GABAA and Glycine Receptor-Mediated Inhibitory Synaptic Transmission onto Adult Rat Lamina IIi PKCγ-Interneurons: Pharmacological but Not Anatomical Specialization.

Mechanical allodynia (pain to normally innocuous tactile stimuli) is a widespread symptom of inflammatory and neuropathic pain. Spinal or medullary dorsal horn (SDH or MDH) circuits mediating tactile sensation and pain need to interact in order to evoke mechanical allodynia. PKCγ-expressing (PKCγ+) interneurons and inhibitory controls within SDH/MDH inner lamina II (IIi) are pivotal in connecting touch and pain circuits. However, the relative contribution of GABA and glycine to PKCγ+ interneuron inhibition remains unknown. We characterized inhibitory inputs onto PKCγ+ interneurons by combining electrophysiology to record spontaneous and miniature IPSCs (sIPSCs, mIPSCs) and immunohistochemical detection of GABAARα2 and GlyRα1 subunits in adult rat MDH. While GlyR-only- and GABAAR-only-mediated mIPSCs/sIPSCs are predominantly recorded from PKCγ+ interneurons, immunohistochemistry reveals that ~80% of their inhibitory synapses possess both GABAARα2 and GlyRα1. Moreover, nearly all inhibitory boutons at gephyrin-expressing synapses on these cells contain glutamate decarboxylase and are therefore GABAergic, with around half possessing the neuronal glycine transporter (GlyT2) and therefore being glycinergic. Thus, while GABA and glycine are presumably co-released and GABAARs and GlyRs are present at most inhibitory synapses on PKCγ+ interneurons, these interneurons exhibit almost exclusively GABAAR-only and GlyR-only quantal postsynaptic inhibitory currents, suggesting a pharmacological specialization of their inhibitory synapses.

Postnatal development of inner lamina II interneurons of the rat medullary dorsal horn.

ABSTRACT: Pain processing in young mammals is immature. Despite the central role of the medullary dorsal horn (MDH) in processing orofacial sensory information, the maturation of the neurons within the MDH has been largely overlooked. Combining in vitro electrophysiological recordings and 3D morphological analysis over the first postnatal month in rats, we investigated the age-dependent development of the neurons within the inner lamina II (IIi) of the MDH. We show the lamina IIi neuronal population transition into a more hyperpolarized state, with modification of the action potential waveform, and a shift from single spiking, at early postnatal ages, to tonic firing and initial bursting at later stages. These physiological changes are associated with a strong structural remodelling of the neuronal morphology with most of the modifications occurring after the third postnatal week. Among the lamina IIi neuronal population, the subpopulation of interneurons expressing the γ isoform of the protein kinase C (PKCγ+) are key elements for the circuits underlying facial mechanical allodynia. How do they develop from the rest of the lamina IIi constitute an important question that remained to be addressed. Here, we show that PKCγ+ interneurons display electrophysiological changes over time comparable with the PKCγ- population. However, they show a distinctive increase of the soma volume and primary branches length, as opposed to the PKCγ- population. Together, our data demonstrate a novel pattern of late postnatal maturation of lamina IIi interneurons, with a spotlight on PKCγ+ interneurons, that may be relevant for the development of orofacial sensitivity.

Neuropathic pain increases spontaneous and noxious-evoked activity of locus coeruleus neurons.

