Functional imaging evidence for task-induced deactivation and disconnection of a major default mode network hub in the mouse brain.

The default mode network (DMN) has been defined in functional brain imaging studies as a set of highly connected brain areas, which are active during wakeful rest and inactivated during task-based stimulation. DMN function is characteristically impaired in major neuropsychiatric diseases, emphasizing its interest for translational research. However, in the mouse, a major preclinical rodent model, there is still no functional imaging evidence supporting DMN deactivation and deconnection during high-demanding cognitive/sensory tasks. Here we have developed functional ultrasound (fUS) imaging to properly visualize both activation levels and functional connectivity patterns, in head-restrained awake and behaving mice, and investigated their modulation during a sensory-task, whisker stimulation. We identified reproducible and highly symmetric resting-state networks, with overall connectivity strength directly proportional to the wakefulness level of the animal. We show that unilateral whisker stimulation leads to the expected activation of the contralateral barrel cortex in lightly sedated mice, while interhemispheric inhibition reduces activity in the ipsilateral barrel cortex. Whisker stimulation also leads to elevated bilateral connectivity in the hippocampus. Importantly, in addition to functional changes in these major hubs of tactile information processing, whisker stimulation during genuine awake resting-state periods leads to highly specific reductions both in activation and interhemispheric correlation within the restrosplenial cortex, a major hub of the DMN. These results validate an imaging technique for the study of activation and connectivity in the lightly sedated awake mouse brain and provide evidence supporting an evolutionary preserved function of the DMN, putatively improving translational relevance of preclinical models of neuropsychiatric diseases.

Pharmaco-fUS: Quantification of pharmacologically-induced dynamic changes in brain perfusion and connectivity by functional ultrasound imaging in awake mice.

There is a critical need for reliable quantitative biomarkers to assess functional brain alterations in mouse models of neuropsychiatric diseases, but current imaging methods measuring drug effects through the neurovascular coupling, face issues including poor sensitivity, drug-induced changes in global brain perfusion and the effects of anesthesia. Here we demonstrate the proof-of-concept of a minimally-invasive fUS imaging approach to detect the acute cholinergic modulatory effects of Scopolamine (ScoP) on functional brain connectivity in awake and behaving mice, through the intact skull. A machine-learning algorithm constructed an ad-hoc pharmacological score from the ScoP-induced changes in connectivity patterns of five mice. The discrimination model shows important ScoP-induced increase of the hippocampo-cortical connectivity. The pharmacological score led to robust discrimination of ScoP treatment from baseline in an independent dataset and showed, in another independent group, dose-dependent specific effects of central cholinergic modulation of functional connectivity, independent from global brain perfusion changes. In conclusion, we introduce pharmaco-fUS as a simple, robust, specific and sensitive modality to monitor drug effects on perfusion and functional connectivity in the awake mouse brain.

Cholinergic Neurotransmission in the Posterior Insular Cortex Is Altered in Preclinical Models of Neuropathic Pain: Key Role of Muscarinic M2 Receptors in Donepezil-Induced Antinociception.

Neuropathic pain is one of the most debilitating pain conditions, yet no therapeutic strategy has been really effective for its treatment. Hence, a better understanding of its pathophysiological mechanisms is necessary to identify new pharmacological targets. Here, we report important metabolic variations in brain areas involved in pain processing in a rat model of oxaliplatin-induced neuropathy using HRMAS (1)H-NMR spectroscopy. An increased concentration of choline has been evidenced in the posterior insular cortex (pIC) of neuropathic animal, which was significantly correlated with animals' pain thresholds. The screening of 34 genes mRNA involved in the pIC cholinergic system showed an increased expression of the high-affinity choline transporter and especially the muscarinic M2 receptors, which was confirmed by Western blot analysis in oxaliplatin-treated rats and the spared nerve injury model (SNI). Furthermore, pharmacological activation of M2 receptors in the pIC using oxotremorine completely reversed oxaliplatin-induced mechanical allodynia. Consistently, systemic treatment with donepezil, a centrally active acetylcholinesterase inhibitor, prevented and reversed oxaliplatin-induced cold and mechanical allodynia as well as social interaction impairment. Intracerebral microdialysis revealed a lower level of acetylcholine in the pIC of oxaliplatin-treated rats, which was significantly increased by donepezil. Finally, the analgesic effect of donepezil was markedly reduced by a microinjection of the M2 antagonist, methoctramine, within the pIC, in both oxaliplatin-treated rats and spared nerve injury rats. These findings highlight the crucial role of cortical cholinergic neurotransmission as a critical mechanism of neuropathic pain, and suggest that targeting insular M2 receptors using central cholinomimetics could be used for neuropathic pain treatment. SIGNIFICANCE STATEMENT: Our study describes a decrease in cholinergic neurotransmission in the posterior insular cortex in neuropathic pain condition and the involvement of M2 receptors. Targeting these cortical muscarinic M2 receptors using central cholinomimetics could be an effective therapy for neuropathic pain treatment.

