Neuronal and Molecular Mechanisms Underlying Chronic Pain and Depression Comorbidity in the Paraventricular Thalamus.

Patients with chronic pain often develop comorbid depressive symptoms, which makes the pain symptoms more complicated and refractory. However, the underlying mechanisms are poorly known. Here, in a repeated complete Freund's adjuvant (CFA) male mouse model, we reported a specific regulatory role of the paraventricular thalamic nucleus (PVT) glutamatergic neurons, particularly the anterior PVT (PVA) neurons, in mediating chronic pain and depression comorbidity (CDC). Our c-Fos protein staining observed increased PVA neuronal activity in CFA-CDC mice. In wild-type mice, chemogenetic activation of PVA glutamatergic neurons was sufficient to decrease the 50% paw withdrawal thresholds (50% PWTs), while depressive-like behaviors evaluated with immobile time in tail suspension test (TST) and forced swim test (FST) could only be achieved by repeated chemogenetic activation. Chemogenetic inhibition of PVA glutamatergic neurons reversed the decreased 50% PWTs in CFA mice without depressive-like symptoms and the increased TST and FST immobility in CFA-CDC mice. Surprisingly, in CFA-CDC mice, chemogenetically inhibiting PVA glutamatergic neurons failed to reverse the decrease of 50% PWTs, which could be restored by rapid-onset antidepressant S-ketamine. Further behavioral tests in chronic restraint stress mice and CFA pain mice indicated that PVA glutamatergic neuron inhibition and S-ketamine independently alleviate sensory and affective pain. Molecular profiling and pharmacological studies revealed the 5-hydroxytryptamine receptor 1D (Htr1d) in CFA pain-related PVT engram neurons as a potential target for treating CDC. These findings identified novel CDC neuronal and molecular mechanisms in the PVT and provided insight into the complicated pain neuropathology under a comorbid state with depression and related drug development.

Disinhibition of Mesolimbic Dopamine Circuit by the Lateral Hypothalamus Regulates Pain Sensation.

Our recent study demonstrated the critical role of the mesolimbic dopamine (DA) circuit and its brain-derived neurotropic factor (BDNF) signaling in mediating neuropathic pain. The present study aims to investigate the functional role of GABAergic inputs from the lateral hypothalamus (LH) to the ventral tegmental area (VTA; LHGABA→VTA) in regulating the mesolimbic DA circuit and its BDNF signaling underlying physiological and pathologic pain. We demonstrated that optogenetic manipulation of the LHGABA→VTA projection bidirectionally regulated pain sensation in naive male mice. Optogenetic inhibition of this projection generated an analgesic effect in mice with pathologic pain induced by chronic constrictive injury (CCI) of the sciatic nerve and persistent inflammatory pain by complete Freund's adjuvant (CFA). Trans-synaptic viral tracing revealed a monosynaptic connection between LH GABAergic neurons and VTA GABAergic neurons. Functionally, in vivo calcium/neurotransmitter imaging showed an increased DA neuronal activity, decreased GABAergic neuronal activity in the VTA, and increased dopamine release in the NAc, in response to optogenetic activation of the LHGABA→VTA projection. Furthermore, repeated activation of the LHGABA→VTA projection was sufficient to increase the expression of mesolimbic BDNF protein, an effect seen in mice with neuropathic pain. Inhibition of this circuit induced a decrease in mesolimbic BDNF expression in CCI mice. Interestingly, the pain behaviors induced by activation of the LHGABA→VTA projection could be prevented by pretreatment with intra-NAc administration of ANA-12, a TrkB receptor antagonist. These results demonstrated that LHGABA→VTA projection regulated pain sensation by targeting local GABAergic interneurons to disinhibit the mesolimbic DA circuit and regulating accumbal BDNF release.SIGNIFICANCE STATEMENT The mesolimbic dopamine (DA) system and its brain-derived neurotropic factor (BDNF) signaling have been implicated in pain regulation, however, underlying mechanisms remain poorly understood. The lateral hypothalamus (LH) sends different afferent fibers into and strongly influences the function of mesolimbic DA system. Here, utilizing cell type- and projection-specific viral tracing, optogenetics, in vivo calcium and neurotransmitter imaging, our current study identified the LHGABA→VTA projection as a novel neural circuit for pain regulation, possibly by targeting the VTA GABA-ergic neurons to disinhibit mesolimbic pathway-specific DA release and BDNF signaling. This study provides a better understanding of the role of the LH and mesolimbic DA system in physiological and pathological pain.

Neural and molecular investigation into the paraventricular thalamic-nucleus accumbens circuit for pain sensation and non-opioid analgesia.

