Manganese-enhanced magnetic resonance imaging depicts brain activity in models of acute and chronic pain: A new window to study experimental spontaneous pain?

Application of functional imaging techniques to animal models is vital to understand pain mechanisms, but is often confounded by the need to limit movement artefacts with anaesthesia, and a focus on evoked responses rather than clinically relevant spontaneous pain and related hyperalgesia. The aim of the present study was to investigate the potential of manganese-enhanced magnetic resonance imaging (MEMRI) to measure neural responses during on-going pain that underpins hyperalgesia in pre-clinical models of nociception. As a proof of concept that MEMRI is sensitive to the neural activity of spontaneous, intermittent behaviour, we studied a separate positive control group undergoing a voluntary running wheel experiment. In the pain models, pain behaviour (weight bearing asymmetry and hindpaw withdrawal thresholds (PWTs)) was measured at baseline and following either intra-articular injection of nerve growth factor (NGF, 10µg/50µl; acute pain model, n=4 rats per group), or the chondrocyte toxin monosodium iodoacetate (MIA, 1mg/50µl; chronic model, n=8 rats per group), or control injection. Separate groups of rats underwent a voluntary wheel running protocol (n=8 rats per group). Rats were administered with paramagnetic ion Mn2+ as soluble MnCl2 over seven days (subcutaneous osmotic pump) to allow cumulative activity-dependent neural accumulation in the models of pain, or over a period of running. T1-weighted MR imaging at 7T was performed under isoflurane anaesthesia using a receive-only rat head coil in combination with a 72mm volume coil for excitation. The pain models resulted in weight bearing asymmetry (NGF: 20.0 ± 5.2%, MIA: 15 ± 3%), and a reduction in PWT in the MIA model (8.3 ± 1.5g) on the final day of assessment before undergoing MR imaging. Voxel-wise and region-based analysis of MEMRI data did not identify group differences in T1 signal. However, MnCl2 accumulation in the VTA, right Ce amygdala, and left cingulate was negatively correlated with pain responses (greater differences in weight bearing), similarly MnCl2 accumulation was reduced in the VTA in line with hyperalgesia (lower PWTs), which suggests reduced regional activation as a result of the intensity and duration of pain experienced during the 7 days of MnCl2 exposure. Motor cortex T1-weighted signal increase was associated with the distance ran in the wheel running study, while no between group difference was seen. Our data suggest that on-going pain related signal changes identified using MEMRI offers a new window to study the neural underpinnings of spontaneous pain in rats.

Developmental alterations in noxious-evoked EEG activity recorded from rat primary somatosensory cortex.

Primary somatosensory cortex (S1) contains a nociceptive map that localizes potential tissue damage on the body and encodes stimulus intensity. An objective and specific biomarker of pain however is currently lacking and is urgently required for use in non-verbal clinical populations as well as in the validation of pre-clinical pain models. Here we describe studies to see if the responses of the S1 in juvenile rats are different to those in the adult. We recorded electroencephalogram (EEG) responses from S1 of lightly-anesthetized Sprague-Dawley rats at either postnatal day 21 or postnatal day 40 during the presentation of noxious (55 °C) or innocuous (30 °C) thermal stimuli applied to the plantar surface of the left hindpaw. The total EEG power across the recording period was the same in both ages after stimulation but the frequency distribution was significantly affected by age. Noxious heat evoked a significant increase in theta band (4-8 Hz) activity in adults only (P<0.0001 compared to baseline; P<0.0001 compared to juveniles). There were no significant differences in EEG responses to innocuous thermal stimuli. These data show that there are significant alterations in the processing of nociceptive inputs within the maturing cortex and that cortical theta activity is involved only in the adult cortical response to noxious stimulation.

A quantification of the relationship between neuronal responses in the rat rostral ventromedial medulla and noxious stimulation-evoked withdrawal reflexes.