The noradrenergic locus coeruleus nucleus is an important station in both the ascending and descending pain regulatory pathways. These neurons discharge in tonic and phasic modes in response to sensory stimuli. However, few studies have set out to characterize the electrophysiological response of the locus coeruleus to noxious stimuli in conditions of neuropathic pain. Thus, the effects of mechanical nociceptive stimulation of the sciatic nerve area on spontaneous (tonic) and sensory-evoked (phasic) locus coeruleus discharge were studied by extracellular recording in anesthetized rats seven, fourteen and twenty-eight days after chronic constriction injury. Minor significant electrophysiological changes were found seven and fourteen days after nerve injury. However, alterations to the spontaneous activity in both the ipsilateral and contralateral locus coeruleus were found twenty-eight days after nerve constriction, as witnessed by an increase of burst firing incidence and irregular firing patterns. Furthermore, noxious-evoked responses were exacerbated in the contralateral and ipsilateral nucleus at twenty-eight days after injury, as were the responses evoked when stimulating the uninjured paw. In addition, mechanical stimulation of the hindpaw produced a significant sensitization of neuronal tonic activity after 28 days of neuropathy. In summary, long-term nerve injury led to higher spontaneous activity and exacerbated noxious-evoked responses in the locus coeruleus to stimulation of nerve-injured and even uninjured hindpaws, coinciding temporally with the development of depressive and anxiogenic-like behavior.

5-HT2A Receptor-Induced Morphological Reorganization of PKCγ-Expressing Interneurons Gates Inflammatory Mechanical Allodynia in Rat.

Mechanical allodynia, a widespread pain symptom that still lacks effective therapy, is associated with the activation of a dorsally directed polysynaptic circuit within the spinal dorsal horn (SDH) or medullary dorsal horn (MDH), whereby tactile inputs into deep SDH/MDH can gain access to superficial SDH/MDH, eliciting pain. Inner lamina II (IIi) interneurons expressing the γ isoform of protein kinase C (PKCγ+) are key elements for allodynia circuits, but how they operate is still unclear. Combining behavioral, ex vivo electrophysiological, and morphological approaches in an adult rat model of facial inflammatory pain (complete Freund's adjuvant, CFA), we show that the mechanical allodynia observed 1 h after CFA injection is associated with the following (1) sensitization (using ERK1/2 phosphorylation as a marker) and (2) reduced dendritic arborizations and enhanced spine density in exclusively PKCγ+ interneurons, but (3) depolarized resting membrane potential (RMP) in all lamina IIi PKCγ+/PKCγ- interneurons. Blocking MDH 5HT2A receptors (5-HT2AR) prevents facial mechanical allodynia and associated changes in the morphology of PKCγ+ interneurons, but not depolarized RMP in lamina IIi interneurons. Finally, activation of MDH 5-HT2AR in naive animals is enough to reproduce the behavioral allodynia and morphological changes in PKCγ+ interneurons, but not the electrophysiological changes in lamina IIi interneurons, induced by facial inflammation. This suggests that inflammation-induced mechanical allodynia involves strong morphological reorganization of PKCγ+ interneurons via 5-HT2AR activation that contributes to open the gate for transmission of innocuous mechanical inputs to superficial SDH/MDH pain circuitry. Preventing 5-HT2AR-induced structural plasticity in PKCγ+ interneurons might represent new avenues for the specific treatment of inflammation-induced mechanical hypersensitivity.SIGNIFICANCE STATEMENT Inflammatory or neuropathic pain syndromes are characterized by pain hypersensitivity such as mechanical allodynia (pain induced by innocuous mechanical stimuli). It is generally assumed that mechanisms underlying mechanical allodynia, because they are rapid, must operate at only the level of functional reorganization of spinal or medullary dorsal horn (MDH) circuits. We discovered that facial inflammation-induced mechanical allodynia is associated with rapid and strong structural remodeling of specifically interneurons expressing the γ isoform of protein kinase C (PKCγ) within MDH inner lamina II. Moreover, we elucidated a 5-HT2A receptor to PKCγ/ERK1/2 pathway leading to the behavioral allodynia and correlated morphological changes in PKCγ interneurons. Therefore, descending 5-HT sensitize PKCγ interneurons, a putative "gate" in allodynia circuits, via 5-HT2A receptor-induced structural reorganization.

The onset of treatment with the antidepressant desipramine is critical for the emotional consequences of neuropathic pain.