The paucity of medications with novel mechanisms for pain treatment combined with the severe adverse effects of opioid analgesics has led to an imperative pursuit of non-opioid analgesia and a better understanding of pain mechanisms. Here, we identify the putative glutamatergic inputs from the paraventricular thalamic nucleus to the nucleus accumbens (PVTGlut→NAc) as a novel neural circuit for pain sensation and non-opioid analgesia. Our in vivo fiber photometry and in vitro electrophysiology experiments found that PVTGlut→NAc neuronal activity increased in response to acute thermal/mechanical stimuli and persistent inflammatory pain. Direct optogenetic activation of these neurons in the PVT or their terminals in the NAc induced pain-like behaviors. Conversely, inhibition of PVTGlut→NAc neurons or their NAc terminals exhibited a potent analgesic effect in both naïve and pathological pain mice, which could not be prevented by pretreatment of naloxone, an opioid receptor antagonist. Anterograde trans-synaptic optogenetic experiments consistently demonstrated that the PVTGlut→NAc circuit bi-directionally modulates pain behaviors. Furthermore, circuit-specific molecular profiling and pharmacological studies revealed dopamine receptor 3 as a candidate target for pain modulation and non-opioid analgesic development. Taken together, these findings provide a previously unknown neural circuit for pain sensation and non-opioid analgesia and a valuable molecular target for developing future safer medication.

Layer-specific impairment in the developing lateral entorhinal cortex of immune-challenged Disc1+/- mice.

Cognitive deficits in mental disorders result from dysfunctional activity in large-scale brain networks centred around the hippocampus and the prefrontal cortex. Dysfunctional activity emerges early during development and precedes the cognitive disabilities. The prefrontal-hippocampal network is driven by a prominent input from the lateral entorhinal cortex. We have previously shown that during early development, the entorhinal drive of the prefrontal-hippocampal network is impaired in a mouse model of mental disorders, yet the cellular substrate of this impairment is still poorly understood. Here, we address this question by a detailed characterization of projection neurons across the layers of the lateral entorhinal cortex in immune-challenged Disc1+/- mice at the beginning of the second postnatal week. We found that the activity and morphology of neurons in layers 2b and 3, which project to the hippocampus, are impaired. Neurons in layer 2b show increased spike-frequency adaptation, whereas neurons in layer 3 have reduced dendritic complexity but increased spike density. These findings identify the developmental alterations of entorhinal-hippocampal communication that underlie network dysfunction in immune-challenged Disc1+/- mice. KEY POINTS: Neonatal immune-challenged Disc1+/- mice show layer-specific changes in the lateral entorhinal cortex. Entorhinal layer 2b pyramidal neurons have increased spike-frequency adaptation. Reduced dendritic complexity but increased spine density characterize layer 3 pyramidal neurons.

Phosphorylation of Focal Adhesion Kinase at Y925: Role in Glia-Dependent and Independent Migration through Regulating Cofilin and N-Cadherin.

The adult neocortex is a six-layered structure, consisting of nearly continuous layers of neurons that are generated in a temporally strictly coordinated order. During development, cortical neurons originating from the ventricular zone migrate toward the Reelin-containing marginal zone in an inside-out arrangement. Focal adhesion kinase (FAK), one tyrosine kinase localizing to focal adhesions, has been shown to be phosphorylated at tyrosine 925 (Y925) by Src, an important downstream molecule of Reelin signaling. Up to date, the precise molecular mechanisms of FAK and its phosphorylation at Y925 during neuronal migration are still unclear. Combining in utero electroporation with immunohistochemistry and live imaging, we examined the function of FAK in regulating neuronal migration. We show that phosphorylated FAK is colocalized with Reelin positive Cajal-Retzius cells in the developing neocortex and hippocampus. Phosphorylation of FAK at Y925 is significantly reduced in reeler mice. Overexpression and dephosphorylation of FAK impair locomotion and translocation, resulting in migration inhibition and dislocation of both late-born and early-born neurons. These migration defects are highly correlated to the function of FAK in regulating cofilin phosphorylation and N-Cadherin expression, both are involved in Reelin signaling pathway. Thus, fine-tuned phosphorylation of focal adhesion kinase at Y925 is crucial for both glia-dependent and independent neuronal migration.

Sparser and Less Efficient Hippocampal-Prefrontal Projections account for Developmental Network Dysfunction in a Model of Psychiatric Risk Mediated by Gene-Environment Interaction.

