GABAB Receptors Tonically Inhibit Motoneurons and Neurotransmitter Release from Descending and Primary Afferent Fibers.

Motoneurons receive thousands of excitatory and inhibitory synapses from descending tracts and primary afferent fibers. The excitability of these neurons must be precisely regulated to respond adequately to the requirements of the environment. In this context, GABAA and GABAB receptors regulate motoneuron synaptic strength. GABAA and GABAB receptors are expressed on primary afferent fibers and motoneurons, while in the descending afferent fibers, only the GABAB receptors are expressed. However, it remains to be known where the GABA that activates them comes from since the GABAergic interneurons that make axo-axonic contacts with primary afferents have yet to be identified in the descending afferent terminals. Thus, the main aim of the present report was to investigate how GABAB receptors functionally modulate synaptic strength between Ia afferent fibers, excitatory and inhibitory descending fibers of the dorsolateral funiculus, and spinal motoneurons. Using intracellular recordings from the spinal cord of the turtle, we provide evidence that the GABAB receptor antagonist, CGP55845, not only prevents baclofen-induced depression of EPSPs but also increases motoneuron excitability and enhances the synaptic strength between the afferent fibers and motoneurons. The last action of CGP55845 was similar in excitatory and inhibitory descending afferents. Interestingly, the action of baclofen was more intense in the Ia primary afferents than in the descending afferents. Even more, CGP55845 reversed the EPSP depression induced by the increased concentration of ambient GABA produced by interneuron activation and GABA transporter blockade. Immunofluorescence data corroborated the expression of GABAB receptors in the turtle's spinal cord. These findings suggest that GABAB receptors are extrasynaptic and tonically activated on descending afferent fibers and motoneurons by GABA released from astrocytes and GABAergic interneurons in the cellular microenvironment. Finally, our results also suggest that the antispastic action of baclofen may be due to reduced synaptic strength between descending fibers and motoneurons.

Activation of α 6 -containing GABA A receptors induces antinociception under physiological and pathological conditions.

ABSTRACT: The loss of GABAergic inhibition is a mechanism that underlies neuropathic pain. Therefore, rescuing the GABAergic inhibitory tone through the activation of GABA A receptors is a strategy to reduce neuropathic pain. This study was designed to elucidate the function of the spinal α 6 -containing GABA A receptor in physiological conditions and neuropathic pain in female and male rats. Results show that α 6 -containing GABA A receptor blockade or transient α 6 -containing GABA A receptor knockdown induces evoked hypersensitivity and spontaneous pain in naive female rats. The α 6 subunit is expressed in IB4 + and CGRP + primary afferent neurons in the rat spinal dorsal horn and dorsal root ganglia but not astrocytes. Nerve injury reduces α 6 subunit protein expression in the central terminals of the primary afferent neurons and dorsal root ganglia, whereas intrathecal administration of positive allosteric modulators of the α 6 -containing GABA A receptor reduces tactile allodynia and spontaneous nociceptive behaviors in female, but not male, neuropathic rats and mice. Overexpression of the spinal α 6 subunit reduces tactile allodynia and restores α 6 subunit expression in neuropathic rats. Positive allosteric modulators of the α 6 -containing GABA A receptor induces a greater antiallodynic effect in female rats and mice compared with male rats and mice. Finally, α 6 subunit is expressed in humans. This receptor is found in CGRP + and P2X3 + primary afferent fibers but not astrocytes in the human spinal dorsal horn. Our results suggest that the spinal α 6 -containing GABA A receptor has a sex-specific antinociceptive role in neuropathic pain, suggesting that this receptor may represent an interesting target to develop a novel treatment for neuropathic pain.

Role of the Ca2+ channel α2δ-1 auxiliary subunit in proliferation and migration of human glioblastoma cells.