Assessment of thermal sensitivity in rats using the thermal place preference test: description and application in the study of oxaliplatin-induced acute thermal hypersensitivity and inflammatory pain models.

Thermal sensitivity is an essential characteristic of some painful states, including oxaliplatin-induced neuropathy. The thermal place preference test (TPPT) was designed to finely assess thermal sensitivity in rodents. The TPPT monitors the time spent by unrestrained rodents on a test plate at fixed temperatures (5-50°C) compared with an adjacent reference plate at a neutral temperature (25°C). Here, we report the results of a study designed (i) to validate the optimal methodological parameters for measuring thermal sensitivity in rats, (ii) to assess the thermal sensitivity of healthy rats and animal models of pain and (iii) to explore the pharmacological effects of analgesic drugs. The most reproducible conditions occurred when the TPPT was performed in the morning and in the dark for 3 min with the reference plate set to 25°C. The temperature preferences of healthy rats were more than 17°C and less than 40°C. When compared with control animals, oxaliplatin-treated rats showed thermal hypersensitivity at 12, 20 and 35°C, and carrageenan-treated rats showed thermal hypersensitivity at 15 and 45°C. Duloxetine (2.5 mg/kg, intraperitoneal) reversed oxaliplatin-induced cold hypersensitivity (20°C) and morphine (1 mg/kg, intravenous) reversed carrageenan-induced heat hypersensitivity (45°C). We conclude that the TPPT enables a fine-grained assessment of thermal sensitivity that is relevant to the pathophysiological exploration of animal pain models and to the pharmacological assessment of analgesic drugs.

Novel intravital approaches to quantify deep vascular structure and perfusion in the aging mouse brain using ultrasound localization microscopy (ULM).

Intra-vital visualization of deep cerebrovascular structures and blood flow in the aging brain has been a difficult challenge in the field of neurovascular research, especially when considering the key role played by the cerebrovasculature in the pathogenesis of both vascular cognitive impairment and dementia (VCID) and Alzheimer's disease (AD). Traditional imaging methods face difficulties with the thicker skull of older brains, making high-resolution imaging and cerebral blood flow (CBF) assessment challenging. However, functional ultrasound (fUS) imaging, an emerging non-invasive technique, provides real-time CBF insights with notable spatial-temporal resolution. This study introduces an enhanced longitudinal fUS method for aging brains. Using elderly (24-month C57BL/6) mice, we detail replacing the skull with a polymethylpentene window for consistent fUS imaging over extended periods. Ultrasound localization mapping (ULM), involving the injection of a microbubble (<<10 µm) suspension allows for recording of high-resolution microvascular vessels and flows. ULM relies on the localization and tracking of single circulating microbubbles in the blood flow. A FIJI-based analysis interprets these high-quality ULM visuals. Testing on older mouse brains, our method successfully unveils intricate vascular specifics even in-depth, showcasing its utility for longitudinal studies that require ongoing evaluations of CBF and vascular aspects in aging-focused research.

