Acetylcholine release in the rat hippocampus as studied by microdialysis is dependent on axonal impulse flow and increases during behavioural activation

Abstract

Changes in extracellular levels of acetylcholine and choline in the hippocampal formation were measured using intracerebral microdialysis coupled to high performance liquid chromatography with post-column enzyme reaction and electrochemical detection. Various pharmacological and physiological manipulations were applied to awake unrestrained normal rats and rats subjected to a cholinergic denervation of the hippocampus by a complete fimbria-fornix lesion (1–2 weeks previously). Low baseline levels of acetylcholine (about 0.3 pmol15 min sample) could be detected in the absence of acetylcholinesterase inhibition in all animals. However, in order to obtain stable and more readily detectable levels, the acetylcholinesterase inhibitor neostigmine was added to the perfusion medium at a concentration of 5 or 10 μM and was used during all subsequent manipulations. Addition of neostigmine increased acetylcholine levels approximately 10-fold (to 3.7 pmol15 min) in the normal rats, which was about 4-fold higher than the levels recovered from the denervated hippocampi. Depolarization by adding KCl (100 mM) to the perfusion fluid produced a 3-fold increase in the extracellular acetylcholine levels, and the muscarinic antagonist atropine (3 μM) resulted in a 4-fold increase in the normal rats, whereas these drugs induced only small responses in the denervated rats. Neuronal impulse blockade by tetrodotoxin (1 μM) resulted, in normal rats, in a 70% reduction in extracellar acetylcholine levels. Sensory stimulation by handling increased acetylcholine levels by 94% in the normal rats, whereas this response was almost totally abolished in the denervated hippocampi. Behavioural activation by electrical stimulation of the lateral habenula resulted in a 4-fold increase in acetylcholine release in normal animals, and this response was totally blocked by a transection of the lateral habenular efferents running in the fasciculus retroflexus. The levels obtained by lateral habenula stimulation were reduced by about 95% in the rats with fimbria-fornix lesions. Following an acute knife transection of the fimbria-fornix performed during ongoing dialysis, acetylcholine levels dropped instantaneously by 70%, indicating that the extracellular acetylcholine levels in the hippocampus are maintained by a tonic impulse flow in the septohippocampal pathway.The extracellular levels of choline were reduced by about 30% after the addition of neostigmine in the normal rats, and increased by about 50% in both normal and denervated rats after addition of KCl to the perfusion fluid. No changes could be detected after atropine, handling, lateral habenula stimulation, or acute fimbria-fornix or fasciculus retroflexus transection.The results indicate that the hippocampal extracellular acetylcholine levels are at least 75% derived from the septohippocampal pathway and that changes in acetylcholine overflow, as measured by microdialysis under partial acetylcholinesterase inhibition, reflect changes in cholinergic neuronal activity. The substantial changes in acetylcholine overflow observed during handling and lateral habenula stimulation, moreover, suggest that the activity of the septohippocampal cholinergic system undergoes marked changes during behavioural activation. The intracerebral microdialysis technique thus appears to be a useful tool to monitor changes in physiological activity in the septohippocampal system during ongoing behaviour.