The overexpression of α2δ-1 is related to the development and degree of malignancy of diverse types of cancer. This protein is an auxiliary subunit of voltage-gated Ca2+ (CaV) channels, whose expression favors the trafficking of the main pore-forming subunit of the channel complex (α1) to the plasma membrane, thereby generating an increase in Ca2+ entry. Interestingly, TLR-4, a protein belonging to the family of toll-like receptors that participate in the inflammatory response and the transcription factor Sp1, have been linked to the progression of glioblastoma multiforme (GBM). Therefore, this report aimed to evaluate the role of the α2δ-1 subunit in the progression of GBM and investigate whether Sp1 regulates its expression after the activation of TLR-4. To this end, the expression of α2δ-1, TLR-4, and Sp1 was assessed in the U87 human glioblastoma cell line, and proliferation and migration assays were conducted using different agonists and antagonists. The actions of α2δ-1 were also investigated using overexpression and knockdown strategies. Initial luciferase assays and Western blot analyses showed that the activation of TLR-4 favors the transcription and expression of α2δ-1, which promoted the proliferation and migration of the U87 cells. Consistent with this, overexpression of α2δ-1, Sp1, and TLR-4 increased cell proliferation and migration, while their knockdown with specific siRNAs abrogated these actions. Our data also suggest that TLR-4-mediated regulation of α2δ-1 expression occurs through the NF-kB signaling pathway. Together, these findings strongly suggest that the activation of TLR-4 increases the expression of α2δ-1 in U87 cells, favoring their proliferative and migratory potential, which might eventually provide a theoretical basis to examine novel biomarkers and molecular targets for the diagnosis and treatment of GBM.

Impaired rate-dependent depression of the H-reflex in type-2 diabetes, prediabetes, overweight and obesity: A cross-sectional study.

Type-2 diabetes is a chronic metabolic disorder characterized by hyperglycemia, resulting from deficits in insulin secretion or insulin resistance. According to the International Diabetes Federation, approximately 463 million people suffered from this condition in 2019, with a rapidly increasing impact in low-and middle-income countries. Obesity is a well-known risk factor for diabetes, and current data project a continuous increase in diabetes prevalence worldwide in obese individuals. Among the common complications, diabetic peripheral neuropathy (DPN) causes sensory symptoms, including pain that contributes to foot ulceration, and if not controlled, limb amputation may occur. The diagnosis of DPN is a clinical problem. Rate-dependent depression (RDD) of the Hoffmann reflex in the lower limbs has been proposed as a test to determine the presence of neuropathic pain in subjects with type-1 and type-2 diabetes. Recently, impaired RDD has been described in obese and diabetic rodent models. In this study, we characterized the RDD by evaluating the H-reflex at 0.2, 1, 2, 5, and 10 Hz in 39 patients with type-2 Diabetes mellitus (T2DM) and 42 controls without diabetes, subsequently classified as overweight/obese and prediabetic. A significant decrease in the RDD of the H-reflex was found in T2DM subjects at 1, 2, 5, and 10 Hz (P < .001) stimulation frequencies compared to controls, but not at 0.2 Hz (P = .48). A major finding of this study is that impaired RDD was also found in 11/25 overweight and obese subjects in at least 2 stimulation frequencies, being 10 of those classified in prediabetic levels according to their HbA1C values. The RDD of the H-reflex could be used as a quantitative and sensitive tool to study T2DM subpopulations with peripheral neuropathy. RDD could be used as a screening tool in combination with clinical tests to diagnose DPN and evaluate the progression of this condition.

Hypothalamic A11 Nuclei Regulate the Circadian Rhythm of Spinal Mechanonociception through Dopamine Receptors and Clock Gene Expression.

