Bethesda Scientists Use Neurolucida to Map Memories in the Brain

When Pavlov’s dogs started salivating in response to a ringing bell, something happened in their brains—a memory was encoded. Over a century later scientists are still figuring out how memories are physically represented in the brain. One lingering question has been whether or not the same set of neurons is activated when a particular memory is formed. Researchers at the Johnson Lab at the Uniformed Services University of the Health Sciences in Bethesda, Maryland may have found the answer.

In their study, “Pavlovian Fear Conditioning Activates a Common Pattern of Neurons in the Lateral Amygdala of Individual Brains,” (PLoS ONE) the researchers set out to determine what happens when the brain forms a specific memory—does it use the same group of neurons to encode that memory? The brains of a population of rats, which had undergone Pavlovian auditory fear conditioning were examined. The research team zeroed in on the lateral amygdala–a region of the brain where neuron changes are known to occur in this kind of fear memory conditioning. Their results suggest that the formation of a Pavlovian conditioned fear memory is associated with a unique and stable neural topography in the amygdala.

Neurolucida played a significant role in their analysis, providing solutions for a variety of challenges. “From brain section alignment to mapping the 2D coordinates of activated neurons and quantitative analytics, Neurolucida was invaluable to this work,” said Dr. Luke Johnson.

To determine which neurons in the lateral amygdala were involved in the formation and storage of Pavlovian fear conditioned memories, the researchers used a molecular marker of neuron plasticity (pMAPK/ERK). The first step in determining whether or not the neurons involved in fear memory formation were spatially organized was to align different brains and compare common locations—a technically challenging maneuver requiring precise stereotaxic alignment, according to Dr. Johnson.

“We devised a novel solution to this problem by using Neurolucida to trace and measure anatomical landmarks that could serve as a reference point for brain section alignment,” he explained. After successfully matching brain sections, Dr. Johnson and his team established a consistent area for mapping activated neurons across different brains by using Neurolucida to trace anatomical boundaries of the amygdala based on a digital image from a standardized rat brain atlas.

“Pavlovian fear conditioning activates a complex, non-uniform population of neurons in the amygdala,” said Dr. Johnson, “making pattern analysis based on visual inspection alone difficult.” To capture differences in the complex spatial distribution of activated neurons, the research team devised a novel application of principle components analysis (PCA). Dr. Johnson explained that spatial PCA requires the neurons in an area be subdivided into “bins” that serve as variables in the analysis. With Neurolucida, they traced and constructed a virtual grid of bins that was aligned within the anatomical boundaries of the amygdala (Figure 1). XY coordinates of activated neurons were then mapped within the boundaries of the grid, and XY coordinates for each activated neuron were used to generate density maps, which allowed the researchers to visualize spatial distribution across brains.

Bergstrom HC, McDonald CG, Johnson LR (2011) Pavlovian Fear Conditioning Activates a Common Pattern of Neurons in the Lateral Amygdala of Individual Brains. PLoS ONE 6(1):e15698. doi:10.1371/journal.pone.0015698

Fig 1. (A) A grid was traced and carefully aligned over a brain section using Neurolucida. (B) The XY coordinates of activated neurons from the amygdala were used to generate density “heat” maps.

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