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Effects of aspartate (2 · 10 M), substance P (10-10 M), and serotonin (5-hydroxytryptamine, 5-HT; 5 · 10 M) on the background activity of neurons in the central gray substance (CGS) were studied on slices of the rat midbrain. Aspartate and substance P (transmitters of nociceptive signals), and 5-HT (modulator of transmission of nociceptive influences) were found either to facilitate or to depress the activity of CGS neurons. The predominant effect of substance P or 5-HT applications to neurons of the dorsal CGS part was facilitation, and to neurons of the ventral CGS part, inhibition. The effects of aspartate application on studied CGS neurons were of varying nature, but inhibitory effects were found to prevail. The findings support our earlier hypothesis that assigned the studied neurons to spontaneously discharging inhibitory CGS interneurons, which control the activity of efferent CGS neurons. The role of tested substances in the regulation of CGS neuronal activity and the antinociceptive CGS effects is discussed. [ABSTRACT FROM AUTHOR]

Abstract: Cerebellar adaptive plasticity regulates posture and movement in response to changing conditions of sensory stimulation. Study of adaptive plasticity of cerebellar circuitry in vitro confines experimental interest to mechanisms with a time scale of minutes. However, cerebellar plasticity, measured behaviorally or electrophysiologically in vivo, occurs over a time scale of tens of minutes and hours. Here we investigate how optokinetically-evoked increases in climbing fiber activity influence expression of key subcellular signaling proteins that regulate the accumulation of GABAA receptors (GABAARs) in the cytoplasm of Purkinje cells and their insertion into the plasma membrane. We used long-term horizontal optokinetic stimulation (HOKS) to activate climbing fibers that project to the flocculus of mice. Although long-term increases in climbing fiber activity in vivo do not alter the expression of any of the subunits of GABAARs expressed by Purkinje cells, they do influence other subcellular events such as transcription and interaction of signaling proteins. Specifically, increased climbing fiber activity evoked decreased expression of 14-3-3-θ, reduced serine phosphorylation of GABAAg2, and reduced the interaction of 14-3-3-θ with protein kinase C-γ (PKC-γ). Knockdown of 14-3-3-θ in vivo reduced the serine phosphorylation of GABAAγ2. Conversely, treatment of cerebellar lysates with phorbol 12-myristate-13-acetate (PMA), a PKC activator, increased serine phosphorylation of GABAAγ2. Knockdown of 14-3-3-θ or PKC-γ in N2a cells in vitro reduced serine phosphorylation of GABAAγ2 and reduced its cell-surface expression. We interpret these data to mean that a prolonged increase in climbing fiber activity decreases the cell-surface expression of GABAARs in Purkinje cells and thereby reduces their sensitivity to GABAergic inhibition. This provides a homeostatic mechanism by which Purkinje cells become less sensitive to stellate cell inhibition also evoked by climbing fiber activity. [Copyright &y& Elsevier]

Background: Neuroinflammation and mitochondrial dysfunction have been implicated in the progression of neuropathic pain (NP) but can be mitigated by supplementation with gingerol-enriched ginger (GEG). However, the exact benefits of GEG for each sex in treating neuroinflammation and mitochondrial homeostasis in different brain regions and the colon remain to be determined., Objective: Evaluate the effects of GEG on emotional/affective pain and spontaneous pain behaviors, neuroinflammation, as well as mitochondria homeostasis in the amygdala, frontal cortex, hippocampus, and colon of male and female rats in the spinal nerve ligation (SNL) NP model., Methods: One hundred rats (fifty males and fifty females) were randomly assigned to five groups: sham + vehicle, SNL + vehicle, and SNL with three different GEG doses (200, 400, and 600 mg/kg BW) for 5 weeks. A rat grimace scale and vocalizations were used to assess spontaneous and emotional/affective pain behaviors, respectively. mRNA gene and protein expression levels for tight junction protein, neuroinflammation, mitochondria homeostasis, and oxidative stress were measured in the amygdala, frontal cortex, hippocampus, and colon using qRT-PCR and Western blot (colon)., Results: GEG supplementation mitigated spontaneous pain in both male and female rats with NP while decreasing emotional/affective responses only in male NP rats. GEG supplementation increased intestinal integrity (claudin 3) and suppressed neuroinflammation [glial activation (GFAP, CD11b, IBA1) and inflammation (TNFα, NFκB, IL1β)] in the selected brain regions and colon of male and female NP rats. GEG supplementation improved mitochondrial homeostasis [increased biogenesis (TFAM, PGC1α), increased fission (FIS, DRP1), decreased fusion (MFN2, MFN1) and mitophagy (PINK1), and increased Complex III] in the selected brain regions and colon in both sexes. Some GEG dose-response effects in gene expression were observed in NP rats of both sexes., Conclusions: GEG supplementation decreased emotional/affective pain behaviors of males and females via improving gut integrity, suppressing neuroinflammation, and improving mitochondrial homeostasis in the amygdala, frontal cortex, hippocampus, and colon in both male and female SNL rats in an NP model, implicating the gut-brain axis in NP. Sex differences observed in the vocalizations assay may suggest different mechanisms of evoked NP responses in females.