Several types of sensory perception have circadian rhythms. The spinal cord can be considered a center for controlling circadian rhythms by changing clock gene expression. However, to date, it is not known if mechanonociception itself has a circadian rhythm. The hypothalamic A11 area represents the primary source of dopamine (DA) in the spinal cord and has been found to be involved in clock gene expression and circadian rhythmicity. Here, we investigate if the paw withdrawal threshold (PWT) has a circadian rhythm, as well as the role of the dopaminergic A11 nucleus, DA, and DA receptors (DR) in the PWT circadian rhythm and if they modify clock gene expression in the lumbar spinal cord. Naïve rats showed a circadian rhythm of the PWT of almost 24 h, beginning during the night-day interphase and peaking at 14.63 h. Similarly, DA and DOPAC's spinal contents increased at dusk and reached their maximum contents at noon. The injection of 6-hydroxydopamine (6-OHDA) into the A11 nucleus completely abolished the circadian rhythm of the PWT, reduced DA tissue content in the lumbar spinal cord, and induced tactile allodynia. Likewise, the repeated intrathecal administration of D1-like and D2-like DA receptor antagonists blunted the circadian rhythm of PWT. 6-OHDA reduced the expression of Clock and Per1 and increased Per2 gene expression during the day. In contrast, 6-OHDA diminished Clock, Bmal, Per1, Per2, Per3, Cry1, and Cry2 at night. The repeated intrathecal administration of the D1-like antagonist (SCH-23390) reduced clock genes throughout the day (Clock and Per2) and throughout the night (Clock, Per2 and Cry1), whereas it increased Bmal and Per1 throughout the day. In contrast, the intrathecal injection of the D2 receptor antagonists (L-741,626) increased the clock genes Bmal, Per2, and Per3 and decreased Per1 throughout the day. This study provides evidence that the circadian rhythm of the PWT results from the descending dopaminergic modulation of spinal clock genes induced by the differential activation of spinal DR.

The role of spinal cord extrasynaptic α5 GABAA receptors in chronic pain.

Chronic pain is an incapacitating condition that affects a large population worldwide. Until now, there is no drug treatment to relieve it. The impairment of GABAergic inhibition mediated by GABAA receptors (GABAA R) is considered a relevant factor in mediating chronic pain. Even though both synaptic and extrasynaptic GABAA inhibition are present in neurons that process nociceptive information, the latter is not considered relevant as a target for the development of pain treatments. In particular, the extrasynaptic α5 GABAA Rs are expressed in laminae I-II of the spinal cord neurons, sensory neurons, and motoneurons. In this review, we discuss evidence showing that blockade of the extrasynaptic α5 GABAA Rs reduces mechanical allodynia in various models of chronic pain and restores the associated loss of rate-dependent depression of the Hoffmann reflex. Furthermore, in healthy animals, extrasynaptic α5 GABAA R blockade induces both allodynia and hyperalgesia. These results indicate that this receptor may have an antinociceptive and pronociceptive role in healthy and chronic pain-affected animals, respectively. We propose a hypothesis to explain the relevant role of the extrasynaptic α5 GABAA Rs in the processing of nociceptive information. The data discussed here strongly suggest that this receptor could be a valid pharmacological target to treat chronic pain states.

L5-6 Spinal Nerve Ligation-induced Neuropathy Changes the Location and Function of Ca2+ Channels and Cdk5 and Affects the Compound Action Potential in Adjacent Intact L4 Afferent Fibers.

Voltage-gated Ca2+ (CaV) channels regulate multiple cell processes, including neurotransmitter release, and have been associated with several pathological conditions, such as neuropathic pain. Cdk5, a neuron-specific kinase, may phosphorylate CaV channels, altering their functional expression. During peripheral nerve injury, upregulation of CaV channels and Cdk5 in the dorsal root ganglia (DRG) and the spinal cord, has been correlated with allodynia. We recently reported an increase in the amplitude of the C component of the compound action potential (cAP) of afferent fibers in animals with allodynia induced by L5-6 spinal nerve ligation (SNL), recorded in the corresponding dorsal roots. This was related to an increase in T-type (CaV3.2) channels generated by Cdk5-mediated phosphorylation. Here, we show that CaV channel functional expression is also altered in the L4 adjacent intact afferent fibers in rats with allodynia induced by L5-6 SNL. Western blot analysis showed that both Cdk5 and CaV3.2 total levels are not increased in the DRG L3-4, but their subcellular distribution changes by concentrating on the neuronal soma. Likewise, the Cdk5 inhibitor olomoucine affected the rapid and the slow C components of the cAP recorded in the dorsal roots. Patch-clamp recordings revealed an increase in T- and N-type currents recorded in the soma of acute isolated L3-4 sensory neurons after L5-6 SNL, which was prevented by olomoucine. These findings suggest changes in CaV channels location and function in L3-4 afferent fibers associated with Cdk5-mediated phosphorylation after L5-6 SNL, which may contribute to nerve injury-induced allodynia.