Neuropathic pain is a chronic condition that is challenging to treat. It often produces considerable physical disability and emotional distress. Patients with neuropathic pain often experience depression and anxiety both of which are known to be temporally correlated with noradrenergic dysfunction in the locus coeruleus (LC) as pain becomes chronic. Antidepressants are the first-line drug therapy for neuropathic pain, and the LC represents a potential target for such therapy. In this study, we evaluated the efficacy of the tricyclic antidepressant desipramine (DMI, a noradrenaline reuptake inhibitor) in preventing or relieving the noradrenergic impairment induced by neuropathic pain. The treatment started before or after the onset of the anxiodepressive phenotype ("early or late treatment") in adult rats subjected to chronic sciatic constriction. Electrophysiological and western blotting assays showed LC dysfunction (increased bursting activity, alpha2-adrenoceptor sensitivity, tyrosine hydroxylase, and noradrenaline transporter expression) in chronic constriction injury at long term. These noradrenergic changes were concomitant to the progression of anxiety and despair-like features. Desipramine induced efficient analgesia, and it counteracted the despair-like behavior in chronic constriction injury-DMI animals, reducing the burst rate and tyrosine hydroxylase expression. Surprisingly, "early" DMI treatment did not modify pain-induced anxiety, and it dampened pain aversion, although these phenomena were abolished when the treatment commenced after noradrenaline impairment had been established. Hence, DMI seems to produce different outcomes depending when the treatment commences, indicating that the balance between the benefits and adverse effects of DMI therapy may shift as neuropathy progresses.

Comorbid anxiety-like behavior and locus coeruleus impairment in diabetic peripheral neuropathy: A comparative study with the chronic constriction injury model.

Anxiety frequently appears in patients with diabetic neuropathic pain, a highly prevalent clinical condition. However, the neurobiological mechanisms of this comorbidity are poorly known. Anxiogenic phenotype has been associated with alterations of the noradrenergic locus coeruleus (LC) after peripheral nerve entrapment. We have examined the sensorial (pain) and affective (anxiety) behaviors, and the LC activity in streptozotocin (STZ)-induced diabetic rats. A comparative study with the chronic constriction injury (CCI) model of sciatic nerve was also carried out. Diabetic nociceptive hypersensitivity was observed to appear gradually, reaching their maximum at fourth week. In contrast, CCI displayed a sharp decrease in their sensorial threshold at seventh day. In both models, anxiety-like phenotype was evident after four weeks but not earlier, coincident with the LC alterations. Indeed, STZ animals showed reduced LC firing activity, tyrosine hydroxylase, pCREB and noradrenaline transporter levels, contrary to observed in CCI animals. However, in both models, enhanced LC alpha2-adrenoceptor sensitivity was presented at this time point. This study demonstrated that diabetes induced anxiety-like behavior comorbid with LC impairment at long-term. However, the nociceptive sensitivity time-course, as well as the LC functions, showed distinct features compared to the CCI model, indicating that specific neuroplastic mechanisms are at play in every model.

Stress Increases the Negative Effects of Chronic Pain on Hippocampal Neurogenesis.

