Robert A. Nichols, Ph.D.
Associate Professor
Ph.D. (1982) Stanford University
Phone: 215-762-2383
New email: robert.nichols@hawaii.edu
Email: robert.nichols@drexelmed.edu
Professor Nichols has moved to the Dept. of Cell & Molecular Biology at the University of Hawaii.
Nerve cells communicate via specialized contacts known as synapses where nerve impulse activity in one nerve cell causes the release from its nerve ending of a chemical messenger, referred to as a neurotransmitter, which then interacts with and modifies the activity in the target nerve cell. At a subset of synapses in the brain, neurotransmitter receptors are present on the presynaptic nerve endings. The functions of different presynaptic receptors are poorly understood, but it is likely that these receptors are important targets for particular drugs.
Beta-amyloid regulation of nerve terminal Ca2+ via presynaptic nicotinic receptors
One of the prominent characteristics of Alzheimer’s disease is the alteration of cholinergic targets, specifically nicotinic receptors. In fact, fragments of nicotinic receptors have been found in neuritic plaques which are hallmarks in the pathology of this disease. The nicotinic receptor, which has a cation-selective ion channel integral to its structure, has been linked to memory processing in the brain, involving, in particular, the hippocampus. A substantial portion of the nicotinic receptors are located on presynaptic nerve terminals at select synaptic sites, including those of the hippocampus and the prefrontal cortex, among other brain regions. Intriguingly, we have discovered that at very low concentrations (pM to nM) soluble beta amyloid peptide, the major component of neuritic plaques, induces an increase in nerve terminal Ca2+ largely via presynaptic nicotinic receptors. This beta amyloid-induced activation occludes any further activation of the presynaptic nicotinic receptors by nicotine, whereas the opposite is also true: nicotine occludes the action of beta amyloid. In addition, nerve terminal activity appears to attenuate the presynaptic effect of beta amyloid. We propose that as soluble beta amyloid accumulates in the brain during the course of the disease, presynaptic regulation via nicotinic receptors becomes progressively compromised, depending on the extent of nerve impulse activity as well as normal activation of the nicotinic receptors by endogenous acetylcholine.
From Dougherty et al., 2003
Beta amyloid (Ab)-evoked Ca2+ increases in individual nerve endings assayed using confocal imaging of isolated nerve endings from hippocampus. Composites of Ca2+ responses in individual nerve endings to Ab1-42 (A) and Ab12-28 (B) over an extended time course. Top, Representative initial phases of successive Ca2+ responses in an individual nerve ending to 100 nM Ab1-42. C, Concentration-dependence; D, Insensitivity to prior treatment with 50µM ZnCl2 (n=15). E, Average maximal Ca2+ responses to 100 nM or 100pM Ab1-42 before or after filtration of the Ab solutions through 0.2µm filters.
The major questions under consideration are:
- What is the mechanism by which beta amyloid inhibits presynaptic nicotinic responses?
- How does inhibition of presynaptic nicotinic responses relate to the direct action of beta amyloid in stimulating increase nerve terminal Ca2+? Are protein kinase pathways activated in the nerve terminal by beta amyloid?
Approach: confocal microscopy and biochemical assays
Neurotrophic regulation of presynaptic receptors:
Our early work focused on presynaptic ligand-gated ion channels, such as nicotinic receptors and 5-HT3 serotonin receptors, because they will likely have strong regulatory actions on neurotransmission. When located presynaptically, these receptors appear to have functional characteristics that are, on the whole, distinct from those found at somatic sites. In the case of presynaptic 5-HT3 receptors, we have found that their nature depends on the presence of postsynaptic target, likely involving neurotrophic molecules (NTs). At present, which neurotrophic molecules regulate presynaptic receptors are yet not defined. Moreover, why ligand-gated ion channels are on the nerve endings and what their functions are at these sites remain as fundamental questions in our understanding of the biology and pharmacology of the synapse.
The major questions under consideration are:
- How does the target cell at the synapse regulate the nerve ending?