Sex-dependent pronociceptive role of spinal α5 -GABAA receptor and its epigenetic regulation in neuropathic rodents.

Extrasynaptic α5 -subunit containing GABAA (α5 -GABAA ) receptors participate in chronic pain. Previously, we reported a sex difference in the action of α5 -GABAA receptors in dysfunctional pain. However, the underlying mechanisms remain unknown. The aim of this study was to examine this sexual dimorphism in neuropathic rodents and the mechanisms involved. Female and male Wistar rats or ICR mice were subjected to nerve injury followed by α5 -GABAA receptor inverse agonist intrathecal administration, L-655,708. The drug produced an antiallodynic effect in nerve-injured female rats and mice, and a lower effect in males. We hypothesized that changes in α5 -GABAA receptor, probably influenced by hormonal and epigenetic status, might underlie this sex difference. Thus, we performed qPCR and western blot. Nerve injury increased α5 -GABAA mRNA and protein in female dorsal root ganglia (DRG) and decreased them in DRG and spinal cord of males. To investigate the hormonal influence over α5 -GABAA receptor actions, we performed nerve injury to ovariectomized rats and reconstituted them with 17ß-estradiol (E2). Ovariectomy abrogated L-655,708 antiallodynic effect and E2 restored it. Ovariectomy decreased α5 -GABAA receptor and estrogen receptor α protein in DRG of neuropathic female rats, while E2 enhanced them. Since DNA methylation might contribute to α5 -GABAA receptor down-regulation in males, we examined CpG island DNA methylation of α5 -GABAA receptor coding gene through pyrosequencing. Nerve injury increased methylation in male, but not female rats. Pharmacological inhibition of DNA methyltransferases increased α5 -GABAA receptor and enabled L-655,708 antinociceptive effect in male rats. These results suggest that α5 -GABAA receptor is a suitable target to treat chronic pain in females.

γ-Aminobutyric acid (GABA) from satellite glial cells tonically depresses the excitability of primary afferent fibers.

Primary afferent fibers express extrasynaptic GABAA and GABAB receptors in the axons and soma. However, whether these receptors are tonically activated by ambient GABA and the source of the neurotransmitter is presently unknown. Here, we show that GABA release from dorsal root ganglia (DRG) does not depend on extracellular calcium, but depends upon calcium released from intracellular stores, and is mediated by Best1 channels. Using a preparation consisting of the spinal nerve in continuity with the DRG and the dorsal root, we found that endogenous GABA tonically activates GABA receptors, depressing the excitability of the primary afferents. In addition, using HPLC we found that GABA is released in the DRG, and by immunofluorescence microscopy we show the presence of GABA, the Best1 channel, and some enzymes of the putrescine pathway of GABA biosynthesis, in glutamine synthase- and GFAP-positive satellite glial cells. Last, we found that the blockade of the Best1 channel activity reduced the excitability of primary afferents and prevented the activation of the GABA receptors. These results suggest that satellite glial cells may be the source of endogenous GABA released in the DRG via Best1 channels, which tonically activates extrasynaptic GABA receptors.

The role of cyclin-dependent kinase 5 in neuropathic pain.

