Neuroscience concepts and applications

Krimer, L. & Goldman-Rakic, P. (2001) "Prefrontal microcircuits: Membrane proper ties and excitatory input of local, medium, and wide arbor interneurons." The journal of neuroscience 21(11), pp. 3788-96.

This study incorporates advances in mathematical modeling as well as technological improvements in measuring capabilities to identify heretofore unknown differentiations in axon form and functions, specifically in regards to their arbor size.

Research conducted by the authors also confirmed the results and deepened the understanding of other neural identifiers and functions, especially an understanding of the relationship axon differentiations in size/structure to function and transmission speed of axons (Krimer & Goldman-Rakic, 2001). Of overriding importance to the authors of this study was the role that lateral inhibition plays in neural transmission throughout the cortex; to this end, research was focused on the relationship of primary neurons to adjacent local neurons in areas known from previous research to be involved with memory formation, storage, and retrieval in an attempt to determine the overall role of pyramidal-neurons in neural transmission (Krimer & GOlcman-Rakic, 2001).

The role of pyramidal neurons as signal neurons for adjacent neurons to fire in the visual cortex had been previously discovered and described, and the authors of this study wished to determine to what extent the same functions occurred in other cortical functions, specifically memory (Sillito, 1975; Eysel et al., 1998; ctd. In Krimer & Goldman-Rakic, 2001). To achieve this, three groups of inteneurons with predominantly horizontal axonal arbors were classified based on the size of these arbors, and measurements relating to activity were conducted (Krimer & Goldman-Rakic, 2001).

Similar studies had previously been carried out on primates and in rats, but this study relied on ferrets (Lund & Lewis, 1993; Kawaguchi, 1995; ctd. In Krimer & Goldman-Rikac, 2001). This required the decapitation of the ferrets and the quick cold storage of their brains. Preparation of the cortical tissue for measurement and analysis was a complex process; neuron pairs could only be identified visually after removal of the frontal cortex and the cutting of this portion into sagittal sections only four hundred micrometers thick (Krimer & Goldman-Rakic, 2001). Samples were then incubated at approximately thirty-five degrees Celsius for at least one-and-a-half hours before submersion in a perfusion chamber with a complex liquid and gas mixture at a temperature of thirty-one to thirty-two degrees Celsius (Krimer & Goldman-Rakic, 2001). All of this was in preparation for the actual visualization of the neurons.

This was accomplished using infrared differential interference contrast video microscopy, greatly enhancing the contrast of materials in the prepared samples and enabling the researchers to identify and select eighteen usable neural pairs with which to begin their measurements and the real heart of their research (Krimer & Goldman-Rakic, 2001). In order to identify neural pairs that would fit the specific needs of the research question at hand, the authors of this study were examined for certain physiological features. Those with a smaller cell size (in comparison to nearby pyramidal neurons) and lacking apical dendrites were identified as laminae II/III cells and chosen for inclusion in the study (Krimer & Goldman-Rakic, 2001).

Once the cells to be tested had been properly identified, examined, and isolated, the true experimentation phase of the research began. In order to analyze the relationship between pyramidal neuron cells and their adjacent local neurons, it was necessary to deliver isolated and carefully measured and controlled electric charges to the pyramidal cells (Krimer & Goldman-Rakic, 2001). At the same time, very low-resistance electrodes containing a variety of chemical mixtures to aid in identifying morphological differences in conduction were then applied to various adjacent cells in order to measure the direction, strength, and speed of conductivity in neurons with measured and identified variations in the size and direction of their axonal arbors (Krimer & Goldman-Rakic, 2001). The researchers used a whole-cell recording method of electric charges, which reduced the amount of "noise" or interference in the extremely sensitive tools used for measurement and therefore a more reliable recording and description of "evoked postsynaptic events" -- meaning they were able to identify and measure charges passed from the pyramidal neurons onto other neurons with much greater precision and reliability without contaminating their data with other electric charges (Krimer & Goldman-Rakic, 2001).

Based on an earlier study conducted on rats, the researchers determined that the most efficacious way of testing the specific electrical membrane properties of the non-pyramidal neurons being studied was an application of alternating hyper-polarizing and depolarizing rectangular current pulses lasting five hundred milliseconds (Kawaguchi, 1995, ctd. In Krimer & Goldman-Rakic, 2001). The research confirmed the high firing rates of connected local neurons, especially when compared to the relatively low-firing pyramidal neurons, strengthening the observations of in-tandem functional properties of cortical neurons (Krimer & Goldman-Rakic, 2001). For ease of measurement, voltages were amplified but not altered (Krimer & Goldman-Rakic, 2001).

After conducting the physiological and electronic tests on the prepared neuron pairs, morphological analysis was conducted as well. This was the final step in assessing the differences in conduction of different cortical neurons in relationship to their differences in structure and interrelationship with other neurons; now that electronic measurements had been taken, morphological analysis would provide a more detailed visual picture of the neurons -- specifically the axonal arbors and their proximity to neighboring dendrites -- so that the correlation between the two features could be determined (Krimer & Goldman-Rakic, 2001). This analysis could not be done prior to the recording of electronic conduction data due to the destructive nature of the morphological analysis procedure, which necessarily separated neurons from each other.

The pairs were left intact for one to two hours after the recording process, which allowed enough time for the proper saturation of the neurons with dye to ease visualization of axons, dendrites, and inter-neural spaces (Krimer & Goldman-Rakic, 2001). They were then preserved in a cold paraformaldehyde solution for seventy-two hours before being transferred to an anti-freezing solution and storage at seventy-nine degrees below zero Celsius (Krimer & Goldman-Rakic, 2001). Sections were also dehydrated during this process to minimize later shrinkage in terms of thickness; upon removal from cold storage the samples were sliced into sections no more than sixty micrometers thick and further dyed (Krimer & Goldman-Rakic, 2001). Visual analysis was aided by three-dimensional computer reconstruction (Krimer & Goldman-Rakic, 2001).