Nerve Preparation and Recordings for Pharmacological Tests of Sensory and Nociceptive Fiber Conduction Ex Vivo.

Measuring signal propagation through nerves is a classical electrophysiological technique established decades ago to evaluate sensory and motor functions in the nervous system. The whole-nerve preparation provides a valuable model to investigate nerve function ex vivo; however, it requires specific knowledge to ensure successful and stable measurements. Although the methodology for sciatic nerve recordings has long existed, a method for reliable and long-lasting recordings from myelinated and non-myelinated (nociceptive) fibers still needs to be adapted for pharmacological testing. This protocol takes benefits from epineurium sheath removal for pharmacological tests and provides a detailed description of how to make accurate nerve preparations, from the dissection and handling of nerves to epineurium cleaning, fabrication of adaptable suction electrodes for appropriate fiber stimulation and recordings, setting of electrophysiological protocols for compound action potential (CAP) recordings to distinguish between myelinated and non-myelinated (nociceptive) fibers, and finally to the analysis of the datasets of CAP components. We also demonstrate the feasibility of CAP recordings from individual branches in epineurium-free nerve preparations and provide clues to help retain nerve viability and maintain stable recordings over time. Although a sciatic nerve preparation was used here, the methodology can be applied to other nerve-type preparations. Key features • Detailed and simplified protocol for peripheral nerve preparation for recording sensory inputs ex vivo. • Recordings from myelinated and non-myelinated (nociceptive) fibers can be performed hours after nerve preparation. • The protocol involves the epineurium removal to facilitate drug permeability into nerve tissue for pharmacological tests. • The protocol allows physiological and pathological studies (pain/chronic pain conditions).

Neuropathic pain changes the output of rat lamina I spino-parabrachial neurons.

• Spared nerve injury (SNI) altered the action potential (AP) output of lamina I spino-parabrachial neurons (SPNs) without affecting their resting potential or membrane resistance. • In one-third of SPNs, high-threshold dorsal root stimulation elicited persistent AP firing which was never observed in cells from naïve animals. • 38% of SPNs from SNI rats showed spontaneous persistent AP firing. • After SNI low- and high-output SPNs were no longer nociceptive-specific as part of them responded with APs to low-threshold stimulation. • These SNI-induced changes of SPN output might represent cellular mechanisms for neuropathy-associated allodynia, hyperalgesia, and spontaneous pain.

A gain-of-function GRIA2 variant associated with neurodevelopmental delay and seizures: Functional characterization and targeted treatment.

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors (AMPARs) are ligand-gated cationic channels formed from combinations of GluA1-4 subunits. Pathogenic variants of GRIA1-4 have been described in patients with developmental delay, intellectual disability, autism spectrum disorder, and seizures, with GRIA2 variants typically causing AMPAR loss of function. Here, we identify a novel, heterozygous de novo pathogenic missense mutation in GRIA2 (c.1928 C>T, p.A643V, NM_001083619.1) in a 1-year-old boy with epilepsy, developmental delay, and failure to thrive. We made patch-clamp recordings to compare the functional and pharmacological properties of variant and wild-type receptors expressed in HEK293 cells, with and without the transmembrane AMPAR regulatory protein γ2. This showed GluA2 A643V-containing AMPARs to exhibit a novel gain of function, with greatly slowed deactivation, markedly reduced desensitization, and increased glutamate sensitivity. Perampanel, an antiseizure AMPAR negative allosteric modulator, was able to fully block GluA2 A643V/γ2 currents, suggesting potential therapeutic efficacy. The subsequent introduction of perampanel to the patient's treatment regimen was associated with a marked reduction in seizure burden, a resolution of failure to thrive, and clear developmental gains. Our study reveals that GRIA2 disorder can be caused by a gain-of-function variant, and both predicts and suggests the therapeutic efficacy of perampanel. Perampanel may prove beneficial for patients with other gain-of-function GRIA variants.

Phenotypes of Motor Deficit and Pain after Experimental Spinal Cord Injury.