The chronification of pain can be attributed to changes in membrane receptors and channels underlying neuronal plasticity and signal transduction largely within nociceptive neurons that initiate and maintain pathological pain states. These proteins are subject to dynamic modification by posttranslational modifications, creating a code that controls protein function in time and space. Phosphorylation is an important posttranslational modification that affects ∼30% of proteins in vivo. Increased phosphorylation of various nociceptive ion channels and of their modulators underlies sensitization of different pain states. Cyclin-dependent kinases are proline-directed serine/threonine kinases that impact various biological and cellular systems. Cyclin-dependent kinase 5 (Cdk5), one member of this kinase family, and its activators p35 and p39 are expressed in spinal nerves, dorsal root ganglia, and the dorsal horn of the spinal cord. In neuropathic pain conditions, expression and/or activity of Cdk5 is increased, implicating Cdk5 in nociception. Experimental evidence suggests that Cdk5 is regulated through its own phosphorylation, through increasing p35's interaction with Cdk5, and through cleavage of p35 into p25. This narrative review discusses the molecular mechanisms of Cdk5-mediated regulation of target proteins involved in neuropathic pain. We focus on Cdk5 substrates that have been linked to nociceptive pathways, including channels (eg, transient receptor potential cation channel and voltage-gated calcium channel), proteins involved in neurotransmitter release (eg, synaptophysin and collapsin response mediator protein 2), and receptors (eg, glutamate, purinergic, and opioid). By altering the phosphoregulatory "set point" of proteins involved in pain signaling, Cdk5 thus appears to be an attractive target for treating neuropathic pain conditions.

Cdk5-Dependent Phosphorylation of CaV3.2 T-Type Channels: Possible Role in Nerve Ligation-Induced Neuropathic Allodynia and the Compound Action Potential in Primary Afferent C Fibers.

Voltage-gated T-type Ca2+ (CaV3) channels regulate diverse physiological events, including neuronal excitability, and have been linked to several pathological conditions such as absence epilepsy, cardiovascular diseases, and neuropathic pain. It is also acknowledged that calcium/calmodulin-dependent protein kinase II and protein kinases A and C regulate the activity of T-type channels. Interestingly, peripheral nerve injury induces tactile allodynia and upregulates CaV3.2 channels and cyclin-dependent kinase 5 (Cdk5) in dorsal root ganglia (DRG) and spinal dorsal horn. Here, we report that recombinant CaV3.2 channels expressed in HEK293 cells are regulatory targets of Cdk5. Site-directed mutagenesis showed that the relevant sites for this regulation are residues S561 and S1987. We also found that Cdk5 may regulate CaV3.2 channel functional expression in rats with mechanical allodynia induced by spinal nerve ligation (SNL). Consequently, the Cdk5 inhibitor olomoucine affected the compound action potential recorded in the spinal nerves, as well as the paw withdrawal threshold. Likewise, Cdk5 expression was upregulated after SNL in the DRG. These findings unveil a novel mechanism for how phosphorylation may regulate CaV3.2 channels and suggest that increased channel activity by Cdk5-mediated phosphorylation after SNL contributes nerve injury-induced tactile allodynia.SIGNIFICANCE STATEMENT Neuropathic pain is a current public health challenge. It can develop as a result of injury or nerve illness. It is acknowledged that the expression of various ion channels can be altered in neuropathic pain, including T-type Ca2+ channels that are expressed in sensory neurons, where they play a role in the regulation of cellular excitability. The present work shows that the exacerbated expression of Cdk5 in a preclinical model of neuropathic pain increases the functional expression of CaV3.2 channels. This finding is relevant for the understanding of the molecular pathophysiology of the disease. Additionally, this work may have a substantial translational impact, since it describes a novel molecular pathway that could represent an interesting therapeutic alternative for neuropathic pain.

Transcription Factor Sp1 Regulates the Expression of Calcium Channel α2δ-1 Subunit in Neuropathic Pain.

