The olfactory system in vitro


The olfactory epithelium has the remarkable ability of regeneration throughout the adult lifespan. Mature olfactory neurons (the cells that are responsible for detecting odorant stimuli) live for a few months. When they die, they are shed, new olfactory neurons divide and differentiate from neuronal precursors, rewire themselves into the brain (olfactory bulb), and replace the shed olfactory neurons.
This ability of the olfactory epithelium to produce new neuronal cells renders the olfactory system ideal for the study of neuronal growth and differentiation. To study olfactory neurogenesis in the human olfactory epithelium, I have been able to establish cultures from acutely isolated tissue from a number of species (human, cat, bird).  A sample publication (authored by Stephanie Yazinski, Class of '05) can be found here, and a more recent publication on avian olfactory cultures (authored by Grace O'Neill and Christa Musto, Class of '16) can be found here.

The photomicrograph (above, right) shows cultured olfactory neurons in vitro.
A, 10x magnification, neurons are indicated by arrows.
B, 20X magnification of the boxed area in A.
C-E, Neurons have a visually distinct morphology.

My current research focuses on bird olfactory tissue. Cells are mechanically dissociated and are allowed to grow in culture dishes supplemented with culture medium. The neurons continue to grow and proliferate in culture. These cells are visually distinguished by the presence of long processes that distinguish them as neurons. These cells are odorant-sensitive and express marker molecules that are characteristic of olfactory neurons in vitro.

Current research efforts include characterizing the process of maturation of olfactory neurons  using functional (calcium imaging) and immunocytochemical assays.
Many cells respond to stimulation with changes in intracellular calcium.  It is possible to measure these changes under a fluorescence microscope.
Cells are loaded with the fluorescent dye fura-2. Images are taken by computer and  pseudocolored for clarity.  At 340 nm UV excitation (left, top panel), fura-2 that is bound to intracellular calcium fluoresces (emits light at a visible wavelength) brighter as calcium concentrations increase.  At 380 nm UV excitation (left, bottom panel), fluorescence emission by fura-2 decreases as  calcium increases.  The imaging system acquires images of the emission from these two excitation wavelengths.  The computer calculates the ratio between these two images (multiplied by a calibration factor) to measure the calcium concentration inside the cell (right, top panel).  By measuring images over time, it is possible to measure the cell's change in calcium in response to a stimulus such as an odor (right, bottom panel).


Images show an olfactory neuron loaded with the calcium-sensitive ratiometric dye fura 2 (left, image pseudocolored for clarity), then tested  for the presence of neural cell adhesion marker (NCAM, right) using immunocytochemistry techniques.