Psychopharmacology (1997) 130 : 228–234 © Springer-Verlag 1997 ORIGINAL INVESTIGATION Nick Andrews · Sandra E. File · Cathy Fernandes Luis E. Gonzalez · Nicholas M. Barnes Evidence that the median raphé nucleus – dorsal hippocampal pathway mediates diazepam withdrawal-induced anxiety Received : 10 May 1996 / Final version : 15 October 1996 Abstract On the basis of our previous series of exper- iments we had postulated that the increased anxiety that occurred during diazepam withdrawal was medi- ated by increased 5-HT release in the hippocampus. The present series of experiments provide evidence for a major role of the median raphé nucleus (MRN) dor- sal 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 Relative to chronically vehicle treated animals, those withdrawn from diazepam were significantly more anxious and had significantly greater K + -evoked release of [ 3 H]-5-hydroxytryptamine (5-HT) from slices of dorsal and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of 5-HT within the dorsal hippocampus, using in-vivo micro- dialysis, showed doubling in the levels of 5-HT in the rats withdrawn from chronic diazepam treatment.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 It was not possible to test these animals in the social interaction test, but it is proposed that only the diazepam-withdrawn rats with raised extracellular levels of 5-HT would have dis- played increased anxiety. 5-HT 1A receptor agonists injected into the MRN decrease the MRN firing rate, and hence the release of 5-HT in the dorsal hip- pocampus. As a further test of our hypothesis, we exam- ined the effects of MRN injection of the 5-HT 1A receptor agonist, 8-OH DPAT, on animals withdrawn from diazepam and tested in the low light familiar con- dition of the social interaction test. 8-OH DPAT (50 –200 ng) dose-dependently reversed the anxiogenic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 pro- vide 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. Key words Benzodiazepine withdrawal 5-HT release · Hippocampus · Median raphé nucleus · Anxiety · In-vivo microdialysis Introduction Benzodiazepines have a wide spectrum of therapeutic efficacy and are prescribed for the management of a range of disorders. One of their main indications is in the management of anxiety disorders, with many patients receiving several years of continuous treat- ment. However, Hollister et al. (1961) reported that abrupt termination of chronic administration of high doses of chlordiazepoxide resulted in a withdrawal syn- drome. Since these initial observations, the withdrawal syndrome following cessation of chronic benzodi- azepine receptor agonist treatment has been widely documented in animals (for review see File 1990) and man (Peturssen and Lader 1981) and is not limited to cessation from high dose regimes. The mechanism underlying the withdrawal syndrome is unclear but there is growing evidence that differ- ent symptoms are mediated by different neurotransmit- ter systems. For instance, generation of seizures appe- ars to be related to changes at the GABA A /BDZ/ Cl  ionophore (Hitchcott et al. 1989) and also 5-HT 3 receptors (Upton et al. 1993). Changes in the nora- drenergic system appear to be important in the medi- ation of hyperactivity (Kunchandy and Kulkarni 1986) but not anxiety (Abernethy et al. 1981; Baldwin et al. 1989). There is accumulating evidence that increased N. Andrews · S.E. File (*) · C. Fernandes · L.E. Gonzalez Psychopharmacology Research Unit, UMDS Division of Pharmacology, Guy’s Hospital, London SE1 9RT, UK N. Andrews · N.M. Barnes Department of Pharmacology, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK