Electrical Stimulation Of Hind Paw Research Articles

Mapping of the brain hemodynamic responses to sensorimotor stimulation in a rodent model: A BOLD fMRI study.

Blood Oxygenation Level Dependent functional MRI (BOLD fMRI) during electrical paw stimulation has been widely used in studies aimed at the understanding of the somatosensory network in rats. However, despite the well-established anatomical connections between cortical and subcortical structures of the sensorimotor system, most of these functional studies have been concentrated on the cortical effects of sensory electrical stimulation. BOLD fMRI study of the integration of a sensorimotor input across the sensorimotor network requires an appropriate methodology to elicit functional activation in cortical and subcortical areas owing to the regional differences in both neuronal and vascular architectures between these brain regions. Here, using a combination of low level anesthesia, long pulse duration of the electrical stimulation along with improved spatial and temporal signal to noise ratios, we provide a functional description of the main cortical and subcortical structures of the sensorimotor rat brain. With this calibrated fMRI protocol, unilateral non-noxious sensorimotor electrical hindpaw stimulation resulted in robust positive activations in the contralateral sensorimotor cortex and bilaterally in the sensorimotor thalamus nuclei, whereas negative activations were observed bilaterally in the dorsolateral caudate-putamen. These results demonstrate that, once the experimental setup allowing necessary spatial and temporal signal to noise ratios is reached, hemodynamic changes related to neuronal activity, as preserved by the combination of a soft anesthesia with a soft muscle relaxation, can be measured within the sensorimotor network. Moreover, the observed responses suggest that increasing pulse duration of the electrical stimulus adds a proprioceptive component to the sensory input that activates sensorimotor network in the brain, and that these activation patterns are similar to those induced by digits paw’s movements. These findings may find application in fMRI studies of sensorimotor disorders within cortico-basal network in rodents.

Specificity of stimulus-evoked fMRI responses in the mouse: The influence of systemic physiological changes associated with innocuous stimulation under four different anesthetics

Functional magnetic resonance (fMRI) in mice has become an attractive tool for mechanistic studies, for characterizing models of human disease, and for evaluation of novel therapies. Yet, controlling the physiological state of mice is challenging, but nevertheless important as changes in cardiovascular parameters might affect the hemodynamic readout which constitutes the basics of the fMRI signal. In contrast to rats, fMRI studies in mice report less robust brain activation of rather widespread character to innocuous sensory stimulation. Anesthesia is known to influence the characteristics of the fMRI signal. To evaluate modulatory effects imposed by the anesthesia on stimulus-evoked fMRI responses, we compared blood oxygenation level dependent (BOLD) and cerebral blood volume (CBV) signal changes to electrical hindpaw stimulation using the four commonly used anesthetics isoflurane, medetomidine, propofol and urethane. fMRI measurements were complemented by assessing systemic physiological parameters throughout the experiment. Unilateral stimulation of the hindpaw elicited widespread fMRI responses in the mouse brain displaying a bilateral pattern irrespective of the anesthetic used. Analysis of magnitude and temporal profile of BOLD and CBV signals indicated anesthesia-specific modulation of cerebral hemodynamic responses and differences observed for the four anesthetics could be largely explained by their known effects on animal physiology. Strikingly, independent of the anesthetic used our results reveal that fMRI responses are influenced by stimulus-induced cardiovascular changes, which indicate an arousal response, even to innocuous stimulation. This may mask specific fMRI signal associated to the stimulus. Hence, studying the processing of peripheral input in mice using fMRI techniques constitutes a major challenge and adapted paradigms and/or alternative fMRI readouts should also be considered when studying sensory processing in mice.

Calcium imaging of living astrocytes in the mouse spinal cord following sensory stimulation.