The rostral ventromedial medulla (RVM) regulates a range of involuntary behaviours but is most often associated with nociception via the action of pronociceptive ON cells and antinociceptive OFF cells. The phasic responses of ON and OFF cells determine whether or not incoming noxious signals provoke a withdrawal reflex, and previous studies have suggested that reflex RVM activity patterns actively shape motor output. Here we challenged the model by using juvenile rats, which are known to exhibit markedly different reflex responses compared with adults. By recording single-cell activity in the RVM and the electromyography responses of hindlimb flexor muscles to noxious thermal stimulation we found that the juvenile reflex had a shorter onset latency, was larger in amplitude and exhibited a decreased rise time compared with the adult reflex. The responses of ON and OFF cells faithfully tracked the shorter onset latency of the reflex by also responding earlier and, thus, still preceded the reflex. However, neither the reflex amplitude nor the ongoing response profile was predicted by the firing rate of RVM cells in either age group. Instead we found a close correspondence between RVM activity and the reflex only during the initiation of the response. Furthermore, the short rise time of the juvenile reflex was reflected in higher rates of change of both ON and OFF cell firing. Our data suggest that the RVM is associated only with the initiation of reflexes and does not shape ongoing muscle activity, which is more likely to be subserved by downstream spinal processes.

Enhanced visual responses in the superior colliculus in an animal model of attention-deficit hyperactivity disorder and their suppression by D-amphetamine.

Attention-deficit hyperactivity disorder (ADHD) is a prevalent neurodevelopmental disorder characterized by overactivity, impulsiveness and attentional problems, including an increase in distractibility. A structure that is intimately linked with distractibility is the superior colliculus (SC), a midbrain sensory structure which plays a particular role in the production of eye and head movements. Although others have proposed the involvement of such diverse elements as the frontal cortex and forebrain noradrenaline in ADHD, given the role of the colliculus in distractibility and the increased distractibility in ADHD, we have proposed that distractibility in ADHD arises due to collicular sensory hyper-responsiveness. To further investigate this possibility, we recorded the extracellular activity (multi-unit (MUA) and local field potential (LFP)) in the superficial visual layers of the SC in an animal model of ADHD, the New Zealand genetically hypertensive (GH) rat, in response to wholefield light flashes. The MUA and LFP peak amplitude and summed activity within a one-second time window post-stimulus were both significantly greater in GH rats than in Wistar controls, across the full range of stimulus intensities. Given that baseline firing rate did not differ between the strains, this suggests that the signal-to-noise ratio is elevated in GH animals. D-Amphetamine reduced the peak amplitude and summed activity of the multi-unit response in Wistar animals. It also reduced the peak amplitude and summed activity of the multi-unit response in GH animals, at higher doses bringing it down to levels that were equivalent to those of Wistar animals at baseline. The present results provide convergent evidence that a collicular dysfunction (sensory hyper-responsiveness) is present in ADHD, and that it may underlie the enhanced distractibility. In addition, D-amphetamine - a widely used treatment in ADHD - may have one of its loci of therapeutic action at the level of the colliculus.

An evaluation of in vivo voltage-sensitive dyes: pharmacological side effects and signal-to-noise ratios after effective removal of brain-pulsation artifacts.

In the current study, we investigated pharmacological side effects and signal-to-noise ratios (SNRs) of two commonly used voltage-sensitive dyes (VSDs): the blue dye RH-1691 (1 mg/ml) and the red dye di-4-ANEPPS (0.1 mg/ml), applied in vivo to the rat barrel cortex. Blue dyes are often favored over red dyes in in vivo studies due to their apparent superior SNR, partly because their fluorescence spectrum is farther away from the hemoglobin absorption spectrum, making them less prone to heartbeat-associated brain-pulsation artifacts (BPA). We implemented a previously reported template-based BPA removal algorithm and evaluated its applicability to di-4-ANEPPS before comparing characteristics of the two dyes. Somatosensory-evoked potentials (SEPs) were also recorded. Whereas SEPs recorded before and after application of di-4-ANEPPS failed to exhibit demonstrable differences, RH-1691 caused a significant and prolonged increase in SEP amplitude for several hours. In contrast, neither dye influenced the spontaneous cortical activity as assessed by the spectral content of the EEG. Both dyes turned out to be strikingly similar with respect to changes in fractional fluorescence as a function of SEP response amplitude, as well as regarding shot noise characteristics after removal of the BPA. Thus there is strong evidence that the increased SNR for RH-1691 is a consequence of an artificially increased signal. When applying an appropriate BPA removal algorithm, di-4-ANEPPS has proven to be suitable for single-trial in vivo VSD imaging (VSDI) and produces no detectable neurophysiological changes in the system under investigation. Taken together, our data argue for a careful re-evaluation of pharmacological side effects of RH-1691 and support the applicability of di-4-ANEPPS for stable single-trial in vivo VSDI recordings.