Maladaptive plasticity is linked to the chronification of diseases such as pain, but the transition from acute to chronic pain is not well understood mechanistically. Neuroplasticity in the central nucleus of the amygdala (CeA) has emerged as a mechanism for sensory and emotional-affective aspects of injury-induced pain, although evidence comes from studies conducted almost exclusively in acute pain conditions and agnostic to cell type specificity. Here, we report time-dependent changes in genetically distinct and projection-specific CeA neurons in neuropathic pain. Hyperexcitability of CRF projection neurons and synaptic plasticity of parabrachial (PB) input at the acute stage shifted to hyperexcitability without synaptic plasticity in non-CRF neurons at the chronic phase. Accordingly, chemogenetic inhibition of the PB→CeA pathway mitigated pain-related behaviors in acute, but not chronic, neuropathic pain. Cell-type-specific temporal changes in neuroplasticity provide neurobiological evidence for the clinical observation that chronic pain is not simply the prolonged persistence of acute pain., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Neuroplasticity in the amygdala and its central nucleus (CeA) is linked to pain modulation and pain behaviors, but cellular mechanisms are not well understood. Here, we addressed the role of small-conductance Ca 2+ -activated potassium (SK) channels in pain-related amygdala plasticity. The facilitatory effects of the intra-CeA application of an SK channel blocker (apamin) on the pain behaviors of control rats were lost in a neuropathic pain model, whereas an SK channel activator (NS309) inhibited pain behaviors in neuropathic rats but not in sham controls, suggesting the loss of the inhibitory behavioral effects of amygdala SK channels. Brain slice electrophysiology found hyperexcitability of CeA neurons in the neuropathic pain condition due to the loss of SK channel-mediated medium afterhyperpolarization (mAHP), which was accompanied by decreased SK2 channel protein and mRNA expression, consistent with a pretranscriptional mechanisms. The underlying mechanisms involved the epigenetic silencing of the SK2 gene due to the increased DNA methylation of the CpG island of the SK2 promoter region and the change in methylated CpG sites in the CeA in neuropathic pain. This study identified the epigenetic dysregulation of SK channels in the amygdala (CeA) as a novel mechanism of neuropathic pain-related plasticity and behavior that could be targeted to control abnormally enhanced amygdala activity and chronic neuropathic pain.

Emerging evidence shows that the gut microbiota plays an important role in neuropathic pain (NP) via the gut-brain axis. Male rats were divided into sham, spinal nerve ligation (SNL), SNL + 200 mg GEG/kg BW (GEG200), and SNL + 600 mg GEG/kg BW (GEG600) for 5 weeks. The dosages of 200 and 600 mg GEG/kg BW for rats correspond to 45 g and 135 g raw ginger for human daily consumption, respectively. Both GEG groups mitigated SNL-induced NP behavior. GEG-supplemented animals had a decreased abundance of Rikenella , Muribaculaceae , Clostridia UCG-014 , Mucispirillum schaedleri , RF39 , Acetatifactor , and Clostridia UCG-009 , while they had an increased abundance of Flavonifactor , Hungatella , Anaerofustis stercorihominis , and Clostridium innocuum group. Relative to sham rats, Fos and Gadd45g genes were upregulated, while Igf1 , Ccl2 , Hadc2 , Rtn4rl1 , Nfkb2 , Gpr84 , Pik3cg , and Abcc8 genes were downregulated in SNL rats. Compared to the SNL group, the GEG200 group and GEG600 group had increases/decreases in 16 (10/6) genes and 11 (1/10) genes, respectively. GEG downregulated Fos and Gadd45g genes and upregulated Hdac2 genes in the amygdala. In summary, GEG alleviates NP by modulating the gut microbiome and reversing a molecular neuroimmune signature.