Emerging trends in understanding chemotherapy-induced peripheral neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a major concern in oncology practice given the increasing number of cancer survivors and the lack of effective treatment. The incidence of peripheral neuropathy depends upon the anticancer drug used, but is commonly under-reported in clinical trials. Several animal models have been developed in an attempt to better characterize the pathophysiological mechanisms underlying these CIPN and to find more specific treatments. Over the past two decades, three main trends have emerged from preclinical research on CIPN. There is a compelling body of evidence that neurotoxic anticancer drugs affect the peripheral sensory nerve by directly targeting the mitochondria and producing oxidative stress, by functionally impairing the ion channels and/or by triggering immunological mechanisms through the activation of satellite glial cells. These various neurotoxic events may account for the lack of effective treatment, as neuroprotection may probably only be achieved using a polytherapy that targets all of these mechanisms. The aim of this review is to describe the clinical features of CIPN and to summarize the recent trends in understanding its pathophysiology.

Whole-Brain 3D Activation and Functional Connectivity Mapping in Mice using Transcranial Functional Ultrasound Imaging.

Functional ultrasound (fUS) imaging is a novel brain imaging modality that relies on the high-sensitivity measure of the cerebral blood volume achieved by ultrafast doppler angiography. As brain perfusion is strongly linked to local neuronal activity, this technique allows the whole-brain 3D mapping of task-induced regional activation as well as resting-state functional connectivity, non-invasively, with unmatched spatio-temporal resolution and operational simplicity. In comparison with fMRI (functional magnetic resonance imaging), a main advantage of fUS imaging consists in enabling a complete compatibility with awake and behaving animal experiments. Moreover, fMRI brain mapping in mice, the most used preclinical model in Neuroscience, remains technically challenging due to the small size of the brain and the difficulty to maintain stable physiological conditions. Here we present a simple, reliable and robust protocol for whole-brain fUS imaging in anesthetized and awake mice using an off-the-shelf commercial fUS system with a motorized linear transducer, yielding significant cortical activation following sensory stimulation as well as reproducible 3D functional connectivity pattern for network identification.

Transcranial Functional Ultrasound Imaging in Freely Moving Awake Mice and Anesthetized Young Rats without Contrast Agent.

Functional ultrasound (fUS) imaging by ultrasensitive Doppler detection of blood volume was previously reported to measure adult rat brain activation and functional connectivity with unmatched spatiotemporal sampling (100 µm, 1 ms), but skull-induced attenuation of ultrasonic waves imposed skull surgery or contrast agent use. Also, fUS feasibility remains to be validated in mice, a major pre-clinical model organism. In the study described here, we performed full-depth ultrasensitive Doppler imaging and 3-D Doppler tomography of the entire mouse brain under anesthesia, non-invasively through the intact skull and skin, without contrast agents. Similar results were obtained in anesthetized young rats up to postnatal day 35, thus enabling longitudinal studies on postnatal brain development. Using a newly developed ultralight ultrasonic probe and an optimized ultrasonic sequence, we also performed minimally invasive full-transcranial fUS imaging of brain vasculature and whisker stimulation-induced barrel cortex activation in awake and freely moving mice, validating transcranial fUS for brain imaging, without anesthesia-induced bias, for behavioral studies.

Acid-Sensing Ion Channel 1a in the amygdala is involved in pain and anxiety-related behaviours associated with arthritis.

Chronic pain is associated with anxiety and depression episodes. The amygdala plays a key role in the relationship between emotional responses and chronic pain. Here, we investigated the role of Acid-Sensing Ion Channels 1a within the basolateral amygdala (BLA), in pain and associated anxiety in a rat model of monoarthritis (MoAr). Administration within the BLA of PcTx1 or mambalgin-1, two specific inhibitors of ASIC1a-containing channels significantly inhibited pain and anxiety-related behaviours in MoAr rats. The effect of PcTx1 was correlated with a reduction of c-Fos expression in the BLA. We examined the expression profile of ASICs and other genes in the amygdala in MoAr and sham animals, and found no variation of the expression of ASIC1a, which was confirmed at the protein level. However, an increase in the BLA of MoAr rats of both PI3Kinase mRNA and the phosphorylated form of Akt, along with Bdnf mRNA, suggest that the BDNF/PI3-kinase/Akt pathway might regulate ASIC1a in BLA neurons as demonstrated in spinal sensitisation phenomenon. We also observed changes in several kinase mRNAs expression (PICK1, Sgk1) that are potentially involved in ASIC1a regulation. These results show a crucial role of ASIC1a channels in the BLA in pain and anxiety-related behaviours during arthritis.

