Faculty Bibliography

This study presents, to our knowledge, the first on-line measurement of acetylcholinesterase (AcChoEase) release from brain tissue. It is now well established that a soluble form of the enzyme is released from central nervous system neurons, and it has been proposed on indirect grounds that such release may occur not only from presynaptic terminals but also from the dendrites of dopamine-containing nigrostriatal neurons. We have used a chemiluminescent reaction to examine the real-time release of AcChoEase from the substantia nigra in vitro in brainstem slices. The light emission was captured by two fiber optic systems, one in direct contact with the brain slice from below and the second 4-mm above the slice, allowing simultaneous imaging of the emitted light and quantitative photometry. It was determined that the light signals are not due to the spontaneous hydrolysis of acetylcholine or the presence of free choline, but are caused by the enzymatic action of AcChoEase. Using this technique, it can be directly shown that AcChoEase is spontaneously released from the soma or dendrites of nigral neurons. The release of the enzyme, which is stored in the subcisternal dendritic compartment, is resistant to blockade of voltage-dependent sodium conductances, is calcium dependent, and can be increased by addition of potassium to the bathing solution. The procedure we describe here will make it possible to study the release of endogenous AcChoEase on a time-scale close to that over which it functions.

Delayed and anomalous rectification was studied in inferior olivary (I.O.) neurons in guinea pig brain stem slices maintained in vitro. Hyperpolarization of the I.O. cell beyond rest membrane potential was accompanied by anomalous rectification (AR). This consisted of 2 parts: an instantaneous and a time-dependent component. The 'instantaneous' component was blocked by bath addition of Ba2+ or Cs+ and demonstrated inactivation following prolonged hyperpolarization. The time-dependent component, referred to as the gK(ol), was blocked by harmaline in concentrations of 0.1 mg/ml or by substitution of Co2+, Cd2+, or Mn2+ for Ca2+ in the bath. The gK(ol) was blocked by extracellular Cs+ but not by Ba2+. Delayed rectification (DR), consisting of 2 distinct components, was observed after membrane depolarization by more than 10 mV with respect to rest (usually at -65 mV). One of the components of the DR was found to be quite similar to the classical gK. It did not demonstrate significant inactivation with membrane potential change and was reduced by Ba2+ or tetraethylammonium (TEA). A second component of the DR demonstrated voltage-dependent inactivation and was thus referred to as gK(inact). This inactivation determined by current-clamp measurements had a sigmoidal time course, with approximately a 1 sec onset latency and a half-time to peak of 7 sec. The inactivation of gK(inact) outlasted current injection for tens of seconds to several minutes, depending on the duration and amplitude of the preceding depolarization. During this period, I.O. neurons could be easily activated and demonstrated full dendritic spikes following current injection or excitatory synaptic input that had previously been subthreshold for spike initiation. The inactivation component of the DR was removed by prolonged membrane hyperpolarization beyond rest. gK(inact) was blocked by 4-aminopyridine (4-AP; 100 microM) but not by Ba2+. This inactivation was dependent on the presence of extracellular Ca2+ or Ba2+. Addition of Co2+ or Cd2+ to the bath did not block gK(inact) but did prevent its inactivation. The modulatory effects of these different membrane conductances on the integrative properties of I.O. neurons are described. The long duration of the inactivation of DR and AR is considered as the basis for a dynamic long-term modulation of the electroresponsive and integrated properties of I.O. neurons.

The data presented here provide evidence that the study of neuronal phosphoproteins can lead to the identification of previously unknown proteins and that these proteins may play important roles in neuronal communication. Specifically, in the case of synapsin I, direct evidence has been obtained that this phosphoprotein is involved in regulating neurotransmitter release. A tentative explanation of the results obtained in the micro-injection studies is as follows: synapsin I, in the dephosphostate, is bound to the cytoplasmic surface of synaptic vesicles and inhibits the ability of the vesicle to interact with the plasma membrane; increases in intracellular calcium activate calmodulin kinase II which in turn phosphorylates synapsin I and the phosphorylated synapsin I dissociates from the synaptic vesicle thus removing a constraint on the release of neurotransmitter. Clearly, more studies need to be done to rigorously test this hypothesis. Nevertheless these studies of synapsin I suggest that the study of previously unknown phosphoproteins will lead to the elucidation of previously unknown regulatory processes in neurons