Neuroplasticity in cortico-limbic circuits has been implicated in pain persistence and pain modulation in clinical and preclinical studies. The amygdala has emerged as a key player in the emotional-affective dimension of pain and pain modulation. Reciprocal interactions with medial prefrontal cortical regions undergo changes in pain conditions. Other limbic and paralimbic regions have been implicated in pain modulation as well. The cortico-limbic system is rich in opioids and opioid receptors. Preclinical evidence for their pain modulatory effects in different regions of this highly interactive system, potentially opposing functions of different opioid receptors, and knowledge gaps will be described here. There is little information about cell type- and circuit-specific functions of opioid receptor subtypes related to pain processing and pain-related plasticity in the cortico-limbic system. The important role of anterior cingulate cortex (ACC) and amygdala in MOR-dependent analgesia is most well-established, and MOR actions in the mesolimbic system appear to be similar but remain to be determined in mPFC regions other than ACC. Evidence also suggests that KOR signaling generally serves opposing functions whereas DOR signaling in the ACC has similar, if not synergistic effects, to MOR. A unifying picture of pain-related neuronal mechanisms of opioid signaling in different elements of the cortico-limbic circuitry has yet to emerge. This article is part of the Special Issue on "Opioid-induced changes in addiction and pain circuits"., Competing Interests: Declaration of competing interest None., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Objectives: Emerging evidence suggests an important role of the gut-brain axis in the development of neuropathic pain (NP). We investigated the effects of gingerol-enriched ginger (GEG) on pain behaviors, as well as mRNA expressions of inflammation via tight junction proteins in GI tissues (colon) and brain tissues (amygdala, both left and right) in animals with NP. Methods: Seventeen male rats were randomly divided into three groups: Sham, spinal nerve ligation (SNL, pain model), and SNL+0.375% GEG (wt/wt in diet) for 4 weeks. Mechanosensitivity was assessed by von Frey filament tests and hindpaw compression tests. Emotional responsiveness was measured from evoked audible and ultrasonic vocalizations. Ongoing spontaneous pain was measured in rodent grimace tests. Intestinal permeability was assessed by the lactulose/D-mannitol ratio in urine. The mRNA expression levels of neuroinflammation (NF-κB, TNF-α) in the colon and amygdala (right and left) were determined by qRT-PCR. Data was analyzed statistically. Results: Compared to the sham group, the SNL group had significantly greater mechanosensitivity (von Frey and compression tests), emotional responsiveness (audible and ultrasonic vocalizations to innocuous and noxious mechanical stimuli), and spontaneous pain (rodent grimace tests). GEG supplementation significantly reduced mechanosensitivity, emotional responses, and spontaneous pain measures in SNL rats. GEG supplementation also tended to decrease SNL-induced intestinal permeability markers. The SNL group had increased mRNA expression of NF-κB and TNF-α in the right amygdala and colon; GEG supplementation mitigated these changes in SNL-treated rats. Conclusion: This study suggests GEG supplementation palliated a variety of pain spectrum behaviors in a preclinical NP animal model. GEG also decreased SNL-induced intestinal permeability and neuroinflammation, which may explain the behavioral effects of GEG., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Shen, Wang, Yakhnitsa, Santos, Watson, Kiritoshi, Ji, Hamood and Neugebauer.)

