Biology subjects

Harkin, E.

Publications and source records attributed to Harkin, E..

3 recordsLinked to original sources

bioRxiv2023

Evaluation of short-term hair follicle storage conditions for maintenance of RNA integrity.

Hair follicles provide an easily accessible tissue for interrogating gene expression in mammals. RNAlater(R) is a liquid storage solution that stabilises and preserves cellular RNA, eliminating the need to immediately process or freeze tissue specimens. The manufacturer advises storage of samples at 2-8{degrees}C overnight before transfer to -20{degrees}C. This study aimed to evaluate RNA integrity in hair follicle samples collected from horses, stabilized in RNAlater(R), and stored under three short-term storage conditions. Mane hair samples complete with follicles were collected from three horses at a single time point. Approximately 15 hairs were placed in each of three 2 mL tubes containing 0.75ml RNAlater(R) solution. Test group A was stored at 4{degrees}C for 24-h, then decanted and stored at -20{degrees}C. Test groups B and C were stored at 4{degrees}C and 19{degrees}C (room temperature) respectively for 7 days, then decanted and stored at -20{degrees}C. RNA was isolated from all samples and RNA quantity and quality were measured. One-way ANOVA revealed no difference in RNA concentration (A:516 +/-125 ng/ml, B:273+/-93 ng/ml, C:476+/-176 ng/ml;P = 0.4293) or quality (A:9.5 +/-0.19, B:9.8+/-0.09, C:9.2+/-0.35 RIN; P = 0.3193) between the test groups. There were no group differences in mean Cycle Threshold values from qPCR validation assays confirming high-quality template cDNA. The results suggest that storage of hair follicles for one week in RNAlater(R) at cool or room temperature conditions will not compromise RNA integrity and will permit extended transport times from remote sampling locations without the need for freezing.

molecular biology↗

bioRxiv2022

A slow 5-HT1AR-mediated recurrent inhibitory network in raphe computes contextual value through synaptic facilitation

Serotonin (5-HT) neurons in the dorsal raphe nucleus (DRN) receive a constellation of long-range inputs, yet guiding principles of local circuit organization and underlying computations in this nucleus are largely unknown. Using inputs from the lateral habenula (LHb) to interrogate the processing features of the DRN, we uncovered 5-HT1A receptor-mediated recurrent connections between 5-HT neurons, refuting classical theories of autoinhibition. Cellular electrophysiology and imaging of a genetically encoded 5-HT sensor revealed that these recurrent inhibitory connections spanned the raphe, were slow, stochastic, strongly facilitating, and gated spike output. These features collectively conveyed highly non-linear dynamics to this network, generating excitation-driven inhibition and winner-take-all computations. In vivo optogenetic activation of LHb inputs to DRN, at frequencies where these computations are predicted to ignite, transiently disrupted expression of a reward-conditioned response in an auditory conditioning task. Together, these data identify a core computation supported by an unsuspected slow serotonergic recurrent inhibitory network.

neuroscience↗

bioRxiv2021

Cell-Type Specific Responses to Associative Learning in the Primary Motor Cortex

The primary motor cortex (M1) is known to be a critical site for movement initiation and motor learning. Surprisingly, it has also been shown to possess reward-related activity, presumably to facilitate reward-based learning of new movements. However, whether reward-related signals are represented among different cell types in M1, and whether their response properties change after cue-reward conditioning remains unclear. Here, we performed longitudinal in vivo two-photon Ca2+ imaging to monitor the activity of different neuronal cell types in M1 while mice engaged in a classical conditioning task. Our results demonstrate that most of the major neuronal cell types in M1 showed robust but differential responses to both cue and reward stimuli, and their response properties undergo cell-type specific modifications after associative learning. PV-INs responses became more reliable to the cue stimulus, while VIP-INs responses became more reliable to the reward stimulus. PNs only showed robust response to the novel reward stimulus, and they habituated to it after associative learning. Lastly, SOM-IN responses emerged and became more reliable to both conditioned cue and reward stimuli after conditioning. These observations suggest that cue- and reward-related signals are represented among different neuronal cell types in M1, and the distinct modifications they undergo during associative learning could be essential in triggering different aspects of local circuit reorganization in M1 during reward-based motor skill learning.

neuroscience↗