Approach for Electrophysiological Studies of Spinal Lamina X Neurons.

Despite playing diverse physiological roles, the area surrounding the central canal, lamina X, remains one of the least studied spinal cord regions. Technical challenges and limitations of the commonly used experimental approaches are the main difficulties that hamper lamina X research. In the current protocol, we describe a reliable method for functional investigation of lamina X neurons that requires neither time-consuming slicing nor sophisticated in vivo experiments.

Mitochondrial malfunction mediates impaired cholinergic Ca signalling and submandibular salivary gland dysfunction in diabetes.

Xerostomia (dry-mouth syndrome) is a painful and debilitating condition that frequently occurs in individuals with diabetes and is associated with impaired saliva production and salivary gland hypofunction. Saliva fluid production relies on Ca-coupled secretion driven by neurotransmitter stimulation of submandibular acinar cells. Although impairments in intracellular Ca signalling have been reported in various xerostomia models, the specific Ca-dependent mechanisms underlying saliva fluid hypofunction in diabetes remain unclear.

Diabetes-Induced Amplification of Nociceptive DRG Neuron Output by Upregulation of Somatic T-Type Ca Channels.

The development of pain symptoms in peripheral diabetic neuropathy (PDN) is associated with the upregulation of T-type Ca channels (T-channels) in the soma of nociceptive DRG neurons. Moreover, a block of these channels in DRG neurons effectively reversed mechanical and thermal hyperalgesia in animal diabetic models, indicating that T-channel functioning in these neurons is causally linked to PDN. However, no particular mechanisms relating the upregulation of T-channels in the soma of nociceptive DRG neurons to the pathological pain processing in PDN have been suggested.

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.

Ca-Permeable AMPA Receptors Contribute to Changed Dorsal Horn Neuronal Firing and Inflammatory Pain.

The dorsal horn (DH) neurons of the spinal cord play a critical role in nociceptive input integration and processing in the central nervous system. Engaged neuronal classes and cell-specific excitability shape nociceptive computation within the DH. The DH hyperexcitability (central sensitisation) has been considered a fundamental mechanism in mediating nociceptive hypersensitivity, with the proven role of Ca-permeable AMPA receptors (AMPARs).

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 Aβ/δ-fibers.

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.

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

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.

Peripheral Inflammation Results in Increased Excitability of Capsaicin-Insensitive Nociceptive DRG Neurons Mediated by Upregulation of ASICs and Voltage-Gated Ion Channels.

Previously, we have characterized the capsaicin-insensitive low pH-sensitive (capslpH) subtype of small-sized nociceptive dorsal root ganglion (DRG) neurons that express acid-sensing ion channels, T-type Ca channels, and have isolectin B4-negative phenotype. These neurons demonstrated increased excitability in a model of long-term diabetes, contributing to chronic pain sensation. Here we studied changes in the excitability of the capslpH neurons and underlying changes in the functional expression and gating properties of ion channels under complete Freund's adjuvant (CFA)-induced peripheral inflammation.

Spinal AMPA receptors: Amenable players in central sensitization for chronic pain therapy?

The activity-dependent trafficking of AMPA receptors (AMPAR) mediates synaptic strength and plasticity, while the perturbed trafficking of the receptors of different subunit compositions has been linked to memory impairment and to causing neuropathology. In the spinal cord, nociceptive-induced changes in AMPAR trafficking determine the central sensitization of the dorsal horn (DH): changes in AMPAR subunit composition compromise the balance between synaptic excitation and inhibition, rendering interneurons hyperexcitable to afferent inputs, and promoting Ca influx into the DH neurons, thereby amplifying neuronal hyperexcitability. The DH circuits become over-excitable and carry out aberrant sensory processing; this causes an increase in pain sensation in central sensory pathways, giving rise to chronic pain syndrome.

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.

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

Upregulation of Ca-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).

Nano-engineered microcapsules boost the treatment of persistent pain.

