Gap Junction Blocker Carbenoxolone Research Articles

Gap junctions synchronize the firing of inhibitory interneurons in guinea pig hippocampus

The convulsant 4-aminopyridine (4AP) facilitates the synchronous firing of interneurons in the hippocampus, eliciting giant inhibitory postsynaptic potentials (IPSPs) in CA3 pyramidal cells. We used the gap junction blocker carbenoxolone to investigate the role of electrotonic coupling in both the initiation and the maintenance of 4AP-facilitated inhibitory circuit oscillations. Carbenoxolone abolished all synchronized IPSPs in CA3 cells elicited by 4AP in the presence of ionotropic glutamate receptor blockers. Carbenoxolone also blocked the isolated synchronized GABA B IPSPs generated in CA3 cells by a subpopulation of interneurons. These data confirm that: (1) the interneurons producing GABA B responses in CA3 cells are electrotonically coupled, and (2) gap junctions among interneurons are essential for initiating synchronized interneuron oscillatory firing in 4AP.

Optical recording of spreading depression in rat neocortical slices

A spreading depression (SD) was elicited in adult rat neocortical slices by microdrop application of high potassium and the SD propagation pattern was analyzed by recording simultaneously the extracellular DC potential and the changes in the intrinsic optical signal. The electrical SD with an average peak amplitude of 13.2±3.4 mV showed a good spatial and temporal correlation with the optical signal. In 79% of the slices, the SD was characterized by an initial increase of light reflectance by 2.3±1.6%, followed by a reflectance decrease of 0.5±2.4% and finally a larger and long-lasting increase by 5±2.4%. In the remaining slices, the SD revealed an initial decrease in light reflectance by 5.8±1.8% followed by an increase of 1.4±1.2%. In all slices, the recovery in the DC recording was faster as in the optical signal. The SD preferentially propagated within layers I–IV and could be blocked in most experiments by a vertical incision through upper layers or by local glutamate receptor blockade following microdrop application of kynurenic acid in layers II–III. The SD could be also blocked by bath application of kynurenic acid, MK-801 and octanol, but not by the more specific gap junction blocker carbenoxolone. Our results indicate that the high density of dendritic processes and glutamate receptors in layers II–IV promote the horizontal spread of the SD in these cortical layers and that gap junctions are not required for the propagation of SD in neocortical slices.

Neurotransmitter Coupling through Gap Junctions in the Retina

Although all bipolar cells in the retina probably use the excitatory transmitter glutamate, approximately half of the cone bipolar cells also contain elevated levels of the inhibitory transmitter glycine. Some types of cone bipolar cells make heterologous gap junctions with rod amacrine cells, which contain elevated levels of glycine, leading to the hypothesis that the bipolar cells obtain their glycine from amacrine cells. Experimental support for this hypothesis is now provided by three independent lines of evidence. First, the glycine transporter GLYT1 is expressed by the glycine-containing amacrine cells but not by the glycine-containing bipolar cells, suggesting that only the amacrine cells are functionally glycinergic. Second, the gap-junction blocker carbenoxolone greatly reduces exogenous 3H-glycine accumulation into the bipolar cells but not the amacrine cells. Moreover, when the endogenous glycine stores in both cell classes are depleted by incubating the retina with a glycine-uptake inhibitor, carbenoxolone blocks the subsequent glycine replenishment of the bipolar cells but not the amacrine cells. Third, intracellular injection of rod amacrine cells with the gap-junction permeant tracer Neurobiotin secondarily labels a heterogenous population of cone bipolar cells, all of which show glycine immunoreactivity. Taken together, these findings indicate that the elevated glycine in cone bipolar cells is not derived by high-affinity uptake or de novo synthesis but is obtained by neurotransmitter coupling through gap junctions with glycinergic amacrine cells. Thus transmitter content may be an unreliable indicator of transmitter function for neurons that make heterologous gap junctions.