The amygdala plays a critical role in the emotional-affective component of pain and pain modulation. The central nucleus of amygdala (CeA) serves major output functions and has been linked to pain-related behaviors. Corticotropin releasing factor (CRF) in the CeA has emerged as an important modulator of pain and affective disorders. Here we measured the effects of optogenetic manipulation of CeA-CRF neurons on pain-related behaviors in a rat neuropathic pain model and under control conditions. Emotional-affective behaviors (vocalizations), mechanosensitivity (electronic von Frey anesthesiometer and calibrated forceps), and anxiety-like behaviors (open field test and elevated plus maze) were assessed in adult rats 1 week and 4 weeks after spinal nerve ligation (SNL model) and sham surgery (control). For optogenetic silencing or activation of CRF neurons, a Cre-inducible viral vector encoding enhanced halorhodopsin (eNpHR 3.0 ) or channelrhodopsin 2 (ChR 2 ) was injected stereotaxically into the right CeA of transgenic Crh-Cre rats. Light of the appropriate wavelength (590 nm for eNpHR 3.0 ; 473 nm for ChR 2 ) was delivered into the CeA with an LED optic fiber. Optical silencing of CeA-CRF neurons decreased the emotional-affective responses in the acute and chronic phases of the neuropathic pain model but had anxiolytic effects only at the chronic stage and no effect on mechanosensitivity. Optogenetic activation of CeA-CRF neurons increased the emotional-affective responses and induced anxiety-like behaviors but had no effect on mechanosensitivity in control rats. The data show the critical contribution of CeA-CRF neurons to pain-related behaviors under normal conditions and beneficial effects of inhibiting CeA-CRF neurons in neuropathic pain., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Functional pain syndromes (FPS) occur in the absence of identifiable tissue injury or noxious events and include conditions such as migraine, fibromyalgia, and others. Stressors are very common triggers of pain attacks in various FPS conditions. It has been recently demonstrated that kappa opioid receptors (KOR) in the central nucleus of amygdala (CeA) contribute to FPS conditions, but underlying mechanisms remain unclear. The CeA is rich in KOR and encompasses major output pathways involving extra-amygdalar projections of corticotropin releasing factor (CRF) expressing neurons. Here we tested the hypothesis that KOR blockade in the CeA in a rat model of FPS reduces pain-like and nocifensive behaviors by restoring inhibition of CeA-CRF neurons. Intra-CeA administration of a KOR antagonist (nor-BNI) decreased mechanical hypersensitivity and affective and anxiety-like behaviors in a stress-induced FPS model. In systems electrophysiology experiments in anesthetized rats, intra-CeA application of nor-BNI reduced spontaneous firing and responsiveness of CeA neurons to peripheral stimulation. In brain slice whole-cell patch-clamp recordings, nor-BNI increased feedforward inhibitory transmission evoked by optogenetic and electrical stimulation of parabrachial afferents, but had no effect on monosynaptic excitatory transmission. Nor-BNI decreased frequency, but not amplitude, of spontaneous inhibitory synaptic currents, suggesting a presynaptic action. Blocking KOR receptors in stress-induced FPS conditions may therefore represent a novel therapeutic strategy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yakhnitsa, Ji, Hein, Presto, Griffin, Ponomareva, Navratilova, Porreca and Neugebauer.)

Recent evidence suggests that kappa opioid receptors (KOR) in limbic brain regions such as the amygdala contribute to pain conditions, but underlying mechanisms remain to be determined. The amygdala is an important player in averse-affective aspects of pain and pain modulation. The central nucleus (CeA) serves output functions through projection neurons that include corticotropin releasing factor (CRF) expressing neurons. The CeA is also rich in KOR. Here we tested the novel hypothesis that KOR activation in the CeA generates pain-like behaviors through a mechanism that involves inhibition of synaptic inhibition (disinhibition) of CRF neurons. Intra-CeA administration of a KOR agonist (U-69,593) increased vocalizations of naïve rats to noxious stimuli, and induced anxiety-like behaviors in the open field test (OFT) and avoidance in the conditioned place preference test, without affecting mechanosensory thresholds. Optogenetic silencing of CeA-CRF neurons blocked the facilitatory effects of systemically applied U-69,593 in naïve rats. Patch-clamp recordings of CRF neurons in rat brain slices found that U-69,593 decreased feedforward inhibitory transmission evoked by optogenetic stimulation of parabrachial afferents, but had no effect on monosynaptic excitatory transmission. U-69,593 decreased frequency, but not amplitude, of inhibitory synaptic currents, suggesting a presynaptic action. Multiphoton imaging of CeA-CRF neurons in rat brain slices showed that U-69,593 increased calcium signals evoked by electrical stimulation of presumed parabrachial input. This study shows for the first time that KOR activation increases activity of amygdala CRF neurons through synaptic disinhibition, resulting in averse-affective pain-like behaviors. Blocking KOR receptors may therefore represent a novel therapeutic strategy., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Chronic pain is associated with neuroplastic changes in the amygdala that may promote hyper-responsiveness to mechanical and thermal stimuli (allodynia and hyperalgesia) and/or enhance emotional and affective consequences of pain. Stress promotes dynorphin-mediated signaling at the kappa opioid receptor (KOR) in the amygdala and mechanical hypersensitivity in rodent models of functional pain. Here, we tested the hypothesis that KOR circuits in the central nucleus of the amygdala (CeA) undergo neuroplasticity in chronic neuropathic pain resulting in increased sensory and affective pain responses. After spinal nerve ligation (SNL) injury in rats, pretreatment with a long-acting KOR antagonist, nor-binaltorphimine (nor-BNI), subcutaneously or through microinjection into the right CeA, prevented conditioned place preference (CPP) to intravenous gabapentin, suggesting that nor-BNI eliminated the aversiveness of ongoing pain. By contrast, systemic or intra-CeA administration of nor-BNI had no effect on tactile allodynia in SNL animals. Using whole-cell patch-clamp electrophysiology, we found that nor-BNI decreased synaptically evoked spiking of CeA neurons in brain slices from SNL but not sham rats. This effect was mediated through increased inhibitory postsynaptic currents, suggesting tonic disinhibition of CeA output neurons due to increased KOR activity as a possible mechanism promoting ongoing aversive aspects of neuropathic pain. Interestingly, this mechanism is not involved in SNL-induced mechanical allodynia. Kappa opioid receptor antagonists may therefore represent novel therapies for neuropathic pain by targeting aversive aspects of ongoing pain while preserving protective functions of acute pain.