Environmental enrichment differentially modifies specific components of sensory-evoked activity in rat barrel cortex as revealed by simultaneous electrophysiological recordings and optical imaging in vivo.

Environmental enrichment of laboratory animals leads to multi-faceted changes to physiology, health and disease prognosis. An important and under-appreciated factor in enhancing cognition through environmental manipulation may be improved basic sensory function. Previous studies have highlighted changes in cortical sensory map plasticity but have used techniques such as electrophysiology, which suffer from poor spatial resolution, or optical imaging of intrinsic signals, which suffers from low temporal resolution. The current study attempts to overcome these limitations by combining voltage-sensitive dye imaging with somatosensory-evoked potential (SEP) recordings: the specific aim was to investigate sensory function in barrel cortex using multi-frequency whisker stimulation under urethane anaesthesia. Three groups of rats were used that each experienced a different level of behavioural or environmental enrichment. We found that enrichment increased all SEP response components subsequent to the initial thalamocortical input, but only when evoked by single stimuli; the thalamocortical component remained unchanged across all animal groups. The optical signal exhibited no changes in amplitude or latency between groups, resembling the thalamocortical component of the SEP response. Permanent and extensive changes to housing conditions conferred no further enhancement to sensory function above that produced by the milder enrichment of regular handling and behavioural testing, a finding with implications for improvements in animal welfare through practical changes to animal husbandry.

Design and evaluation of a low-cost respiratory monitoring device for use with anaesthetized animals.

This report describes a simple, non-invasive electronic device that employs a compact accelerometer integrated circuit to transduce movements in the chest wall of an anaesthetized animal into an analogue signal that can be used to calculate the rate and relative depth of respiration. The device requires amplification by signal processing hardware/software which are common to most experimental laboratories. We assessed the sensitivity of the device by its ability to detect changes in respiratory patterns produced by modulating the depth of anaesthesia in isoflurane-anaesthetized Wistar rats. It is widely accepted that many anaesthetic agents affect respiratory patterns, especially respiratory rate (RR), which is often used as an important index of anaesthetic depth. Respiratory parameters obtained with the device were compared with concurrently recorded electroencephalographic and cardiac measures. Different concentrations of anaesthetic agent produced four depths of anaesthesia, identified using established electroencephalographic criteria. The accelerometer was attached easily and securely to the location of maximal chest wall movement and produced a strong respiratory signal that was detectable in all four anaesthetic stages. Deepening the anaesthesia produced a gradual decrease in RR, a decrease in dominant spectral frequency of the electroencephalogram (EEG) but no change in the heart rate. There was a significant correlation between RR and the dominant spectral frequency of the EEG, indicating that one useful application of the monitor could be to identify anaesthetic stages. The results demonstrate that respiratory parameters can be recorded using a simply constructed, low-cost device and suggest an application in the monitoring of anaesthetic depth.

D-amphetamine depresses visual responses in the rat superior colliculus: a possible mechanism for amphetamine-induced decreases in distractibility.

Amphetamines can enhance sustained attention, and reduce distractibility, in normal subjects and patients with attentional-deficit/hyperactivity disorder (ADHD). Their mechanism of action in this regard is unknown, however one possibility is that the drugs affect the superior colliculus (SC), a structure with a clearly defined role in distractibility. The aim of the present studies was to explore the effect of systemically and locally administered d-amphetamine on visual responses in the superficial layers of the SC to wholefield light flashes in the rat, using local field potential and multi-unit recording. Systemic and intra-collicular d-amphetamine both produced a dose-related depression of visual activity, which sometimes progressed to inactivation of the multi-unit response at the highest dose. As a consequence, it is possible that amphetamines enhance sustained attention, and reduce distractibility, via an action on the colliculus. A corollary of this is that collicular dysfunction may underlie enhanced distractibility in ADHD.

Cocaine preferentially enhances sensory processing in the upper layers of the primary sensory cortex.

