Laser-evoked cortical responses in freely-moving rats reflect the activation of C-fibre afferent pathways

The limited success of translating basic animal findings into effective clinical treatments of pain can be partly ascribed to the use of sub-optimal models. Murine models of pain often consist in recording (1) threshold responses (like the tail-flick reflex) elicited by (2) non-nociceptive specific inputs in (3) anaesthetized animals. The direct cortical recording of laser-evoked potentials (LEPs) elicited by stimuli of graded energies in freely-moving rodents avoids these three important pitfalls, and has thus the potential of improving such translation. Murine LEPs are classically reported to consist of two distinct components, reflecting the activity of Ad-and C-fibre afferent pathways. However, we have recently demonstrated that the so-called &quot;Ad-LEPs&quot; in fact reflect the activation of the auditory system by laser-generated ultrasounds. Here we used ongoing white noise to avoid the confound represented by the early auditory response, and thereby comprehensively characterized the physiological properties of C-fibre LEPs recorded directly from the exposed surface of the rat brain. Stimulus-response functions indicated that response amplitude is positively related to the stimulus energy, as well as to nocifensive behavioral score. When displayed using average reference, murine LEPs consist of three distinct deflections, whose polarity, order, and topography are surprisingly similar to human LEPs. The scalp topography of the early N1 wave is somatotopically-organized, likely reflecting the activity of the primary somatosensory cortex, while topographies of the later N2 and P2 waves are more centrally distributed. These results indicate that recording LEPs in freely-moving rats is a valid model to improve the translation of animal results to human physiology and pathophysiology. (C) 2016 The Authors. Published by Elsevier Inc.</p

Exploring the electrophysiological responses to sudden sensory events

Living in rapidly changing and potentially dangerous environments has shaped animal nervous systems toward high sensitivity to sudden and intense sensory events - often signalling threats or affordances requiring swift motor reactions. Unsurprisingly, such events can elicit both rapid behavioural responses (e.g. the defensive eye-blink) and one of the largest electrocortical responses recordable from the scalp of several animals: the widespread Vertex Potential (VP). While generally assumed to reflect sensory-specific processing, growing evidence suggests that the VP instead largely reflects supramodal neural activity, sensitive to the behavioural-relevance of the eliciting stimulus. In this thesis, I investigate the relationship between sudden events and the brain responses and behaviours they elicit. In Chapters 1-3, I give a general introduction to the topic. In Chapter 4, I dissect the sensitivity of the VP to stimulus intensity - showing that its amplitude is sensitive only to the relative increase of intensity, and not the absolute intensity. In Chapter 5, I show that both increases and decreases of auditory and somatosensory stimulus intensity elicit the same supramodal VP, demonstrating that the VP is sensitive to any sufficiently abrupt sensory change, regardless of its direction or sensory modality. In Chapter 6, I observe strong correlations between the magnitudes of the VP and the eye-blink elicited by somatosensory stimuli (hand-blink reflex; HBR), demonstrating a tight relationship between cortical activity and behaviour elicited by sudden stimuli. In Chapter 7, I explore this relationship further, showing that the HBR is sensitive to high-level environmental dynamics. In Chapter 8, I propose an account of the underlying neural substrate of the VP, consistent with my results and the literature, which elucidates the relationship between the VP and behaviour. I also detail future experiments using fMRI and intracranial recordings to test this hypothesis, using the knowledge gained from this thesis

Mechanisms of heat-gated nociception in primary and dorsal horn sensory neurons of the rat

Noxious heat is a natural stimulus that activates peripheral sensory neurons expressing heat-gated ion channels. Recently, the TRPM3 channel emerged as a noxious heat sensor independent of TRPV1, which is also sensitive to the neurosteroid Pregnenolone sulphate (PS). Recently, evidence of a direct mechanism that controls the agonist-induced TRPM3 channel activity by activation of the µ-opioid receptor (MOR) has been described, through direct binding of the G-beta-gamma subunit to TRPM3. The submitted thesis investigated mechanisms of heat-induced nociception using near-infrared laser stimulation as a rapid and accurate way to apply noxious heat. Responses to laser-heat were analyzed: in vitro by functional assays on heterologous expression systems and primary culture of sensory neurons, and in vivo by behavioral experiments and electrophysiological recordings at the dorsal horn of the spinal cord. Laser-heat activates TRPV1 and TRPM3 channels in heterologous expression systems with activation thresholds of about 574 µJ and 615 µJ. The response amplitudes of TRPM3 upon activation with PS exceeded those of maximum laser stimulation (1.5 ± 0.003 of the ratio 340/380 versus 0.66 ± 0.011). Chemical- and thermal- induced activity of the TRPM3 channel co-expressing the MOR was reduced with DAMGO by 63.4% and 44.5%. In DRGs, 15-25% of all neurons analyzed (n= 550) functionally co-expressed TRPV1 and TRPM3, 38% expressed TRPV1 independent of TRPM3, 7-8% expressed TRPM3 but not TRPV1. DRG neurons displayed a direct inhibition by 18 ± 4.1% and 23 ± 3% when co-applying the MOR agonist DAMGO with PS. In the dorsal horn of the spinal cord, the processing of peripheral laser stimulation was carried out by a subset of WDR and HTM neurons, which were found at all depths of the dorsal horn (range: 120-820 µm). Laser-heat stimuli induced pain-behavior in vivo. All neurons that responded to suprathreshold laser-heat were nociceptive, including one third of WDR neurons and half of HTM neurons investigated. No laser-heat responses of LTM neurons were found. The peripheral input of the laser sensitive neurons was composed of C- and A- fibers; however, responses to laser-heat were transmitted by C-fibers. The sizes of the heat receptive fields ranged 10% - 60% of the mechanical receptive field and they located always inside them. The number of AP following laser stimulation was higher in HTM neurons compared to WDR neurons (14 ± 0.7 vs 9 ± 4.3), however not significant, and the latencies after onset of the laser stimulation were 266 ± 16 ms and 308.3 ± 55. The estimated temperature threshold for laser sensitive WDR neurons and HTM neurons (40.1 °C and 43.3 °C) was comparable to the mean heat withdrawal threshold in awake rats (41 °C). Differences in the proportions of neurons expressing TRPM3 and/or TRPV1 could be responsible for those differences in receptive field sizes. Since the threshold for laser-heat activation of the TRPM3 channel was higher than the threshold for TRPV1, a greater proportion of peripheral neurons containing TRPM3 might converge in dorsal horn laser sensitive HTM neurons than for laser sensitive WDR neurons