Motor disability is a common outcome of spinal cord injury (SCI). The recovery of motor function after injury depends on the severity of neurotrauma; motor deficit can be reversible, at least partially, due to the innate tissue capability to recover, which, however, deteriorates with age. Pain is often a comorbidity of injury, although its prediction remains poor. It is largely unknown whether pain can attend motor dysfunction. Here, we implemented SCI for modelling severe and moderate neurotrauma and monitored SCI rats for up to 5 months post-injury to determine the profiles of both motor deficit and nociceptive sensitivity. Our data showed that motor dysfunction remained persistent after a moderate SCI in older animals (5-month-old); however, there were two populations among young SCI rats (1 month-old) whose motor deficit either declined or exacerbated even more over 4-5 weeks after identical injury. All young SCI rats displayed changed nociceptive sensitivity in thermal and mechanical modalities. The regression analysis of the changes revealed a population trend with respect to hyper- or hyposensitivity/motor deficit. Together, our data describe the phenotypes of motor deficit and pain, the two severe complications of neurotrauma. Our findings also suggest the predictability of motor dysfunction and pain syndromes following SCI that can be a hallmark for long-term rehabilitation and recovery after injury.

Precision spinal gene delivery-induced functional switch in nociceptive neurons reverses neuropathic pain.

Second-order spinal cord excitatory neurons play a key role in spinal processing and transmission of pain signals to the brain. Exogenously induced change in developmentally imprinted excitatory neurotransmitter phenotypes of these neurons to inhibitory has not yet been achieved. Here, we use a subpial dorsal horn-targeted delivery of AAV (adeno-associated virus) vector(s) encoding GABA (gamma-aminobutyric acid) synthesizing-releasing inhibitory machinery in mice with neuropathic pain. Treated animals showed a progressive and complete reversal of neuropathic pain (tactile and brush-evoked pain behavior) that persisted for a minimum of 2.5 months post-treatment. The mechanism of this treatment effect results from the switch of excitatory to preferential inhibitory neurotransmitter phenotype in dorsal horn nociceptive neurons and a resulting increase in inhibitory activity in regional spinal circuitry after peripheral nociceptive stimulation. No detectable side effects (e.g., sedation, motor weakness, loss of normal sensation) were seen between 2 and 13 months post-treatment in naive adult mice, pigs, and non-human primates. The use of this treatment approach may represent a potent and safe treatment modality in patients suffering from spinal cord or peripheral nerve injury-induced neuropathic pain.

Segmental and descending control of primary afferent input to the spinal lamina X.

ABSTRACT: Despite being involved in a number of functions, such as nociception and locomotion, spinal lamina X remains one of the least studied central nervous system regions. Here, we show that Aδ- and C-afferent inputs to lamina X neurons are presynaptically inhibited by homo- and heterosegmental afferents as well as by descending fibers from the corticospinal tract, dorsolateral funiculus, and anterior funiculus. Activation of descending tracts suppresses primary afferent-evoked action potentials and also elicits excitatory (mono- and polysynaptic) and inhibitory postsynaptic responses in lamina X neurons. Thus, primary afferent input to lamina X is subject to both spinal and supraspinal control being regulated by at least 5 distinct pathways.

Elucidating afferent-driven presynaptic inhibition of primary afferent input to spinal laminae I and X.

Although spinal processing of sensory information greatly relies on afferent-driven (AD) presynaptic inhibition (PI), our knowledge about how it shapes peripheral input to different types of nociceptive neurons remains insufficient. Here we examined the AD-PI of primary afferent input to spinal neurons in the marginal layer, lamina I, and the layer surrounding the central canal, lamina X; two nociceptive-processing regions with similar patterns of direct supply by Aδ- and C-afferents. Unmyelinated C-fibers were selectively activated by electrical stimuli of negative polarity that induced an anodal block of myelinated Aß/δ-fibers. Combining this approach with the patch-clamp recording in an ex vivo spinal cord preparation, we found that attenuation of the AD-PI by the anodal block of Aß/δ-fibers resulted in the appearance of new mono- and polysynaptic C-fiber-mediated excitatory postsynaptic current (EPSC) components. Such homosegmental Aß/δ-AD-PI affected neurons in the segment of the dorsal root entrance as well as in the adjacent rostral segment. In their turn, C-fibers from the L5 dorsal root induced heterosegmental AD-PI of the inputs from the L4 Aδ- and C-afferents to the neurons in the L4 segment. The heterosegmental C-AD-PI was reciprocal since the L4 C-afferents inhibited the L5 Aδ- and C-fiber inputs, as well as some direct L5 Aß-fiber inputs. Moreover, the C-AD-PI was found to control the spike discharge in spinal neurons. Given that the homosegmental Aß/δ-AD-PI and heterosegmental C-AD-PI affected a substantial percentage of lamina I and X neurons, we suggest that these basic mechanisms are important for shaping primary afferent input to the neurons in the spinal nociceptive-processing network.