Persistent pain remains a major health issue: common treatments relying on either repeated local injections or systemic drug administration are prone to concomitant side-effects. It is thought that an alternative could be a multifunctional cargo system to deliver medicine to the target site and release it over a prolonged time window. We nano-engineered microcapsules equipped with adjustable cargo release properties and encapsulated the sodium-channel blocker QX-314 using the layer-by-layer (LbL) technology.

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

Functional Characterization of Lamina X Neurons in 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 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.

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.

Optimized Model of Cerebral Ischemia 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.

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.

Stable, synthetic analogs of diadenosine tetraphosphate inhibit rat and human P2X3 receptors and inflammatory pain.

Background: A growing body of evidence suggests that ATP-gated P2X3 receptors (P2X3Rs) are implicated in chronic pain. We address the possibility that stable, synthetic analogs of diadenosine tetraphosphate (Ap4A) might induce antinociceptive effects by inhibiting P2X3Rs in peripheral sensory neurons.

Results: The effects of two stable, synthetic Ap4A analogs (AppNHppA and AppCH2ppA) are studied firstly in vitro on HEK293 cells expressing recombinant rat P2XRs (P2X2Rs, P2X3Rs, P2X4Rs, and P2X7Rs) and then using native rat brain cells (cultured trigeminal, nodose, or dorsal root ganglion neurons).

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.

HIF-1α-mediated upregulation of SERCA2b: The endogenous mechanism for alleviating the ischemia-induced intracellular Ca(2+) store dysfunction in CA1 and CA3 hippocampal neurons.

Pyramidal neurons of the hippocampus possess differential susceptibility to the ischemia-induced damage with the highest vulnerability of CA1 and the lower sensitivity of CA3 neurons. This damage is triggered by Ca(2+)-dependent excitotoxicity and can result in a delayed cell death that might be potentially suspended through activation of endogenous neuroprotection with the hypoxia-inducible transcription factors (HIF). However, the molecular mechanisms of this neuroprotection remain poorly understood.

Upregulation of T-type Ca2+ channels in long-term diabetes determines increased excitability of a specific type of capsaicin-insensitive DRG neurons.

Background: Previous studies have shown that increased excitability of capsaicin-sensitive DRG neurons and thermal hyperalgesia in rats with short-term (2-4 weeks) streptozotocin-induced diabetes is mediated by upregulation of T-type Ca(2+) current. In longer-term diabetes (after the 8th week) thermal hyperalgesia is changed to hypoalgesia that is accompanied by downregulation of T-type current in capsaicin-sensitive small-sized nociceptors. At the same time pain symptoms of diabetic neuropathy other than thermal persist in STZ-diabetic animals and patients during progression of diabetes into later stages suggesting that other types of DRG neurons may be sensitized and contribute to 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.

Nociceptive neurons differentially express fast and slow T-type Ca²⁺ currents in different types of diabetic neuropathy.

T-type Ca²⁺ channels are known as important participants of nociception and their remodeling contributes to diabetes-induced alterations of pain sensation. In this work we have established that about 30% of rat nonpeptidergic thermal C-type nociceptive (NTCN) neurons of segments L4-L6 express a slow T-type Ca²⁺ current (T-current) while a fast T-current is expressed in the other 70% of these neurons. Streptozotocin-induced diabetes in young rats resulted in thermal hyperalgesia, hypoalgesia, or normalgesia 5-6 weeks after the induction.

Specific functioning of Cav3.2 T-type calcium and TRPV1 channels under different types of STZ-diabetic neuropathy.

Streptozotocin (STZ)-induced type 1 diabetes in rats leads to the development of peripheral diabetic neuropathy (PDN) manifested as thermal hyperalgesia at early stages (4th week) followed by hypoalgesia after 8weeks of diabetes development. Here we found that 6-7 week STZ-diabetic rats developed either thermal hyper- (18%), hypo- (25%) or normalgesic (57%) types of PDN. These developmentally similar diabetic rats were studied in order to analyze mechanisms potentially underlying different thermal nociception.