BACKGROUND: Patients with chronic pain often suffer from affective disorders and cognitive decline, which significantly impairs their quality of life. In addition, many of these patients also experience stress unrelated to their illness, which can aggravate their symptoms. These nociceptive inputs are received by the hippocampus, in which maladaptive neuroplastic changes may occur in the conditions of chronic pain. The hippocampus is a structure involved in emotionality, learning, and memory, and the proliferating cells in the granular layer of the hippocampal dentate gyrus respond to chronic pain by slowing their turnover. However, whether the maturation, survival, and integration of newborn cells in the hippocampus are affected by chronic pain remains unclear. In addition, it is unknown whether an added stress may increase this effect. METHODS: We have evaluated the proliferation, differentiation, and survival of newborn hippocampal cells in a rat model of neuropathic pain (chronic constriction injury), with or without stress (chronic immobilization), by assessing the incorporation of bromodeoxyuridine into proliferating cells and immunostaining. RESULTS: The data obtained indicated that there was a decrease in the number of proliferating cells 8 days after nerve injury in animals subjected to neuropathic pain, an effect that was exacerbated by stress. Moreover, 4 weeks after nerve injury, neuropathic pain was associated with a loss of neuroblasts and the reduced survival of new mature neurons in the hippocampal granular layer, phenomena that also were increased by stress. By contrast, the rate of differentiation was not affected in this paradigm. CONCLUSIONS: Neuropathic pain negatively influences hippocampal neurogenesis (proliferation and survival), and this effect is exacerbated by stress. These neuroplastic changes may account for the affective and cognitive impairment seen in patients with chronic pain.

Subpopulations of PKCγ interneurons within the medullary dorsal horn revealed by electrophysiologic and morphologic approach.

Mechanical allodynia, a cardinal symptom of persistent pain, is associated with the unmasking of usually blocked local circuits within the superficial spinal or medullary dorsal horn (MDH) through which low-threshold mechanical inputs can gain access to the lamina I nociceptive output neurons. Specific interneurons located within inner lamina II (IIi) and expressing the gamma isoform of protein kinase C (PKCγ⁺) have been shown to be key elements for such circuits. However, their morphologic and electrophysiologic features are still unknown. Using whole-cell patch-clamp recordings and immunohistochemical techniques in slices of adult rat MDH, we characterized such lamina IIi PKCγ⁺ interneurons and compared them with neighboring PKCγ⁻ interneurons. Our results reveal that PKCγ⁺ interneurons display very specific activity and response properties. Compared with PKCγ⁻ interneurons, they exhibit a smaller membrane input resistance and rheobase, leading to a lower threshold for action potentials. Consistently, more than half of PKCγ⁺ interneurons respond with tonic firing to step current. They also receive a weaker excitatory synaptic drive. Most PKCγ⁺ interneurons express Ih currents. The neurites of PKCγ⁺ interneurons arborize extensively within lamina IIi, can spread dorsally into lamina IIo, but never reach lamina I. In addition, at least 2 morphologically and functionally different subpopulations of PKCγ⁺ interneurons can be identified: central and radial PKCγ⁺ interneurons. The former exhibit a lower membrane input resistance, rheobase and, thus, action potential threshold, and less PKCγ⁺ immunoreactivity than the latter. These 2 subpopulations might thus differently contribute to the gating of dorsally directed circuits within the MDH underlying mechanical allodynia.

Pain exacerbates chronic mild stress-induced changes in noradrenergic transmission in rats.

Depression can influence pain and vice versa, yet the biological mechanisms underlying how one influences the pathophysiology of the other remains unclear. Dysregulation of locus coeruleus-noradrenergic transmission is implicated in both conditions, although it is not known whether this effect is exacerbated in cases of co-morbid depression and chronic pain. We studied locus coeruleus activity using immunofluorescence and electrophysiological approaches in rats subjected to unpredictable chronic mild stress (CMS, an experimental model of depression) and/or chronic constriction injury (CCI, a model of chronic neuropathic pain) for 2 weeks. CCI alone had no effect on any of the locus coeruleus parameters studied, while CMS led to a slight reduction in the electrophysiological activity of the locus coeruleus. Furthermore, CMS was associated with an increase in the number of tyrosine hydroxylase-positive cells in the locus coeruleus, although they were smaller in size. Interestingly, these effects of CMS were exacerbated when combined with CCI, even though no changes in the α2-adrenoreceptors or the noradrenaline transporter were observed in any group. Together, these findings suggest that CMS triggers several modifications in locus coeruleus-noradrenergic transmission that are exacerbated by co-morbid chronic pain.

Social stress exacerbates the aversion to painful experiences in rats exposed to chronic pain: the role of the locus coeruleus.

