Versatile Undergraduate Neurobiology Course-Based Research Experiences Using Open Access 3D Electron Microscopy Image Volumes

Abstract

Course-based undergraduate research experiences (CUREs) using inquiry-based methodology provide a range of positive benefits to undergraduates and instructors. Yet, the required time and cost in designing and running CUREs with detailed data acquisition steps can lead to barriers in CURE implementation. This report describes an alternative approach to CUREs that utilizes free, open access 3D image volumes as data-rich resources for neurobiology CUREs. These open access image volume CUREs (ivCUREs) effectively combine the data acquisition and analysis steps within the course, allowing more time for students to critically evaluate their hypotheses and results, compare data with peers, and reflect on their experiences. Undergraduates in this 10-week ivCURE analyzed >670 excitatory synapses across two brain areas for the presence and origins of spinules within presynaptic boutons, and fully reconstructed 13 of these synapses in 3D. These data highlight the prevalence of these enigmatic synaptic features within excitatory presynaptic boutons, and their potential importance to neuronal function. Moreover, these results underscore key benefits to ivCURE implementation, including the (1) low-cost of experimental design and implementation, (2) ability to utilize the same data-rich image volume across multiple ivCUREs, (3) potential to generate publishable analyses, and (4) flexibility to scale projects and class sizes up at little to no cost. Opportunities for undergraduates to engage in inquiry-based ivCUREs that examine a host of unexplored questions in neurobiology will continue to grow, in parallel with rapid advances in 3D microscopy techniques and the increased availability and diversity of open access image volumes and analytical tools.

Keywords: CURE; ImageJ; cortex; hippocampus; image analysis; neuroanatomy; presynaptic bouton; serial section electron microscopy (ssEM); spinule; spinule-bearing bouton (SBB); three-dimensional (3D) reconstruction.

Figure 1

ivCURE classroom at the UW Tacoma. This particular computer lab contains 40 student desktop computers and a presentation-capable computer for the instructor. ivCUREs can be scaled up or down based on computer lab constraints and the requirements of the individual instructor and academic unit.

Figure 2

Number of publications per year (1999–2018) using either focused ion beam scanning electron microscopy (FIBSEM) or serial block face scanning electron microscopy (SBFSEM), techniques that generate ssEM image volumes. Data from pubmed.gov.

Figure 3

Open Connectome Project (OCP) Website Interfaces. A. OCP site for the selection of an open access 3D image volume project (https://neurodata.io/ocp). B. OCP tool (ndwebtools) interface for downloading small sections (< 1 GB) of a larger image volume.

Figure 4

ssEM Student Training Image with ROIs. A. Representative area within a ssEM training image, showing two prominent excitatory synapses. B. Identical training image area with ROIs shown around excitatory presynaptic boutons (blue), postsynaptic spines (orange), spinule (purple) within SBB, and putative dendrites (red). Note the large ‘flattened pancake-shaped’ spine apparatus in the larger spine toward the top of the image. Scale bar = 0.25 μm (for A & B); Raw data from Bock et al. (2011).

Figure 5

ivCURE Student 3D Reconstructions of SBBs in CA1 Hippocampus. A – B. A single ssEM section (A) and its pseudo-colored copy (B) showing a postsynaptic spine (gray) invaginating a spinule into its presynaptic bouton partner (purple). Note the postsynaptic density (green) at the edge of the spinule. C and D. Full reconstruction of the synapse in A – B from 311 serial ssEM sections, showing this SBB (purple) engulfing spinules from two postsynaptic spines (gray). E – F. A single ssEM section (E) and its pseudo-colored copy (F) showing a postsynaptic spine (gray) projecting a spinule into its presynaptic bouton (purple). Note the presynaptic vesicles (light blue), and the postsynaptic density (green) at the edge of the spinule. G – H. Full reconstruction of the synapse in E and F from 264 serial ssEM sections, showing this SBB (purple) with a large anchor-like spinule from its postsynaptic spine (gray). Note the mitochondria (red) and presynaptic vesicles (light blue) within this SBB. Scale bars for A, B, E, and F = 0.5 μm; Scale cubes for C, D, G, and H = 0.5 μm/side (0.125 μm³). Raw data from EPFL (https://www.epfl.ch/labs/cvlab/data/data-em/).

Figure 6

Averaged Student Evaluations from two ivCUREs. End of term student evaluations on representative measures of overall course effectiveness, lab efficiency, and intellectual challenge.