Astrocytic Ca2+ dynamics have been extensively studied in ex vivo models; however, the recent development of two-photon microscopy and astrocyte-specific labeling has allowed the study of Ca2+ signaling in living central nervous system. Ca2+ waves in astrocytes have been described in cultured cells and slice preparations, but evidence for astrocytic activation during sensory activity is lacking. There are currently few methods to image living spinal cord: breathing and heart-beating artifacts have impeded the widespread application of this technique. We here imaged the living spinal cord by two-photon microscopy in C57BL6/J mice. Through pressurized injection, we specifically loaded spinal astrocytes using the red fluorescent dye sulforhodamine 101 (SR101) and imaged astrocytic Ca2+ levels with Oregon-Green BAPTA-1 (OGB). Then, we studied astrocytic Ca2+ levels at rest and after right electrical hind paw stimulation. Sensory stimulation significantly increased astrocytic Ca2+ levels within the superficial dorsal horn of the spinal cord compared to rest. In conclusion, in vivo morphofunctional imaging of living astrocytes in spinal cord revealed that astrocytes actively participate to sensory stimulation.

Functional laser speckle imaging of cerebral blood flow under hypothermia

Hypothermia can unintentionally occur in daily life, e.g., in cardiovascular surgery or applied as therapeutics in the neurosciences critical care unit. So far, the temperature-induced spatiotemporal responses of the neural function have not been fully understood. In this study, we investigated the functional change in cerebral blood flow (CBF), accompanied with neuronal activation, by laser speckle imaging (LSI) during hypothermia. Laser speckle images from Sprague-Dawley rats (n = 8, male) were acquired under normothermia (37°C) and moderate hypothermia (32°C). For each animal, 10 trials of electrical hindpaw stimulation were delivered under both temperatures. Using registered laser speckle contrast analysis and temporal clustering analysis (TCA), we found a delayed response peak and a prolonged response window under hypothermia. Hypothermia also decreased the activation area and the amplitude of the peak CBF. The combination of LSI and TCA is a high-resolution functional imaging method to investigate the spatiotemporal neurovascular coupling in both normal and pathological brain functions.

BOLD and blood volume-weighted fMRI of rat lumbar spinal cord during non-noxious and noxious electrical hindpaw stimulation

Spinal cord fMRI is a useful tool for studying spinal mechanisms of pain, hence for analgesic drug development. Its technical feasibility in both humans and rats has been demonstrated. This study investigates the reproducibility, robustness, and spatial accuracy of fMRI of lumbar spinal cord activation due to transcutaneous noxious and non-noxious electrical stimulation of the hindpaw in α-chloralose-anesthetized rats. Blood oxygenation level-dependent (BOLD) and blood volume-weighted fMRI data were acquired without and with intravenous injection of ultra small superparamagnetic iron oxide particles (USPIO), respectively, using a gradient echo (GE) echo planar imaging (EPI) technique at 4.7 T. Neuronal activation in the spinal cord induced by noxious stimulation to the hindpaw (2 ms wide, 5 mA amplitude, known to activate C-fibers) can be robustly detected by both fMRI techniques with excellent reproducibility and peaked at the stimulus frequency of 40 Hz. However, both fMRI techniques were not sensitive to neuronal activation in spinal cord induced by non-noxious stimulation (0.3 ms, 1.5 mA, known only to activate A-fibers). Spatially, the fMRI signal extended ∼ 5 mm in the longitudinal direction, covering L 3–L 5 segments. In the cross-sectional direction, the highest signal change of blood volume-weighted fMRI was in the middle of the ipsilateral dorsal horn, which roughly corresponds to laminae V and VI, while the highest signal change of BOLD fMRI was in the ipsilateral dorsal surface. This study demonstrates that spinal cord fMRI can be performed in anesthetized rats reliably and reproducibly offering it as a potential tool for analgesic drug discovery.