Stressful experiences seem to negatively influence pain perception through as yet unknown mechanisms. As the noradrenergic locus coeruleus (LC) nucleus coordinates many components of the stress response, as well as nociceptive transmission, we evaluated whether the sensory and affective dimension of chronic neuropathic pain worsens in situations of stress due to adaptive changes of LC neurons. Accordingly, male rats were socially isolated for 5 weeks, and in the last 2 weeks, neuropathic pain was induced by chronic constriction injury. In this situation of stress, chronic pain selectively heightened the animal's aversion to painful experiences (affective pain), as measured in the place escape/avoidance test, although no changes were observed in the sensory dimension of pain. In addition, electrophysiological recordings of LC neurons showed a low tonic but exacerbated nociceptive-evoked activity when the injured paw was stimulated. These changes were accompanied by an increase in tyrosine hydroxylase and gephyrin expression in the LC. Furthermore, intra-LC administration of bicuculline, a γ-aminobutyric acid-A receptor antagonist, attenuated the negative affective effects of pain. These data show that changes in the LC are greater than those expected from the simple summation of each independent factor (pain and stress), revealing mechanisms through which stressors may exacerbate pain perception without affecting the sensorial dimension.

Effect of tapentadol on neurons in the locus coeruleus.

Tapentadol is a novel centrally acting drug that combines mu-opioid receptor (MOR) agonism and noradrenaline reuptake inhibition (NRI), producing analgesic effects in various painful conditions. We investigated the acute effects of tapentadol in the locus coeruleus (LC), a central nucleus regulated by the noradrenergic and opioid systems that is critical in pain modulation. In single-unit extracellular recordings of LC neurons from anaesthetized male Sprague-Dawley rats, tapentadol clearly inhibited the spontaneous electrophysiological activity of LC neurons in a dose-dependent manner (ED50 = 0.8 mg/kg). This inhibitory effect was reversed by RX821002 (an alpha2-adrenoceptor antagonist) and naloxone (a mu-opioid receptor antagonist) by 96.7% and 28.2%, respectively. Pretreatment with RX821002, N-ethoxycarbonyl-2-ethoxy-1-2-dihydroquinoline (EEDQ, an irreversible alpha2-adrenoceptor antagonist) or naloxone shifted the tapentadol dose-effect curve to the right (ED50 = 2.2 mg/kg, 2.0 mg/kg and 2.1 mg/kg, respectively). Furthermore, tapentadol inhibited the LC response to mechanical stimulation of the hindpaw in a dose-dependent manner. In summary, we demonstrate that acute administration of tapentadol inhibits LC neurons in vivo, mainly due to the activation of alpha2-adrenoceptors. These data suggest that both the noradrenergic and opioid systems participate in the inhibitory effect of tapentadol on LC neurons, albeit to different extents, which may account for its potent analgesic effect and mild opioidergic side-effects.

Chronic pain leads to concomitant noradrenergic impairment and mood disorders.

BACKGROUND: Patients suffering chronic pain are at high risk of suffering long-lasting emotional disturbances characterized by persistent low mood and anxiety. We propose that this might be the result of a functional impairment in noradrenergic circuits associated with locus coeruleus (LC) and prefrontal cortex, where emotional and sensorial pain processes overlap. METHODS: We used a chronic constriction injury of sciatic nerve as a model of neuropathic pain in male Sprague-Dawley rats to assess the time-dependent changes that might potentially precipitate mood disorders (2, 7, 14, and 28 days after injury). This was measured through a combination of behavioral, electrophysiological, microdialysis, immunohistochemical, and Western blot assays. RESULTS: As expected, nerve injury produced an early and stable decrease in sensorial pain threshold over the testing period. By contrast, long-term neuropathic pain (28 days after injury) resulted in an inability to cope with stressful situations, provoking depressive and anxiogenic-like behaviors, even more intense than the aversiveness associated with pain perception. The onset of these behavioral changes coincided with irruption of noradrenergic dysfunction, evident as: an increase in LC bursting activity; in tyrosine hydroxylase expression and that of the noradrenaline transporter; and enhanced expression and sensitivity of α2-adrenoceptors in the LC. CONCLUSIONS: Long-term neuropathic pain leads to anxio-depressive-like behaviors that are more predominant than the aversion of a painful experience. These changes are consistent with the impairment of noradrenergic system described in depressive disorders.