- What specifies the character of presynaptic receptors on the nerve ending?
- How do presynaptic receptors regulate the transmission of signals across the synapse?
For the 5-HT3 receptors, these questions are being directly addressed using a model tissue culture system consisting of a nerve cell-like clonal cell line (NG108-15), which elaborates an extensive network of presynaptic-like varicosities, and muscle cells, which serve as targets. When cultured together, the presynaptic varicosities from the nerve cell line will indeed form functional synapses with the muscle cells, but not with each other. In addition, the components of the synapses - the presynaptic varicosities and the muscle cells - are quite large in comparison with typical synaptic components found in the nervous system. Moreover, the presynaptic varicosities in these cultures express 5-HT3 serotonin receptors. Thus, this well-defined co-culture system offers all of the features necessary to begin to address the questions posed.
Approaches employed are: microscopic imaging of changes in [Ca2+]i in individual varicosities and patch-clamp recording of ionic currents in isolated varicosities and in the target muscle cells.
Co-localization of 5-HT3 serotonin and nicotinic acetylcholine receptors at presynaptic sites:
Convergence of neurotransmitter pathways is a common theme in the nervous system. However, convergence at the level of the presynaptic nerve ending has not been well characterized. In the course of our studies of presynaptic receptors on individual isolated nerve endings from rat corpus striatum, we discovered substantial co-localization of 5-HT3 and nicotinic receptors, suggesting the possible convergence of the serotonergic and cholinergic systems on presynaptic endings in the mammalian brain. It is intriguing to consider why both receptors are expressed on the same presynaptic ending and what the resultant consequences may be. We are establishing transfectant NG108-15 cells containing both 5-HT3 receptors and specific subtypes of nicotinic receptors to better address the functional consequence of this colocalization.
The major questions under consideration are:
- Does activation of one presynaptic receptor system alter the activation and/or down-stream signaling of the other co-localized receptor system?
- Do the different presynaptic receptor systems differentially regulate the transmission of signals across the synapse? Is the result of simultaneous activation distinct from activation of each individual presynaptic receptor system?
Individual nerve endings from brain, loaded with a fluorescent dye that detects Ca2+, were viewed with a confocal microscope. Arrows indicate two nerve endings that responded to nicotine with dramatic increases in [Ca2+],evident as shift in color from blue/green to yellow/red. [from Nayak et al., 2001]
Approach: microscopic imaging of changes in [Ca2+]i in individual isolated nerve endings; molecular cell biological methods.
Recently, we have begun to characterize presynaptic dopamine receptors in the striatum, a key region in the control of movement where presynaptic nicotinic receptors, as well as 5-HT3 receptors, regulate the release of dopamine. Interestingly, activation of presynaptic D1-like and D2-like dopamine receptors results in robust increases in nerve terminal Ca2+, most likely via coupling to voltage-gated ion channels. In addition, prolonged activation of these presynaptic dopamine receptors leads to profound desensitization.
Selected Recent Publications:
Wu, J., Dougherty, J.J. and Nichols, R.A. Dopamine receptor regulation of Ca2+ levels in individual nerve terminals from rat striatum: comparison of presynaptic D1-like and D2-like receptors. Submitted to Neuroscience.
Dougherty, J.J., Wu, J. and Nichols, R.A. (2003) Beta amyloid regulation of presynaptic nicotinic receptors in rat hippocampus and neocortex. Journal of Neuroscience 23, 6740-6747.
Rondé, P. and Nichols, R.A. (2001) Postsynaptic target regulates functional responses induced by 5-HT3 serotonin receptors on axonal varicosities of NG108-15 hybrid neuroblastoma cells. Neuroscience, 102, 979-987.
Nayak, S.V., Dougherty, J.J., McIntosh, J.M. and Nichols, R.A. (2001) Ca2+ changes induced by different presynaptic nicotinic receptors in separate populations of individual striatal nerve terminals. Journal of Neurochemistry, 76, 1860-1870.
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