Background and Purpose: Chronic neuropathic pain is an important healthcare issue with significant emotional components. The amygdala is a brain region involved in pain and emotional-affective states and disorders. The central amygdala output nucleus (CeA) contains small-conductance calcium-activated potassium (SK) channels that can control neuronal activity. A clinically available therapeutic, riluzole can activate SK channels and may have antinociceptive effects through a supraspinal action. We tested the hypothesis that riluzole inhibits neuropathic pain behaviors by inhibiting pain-related changes in CeA neurons, in part at least through SK channel activation., Experimental Approach: Brain slice physiology and behavioral assays were done in adult Sprague Dawley rats. Audible and ultrasonic vocalizations and von Frey thresholds were measured in sham and neuropathic rats 4 weeks after left L5 spinal nerve ligation (SNL model). Whole cell patch-clamp recordings of regular firing CeA neurons in brain slices were used to measure synaptic transmission and neuronal excitability., Key Results: In brain slices, riluzole increased the SK channel-mediated afterhyperpolarization and synaptic inhibition, but inhibited neuronal excitability through an SK channel independent action. SNL rats had increased vocalizations and decreased withdrawal thresholds compared to sham rats, and intra-CeA administration of riluzole inhibited vocalizations and depression-like behaviors but did not affect withdrawal thresholds. Systemic riluzole administration also inhibited these changes, demonstrating the clinical utility of this strategy. SK channel blockade in the CeA attenuated the inhibitory effects of systemic riluzole on vocalizations, confirming SK channel involvement in these effects., Conclusions and Implications: The results suggest that riluzole has beneficial effects on neuropathic pain behaviors through SK channel dependent and independent mechanisms in the amygdala., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background The amygdala plays a key role in fear learning and extinction and has emerged as an important node of emotional-affective aspects of pain and pain modulation. Impaired fear extinction learning, which involves prefrontal cortical control of amygdala processing, has been linked to neuropsychiatric disorders. Here, we tested the hypothesis that fear extinction learning ability can predict the magnitude of neuropathic pain. Results We correlated fear extinction learning in naive adult male rats with sensory and affective behavioral outcome measures (mechanical thresholds, vocalizations, and anxiety- and depression-like behaviors) before and after the induction of the spinal nerve ligation model of neuropathic pain compared to sham controls. Auditory fear conditioning, extinction learning, and extinction retention tests were conducted after baseline testing. All rats showed increased freezing responses after fear conditioning. During extinction training, the majority (75%) of rats showed a decline in freezing level to 50% in 5 min (fear extinction+), whereas 25% of the rats maintained a high freezing level (>50%, fear extinction-). Fear extinction- rats showed decreased open-arm preference in the elevated plus maze, reflecting anxiety-like behavior, but there were no significant differences in sensory thresholds, vocalizations, or depression-like behavior (forced swim test) between fear extinction+ and fear extinction- types. In the neuropathic pain model (four weeks after spinal nerve ligation), fear extinction- rats showed a greater increase in vocalizations and anxiety-like behavior than fear extinction+ rats. Fear extinction- rats, but not fear extinction+ rats, also developed depression-like behavior. Extracellular single unit recordings of amygdala (central nucleus) neurons in behaviorally tested rats (anesthetized with isoflurane) found greater increases in background activity, bursting, and evoked activity in fear extinction- rats than fear extinction+ rats in the spinal nerve ligation model compared to sham controls. Conclusion The data may suggest that fear extinction learning ability predicts the magnitude of neuropathic pain-related affective rather than sensory behaviors, which correlates with differences in amygdala activity changes.