High-threshold primary afferent supply of spinal lamina X neurons.

The spinal gray matter region around the central canal, lamina X, is critically involved in somatosensory processing and visceral nociception. Although several classes of primary afferent fibers terminate or decussate in this area, little is known about organization and functional significance of the afferent supply of lamina X neurons. Using the hemisected ex vivo spinal cord preparation, we show that virtually all lamina X neurons receive primary afferent inputs, which are predominantly mediated by the high-threshold Aδ- fibers and C-fibers. In two-thirds of the neurons tested, the inputs were monosynaptic, implying a direct targeting of the population of lamina X neurons by the primary nociceptors. Beside the excitatory inputs, 48% of the neurons also received polysynaptic inhibitory inputs. A complex pattern of interactions between the excitatory and inhibitory components determined the output properties of the neurons, one-third of which fired spikes in response to the nociceptive dorsal root stimulation. In this respect, the spinal gray matter region around the central canal is similar to the superficial dorsal horn, the major spinal nociceptive processing area. We conclude that lamina X neurons integrate direct and indirect inputs from several types of thin primary afferent fibers and play an important role in nociception.

Spinal PKCα inhibition and gene-silencing for pain relief: AMPAR trafficking at the synapses between primary afferents and sensory interneurons.

Upregulation of Ca2+-permeable AMPA receptors (CP-AMPARs) in dorsal horn (DH) neurons has been causally linked to persistent inflammatory pain. This upregulation, demonstrated for both synaptic and extrasynaptic AMPARs, depends on the protein kinase C alpha (PKCα) activation; hence, spinal PKC inhibition has alleviated peripheral nociceptive hypersensitivity. However, whether targeting the spinal PKCα would alleviate both pain development and maintenance has not been explored yet (essential to pharmacological translation). Similarly, if it could balance the upregulated postsynaptic CP-AMPARs also remains unknown. Here, we utilized pharmacological and genetic inhibition of spinal PKCα in various schemes of pain treatment in an animal model of long-lasting peripheral inflammation. Pharmacological inhibition (pre- or post-treatment) reduced the peripheral nociceptive hypersensitivity and accompanying locomotive deficit and anxiety in rats with induced inflammation. These effects were dose-dependent and observed for both pain development and maintenance. Gene-therapy (knockdown of PKCα) was also found to relieve inflammatory pain when applied as pre- or post-treatment. Moreover, the revealed therapeutic effects were accompanied with the declined upregulation of CP-AMPARs at the DH synapses between primary afferents and sensory interneurons. Our results provide a new focus on the mechanism-based pain treatment through interference with molecular mechanisms of AMPAR trafficking in central pain pathways.

Maturation of neural stem cells and integration into hippocampal circuits - a functional study in an in situ model of cerebral ischemia.

The hippocampus is the region of the brain that is most susceptible to ischemic lesion because it contains pyramidal neurons that are highly vulnerable to ischemic cell death. A restricted brain neurogenesis limits the possibility of reversing massive cell death after stroke and, hence, endorses cell-based therapies for neuronal replacement strategies following cerebral ischemia. Neurons differentiated from neural stem/progenitor cells (NSPCs) can mature and integrate into host circuitry, improving recovery after stroke. However, how the host environment regulates the NSPC behavior in post-ischemic tissue remains unknown. Here, we studied functional maturation of NSPCs in control and post-ischemic hippocampal tissue after modelling cerebral ischemia in situ We traced the maturation of electrophysiological properties and integration of the NSPC-derived neurons into the host circuits, with these cells developing appropriate activity 3 weeks or less after engraftment. In the tissue subjected to ischemia, the NSPC-derived neurons exhibited functional deficits, and differentiation of embryonic NSPCs to glial types - oligodendrocytes and astrocytes - was boosted. Our findings of the delayed neuronal maturation in post-ischemic conditions, while the NSPC differentiation was promoted towards glial cell types, provide new insights that could be applicable to stem cell therapy replacement strategies used after cerebral ischemia.

Functional Characterization of Lamina X Neurons in ex-Vivo Spinal Cord Preparation.