High voltage-activated (HVA) Ca2+ (CaV) channels are oligomeric complexes formed by an ion-conducting main subunit (Cavα1) and at least two auxiliary subunits (Cavß and CaVα2δ). It has been reported that the expression of CaVα2δ1 increases in the dorsal root ganglia (DRGs) of animals with mechanical allodynia, and that the transcription factor Sp1 regulates the expression of the auxiliary subunit. Hence, the main aim of this work was to investigate the role of Sp1 as a molecular determinant of the exacerbated expression of CaVα2δ-1 in the nerve ligation-induced model of mechanical allodynia. Our results show that ligation of L5/L6 spinal nerves (SNL) produced allodynia and increased the expression of Sp1 and CaVα2δ-1 in the DRGs. Interestingly, intrathecal administration of the Sp1 inhibitor mithramycin A (Mth) prevented allodynia and decreased the expression of Sp1 and CaVα2δ-1. Likewise, electrophysiological recordings showed that incubation with Mth decreased Ca2+ current density in the DRG neurons, acting mostly on HVA channels. These results suggest that L5/L6 SNL produces mechanical allodynia and increases the expression of the transcription factor Sp1 and the subunit CaVα2δ-1 in the DRGs, while Mth decreases mechanical allodynia and Ca2+ currents through HVA channels in sensory neurons by reducing the functional expression of the CaVα2δ-1 subunit.

Blockade of spinal α5-GABAA receptors differentially reduces reserpine-induced fibromyalgia-type pain in female rats.

The role of spinal α5 subunit-containing GABAA (α5-GABAA) receptors in chronic pain is controversial. The purpose of this study was to investigate the participation of spinal α5-GABAA receptors in the reserpine-induced pain model. Reserpine administration induced tactile allodynia and muscle hyperalgesia in female and male rats. Intrathecal injection of L-655,708 and TB 21007 (7 days after the last reserpine injection) decreased tactile allodynia and, at a lesser extent, muscle hyperalgesia in female rats. The effects of these drugs produced a lower antiallodynic and antihyperalgesic effect in male than in female rats. Contrariwise, these drugs produced tactile allodynia and muscle hyperalgesia in naïve rats and these effects were lower in naïve male than female rats. Intrathecal L-838,417 prevented or reversed L-655,708-induced antiallodynia in reserpine-treated female rats. Repeated treatment with α5-GABAA receptor small interfering RNA (siRNA), but not scramble siRNA, reduced reserpine-induced allodynia in female rats. Accordingly, α5-GABAA receptor siRNA induced nociceptive hypersensitivity in naïve female rats. Reserpine enhanced α5-GABAA receptors expression in spinal cord and dorsal root ganglia (DRG), while it increased CD11b (OX-42) and glial fibrillary acidic protein (GFAP) fluorescence intensity in the lumbar spinal cord. In contrast, reserpine diminished K+-Cl- co-transporter 2 (KCC2) protein in the lumbar spinal cord. Data suggest that spinal α5-GABAA receptors play a sex-dependent proallodynic effect in reserpine-treated rats. In contrast, these receptors have a sex-dependent antiallodynic role in naïve rats.

α5GABAA receptors play a pronociceptive role and avoid the rate-dependent depression of the Hoffmann reflex in diabetic neuropathic pain and reduce primary afferent excitability.

Diabetic neuropathy is an incapacitating complication in diabetic patients. The cellular and molecular mechanisms involved in this pathology are poorly understood. Previous studies have suggested that the loss of spinal GABAergic inhibition participate in painful diabetic neuropathy. However, the role of extrasynaptic α5 subunit-containing GABAA (α5GABAA) receptors in this process is not known. The purpose of this study was to investigate the role of α5GABAA receptors in diabetes-induced tactile allodynia, loss of rate-dependent depression (RDD) of the Hoffmann reflex (HR), and modulation of primary afferent excitability. Intraperitoneal administration of streptozotocin induced tactile allodynia. Intrathecal injection of α5GABAA receptor inverse agonist, L-655,708, produced tactile allodynia in naive rats, whereas it reduced allodynia in diabetic rats. In healthy rats, electrical stimulation of the tibial nerve at 5 Hz induced RDD of the HR, although intrathecal treatment with L-655,708 (15 nmol) abolished RDD of the HR. Streptozotocin induced the loss of RDD of the HR, while intrathecal L-655,708 (15 nmol) restored RDD of the HR. L-655,708 (15 nmol) increased tonic excitability of the primary afferents without affecting the phasic excitability produced by the primary afferent depolarization. α5GABAA receptors were immunolocalized in superficial laminae of the dorsal horn and L4 to L6 dorsal root ganglion. Streptozotocin increased mean fluorescence intensity and percentage of neurons expressing α5GABAA receptors in dorsal horn and L4 to L6 dorsal root ganglia in 10-week diabetic rats. Our results suggest that spinal α5GABAA receptors modulate the HR, play an antinociceptive and pronociceptive role in healthy and diabetic rats, respectively, and are tonically active in primary afferents.

