When two stimuli occur consistently close together in time, they come to be “associated” with one another and over time the response to one stimulus can influence, or even modify, the response to the other stimulus (e.g, Pavlov’s dog experiment). To study mechanisms that underlie this association between two inputs, the Rose Lab employs the microscopic nematode Caenorhabditis elegans. This worm measures ~1 mm in length, with exactly 302 neurons in the hermaphrodite animal. The connections between these identified neurons have been described (the Worm “Connectome”) and importantly, the putative neurotransmitters and mode of signaling (chemical vs electrical) between these neurons have also been documented (see WormAtlas). Finally, stimuli that trigger activity in these known circuits have also been described. Knowing which neurons are activated by what type of sensory input allows for the prediction of where signalling changes are taking place, and by extension, putative mechanisms involved based on the documented gene expression locales. In the Rose Lab, to study Associative Conditioning two stimuli are presented together and the resultant change in locomotion in response to a later presentation of one of the stimuli is measured. Worm Tracker software (Tierpsy) then extracts behavioral data for analysis.
Tierpsy Worm Behaviour Tracker allows for identification of worms and analyses of behavior.
Direction of locomotion is altered after paired stimulus presentation. Naive (blue bars) show predominately forward locomotion while trained (red bars) more often pause locomotion in response to a single test stimulus.
To test mechanisms of Associative Conditioning, a variety of mutant and transgenic C. elegans strains undergo the same behavioral test.
In addition to behavioral tests, students have also examined changes in protein expression following Associative Conditioning using confocal microscopy.
Top: Image analysis program Shiny (Dr. David Leaf & Dr. Benjamin Minor, Dept of Biology, WWU) measures relative fluorescence and puncta lengths; Bottom: Green Fluorescent Protein (GFP) expressed in the C. elegans ventral nerve cord.
To investigate the effects of mild chronic stress, the Rose Lab employs the microscopic nematode Caenorhabditis elegans. Most rodent research use some form of inescapable environmental condition for chronic stress trials. When adult worms are exposed to an inescapable but mild stressor for a 4-hour period, subsequent generations of worms show increased reversal locomotion time.
Mean (+/- SEM) time performing reversal locomotion during a 10-minute observation period of spontaneous behavior. F0 = experimental worm generation; F1 = next generation; F2 = second generation.
Students also examine protein expression following each of these conditions using confocal microscopy.
Left: GLR-1::GFP image highlighting GLR-1 receptor subunits along the worm ventral nerve cord; Right: DIC image of the same worm.
Fluorescence image analysis reveals brighter puncta in stressed worms of the Original generation (F0).
Mean and semi-quartile range of total GFP fluorescence in the worm ventral nerve cord.
To observe direct changes in CaMKII expression following excitatory stimulation, students in the Rose Lab have observed and captured images from dissociated hippocampal neuron cultures. Immunocytochemistry (immunostaining) to CaMKII and the synaptic protein PSD-95 indicate overlapping expression in stimulated neurons.
Whole Neuron Stimulation Results in Synaptic CaMKII Accumulation. Immunocytochemistry trials in control non-stimulated neurons (A-C); A) CaMKII in red and PSD-95 in green, B) inset CaMKII alone, C) inset overlay with CaMKII in red and PSD-95 in green. A neuron exposed to 100 μM glutamate and 10 μM glycine for 1 minute followed by a 15-minute wash-out in extracellular solution (D-F); D) CaMKII in red and PSD-95 in green, B) inset CaMKII alone, C) inset overlay with CaMKII in red and PSD-95 in green.
When excitatory stimulation is restricted to a narrow region of dendrite, synaptic translocation of CaMKII still occurs but slowly, over time.
Time-lapse image capture of electroporated neuron expressing GFP-CaMKII. Number represent time from global stimulation; white arrow indicates pipette location. Top panel is 30s, second panel is 120s, third panel is 270s, bottom panel is 420s after stimulation, respectively. Time-lapse imaging conducted by former graduate student Robert Bragg