Microfluidic approach to correlate C. elegans neuronal functional aging and underlying changes of gene expression in mechanosensation

Abstract

The aging process has broad physiological impacts, including a significant decline in sensory function, which threatens both physical health and quality of life. One ideal model to study aging, neuronal function, and gene expression is the nematode Caenorhabditis elegans, which has a short lifespan and relatively simple, thoroughly mapped nervous system and genome. Previous works have identified that mechanosensory neuronal structure changes with age, but importantly, the actual age-related changes in the function and health of neurons, as well as the underlying genetic mechanisms responsible for these declines, are not fully understood. While advanced techniques such as single-cell RNA-sequencing have been developed to quantify gene expression, it is difficult to relate this information to functional changes in aging due to a lack of tools available. To address these limitations, we present a platform capable of measuring both physiological function and its associated gene expression throughout the aging process in individuals. Using our pipeline, we investigate the age-related changes in function of the mechanosensing ALM neuron in C. elegans, as well as some relevant gene expression patterns (mec-4 and mec-10). Using a series of devices for animals of different ages, we examined subtle changes in neuronal function and found that while the magnitude of neuronal response to a large stimulus declines with age, sensory capability does not significantly decline with age; further, gene expression is well maintained throughout aging. Additionally, we examine PVD, a harsh-touch mechanosensory neuron, and find that it exhibits a similar age-related decline in magnitude of neuronal response. Together, our data demonstrate that our strategy is useful for identifying genetic factors involved in the decline in neuronal health. We envision that this framework could be applied to other systems as a useful tool for discovering new biology.

Fig. 1. Microfluidic-based strategy to correlate age-related changes in gene expression and neuronal function. a) Information from the environment is processed within the animal to elicit a response. b) The DEG/ENaC channels have a transmembrane portion made of MEC-4 and MEC-10, which are responsible for mechanosensation in C. elegans gentle touch neurons. c) Measuring both gene expression and neuronal function of animals of different ages allows the two trends to be correlated. d) Our microfluidic device enables us to capture both gene expression and functional neuron activity. e) Imaging fluorescent gene reporters quantifies gene expression patterns in fluorescence intensity. f) Delivering precise, robust, high-frequency stimuli to differently aged animals allows measurement of neuronal function.

Fig. 2. Computational modeling optimizes device design and operation for consistent stimulus delivery to animals of different ages and sizes. a–d) Resulting deformation of the stimulus membrane and animal at increasing pressures calculated using COMSOL simulation. The values L and L₀ used to calculate percent deformation are shown in c) where L is the shortest distance between the actuators at time t and L₀ is the distance at t = 0. e) Using a range of PDMS properties, a range of possible deformation values in the device was calculated with a 50 μm wide imaging channel. The presence of the animal (black filled circles and dark grey background) affects the curve compared to an empty channel (open circles with light grey background) f) the trend is the same for the device with a 60 μm imaging channel. Empirical calibration (filled triangles) results in a close match with the computational model for both the device with g) 50 μm and h) 60 μm wide imaging channels. Green dotted line indicates 30% deformation, the target for gentle touch stimulus.

Fig. 3. Microfluidic-based platform can measure subtle changes of neuronal function in vivo in a mock drug application. a) Averaged neuronal activity traces. Amiloride blocks DEG/ENaC channels. When applied to the animals, the animals have decreased neuronal function. b) Comparing peak heights of the untreated and amiloride-treated groups, the amiloride-treated group is significantly lower. An unpaired t-test was performed (*P < 0.05).

Fig. 4. Aging leads to a decline in neuronal function in mechanosensation. a) Each animal was loaded into the microfluidic chip (shown in inset), which applied stimulus pulses (8 gentle touch presses at 2.7 ± 0.1 Hz) for 3 seconds followed by a 40 second gap. This was repeated 9 times, followed by a longer 30 second stimulus also delivered at 2.7 ± 0.1 Hz. b–d) The first, 9th, and final stimulus results enlarged. e) First peaks from individual animals reveal there is no significant difference in neuronal response to pulse stimulus between age groups. f) Long stimulus reveals a significant decline in response as populations age from day 2 to day 10, and a large variance of response magnitudes at day 15. The Kruskal–Wallis test was performed, followed by Dunn's multiple comparison test (*P < 0.05, **P < 0.01, ***P < 0.001), comparisons that were found to be not significant are not shown.

Fig. 5. Aging leads to changes in gene expression patterns while regulation is well maintained. a) Gene expression patterns across different ages reveals significant increases in MEC-4::mNeonGreen and MEC-10::mScarlet-I expression with age up to day 10, followed by a decrease at day 15. b–e) With individual-level data, we could examine gene expressions within the same animal across different ages. There is a strong positive correlation between the two genes across the different ages, indicating that the regulation of the genes is well maintained regardless of age. The Kruskal–Wallis test was performed, followed by Dunn's multiple comparison test (***P < 0.001), only comparisons found to be significant are shown.

Fig. 6. Different neuron class also demonstrates aging related decline in function. a) A single 1 s harsh stimulus is used to examine PVD neuronal function for populations of different ages. b) The peak amplitude for each individual for each age group demonstrates a gradual decline in neuronal function from day 2 to day 10. An increasing individual variability of function is also demonstrated, with extreme outliers appearing in day 15. The Kruskal–Wallis test was performed, followed by Dunn's multiple comparison test (*P < 0.05, **P < 0.01).