Synaptic activation of central neurons is often evoked by electrical stimulation leading to post-tetanic potentiation, long-term potentiation or long-term depression. Even a brief electrical tetanus can induce changes in as many as 100 proteins. Since climbing fibre activity is often associated with cerebellar behavioural plasticity, we used horizontal optokinetic stimulation (HOKS) to naturally increase synaptic input to floccular Purkinje cells in mice for hours, not minutes, and investigated how this activity influenced the transcription of microRNAs, small non-coding nucleotides that reduce transcripts of multiple, complementary mRNAs. A single microRNA can reduce the translation of as many as 30 proteins. HOKS evoked increases in 12 microRNA transcripts in floccular Purkinje cells. One of these microRNAs, miR335, increased 18-fold after 24 h of HOKS. After HOKS stopped, miR335 transcripts decayed with a time constant of approximately 2.5 h. HOKS evoked a 28-fold increase in pri-miR335 transcripts compared with an 18-fold increase in mature miR335 transcripts, confirming that climbing fibre-evoked increases in miR335 could be attributed to increases in transcription. We used three screens to identify potential mRNA targets for miR335 transcripts: (i) nucleotide complementarity, (ii) detection of increased mRNAs following microinjection of miR335 inhibitors into the cerebellum, and (iii) detection of decreased mRNAs following HOKS. Two genes, calbindin and 14-3-3-θ, passed these screens. Transfection of N2a cells with miR335 inhibitors or precursors inversely regulated 14-3-3-θ transcripts. Immunoprecipitation of 14-3-3-θ co-immunoprecipitated PKC-γ and GABAAγ2. Knockdown of either 14-3-3-θ or PKC-γ decreased the serine phosphorylation of GABAAγ2, suggesting that 14-3-3-θ and PKC-γ under the control of miR335 homeostatically regulate the phosphorylation and insertion of GABAAγ2 into the Purkinje cell post-synaptic membrane., (© 2014 The Author(s) Published by the Royal Society. All rights reserved.)

Sustained neuronal activity induces synaptic remodeling, in part, by altering gene expression. We have used a major climbing fiber pathway onto cerebellar Purkinje cells to investigate the effects of sustained climbing fiber-evoked glutamatergic synaptic transmission on transcription, expression and phosphorylation of proteins related to the regulation of inhibitory GABA(A) receptor function. Binocular horizontal optokinetic stimulation was used to modulate climbing fiber signals to Purkinje cells in the flocculus and nodulus of rabbits and mice. Purkinje cells in the flocculus and nodulus ipsilateral to the eye stimulated in the Posterior→Anterior direction received increased climbing fiber activity. Purkinje cells in flocculus and nodulus ipsilateral to the eye stimulated in the Anterior→Posterior direction received decreased climbing fiber activity. We identified changes in levels of gene transcripts in floccular and nodular Purkinje cells with the technique of differential display RT-PCR. Increased climbing fiber input reduced transcript levels and expression of GABA receptor-associated protein (GABARAP). Using a protein 'pull down' technique, we showed that GABARAP interacts with serine phosphorylated GABA(A)γ2, gephyrin and β-tubulin. Serine de-phosphorylation of GABA(A)γ2 reduced association of GABARAP with GABA(A)γ2. Climbing fiber activity did not influence the expression of GABA(A)γ2. Rather, it decreased its serine phosphorylation. Climbing fiber discharge decreased both expression of GABARAP and serine phosphorylation of GABA(A)γ2. Consequently, climbing fiber activity may reduce the surface expression of GABA(A) receptors in Purkinje cells rendering Purkinje cells less susceptible to interneuronal GABAergic inhibition., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

The cerebellum's role in sensory-motor control and adaptation is undisputed. However, a key hypothesis pertaining to the function of cerebellar circuitry lacks experimental support. It is universally assumed that the discharge of mossy fibers accounts for modulation of Purkinje cell "simple spikes" (SSs). This assumption acts as a prism through which all other functions of cerebellar circuitry are viewed. The vestibulo-cerebellum (nodulus and uvula) receives a large, unilateral, vestibular primary afferent mossy fiber projection. We can test its role in modulating Purkinje cell SSs by recording the modulated activity of both mossy fiber terminals and Purkinje cell SSs evoked by identical natural vestibular stimulation. Sinusoidal rotation about the longitudinal axis (roll) modulates the activity of vestibular primary afferent mossy and climbing fibers as well as Purkinje cell SSs and complex spikes (CSs). Remarkably, vestibular primary afferent mossy fibers discharge 180 degrees out of phase with SSs. This indicates that mossy fibers cannot account for SS modulation unless an inhibitory synapse is interposed between mossy fibers or vestibular climbing fibers and Purkinje cells. The authors review several experiments that address the relative contributions of mossy and climbing fiber afferents to the modulation of SSs. They conclude that climbing fibers, not mossy fibers, are primarily responsible for the modulation of SSs as well as CSs and they propose revised functions for these two afferent systems.