Functional properties of lamina X neurons in the spinal cord remain unknown despite the established role of this area for somatosensory integration, visceral nociception, autonomic regulation and motoneuron output modulation. Investigations of neuronal functioning in the lamina X have been hampered by technical challenges. Here we introduce an ex-vivo spinal cord preparation with both dorsal and ventral roots still attached for functional studies of the lamina X neurons and their connectivity using an oblique LED illumination for resolved visualization of lamina X neurons in a thick tissue. With the elaborated approach, we demonstrate electrophysiological characteristics of lamina X neurons by their membrane properties, firing pattern discharge and fiber innervation (either afferent or efferent). The tissue preparation has been also probed using Ca2+ imaging with fluorescent Ca2+ dyes (membrane-impermeable or -permeable) to demonstrate the depolarization-induced changes in intracellular calcium concentration in lamina X neurons. Finally, we performed visualization of subpopulations of lamina X neurons stained by retrograde labeling with aminostilbamidine dye to identify sympathetic preganglionic and projection neurons in the lamina X. Thus, the elaborated approach provides a reliable tool for investigation of functional properties and connectivity in specific neuronal subpopulations, boosting research of lamina X of the spinal cord.

Atlanto-occipital catheterization of young rats for long-term drug delivery into the lumbar subarachnoid space combined with in vivo testing and electrophysiology in situ.

BACKGROUND: Catheterization has been widely used in neuroscience and pain research for local drug delivery. Though different modifications were developed, the use of young animals for spinal catheterization remains limited because of a little success rate. A reliable technique is needed to catheterize young animals aimed for in vivo testing combined with spinal cord electrophysiology, often limited by animal age, to facilitate pain research. NEW METHODS: We describe intrathecal catheterization of young rats (3-week-old) through atlanto-occipical approach for long-lasting drug delivery into the lumbar subarachnoid space. The technique represents a surgical approach of minimized invasiveness that requires PE-10 catheter and few equipment of standard laboratory use. RESULTS: Behavioral assessments revealed that spinal catheterization does not change peripheral sensitivity of different modalities (thermal and mechanical) and gives no rise to locomotive deficit or anxiety-like behavior in young rats. The long-term administration of genetic material (oligodeoxynucleotides given up to 4days), examined both in vivo and in situ, produced no adverse effects on basal peripheral sensitivity, but changed the AMPA receptor-mediated currents in sensory interneurons of the spinal cord. COMPARISON WITH EXISTING METHODS: Dissimilar to already described methods, the method is designed for the use of young rats for behavioral testing in vivo and/or spinal cord electrophysiology in situ. CONCLUSIONS: A practical method for spinal catheterization of young animals designed for studies in vivo and in situ is proposed. The method is rapid and effective and should facilitate investigation of therapeutic effects on both systemic and subcellular levels, as an advantage over the existing methods.

Opposite, bidirectional shifts in excitation and inhibition in specific types of dorsal horn interneurons are associated with spasticity and pain post-SCI.

Spasticity, a common complication after spinal cord injury (SCI), is frequently accompanied by chronic pain. The physiological origin of this pain (critical to its treatment) remains unknown, although spastic motor dysfunction has been related to the hyperexcitability of motoneurons and to changes in spinal sensory processing. Here we show that the pain mechanism involves changes in sensory circuits of the dorsal horn (DH) where nociceptive inputs integrate for pain processing. Spasticity is associated with the DH hyperexcitability resulting from an increase in excitation and disinhibition occurring in two respective types of sensory interneurons. In the tonic-firing inhibitory lamina II interneurons, glutamatergic drive was reduced while glycinergic inhibition was potentiated. In contrast, excitatory drive was boosted to the adapting-firing excitatory lamina II interneurons while GABAergic and glycinergic inhibition were reduced. Thus, increased activity of excitatory DH interneurons coupled with the reduced excitability of inhibitory DH interneurons post-SCI could provide a neurophysiological mechanism of central sensitization and chronic pain associated with spasticity.

Optimized Model of Cerebral Ischemia In situ for the Long-Lasting Assessment of Hippocampal Cell Death.

