Kurt Potgieter's Research

Pleurobranchaea’s CNS is composed of roughly 10, 000 neurons ranging from 40 to 800 mm in diameter, many of which are physiologically identified and well-characterized. The giant Metacerebral neurons (MCCs) of Pleurobranchaea’s CNS have been well characterized both physiologically and for their roles in neural networks generating behavior. Giant neurons containing both 5-HT and NO separately and together have been identified and mapped, and studied both biochemically. The investigative contexts for studying regulation of 5-HT and NO are well established. Serotonin acts as a major arousal factor in Pleurobranchaea, where increased 5-HT mediates readiness to feed, and at the cellular level activates or potentiates cyclic AMP-gated Na+ current and up-regulates Ca2+-activated nonspecific cation currents in neurons of the feeding motor network. In this animal, 5-HT regulates the arousal state, or general excitability, of the feeding network and thereby is a central organizing factor in the expression of the animal’s behavioral repertory. Capillary electrophoresis measures of 5-HT in single serotonergic neurons of the feeding network have shown that 5-HT levels vary directly with satiation state, being high in hungry animals and low in satiated animals (Work done in collaboration with Dr. Sweedler's group). Current issues of particular interest are as follows:

Intracellular measures of 5-HT using Fast Voltammetry

 

  • Does 5-HT neuropil concentration increase in the Buccal Ganglion when the MCC is stimulated?
  • Can we detect neuropil 5-HT differences in hungry versus satiated animals?
CLICK ON IMAGES TO ENLARGE

Oxidation of Serotonin

Serotonin is extremely unstable and degrades rather quickly when exposed to either heat or light. Fortunately this biogenic amine is easily oxidized. The faradaic current specifically due to the oxidation of 5-HT can be selectively isolated and measured. Selective oxidation of 5HT and NO is accomplished by bracketing the electrode potential around the maximum oxidation potential for the chemical species of interest and/or by coating it with a selective membrane. High-speed voltammetry techniques use biologically compatible carbon fiber electrodes, but there is no reason why compatible metals could not also work. We have experience in electrode construction, selective membrane application (both commercial and in-house), and 5HT/NO dual measurement using the bracketed potential system to isolate the small amounts of faradaic current produced by oxidation of biological chemical species of interest. We use the following electrode potential windows to detect 5HT (300 to 450 mV) and NO (650 – 750 mV). A modified DAGAN patch-clamp system in voltage clamp mode and with an Ag/AgCl2 reference electrode can be used to measure I for a given V(t), which is time dependent on a voltage profile generated by computer software (Axoclamp 6). Background subtraction of I at the lower bracketed potential is typically performed in MatLab. Electrochemical measurements are typically preceded by short anodic-cathodic pulses (± 1275 mV) to recondition/polish the active electrode surface. Multiple measurements are taken and averaged to reduce noise. The sampling time-scale is short (10 usec), useful for monitoring the kinetics of 5-HT release.

 

 

 

Carbon Fiber Electrode Construction

These electrodes are relatively easy to make. Five cm lengths of 7 - 30 um diameter carbon fiber is attached to copper wire using silver paint. The carbon tip is pushed through a micropipette. Epoxy is then used to seal the pipette tip via capillary action. The end of the copper wire is soldered to a gold pin which is then secured to the pipette body with heat-shrink tubing. Once the epoxy has finished curing at room temperature overnight or briefly in a 50 C oven, the tip is coated with the ion-selective resin, Nafion, and allowed to dry.

Raw Data of a Single Voltammetric Scan

The voltage profile (white trace) is superimposed on the measured total current (in red); this scan represents 102 msec. The letters (A - D) represent regions (representing 200 samples) that are imported into Matlab for further analysis. As demonstrated in the image, it is possible to simultaneously measure two oxidizable chemical species . In this particular case, C and D represent the limits for the bracketed voltage potential of 5-HT. The current from the oxidation of serotonin is calculated by the difference between the average C and D.

Matlab processing software was written by Gilbert Feng and Liudmila Yafremava

Serotonergic Somas: Cerebral and Buccal Ganglia

The major processes of the large MCC neurons (Cerebral ganglion; top right) extensively innervates the Buccal ganglion (bottom right) via the CBC, and represent the sole source of 5-HT (note the lack of serotonergic somas in the Buccal ganglion). Many of the feeding network neurons, including the command neuron, are located in the Buccal ganglion; this neural network drives the buccal mass movements and hence feeding. This layout provides an ideal sytem in which to study the feeding network. By isolating the Buccal ganglion and stimulating the MCCs by suction electrodes on the CBCs, 5-HT release into the neuropil could be measured using the rapid voltammetry technique described above.

Buccal Ganglion Preparation

Pinned Buccal ganglion in Pleurobranchaea saline with sheath still intact. Roots R1, R2 and R3 originate in the buccal ganglion and innervate the buccal mass, a part of the feeding apparatus performing rhythmic protracting and retracting movements during ingestion. In particular, R1 receives axons from the protractor motor neurons (denoted P in the model), thus governing protraction movement, however for some reason R1 is often not active in isolated CNS; R3 receives axons from the retractor motor neurons (denoted R in the model); R2 receives axons from both the protractor and retractor motor neurons, which is used in electrophysiological experience as an advantage of following the activity of both P and R populations from the same nerve. Every root R-1,2,3 has a symmetrical pair on the contralateral side of the buccal mass.
Cerebrobuccal connectives (CBC) are a pair of nerves that connect the buccal ganglion with the cerebropleural ganglion, thus carrying axons from paracerebral command neurons (PCN), located in the brain, to the feeding motor network, located in the buccal ganglion. PCNs are so called for their ability to excite feeding motor output when being stimulated in experiment.
CBC also receives axons from metacerebral giant neurons (MCC) which give the largest spikes in that nerve, and probably in the whole nervous system.
Stomatogastric nerve (SgN) originates in the buccal mass and innervates esophagus; it carries axons from the bilaterally paired ventral white cells (VWC) located in the buccal ganglion. VWCs are putative command neurons which in part derive their ability to drive neural network controlling the buccal mass from the progressive broadening of action potentials during repetitive firing.
SgN also carries R axons and sensory afference, which is why SgN is the only nerve used in experiments to drive feeding.

Sampling Strategy Based on 5-HT Histostaining

CFE insertion points are indicated. Changes in 5-HT with CBC simulation or differences in hunger-state are most likely to show up in the chosen sampling areas.

Changes in Buccal Ganglion Neuropil 5-HT

Stimulation of the CBC results in an initial decrease in 5-HT, followed by a general slow increase over the next 20 min. Difficulty in reproducing these results indicate that 5-HT release is localized and not general throughout the neuropil. CE-LIF analysis of of extracellular samples did not detect 5-HT, but this may have been due excessive dilution.

 

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