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Modulation of Synaptic Properties in Central Pattern Generator Neuronal Networks
Peter Kloppenburg, Professor of Cellular Neurophysiology, Universität zu Köln.
Ronald M. Harris-Warrick, Professor of Neurobiology and Behavior, Cornell University.
Funding: NINDS R37 NS17323-21 (1/1/06-12/31/12).

Neurotransmitter release and synaptic strength are regulated by localized intracellular Ca2+ levels in the nerve terminals. Ca2+ also plays an essential role in controlling intrinsic firing properties and gene expression, and therefore cytosolic Ca2+ levels must be functionally compartmentalized. The primary source of Ca2+ that triggers synaptic release originates from functionally specialized voltage-gated channels close to the transmitter release sites. Using a combination of voltage clamp recording and multiphoton microscopy, we are studying localized voltage-induced Ca2+ accumulation and its modulation by dopamine (DA) in identified neurons of the crustacean pyloric network pacemaker model system.

Previously, we demonstrated that voltage-activated Ca2+ accumulation in Pyloric Dilator (PD) neurons in the lobster stomatogastric ganglion arises mostly from small, spatially restricted varicosities, which are thought to be presynaptic sites (Kloppenburg et al, 2000). Dopamine (DA) reduces calcium accumulation at these localized sites, consistent with our previous work showing that DA reduces release from PD synapses. In contrast, DA enhances release from lateral Pyloric (LP) neuron terminals, which is accompanied by an increase in voltage-dependent calcium accumulation in LP varicosities. In the Pyloric Constrictor (PY) neurons, DA increases synaptic output, and also increased Ca2+ accumulation at the majority of synaptic varicosities. However, in about a third of the varicosities, DA decreased the voltage activated Ca2+ accumulation. We have now demonstrated opposing effects of DA on two varicosities within the same cell at the same time (Kloppenburg et al, 2007), demonstrating a spatially differential effect of DA within a single neuron (Fig. 1). Taken together with our earlier studies, these results suggest that modulation of Ca2+ entry is regulated at the level of the individual varicosity, and that the net effect of DA on the functional synapse may reflect a “majority vote” of opposing effects at individual varicosities.

Figure 1.  PY neuron. Dopamine has opposite effects on voltage activated Ca2+ accumulation in two synaptic varicosities on a neurite from a PY neuron.  A1-A3  Neurite of a Calcium Green-1 loaded PY neuron shown in 3 consecutive focal planes.  Ca2+ measurements were performed simultaneously at the varicosities α and β.  Scale: 10 µm.  A4 Computer reconstruction for better visualization of the neurite shown in A1-3.  The varicosities a and b are labeled yellow.  B1, B2  Line scans of sites α and β before, during and after bath application of DA (10-4M). C1, C2 Quantified changes in fluorescence extracted from the line scans in B1 and B2, calculated as the ratio of the fluorescence during the scan (F) to the average baseline fluorescence at -45 mV (F0).

We are continuing with our analysis of calcium accumulation in STG neurons. The Anterior Burster (AB) neuron is the prime pacemaker of the pyloric network, and DA enhances release from all its synapses. However, voltage clamp from the soma shows clearly that DA reduces voltage-activated calcium currents. It is possible that DA has differential effects at the soma (reducing the calcium current) and at synaptic terminals (increasing calcium accumulation and enhancing release of transmitter). 

It is usually assumed that the voltage change in the soma reflects what is occurring at the distant varicosities where we measure calcium changes. This relationship is crucial to understanding local calcium responses.  In the this continuing project, we will attempt to measure the relationship between soma potential measured by two-electrode voltage clamp and the potential measured in the neurites using second harmonic imaging of FM4-64 (Dombeck et al, 2005), in which Core R&D project IV, Aim 3 may provide a crucial strategy for reducing the toxicity that has plagued the the technique (Sacconi, et al, 2006).


Kloppenburg, P, Zipfel, WR, Webb, WW and Harris-Warrick, RM. 2000. Highly localized Ca2+ accumulation revealed by multiphoton microscopy in an identified motoneuron and its modulation by dopamine. J. Neurosci. 20:2523-2533.

Kloppenburg P, Johnson BR, Zipfel, WR, Webb, WW and Harris-Warrick, RM. 2003. Multiple effects of a single neuromodulator on an identified motoneuron revealed by combined electrophysiological and imaging techniques.In: Frontiers in Crustacean Neurobiology. Springer Verlag, Berlin, Heidelberg, pp 204-216.

Kloppenburg, P, Zipfel, WR, Webb, WW and Harris-Warrick, RM. 2007. Heterogeneous Effects of Dopamine on Highly Localized Voltage-Induced Calcium Accumulation in Identified Motoneurons. In Press: Journal of Neurophysiology.

Dombeck, D. A., L. Sacconi, M. Blanchard-Desce and W. W. Webb, “Optical recording of fast neuronal membrane potential transients in acute mammalian brain slices by second-harmonic generation microscopy,” Journal of Neurophysiology 94, 3628–3636, 2005

Sacconi, L., D. A. Dombeck and W. W. Webb, "Overcoming photodamage in second-harmonic generation microscopy: Real-time optical recording of neuronal action potentials," PNAS 103(9), 3124-3129, 2006.




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