Analgesic antidepressants promote the responsiveness of locus coeruleus neurons to noxious stimulation: implications for neuropathic pain.

Antidepressants that block the reuptake of noradrenaline and/or serotonin are among the first-line treatments for neuropathic pain, although the mechanisms underlying this analgesia remain unclear. The noradrenergic locus coeruleus is an essential element of both the ascending and descending pain modulator systems regulated by these antidepressants. Hence, we investigated the effect of analgesic antidepressants on locus coeruleus activity in Sprague-Dawley rats subjected to chronic constriction injury (CCI), a model of neuropathic pain. In vivo extracellular recordings of locus coeruleus revealed that CCI did not modify the basal tonic activity of this nucleus, although its sensory-evoked response to noxious stimuli was significantly altered. Under normal conditions, noxious stimulation evokes an early response, corresponding to the activation of myelinated A fibers, which is followed by an inhibitory period and a subsequent late capsaicin-sensitive response, consistent with the activation of unmyelinated C fibers. CCI provokes an enhanced excitatory early response in the animals and the loss of the late response. Antidepressant administration over 7 days (desipramine, 10mg/kg/day or duloxetine, 5mg/kg/day, delivered by osmotic minipumps) decreased the excitatory firing rate of the early response in the CCI group. Moreover, in all animals, these antidepressants reduced the inhibitory period and augmented the late response. We propose that N-methyl-d-aspartate and alpha-2-adrenoceptors are involved in the analgesic effect of antidepressants. Antidepressant-mediated changes were correlated with behavioral effects indicative of analgesia in healthy and neuropathic rats.

The function of alpha-2-adrenoceptors in the rat locus coeruleus is preserved in the chronic constriction injury model of neuropathic pain.

RATIONALE: Peripheral neuropathic pain is a chronic condition that may produce plastic changes in several brain regions. The noradrenergic locus coeruleus (LC) is a crucial component of ascending and descending pain pathways, both of which are frequently compromised after nerve injury. OBJECTIVES: The objective of the study was to examine whether chronic constriction injury (CCI), a model of neuropathic pain, alters noradrenergic activity in the rat LC. METHODS: Activity in the LC was assessed by electrophysiology and microdialysis, while protein expression was monitored in western blots and by immunohistochemistry. RESULTS: The pain threshold had dropped in injured rats 7 days after inducing neuropathy. While alpha-2-adrenoceptors mediate activity in the LC and in its terminal areas, no alterations in either spontaneous neuronal activity or extracellular noradrenaline levels were observed following CCI. Moreover, alpha-2-adrenoceptor activity in the LC of CCI rats remained unchanged after systemic administration of UK14,304, RX821002 or desipramine. Accordingly, extracellular noradrenaline levels in the LC were similar in CCI and control animals following local administration of clonidine or RX821002. In addition, there were no changes in the expression of the alpha-2-adrenoceptors, Gαi/z subunits or the regulators of G-protein signaling. However, pERK1/2 (phosphorylated extracellular signal-regulated kinases 1/2) expression augmented in the spinal cord, paragigantocellularis nucleus (PGi) and dorsal raphe nucleus (DRN) following CCI. CONCLUSIONS: Neuropathic pain is not accompanied by modifications in tonic LC activity after the onset of pain. This may indicate that the signals from the PGi and DRN, the excitatory and inhibitory afferents of the LC, cancel one another out.