Spatiotemporal Evolution of Functional Hemodynamic Changes and Their Relationship to Neuronal Activity

Brain imaging techniques such as functional magnetic resonance imaging (fMRI) have provided a wealth of information about brain organization, but their ability to investigate fine-scale functional architecture is limited by the spatial specificity of the hemodynamic responses upon which they are based. We investigated the spatiotemporal evolution of hemodynamic responses in rat somatosensory cortex to electrical hindpaw stimulation. We combined the advantages of optical intrinsic signal imaging and spectroscopy to produce high-resolution two-dimensional maps of functional changes in tissue oxygenation and blood volume. Cerebral blood flow changes were measured with laser-Doppler flowmetry, and simultaneously recorded field potentials allowed comparison between hemodynamic changes and underlying neuronal activity. For the first 2 to 3 secs of activation, hemodynamic responses overlapped in a central parenchymal focus. Over the next several seconds, cerebral blood volume changes propagated retrograde into feeding arterioles, and oxygenation changes anterograde into draining veins. By 5 to 6 secs, responses localized primarily in vascular structures distant from the central focus. The peak spatial extent of the hemodynamic response increased linearly with synaptic activity. This spatial spread might be because of lateral subthreshold activation or passive vascular overspill. These results imply early microvascular changes in volume and oxygenation localize to activated neural columns, and that spatial specificity will be optimal within a 2- to 3-sec window after neuronal activation.

Evaluation of coupling between optical intrinsic signals and neuronal activity in rat somatosensory cortex

We investigated the coupling between perfusion-related brain imaging signals and evoked neuronal activity using optical imaging of intrinsic signals (OIS) at 570 and 610 nm. OIS at 570 nm reflects changes in cerebral blood volume (CBV), and the 610 nm response is related to hemoglobin oxygenation changes. We assessed the degree to which these components of the hemodynamic response were coupled to neuronal activity in rat barrel, hindpaw, and forepaw somatosensory cortex by simultaneously recording extracellular evoked field potentials (EPs) and OIS while varying stimulation frequency. In all stimulation paradigms, 10 Hz stimulation evoked the largest optical and electrophysiological responses. Across all animals, the 610 late phase and 570 responses correlated linearly with ΣEP ( P < 0.05) during both whisker deflection and electrical hindpaw stimulation, but the 610 early phase did not (whisker P = 0.27, hindpaw P = 0.28). The signal-to-noise ratio (SNR) of the 610 early phase (whisker 3.1, hindpaw 5.3) was much less than that for the late phase (whisker 14, hindpaw 51) and 570 response (whisker 11, hindpaw 46). During forepaw stimulation, however, the 610 early phase had a SNR (17) higher than that during hindpaw stimulation and correlated well with neuronal activity ( P < 0.05). We conclude that the early deoxygenation change does not correlate consistently with neuronal activity, possibly because of its low SNR. The robust CBV-related response, however, has a high SNR and correlates well with evoked cortical activity.

Cyclooxygenase-pathway participates in the regulation of regional cerebral blood flow in response to neuronal activation under normo- and hypercapnia

The present study was designed to investigate whether cyclooxygenase products are involved in the regulation of the regional cerebral blood flow, evoked by somatosensory activation (evoked rCBF) under normo- and hypercapnia. Indomethacin (IMC) was used as cyclooxygenase inhibitor. It was applied intravenously (i.v., 10 mg/kg/h) in two experimental protocols—before hypercapnia (i) and after hypercapnia (ii). Somatosensory activation was induced by electrical hind paw stimulation (5 Hz frequency, 5 s duration, 1.5 mA). The evoked rCBF-response was measured in α -chloralose anesthetized rats using laser-Doppler flowmetry. IMC abolished completely the effect of hypercapnia on the baseline level of CBF. The drug reduced significantly evoked rCBF-response also. The inhibitory effect of IMC on evoked rCBF-response is better expressed under normocapnia (∼70%) than that under hypercapnia (∼40%). After IMC application, the normalized evoked rCBF curves peaked earlier as compared to that before its application ( P<0.05), although the rise time of 0.5 s was nearly constant regardless of stimulus frequency. In conclusion, the results suggest a participation of IMC-sensitive and cyclooxygenase-dependent mechanisms in the regulation of evoked rCBF, induced by somatosensory stimulation.

