateral fibers, supporting that SR101 enhances activation of synaptic NMDARs. The long-lasting increase in synaptic NMDAR currents () suggests that continuing activation of NMDARs induces slowly and progressively developed LTP-IE. Since activation of NMDARs requires synaptic release of glutamate, the SR101 effect depends on spontaneous synaptic activity. Thus, SR101 potentiates activation of NMDARs in a long-lasting manner, leading to progressive development of LTP-IE. The blockade of the late portion of LTP-IE by AP-5 applied after SR101 application () and the induction of the late portion of LTP-IE by washing out AP-5 () support this idea. These results also suggest that SR101 potentiates activation of NMDARs after washing out SR101, implying that SR101 may affect NMDARs intracellularly. SR101 may enhance synaptic NMDAR currents by directly affecting gating property of NMDARs. The results that the rising time of NMDAR currents was increased by SR101 and the SR101 analog, Texas Red-hydrazide, induced a similar LTP-IE support this idea. Astrocytic release of gliotransmitters induced by loading SR101 seems not involved because patching astrocytes with SR101 did not induce LTP-IE (). The intracellular pathway that couples activation of NMDARs to voltage-gated sodium channels is unknown yet. The possible pathway is that NMDAR-mediated increases in intracellular Ca2+ activate Ca2+-dependent Cam/Kinases and/or PKA pathways, which in turn modulate voltage-gated sodium channels.
The mechanism underlying SR101-induced potentiation of eEPSCs is more complicated, because glutamate receptor antagonists only partially inhibited the major portion of the potentiation (). The major portion of SR101-induced potentiation of eEPSCs involves activation of NMDARs because AP-5 inhibited this portion of the potentiation (). The pathways involved in glutamate receptor-independent portion were not fully explored in this study. It is possible that SR101 also directly enhancing activation of AMPA receptors in the dendritic membrane of pyramidal neurons by affecting either gating or phosphorylation of AMPARs. Thus, additional studies are required to dissect how SR101 induces the glutamate receptor-independent portion of the potentiation.
It is in this study documented that SR101 induced LTP-IE, increases in synaptic efficacy, and epileptic seizures. The bioactive properties of SR101 may permit its use in creating animal models of epileptic seizures. However, the bioactive effects of SR101 also raise concerns with regard to the use of SR101 as a specific marker of astrocytes where neuronal functions are studied.