Evidence that the median raphe nucleus - Dorsal hippocampal pathway mediates diazepam withdrawal induced anxiety

On the basis of our previous series of experiments we had postulated that the increased anxiety that occurred during diazepam withdrawal was mediated by increased 5-HT release in the hippocampus. The present series of experiments provide evidence for a major role of the median raphe nucleus (MRN) dorsal hippocampal pathway. Rats were treated once daily for 21 days with diazepam (2 mg/kg IP) and then tested after 24 h withdrawal in the social interaction test of anxiety. Relative to chronically vehicle treated animals, those withdrawn from diazepam were significantly more anxious and had significantly greater K+-evoked release of [H-3]-5-hydroxytryptamine (5-HT) from slices of dorsal and of ventral regions of the hippocampus. Estimation of extracellular concentrations of 5-HT within the dorsal hippocampus, using in-vivo microdialysis, showed doubling in the levels of 5-HT in the rats withdrawn from chronic diazepam treatment. This just failed to reach significance, but 33% of the rats showed dramatic increases (650%). It was not possible to test these animals in the social interaction test, but it is proposed that only the diazepam-withdrawn rats with raised extracellular levels of 5-HT would have displayed increased anxiety. 5-HT1A receptor agonists injected into the MRN decrease the MRN firing rate, and hence the release of 5-HT in the dorsal hippocampus. As a further test of our hypothesis, we examined the effects of MRN injection of the 5-HT1A receptor agonist, 8-OH DPAT, on animals withdrawn from diazepam and tested in the low light familiar condition of the social interaction test. 8-OH DPAT (50-200 ng) dose-dependently reversed the anxiogenic effect of diazepam withdrawal, while having no effects in chronic vehicle-treated animals. These results provide clear evidence that the MRN-dorsal hippocampal 5-HT pathway is at least one of the pathways playing an important role in mediating diazepam withdrawal-induced anxiety.