Sensory systems are believed to play an important role in drug addiction, particularly in triggering craving and relapse, and it has been shown in previous studies that administration of cocaine can enhance evoked responses in the primary sensory cortex of experimental animals. Primary sensory cortex comprises a multi-layered structure to which a variety of roles have been assigned; an understanding of how cocaine affects evoked activity in these different layers may shed light on how drug-associated sensory cues gain control over behavior. The aim of the present study was to examine how cocaine affects whisker sensory responses in different layers of the primary sensory (barrel) cortex. Field potential and multi-unit activity were recorded from the cortex of anesthetized rats using 16 channel linear probes during repetitive (air puff) stimulation of the whiskers. In control conditions (under saline, i.v.), responses strongly adapted to the repeated sensory stimulation. Following an i.v. injection of cocaine (0.5 mg/kg, i.v.), this adaptation was strongly attenuated, giving each stimulus a more equal representation and weight. Attenuation of adaptation was more marked in the upper cortical layers in both field potential and multi-unit data. Indeed, in these layers, not only was adaptation attenuated but multi-unit response amplitudes under cocaine exceeded those under saline for stimuli occurring early in the train. The results extend our previous findings concerning the enhancement by cocaine of primary sensory responses. Insofar as enhanced neural responses equate to enhanced stimulus salience, the results indicate that cocaine may play a previously under-appreciated role in the formation of associations between drug and drug-related environmental cues by enhancing stimulus salience. The associative process itself may be assisted by a preferential action in the upper cortical layers, thought to be involved in learning and plasticity.

Cocaine administration produces a protracted decoupling of neural and haemodynamic responses to intense sensory stimuli.

Evidence suggests that for relatively weak sensory stimuli, cocaine elevates background haemodynamic parameters but still allows enhanced neural responses to be reflected in enhanced haemodynamic responses. The current study investigated the possibility that for more intense stimuli, the raised background may produce a protracted attenuation of the haemodynamic response. Three experiments were performed to measure effects of i.v. cocaine administration (0.5 mg/kg) or saline on responses in rat barrel cortex to electrical stimulation of the whisker pad. The first experiment used optical imaging spectroscopy (OIS) and laser Doppler flowmetry (LDF) to measure haemodynamic changes. Cocaine caused an increase in baseline blood flow (peak approximately 90%), which lasted for the duration of the test period (25 min). Haemodynamic responses to whisker stimulation were substantially reduced throughout. The second experiment used a 16-channel multi-electrode to measure evoked potentials at 100 mum intervals through the barrel cortex. Summed neural responses (collapsed across the spatial dimension) after cocaine administration were similar to those after saline. The third experiment extended experiment 1 by examining the effects of cocaine on whisker sensory responses using functional magnetic resonance imaging (and concurrent OIS or LDF). Cocaine caused a similar increase in baseline and reduction in the evoked response to that seen in experiment 1. Together, the results of these three experiments show that cocaine produces a protracted decoupling of neural activity and haemodynamic responses to intense sensory stimulation, which suggests that imaging techniques based on changes in haemodynamic parameters may be unsuitable for studying the effects of cocaine on sensory processing in humans.

Haemodynamic responses to sensory stimulation are enhanced following acute cocaine administration.

Cocaine enhances neural activity in response to sensory stimulation, an effect that may play a role in the development of drug craving. However, cocaine-induced sensory enhancement may be difficult to study in humans using neuroimaging if the global increases in baseline haemodynamic parameters, which cocaine produces, interfere with the ability of enhanced sensory-related neural activity to lead to enhanced haemodynamic responses. To investigate the effect of cocaine-induced baseline haemodynamic changes on sensory-related haemodynamic (and electrophysiological) responses, field potential (FP) and haemodynamic responses (obtained using optical imaging spectroscopy and laser-Doppler flowmetry) in the barrel cortex of the anaesthetised rat were measured during mechanical whisker stimulation following cocaine (0.5 mg/kg) or saline administration. During cocaine infusion, the relationship between blood flow and volume transiently decoupled. Following this, cocaine caused large baseline increases in blood flow (133%) and volume (33%), which peaked after approximately 6 min and approached normal levels again after 25 min. During the peak baseline increases, FP responses to whisker stimulation were similar to saline whereas several haemodynamic response parameters were slightly reduced. After the peak, significant increases in FP responses were observed, accompanied by significantly enhanced haemodynamic responses, even though the haemodynamic baselines remained elevated. Hence, the haemodynamic response to sensory stimulation is transiently reduced in the presence of large increases in baseline but, after the baseline peak, enhanced neural responses are faithfully accompanied by enhanced haemodynamic responses. The findings suggest that any cocaine-induced enhancement of sensory-related neural activity in humans is likely to be detectable by neuroimaging.