Nerve growth factor modulation of basal forebrain neuron acetylcholine release : new perspectives on a classic interaction

This thesis addresses long- and short-term neurotrophin modulation of basal forebrain cholinergic neurons (BFCNs), focusing on acetylcholine (ACh) release and choline acetyltransferase (ChAT; the enzyme responsible for ACh synthesis) activity. (1) Initially, ACh release was characterized from primary embryonic basal forebrain cultures. Release was vesicular, dependent upon extracellular choline and regulated by negative feedback via M2-like muscarinic receptors. (2) The next step was to determined the long-term (24--96 hour) effects of nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4) and neurotrophin-3 (NT-3) on ChAT activity, ACh content, high-affinity choline uptake, constitutive and K+ stimulated ACh release. These experiments showed differential capacities of NTs to enhance cholinergic markers (NGF > BDNF > NT-4 > NT-3) and that the various markers, although related, respond differentially to NGF. This is important because previously, direct relationships between these markers were largely assumed. (3) The acute effects of NTs on BFCN functional markers were also determined, and there was an activity dependent increase in ACh release. Short exposures (5--60 min) to NGF (0.1--100 ng/ml) robustly enhanced spontaneous ACh release (up to over 3-fold of control) without a persistent increase in ChAT activity. To our knowledge, this is the most potent enhancement of basal forebrain ACh release ever reported. The increases were TrkA NGF receptor and calcium dependent, but were not dependent upon de novo protein synthesis or cAMP. Further, even a brief (5--60 min) exposure to NGF increased ACh release (approximately 2-fold) for at least 4 h following its removal in a long term potentiation-like fashion. This property of inducing prolonged ACh release was shared with the other NTs (NGF > BDNF > NT-4 > NT-3). (4) The relationship between the fast and long-term actions of NGF on ACh release/ChAT activity. Inhibition of protein