Laser-doppler imaging of activation-flow coupling in the somatosensory cortex: Normalization of signal when the baseline changes significantly

This study describes two approaches used to normalize the laser-Doppler flowmetry (LDF) signal, corresponding to the regional cerebral blood flow (rCBF) response after electrical hind-paw stimulation. The first approach divides the LDF signal to the baseline and subsequently integrates the response curve from the rise point to the termination point (defined formally as "normalized rCBF response", and the second subtracts the baseline from the LDF signal and subsequently integrates the response curve from the rise point to the termination point (defined as "absolute rCBF response"). Both parameters are given in arbitrary units. A comparative analysis of the changes in the "normalized" and "absolute" LDrCBF response is presented both for when the baseline does not change significantly, and for when the baseline changes significantly under the influence of different factors. In summary, when the baseline changes significantly it is preferable to normalize the LDrCBF response towards the baseline by subtraction, not by division.

Nociceptive inputs into rostral ventrolateral medulla‐spinal vasomotor neurones in rats.

1. In anaesthetized rats recordings were made from thirty-eight neurones in the rostral ventrolateral medulla (RVL) with spinal-projecting axons. Their responses to mechanical, thermal and/or electrical stimulation were examined as were the accompanying changes in arterial pressure. 2. Mechanical, thermal and electrical stimulation of either hindpaw at a strength that can be regarded as noxious produced a consistent rise in arterial pressure. RVL-spinal-projecting 'vasomotor' neurones were excited by the noxious mechanical and thermal (52 degrees C) stimulation at a latency that was shorter than that of the evoked pressor response. 3. Percutaneous electrical stimulation of either hindlimb extremity resulted in an early peak of excitation (fourteen out of fourteen), an early trough of inhibition (twelve out fourteen), and a later peak of excitation (two out of fourteen). This response pattern to stimulation of either limb was independent of which limb was activated, but contralateral hindpaw stimulation elicited excitation at a shorter latency. The differences in latency of responses to stimulating two locations along the tail suggested that the early excitation and inhibition of RVL-spinal 'vasomotor' neurones were evoked by activation of peripheral fibres with a mean conduction velocity in the A delta range. 4. Short-latency excitatory and inhibitory responses in RVL-spinal 'vasomotor' neurones were observed also when single-pulse stimuli were delivered within the lateral part of the spinal cord. 5. Ionophoretic application of bicuculline, a GABAA receptor antagonist, blocked the evoked inhibition of these neurones on electrical stimulation of the hindpaw without attenuating the excitatory input from the same stimulus. 6. These results indicate that RVL-spinal 'vasomotor' neurones receive an input from cutaneous nociceptive afferents. This suggests that these neurones mediate, at least partly, the cardiovascular responses related to nociceptor stimulation.

Suppression of the hindlimb flexor reflex by stimulation of the medial hypothalamus and thalamus in the rat

Pentobarbital-anestetized rats received electrical hindpaw stimulation every 10 s to elicit a maximal hindlimb withdrawal reflex. The integrated EMG response in the ipsilateral tibialis anterior was sampled by a computer which also controlled the timing of electrical stimuli applied to the brain. A suppression of the evoked flexor activity was obtained with currents below 0.05 mA for stimuli applied in the medial hypothalamic region. A second effective site was located in the paraventricular area of the thalamus. The suppression had an onset latency of 30 ms, increased over a period of 500 ms and was followed by a postinhibitory facilitation (rebound). When the noxious electrical shocks were given over prolonged periods (140 s) the suppression of the flexor reflex was seen to outlast the central stimulation by more than 100ss. Intravenous injection of naloxone or methysergide failed to reverse the effects of the brain stimuli. It is suggested that the hypothalamic induced inhibition of withdrawal reflexes is functionally meaningful in view of the incompatibility between these reflexes and the locomotor behavior which is part of the behavioral responses (i.e. fight or flight) controlled by this area.