Prevention of oxaliplatin-induced peripheral neuropathy by a polyamine-reduced diet-NEUROXAPOL: protocol of a prospective, randomised, controlled, single-blind and monocentric trial.

INTRODUCTION: Oxaliplatin remains the most widely used chemotherapeutic agent for treating advanced colorectal cancer but its efficacy is hampered by dose-limiting neurotoxicity manifested by a painful polyneuropathy. Oxaliplatin-induced peripheral neuropathy (OIPN) is characterised by acute and transient cold hyperaesthesia in the hours and days following oxaliplatin infusion (>90% of patients), but also by retarded chronic neuropathy due to the repetition of chemotherapy cycles (30-50% of patients). OIPN impairs the health-related quality of life (HRQOL) of patients and no preventive or curative strategies have as yet proven effective. A polyamine-reduced diet (PRD) has recently demonstrated its efficacy to prevent OIPN in animals without adverse effects. METHODS AND ANALYSIS: The NEUROXAPOL trial is a prospective, randomised, controlled, single-blind, monocentric and interventional study. This trial is aimed at evaluating the efficacy and feasibility of a PRD compared to a normal polyamine containing diet to prevent OIPN in patients treated by oxaliplatin-based chemotherapy. Patients (n=40 per group) will be randomly assigned to receive either a PRD or a normal diet before and during the chemotherapy regimen. The main objectives are to improve the cold pain thresholds, neuropathic pain symptoms, comorbidities (anxiety and depression) and HRQOL of patients. The primary end point is the assessment of cold pain thresholds 2 weeks after the third cycle of chemotherapy. The secondary end points are the evaluation of thermal pain thresholds, the grade of neuropathy, neuropathic pain, symptoms of anxiety and depression and HRQOL, until the 12th cycle of chemotherapy. ETHICS AND DISSEMINATION: The study was approved by an independent medical ethics committee 1 (CPP Sud Est 1, Saint Etienne, France) and registered by the competent French authority (ANSM, Saint Denis, France). The results will be disseminated in a peer-reviewed journal and presented at international congresses. TRIAL REGISTRATION NUMBER: NCT01775449.

The Nav1.9 channel is a key determinant of cold pain sensation and cold allodynia.

Cold-triggered pain is essential to avoid prolonged exposure to harmfully low temperatures. However, the molecular basis of noxious cold sensing in mammals is still not completely understood. Here, we show that the voltage-gated Nav1.9 sodium channel is important for the perception of pain in response to noxious cold. Nav1.9 activity is upregulated in a subpopulation of damage-sensing sensory neurons responding to cooling, which allows the channel to amplify subthreshold depolarizations generated by the activation of cold transducers. Consequently, cold-triggered firing is impaired in Nav1.9(-/-) neurons, and Nav1.9 null mice and knockdown rats show increased cold pain thresholds. Disrupting Nav1.9 expression in rodents also alleviates cold pain hypersensitivity induced by the antineoplastic agent oxaliplatin. We conclude that Nav1.9 acts as a subthreshold amplifier in cold-sensitive nociceptive neurons and is required for the perception of cold pain under normal and pathological conditions.

Cannabinoid-induced actomyosin contractility shapes neuronal morphology and growth.