Long-term horizontal optokinetic stimulation (HOKS) decreases the gain of the horizontal optokinetic reflex and evokes the second phase of optokinetic afternystagmus (OKAN-II). We investigated the possible molecular constituents of this adaptation. We used a differential display reverse transcriptase-PCR screen for mRNAs isolated from retinas of rabbits that received HOKS. In each rabbit, we compared mRNAs from the retina stimulated in the posterior-->anterior (preferred) direction with mRNAs from the retina stimulated in the anterior-->posterior (null) direction. Acyl-CoA-binding protein (ACBP) mRNA was one of four mRNAs selected by this screen, the proteins of which interact with GABA receptors. HOKS in the preferred direction increased ACBP mRNA transcription and ACBP protein expression. ACBP was localized to Muller glial cells by hybridization histochemistry and by immunohistochemistry. ACBP interacts with the alpha1-subunit of the GABA(A) receptor, as determined by a yeast two-hybrid technique. This interaction was confirmed by coimmunoprecipitation of ACBP and the alpha1-subunit of the GABA(A) receptor using an antibody to GABA(A)alpha1. The interaction was also confirmed by a "pull-down" assay in which histidine-tagged ACBP was used to pull down the GABA(A)alpha1. ACBP does not cross the blood-brain barrier. However, smaller truncated proteolytic fragments of ACBP do, increasing the excitability of central cortical neurons. Muller cells may secrete ACBP in the inner plexiform layer, thereby decreasing the sensitivity of GABA(A) receptors expressed on the surface of ganglion cell dendrites. Because retinal directional sensitivity is linked to GABAergic transmission, HOKS-induced expression of ACBP could provide a molecular basis for adaptation to HOKS and for the genesis of OKAN-II.

Purkinje cells have two action potentials: Climbing fiber responses (CFRs) and simple spikes (SSs). CFRs reflect the discharge of a single climbing fiber at multiple synaptic sites on the proximal dendrite of the Purkinje cell. SSs reflect the summed action of a subset of parallel fiber synapses on Purkinje cell dendritic spines. Because mossy fiber afferents terminate on granule cells, the ascending axons of which bifurcate, giving rise to parallel fibers, the modulation of SSs has been attributed to mossy fiber afferent signals. This inference has never been tested. Conversely, the low discharge frequency of CFRs has led many to conclude that they have a unique and intermittent role in cerebellar signal processing. We examine the relative potency of vestibularly modulated mossy fiber and climbing fiber signals in evoking CFRs and SSs in Purkinje cells of the uvula-nodulus in chloralose-urethane-anesthetized rabbits. Vestibular primary afferents were blocked by unilateral labyrinthectomy (UL). A UL destroys the vestibular primary afferent signal to the ipsilateral uvula-nodulus, while leaving intact the vestibular climbing fiber signal from the contralateral inferior olive. After UL, vestibular stimulation modulated CFRs and SSs in ipsilateral uvula-nodular Purkinje cells, demonstrating that the primary vestibular afferent mossy fiber input to the ipsilateral uvula-nodulus was not necessary for SS modulation. Unilateral microlesions of the caudal half of the beta-nucleus of the inferior olive reduced a modulated climbing fiber signal to the contralateral uvula-nodulus, causing loss of both vestibularly modulated CFRs and SSs in contralateral Purkinje cells. Vestibular climbing fibers not only evoke low-frequency CFRs, but also indirectly modulate higher-frequency SSs. This modulation must be attributed to cerebellar interneurons. Golgi cell inhibition of granule cells may provide the interneuronal mechanism for CFR-induced SS modulation.