Among all the brain, the hippocampus is the most susceptible region to ischemic lesion, with the highest vulnerability of CA1 pyramidal neurons to ischemic damage. This damage may cause either prompt neuronal death (within hours) or with a delayed appearance (over days), providing a window for applying potential therapies to reduce or prevent ischemic impairments. However, the time course when ischemic damage turns to neuronal death strictly depends on experimental modeling of cerebral ischemia and, up to now, studies were predominantly focused on a short time-window-from hours to up to a few days post-lesion. Using different schemes of oxygen-glucose deprivation (OGD), the conditions taking place upon cerebral ischemia, we optimized a model of mimicking ischemic conditions in organotypical hippocampal slices for the long-lasting assessment of CA1 neuronal death (at least 3 weeks). By combining morphology and electrophysiology, we show that prolonged (30-min duration) OGD results in a massive neuronal death and overwhelmed astrogliosis within a week post-OGD whereas OGD of a shorter duration (10-min) triggered programmed CA1 neuronal death with a significant delay-within 2 weeks-accompanied with drastically impaired CA1 neuron functions. Our results provide a rationale toward optimized modeling of cerebral ischemia for reliable examination of potential treatments for brain neuroprotection, neuro-regeneration, or testing neuroprotective compounds in situ.

Inhibition of Spinal Ca(2+)-Permeable AMPA Receptors with Dicationic Compounds Alleviates Persistent Inflammatory Pain without Adverse Effects.

Upregulation of Ca(2+)-permeable AMPA receptors (CP-AMPARs) in the dorsal horn (DH) neurons of the spinal cord has been causally linked to the maintenance of persistent inflammatory pain. Therefore, inhibition of CP-AMPARs could potentially alleviate an, otherwise, poorly treatable chronic pain. However, a loss of CP-AMPARs could produce considerable side effects because of the crucial role of CP-AMPARs in synaptic plasticity. Here we have tested whether the inhibition of spinal CP-AMPARs with dicationic compounds, the open-channel antagonists acting in an activity-dependent manner, can relieve inflammatory pain without adverse effects being developed. Dicationic compounds, N1-(1-phenylcyclohexyl)pentane-1,5-diaminium bromide (IEM-1925) and 1-trimethylammonio-5-1-adamantane-methyl-ammoniopentane dibromide (IEM-1460) were applied intrathecally (i.t.) as a post-treatment for inflammatory pain in the model of complete Freund's adjuvant (CFA)-induced long-lasting peripheral inflammation. The capability of dicationic compounds to ameliorate inflammatory pain was tested in rats in vivo using the Hargreaves, the von Frey and the open-field tests. Treatment with IEM-1460 or IEM-1925 resulted in profound alleviation of inflammatory pain. The pain relief appeared shortly after compound administration. The effects were concentration-dependent, displaying a high potency of dicationic compounds for alleviation of inflammatory hyperalgesia in the micromolar range, for both acute and long-lasting responses. The period of pain maintenance was shortened following treatment. Treatment with IEM-1460 or IEM-1925 changed neither thermal and mechanical basal sensitivities nor animal locomotion, suggesting that inhibition of CP-AMPARs with dicationic compounds does not give rise to detectable side effects. Thus, the ability of dicationic compounds to alleviate persistent inflammatory pain may provide new routes in the treatment of chronic pain.

Inflammatory-induced changes in synaptic drive and postsynaptic AMPARs in lamina II dorsal horn neurons are cell-type specific.

Persistent peripheral inflammation alters trafficking of AMPA receptors (AMPARs) at the synapses between primary afferents and dorsal horn (DH) neurons that contribute to the maintenance of inflammatory pain. However, whether peripheral inflammation changes the synaptic activity within the DH circuitry and how it modulates synaptic AMPARs in different neuronal types still remain unknown. We find that complete Freund adjuvant (CFA)-induced peripheral inflammation prominently augments excitatory neurotransmission in rat lamina II neurons characterized by intrinsic adapting firing properties and apparently decreases it in the tonic firing lamina II neurons, suggesting different roles of these types of interneurons in pain processing. Peripheral inflammation also differentially changes inhibitory neurotransmission in these neuronal types, shifting the balance between neuronal excitation and inhibition toward excitation of the adapting firing, but toward inhibition of the tonic firing lamina II neurons. Synaptic AMPARs were differentially changed in the adapting firing and the tonic firing neurons, implying different mechanisms of AMPAR adjustment at the synapses in these types of interneurons during persistent inflammation. The inflammatory-induced, neuron-type specific changes in synaptic drive within the DH circuitry and synaptic AMPAR functioning in lamina II neurons may contribute to the persistent pain maintenance.