Biology subjects

Ingram, S. L.

Publications and source records attributed to Ingram, S. L..

3 recordsLinked to original sources

bioRxiv2023

Assessing spontaneous sensory neuron activity using in vivo calcium imaging

Heightened spontaneous activity in sensory neurons is often reported in individuals living with chronic pain. It is possible to study this activity in rodents using electrophysiology, but these experiments require great skill and can be prone to bias. Here, we have examined whether in vivo calcium imaging with GCaMP6s can be used as an alternative approach. We show that spontaneously active calcium transients can be visualised in the fourth lumbar dorsal root ganglion (L4 DRG) via in vivo imaging in a mouse model of pain. Application of lidocaine to the nerve, between the inflamed site and the DRG, silenced spontaneous firing and revealed the true baseline level of calcium for spontaneously active neurons. We used this data to train a machine leaning algorithm to predict when a neuron is spontaneously active. We show that our algorithm is accurate in two different models of pain: intraplantar Complete Freunds Adjuvant and antigen-induced arthritis, with accuracies of 90.0% +/-1.2 and 85.9 % +/-2.1, respectively, assessed against visual inspection by an experienced observer. The algorithm can also detect neuronal activity in imaging experiments generated in a different lab using a different microscope configuration (Accuracy = 94.0 % +/2.2). We provide a Google Colaboratory Notebook to allow anyone easy access to this novel tool, for assessment of peripheral neuron activity in their own calcium imaging setups.

neuroscience↗

bioRxiv2022

Persistent inflammation promotes endocannabinoid release and presynaptic cannabinoid 1 receptor desensitization

Pain therapies targeting the cannabinoid system are increasing with expansion of cannabis legalization but adaptations in the endogenous cannabinoid system during inflammatory pain could limit their efficacy. Presynaptic inhibition of GABA release mediated by cannabinoid 1 receptor (CB1R) agonists in the ventrolateral periaqueductal gray (vlPAG) is markedly reduced in male and female Sprague Dawley rats after persistent inflammation induced by Complete Freunds Adjuvant (CFA). Inflammation results in increased endocannabinoid (eCB) synthesis and desensitization of presynaptic CB1Rs that is reversed by a GRK2/3 inhibitor, Compound 101. Despite CB1R desensitization, eCB activation of CB1Rs is maintained after inflammation. Depolarization-induced suppression of inhibition (DSI) in naive animals is rapid and transient, but is prolonged in recordings after inflammation. Prolonged DSI is mediated by 2-arachidonoylglycerol (2-AG) indicating reduced monoacylglycerol lipase (MAGL) activity. These adaptations within the endogenous cannabinoid system have important implications for the development of future pain therapies targeting CB1Rs or MAGL.

neuroscience↗

bioRxiv2021

Physiologically distinct neurons within the ventrolateral periaqueductal gray are not defined by mu-opioid receptor expression but are differentially activated by persistent inflammation

The ventrolateral periaqueductal gray (vlPAG) is a key structure within the descending pain modulatory pathway and an important target for opioid-induced analgesia. This area contains heterogeneous neurons with respect to neurotransmitter and receptor expression so it is difficult to define vlPAG neurons that contribute to pain and analgesia. Characterization of intrinsic membrane properties of 371 vlPAG neurons from female and male Long-Evans rats identified 4 neuron types with distinct intrinsic firing patterns: Phasic, Tonic, Onset, and Random. Phasic and Tonic neurons comprise the majority of the neurons sampled. Mu-opioid receptor (MOR) expression was determined by the ability of the selective MOR agonist DAMGO to activate G protein-coupled inwardly-rectifying potassium channel (GIRK) currents. Opioid-sensitive and -insensitive neurons were observed within each neuron type in naive rats and in rats pretreated with Complete Freunds adjuvant in a hindpaw to produce persistent inflammation. The presence of low threshold spikes (LTS) did not correlate with MOR-mediated GIRK currents indicating that MOR expression alone does not define a physiologically distinct neuron type in the vlPAG. MOR activation inhibited firing in nearly all spontaneously active neurons, both in naive and persistent inflammation conditions. CFA-induced inflammation increased Fos expression at both acute (2 h) and persistent inflammation (5-7 d) time points. However, persistent, but not acute, inflammation selectively enhanced spontaneous firing and lowered firing thresholds of Phasic neurons which was maintained in the absence of synaptic inputs. Taken together, persistent inflammation selectively activates Phasic neurons, of which only a subset was opioid-sensitive. Significance statementIntrinsic membrane properties define separate vlPAG neurons types that are functionally important. Persistent, and not acute, inflammation selectively activates Phasic firing vlPAG neurons that are not defined by MOR expression. Although the vlPAG is known to contribute to the descending inhibition of pain, the activation of a single physiologically-defined neuron type in the presence of inflammation may represent a mechanism by which the vlPAG participates in descending facilitation of pain.

neuroscience↗