The nodulus receives a primary vestibular afferent input from the ipsilateral labyrinth and a vestibularly related climbing-fiber input originating from the contralateral labyrinth. Previously we demonstrated that increased discharge of vestibularly evoked climbing-fiber responses (CFRs) in nodular Purkinje cells was correlated with decreased discharge of simple spikes (SSs). This left unresolved the question of whether vestibularly evoked antiphasic behavior of CFRs and SSs reflects a common neural mechanism or the activation of two separate parallel pathways. We answered this question using natural vestibular stimulation to modulate the discharge of uvula-nodular Purkinje cells recorded extracellularly in unilaterally labyrinthectomized, chloralose urethane-anesthetized rabbits. In such animals, vestibular primary afferents projecting to the uvula-nodulus as mossy fibers remained intact on the side contralateral to the unilateral labyrinthectomy. The discharge of CFRs recorded in ipsilateral nodular Purkinje cells was increased by ipsilateral roll-tilt while the discharge of SSs was increased by contralateral roll-tilt. These polarities were reversed for Purkinje cells recorded in the contralateral uvula-nodulus. The polarity of SS discharge recorded from Purkinje cells on both sides of the nodulus was opposite to that of the vestibular primary mossy-fiber afferents. SSs continued to respond to contralateral roll-tilt even when the primary vestibular afferent mossy-fiber pathway was destroyed by the unilateral labyrinthectomy. Although the discharge of SSs recorded in the contralateral uvula-nodulus was increased by contralateral roll-tilt, this modulation was reduced relative to that observed in Purkinje cells recorded in the ipsilateral uvula-nodulus. We conclude that vestibularly evoked CFRs caused the modulation of SS discharge.

Spinal cord stimulation (SCS) is an effective tool in alleviating neuropathic pain. However, a number of well-selected patients fail to obtain satisfactory pain relief. Previous studies have demonstrated that i.t. baclofen and/or adenosine can enhance the SCS effect, but this combined therapy has been shown to be useful in less than half of the cases and more effective substances are therefore needed. The aim of this experimental study in rats was to examine whether gabapentin or pregabalin attenuates tactile allodynia following partial sciatic nerve injury and whether subeffective doses of these drugs can potentiate the effects of SCS in rats which do not respond to SCS. Mononeuropathy was produced by a photochemically induced ischaemic lesion of the sciatic nerve. Tactile withdrawal thresholds were assessed with von Frey filaments. Effects of increasing doses of gabapentin and pregabalin (i.t. and i.v.) on the withdrawal thresholds were analysed. These drugs were found to reduce tactile allodynia in a dose-dependent manner. In SCS non-responding rats, i.e. where stimulation per se failed to suppress allodynia, a combination of SCS and subeffective doses of the drugs markedly attenuated allodynia. In subsequent acute experiments, extracellular recordings from wide dynamic range neurones in the dorsal horn showed prominent hyperexcitability. The combination of SCS and gabapentin, at the same subeffective dose, clearly enhanced suppression of this hyperexcitability. In conclusion, electrical therapy and pharmacological therapy in neuropathic pain can, when they are inefficient individually, become effective when combined., (Copyright 2002 European Federation of Chapters of the International Association for the Study of Pain. Published by Elsevier Science Ltd.)

Experimental studies indicate that the effects of spinal cord stimulation (SCS) on 'hypersymptoms' in neuropathic pain conditions may at least partly be mediated via GABAergic and adenosine-dependent mechanisms. Concomitant intrathecal administration of receptor-active drugs modulating the function of the GABA and adenosine systems may both depress and enhance the effects of SCS. The first few patients with simultaneous intrathecal administration of the GABAB agonist baclofen and/or adenosine together with SCS, when the stimulation alone proved insufficient, are reported.

Changes in the background impulse activity of midbrain central gray substance neurons have been studied on slice preparations from the rat midbrain upon application of calcium-free solution, an activator of calcium channels, BAY-K 8644 (10 nM), organic (verapamil, 40 microM; D600, 10 microM; nifedipine, 1-10 microM; amiloride, 1 microM) and inorganic (Co2+, 1.5 mM) calcium channel blockers. Besides BAY-K 8644, all the substances inhibited most of the neurons studied. Verapamil, BAY-K 8644 and Co2+ also revealed facilitatory effects. Facilitatory action of BAY-K was most effective in silent neurons and in those previously inhibited by amiloride. Latent period values of inhibition in calcium-free solution and upon application of organic and inorganic blockers have the following sequence: D600 > amiloride > verapamil > Co2+ > nifedipine > calcium-free solution. Maximum rise time had the following order: amiloride > D600 > nifedipine > verapamil > Co2+ > calcium-free solution. Complete suppression of the neuronal activity induced by amiloride lasted twice as long as that induced by calcium-free solution, Co2+ and nifedipine, and six times as long as verapamil-induced suppression. Preliminary application of calcium channel blockers reduced facilitatory and increased inhibitory effects of serotonin and substance P. Data obtained are discussed with the supposition in mind that inhibition of the function of calcium channels in central gray substance neurons could be one of the mechanisms underlying the analgesic effect of a series of neurotropic agents after their introduction into this structure.