Spinal dorsal horn astrocytes release GABA in response to synaptic activation.

KEY POINTS: GABA is an essential molecule for sensory information processing. It is usually assumed to be released by neurons. Here we show that in the dorsal horn of the spinal cord, astrocytes respond to glutamate by releasing GABA. Our findings suggest a novel role for astrocytes in somatosensory information processing. ABSTRACT: Astrocytes participate in neuronal signalling by releasing gliotransmitters in response to neurotransmitters. We investigated if astrocytes from the dorsal horn of the spinal cord of adult red-eared turtles (Trachemys scripta elegans) release GABA in response to glutamatergic receptor activation. For this, we developed a GABA sensor consisting of HEK cells expressing GABAA receptors. By positioning the sensor recorded in the whole-cell patch-clamp configuration within the dorsal horn of a spinal cord slice, we could detect GABA in the extracellular space. Puff application of glutamate induced GABA release events with time courses that exceeded the duration of inhibitory postsynaptic currents by one order of magnitude. Because the events were neither affected by extracellular addition of nickel, cadmium and tetrodotoxin nor by removal of Ca2+ , we concluded that they originated from non-neuronal cells. Immunohistochemical staining allowed the detection of GABA in a fraction of dorsal horn astrocytes. The selective stimulation of A∂ and C fibres in a dorsal root filament induced a Ca2+ increase in astrocytes loaded with Oregon Green BAPTA. Finally, chelating Ca2+ in a single astrocyte was sufficient to prevent the GABA release evoked by glutamate. Our results indicate that glutamate triggers the release of GABA from dorsal horn astrocytes with a time course compatible with the integration of sensory inputs.

The Spinal Neurons Exhibit an ON-OFF and OFF-ON Firing Activity Around the Onset of Fictive Scratching Episodes in the Cat.

In a previous report, we found neurons with ON-OFF and OFF-ON firing activity in the obex reticular formation during scratching. The aim of the present study was to examine whether the spinal neurons also exhibit this type of activity in relation to the "postural stage" of fictive scratching in the cat. We found that the extensor and intermediate scratching neurons exhibit an ON-OFF firing rate; conversely, the flexor neurons show an OFF-ON activity, relative to every scratching episode. These patterns of spiking activity are similar to those found in neurons from the obex reticular formation during scratching. Our findings provide support to the following hypotheses. First, there is a possible functional link between supraspinal and spinal, ON-OFF and OFF-ON neuronal groups. Second, the fictive goal-directed motor action to maintain the fictive "postural stage" of the hindlimb during fictive scratching is associated with the neuronal tonic activity of the OFF-ON spinal neurons, whereas the ON-OFF spinal neurons are associated with an extensor tone that occurred prior the postural stage.

Evaluation of the neonatal streptozotocin model of diabetes in rats: Evidence for a model of neuropathic pain.