Endocannabinoids are recently recognized regulators of brain development, but molecular effectors downstream of type-1 cannabinoid receptor (CB1R)-activation remain incompletely understood. We report atypical coupling of neuronal CB1Rs, after activation by endo- or exocannabinoids such as the marijuana component ∆(9)-tetrahydrocannabinol, to heterotrimeric G12/G13 proteins that triggers rapid and reversible non-muscle myosin II (NM II) dependent contraction of the actomyosin cytoskeleton, through a Rho-GTPase and Rho-associated kinase (ROCK). This induces rapid neuronal remodeling, such as retraction of neurites and axonal growth cones, elevated neuronal rigidity, and reshaping of somatodendritic morphology. Chronic pharmacological inhibition of NM II prevents cannabinoid-induced reduction of dendritic development in vitro and leads, similarly to blockade of endocannabinoid action, to excessive growth of corticofugal axons into the sub-ventricular zone in vivo. Our results suggest that CB1R can rapidly transform the neuronal cytoskeleton through actomyosin contractility, resulting in cellular remodeling events ultimately able to affect the brain architecture and wiring.

Bilateral lesions of the nigrostriatal pathways are associated with chronic mechanical pain hypersensitivity in rats.

Some patients with Parkinson's disease (PD) suffer from pain before motor symptoms. To model this, we proposed to assess the mechanical hypersensitivity in an animal model of PD with a bilateral lesion of the nigrostriatal pathway. PD model was validated by a decrease of locomotor activity and a 76% dopamine cell loss in the substantia nigra pars compacta. PD animals displayed a decrease of mechanical thresholds, correlated with the degree of the dopamine lesion. This animal model displays nociceptive disorders as found in PD patients and could be useful to assess the effects of new antiparkinsonian drugs.

A polyamine-deficient diet prevents oxaliplatin-induced acute cold and mechanical hypersensitivity in rats.

BACKGROUND: Oxaliplatin is an anticancer drug used for the treatment of advanced colorectal cancer, but it can also cause painful peripheral neuropathies. The pathophysiology of these neuropathies has not been yet fully elucidated, but may involve spinal N-methyl-D-aspartate (NMDA) receptors, particularly the NR2B subunit. As polyamines are positive modulators of NMDA-NR2B receptors and mainly originate from dietary intake, the modulation of polyamines intake could represent an interesting way to prevent/modulate neuropathic pain symptoms by opposing glutamate neurotransmission. METHODS: The effect of a polyamine deficient diet was investigated in an animal model of oxaliplatin-induced acute pain hypersensitivity using behavioral tests (mechanical and cold hypersensitivity). The involvement of spinal glutamate neurotransmission was monitored by using a proton nuclear magnetic resonance spectroscopy based metabolomic approach and by assessing the expression and phosphorylation of the NR2B subunit of the NMDA receptor. RESULTS: A 7-day polyamine deficient diet totally prevented oxaliplatin-induced acute cold hypersensitivity and mechanical allodynia. Oxaliplatin-induced pain hypersensitivity was not associated with an increase in NR2B subunit expression or phosphorylation, but with an increase of glutamate level in the spinal dorsal horn which was completely prevented by a polyamine deficient diet. As a validation that the oxaliplatin-induced hypersensitivity could be due to an increased activity of the spinal glutamate system, an intrathecal administration of the specific NR2B antagonist, ifenprodil, totally reversed oxaliplatin-induced mechanical and cold hypersensitivity. CONCLUSION: A polyamine deficient diet could represent a promising and valuable nutritional therapy to prevent oxaliplatin-induced acute pain hypersensitivity.

Chemotherapy-induced peripheral neuropathies: from clinical relevance to preclinical evidence.

INTRODUCTION: Chemotherapy-induced peripheral neuropathies (CIPN) are major dose-limiting side effects of many anticancer drugs. The incidence of CIPN varies from 10 to 100% depending on the anticancer drug. The characteristics of CIPN are related to dose intensity, cumulative dose and anticancer drug. CIPN can profoundly affect the quality-of-life, often compelling clinicians to lower the chemotherapy regimen, consequently limiting therapeutic efficacy. AREAS COVERED: Relevant literature in the field is identified through a Medline search for articles published up to August 2010 with the keywords 'neuropathy', 'anticancer drugs' and 'pain'. This study considers original papers and reviews. EXPERT OPINION: Neurotoxic anticancer drugs can affect specific peripheral nervous system structures (neuronopathy, axonopathy or myelinopathy) leading to CIPN, often with pain. Gaining deeper insights into neurotoxic mechanisms is critical to the development of new CIPN treatment and prevention strategies.