BACKGROUND: The purpose of this study was to evaluate the participation of satellite glial cells (SGC), microglia and astrocytes in a model of streptozotocin-induced diabetes initiated in neonatal rats (nSTZ) and to determine the pharmacological profile for pain relief. METHODS: nSTZ was used to induce experimental diabetes. Von Frey filaments were used to assess tactile allodynia. Drugs were given by systemic administration. Western blotting and immunohistochemistry were used to determine protein expression and cellular localization. RESULTS: nSTZ produced mild hyperglycemia, weight loss, glucose intolerance, and reduction of nerve conduction velocity of C fibers. Moreover, nSTZ enhanced activating transcription factor 3 (ATF3) immunoreactivity in dorsal root ganglia (DRG) and sciatic nerve of adult rats. ATF3 was found in SGC (GFAP+ cells) surrounding DRG at week 16. Late changes in ATF3 immunoreactivity in DRG correlated with up-regulation of ATF3 and GFAP protein expression. nSTZ increased GFAP and OX-42 immunoreactivity and percentage of hypertrophied and ameboid microglia in the spinal dorsal horn. These changes correlated with the presence of mechanical hypersensitivity (tactile allodynia). Administration of gabapentin (30-100mg/kg, po) and metformin (200mg/kg/day, po for 2 weeks) alleviated tactile allodynia, whereas morphine (1-3mg/kg, ip) had a modest effect. CONCLUSIONS: Results suggest that nSTZ leads to activation of SGC, microglia and astrocytes in DRG and spinal cord. Pharmacological profile in the nSTZ model resembles diabetic neuropathic pain in humans. Our findings support the conclusion that the nSTZ rat model has utility for the study of a long-lasting diabetic neuropathic pain.

Tonically Active α5GABAA Receptors Reduce Motoneuron Excitability and Decrease the Monosynaptic Reflex.

Motoneurons, the final common path of the Central Nervous System (CNS), are under a complex control of its excitability in order to precisely translate the interneuronal pattern of activity into skeletal muscle contraction and relaxation. To fulfill this relevant function, motoneurons are provided with a vast repertoire of receptors and channels, including the extrasynaptic GABAA receptors which have been poorly investigated. Here, we confirmed that extrasynaptic α5 subunit-containing GABAA receptors localize with choline acetyltransferase (ChAT) positive cells, suggesting that these receptors are expressed in turtle motoneurons as previously reported in rodents. In these cells, α5GABAA receptors are activated by ambient GABA, producing a tonic shunt that reduces motoneurons' membrane resistance and affects their action potential firing properties. In addition, α5GABAA receptors shunted the synaptic excitatory inputs depressing the monosynaptic reflex (MSR) induced by activation of primary afferents. Therefore, our results suggest that α5GABAA receptors may play a relevant physiological role in motor control.

The α5 subunit containing GABAA receptors contribute to chronic pain.

It has been recently proposed that α5-subunit containing GABAA receptors (α5-GABAA receptors) that mediate tonic inhibition might be involved in pain. The purpose of this study was to investigate the contribution of α5-GABAA receptors in the loss of GABAergic inhibition and in formalin-induced, complete Freund's adjuvant (CFA)-induced and L5 and L6 spinal nerve ligation-induced long-lasting hypersensitivity. Formalin or CFA injection and L5 and L6 spinal nerve ligation produced long-lasting allodynia and hyperalgesia. Moreover, formalin injection impaired the rate-dependent depression of the Hofmann reflex. Peripheral and intrathecal pretreatment or post-treatment with the α5-GABAA receptor antagonist, L-655,708 (0.15-15 nmol), prevented and reversed, respectively, these long-lasting behaviors. Formalin injection increased α5-GABAA receptor mRNA expression in the spinal cord and dorsal root ganglia (DRG) mainly at 3 days. The α5-GABAA receptors were localized in the dorsal spinal cord and DRG colabeling with NeuN, CGRP, and IB4 which suggests their presence in peptidergic and nonpeptidergic neurons. These receptors were found mainly in small and medium sized neurons. Formalin injection enhanced α5-GABAA receptor fluorescence intensity in spinal cord and DRG at 3 and 6 days. Intrathecal administration of L-655,708 (15 nmol) prevented and reversed formalin-induced impairment of rate-dependent depression. These results suggest that α5-GABAA receptors play a role in the loss of GABAergic inhibition and contribute to long-lasting secondary allodynia and hyperalgesia.