Precise information flow from the hippocampus (HP) to prefrontal cortex (PFC) emerges during early development and accounts for cognitive processing throughout life. On flip side, this flow is selectively impaired in mental illness. In mouse models of psychiatric risk mediated by gene-environment interaction (GE), the prefrontal-hippocampal coupling is disrupted already shortly after birth. While this impairment relates to local miswiring in PFC and HP, it might be also because of abnormal connectivity between the two brain areas. Here, we test this hypothesis by combining in vivo electrophysiology and optogenetics with in-depth tracing of projections and monitor the morphology and function of hippocampal afferents in the PFC of control and GE mice of either sex throughout development. We show that projections from the hippocampal CA1 area preferentially target layer 5/6 pyramidal neurons and interneurons, and to a lesser extent layer 2/3 neurons of prelimbic cortex (PL), a subdivision of PFC. In neonatal GE mice, sparser axonal projections from CA1 pyramidal neurons with decreased release probability reach the PL. Their ability to entrain layer 5/6 oscillatory activity and firing is decreased. These structural and functional deficits of hippocampal-prelimbic connectivity persist, yet are less prominent in prejuvenile GE mice. Thus, besides local dysfunction of HP and PL, weaker connectivity between the two brain areas is present in GE mice throughout development.SIGNIFICANCE STATEMENT Poor cognitive performance in mental disorders comes along with prefrontal-hippocampal dysfunction. Recent data from mice that model the psychiatric risk mediated by gene-environment (GE) interaction identified the origin of deficits during early development, when the local circuits in both areas are compromised. Here, we show that sparser and less efficient connectivity as well as cellular dysfunction are the substrate of the weaker excitatory drive from hippocampus (HP) to prefrontal cortex (PFC) as well as of poorer oscillatory coupling between the two brain areas in these mice. While the structural and functional connectivity deficits persist during the entire development, their magnitude decreases with age. The results add experimental evidence for the developmental miswiring hypothesis of psychiatric disorders.

Developmental decrease of entorhinal-hippocampal communication in immune-challenged DISC1 knockdown mice.

The prefrontal-hippocampal dysfunction that underlies cognitive deficits in mental disorders emerges during early development. The lateral entorhinal cortex (LEC) is tightly interconnected with both prefrontal cortex (PFC) and hippocampus (HP), yet its contribution to the early dysfunction is fully unknown. Here we show that mice that mimic the dual genetic (G) -environmental (E) etiology (GE mice) of psychiatric risk have poor LEC-dependent recognition memory at pre-juvenile age and abnormal communication within LEC-HP-PFC networks throughout development. These functional and behavioral deficits relate to sparser projections from LEC to CA1 and decreased efficiency of axonal terminals to activate the hippocampal circuits in neonatal GE mice. In contrast, the direct entorhinal drive to PFC is not affected, yet the PFC is indirectly compromised, as target of the under-activated HP. Thus, the entorhinal-hippocampal circuit is already impaired from neonatal age on in GE mice.

Knock-Down of Hippocampal DISC1 in Immune-Challenged Mice Impairs the Prefrontal-Hippocampal Coupling and the Cognitive Performance Throughout Development.

Disrupted-in-schizophrenia 1 (DISC1) gene represents an intracellular hub of developmental processes. When combined with early environmental stressors, such as maternal immune activation, but not in the absence of thereof, whole-brain DISC1 knock-down leads to memory and executive deficits as result of impaired prefrontal-hippocampal communication throughout development. While synaptic dysfunction in neonatal prefrontal cortex (PFC) has been recently identified as one source of abnormal long-range coupling, the contribution of hippocampus (HP) is still unknown. Here, we aim to fill this knowledge gap by combining in vivo electrophysiology and optogenetics with morphological and behavioral assessment of immune-challenged mice with DISC1 knock-down either in the whole brain (GE) or restricted to pyramidal neurons in hippocampal CA1 area (GHPE). We found abnormal network activity, sharp-waves, and neuronal firing in CA1 that complement the deficits in upper layer of PFC. Moreover, optogenetic activating CA1 pyramidal neurons fails to activate the prefrontal local circuits. These deficits that persist till prejuvenile age relate to dendrite sparsification and loss of spines of CA1 pyramidal neurons. As a long-term consequence, DISC1 knock-down in HP leads to poorer recognition memory at prejuvenile age. Thus, DISC1-controlled developmental processes in HP in immune-challenged mice are critical for circuit function and cognitive behavior.

Glutamatergic drive along the septo-temporal axis of hippocampus boosts prelimbic oscillations in the neonatal mouse.

The long-range coupling within prefrontal-hippocampal networks that account for cognitive performance emerges early in life. The discontinuous hippocampal theta bursts have been proposed to drive the generation of neonatal prefrontal oscillations, yet the cellular substrate of these early interactions is still unresolved. Here, we selectively target optogenetic manipulation of glutamatergic projection neurons in the CA1 area of either dorsal or intermediate/ventral hippocampus at neonatal age to elucidate their contribution to the emergence of prefrontal oscillatory entrainment. We show that despite stronger theta and ripples power in dorsal hippocampus, the prefrontal cortex is mainly coupled with intermediate/ventral hippocampus by phase-locking of neuronal firing via dense direct axonal projections. Theta band-confined activation by light of pyramidal neurons in intermediate/ventral but not dorsal CA1 that were transfected by in utero electroporation with high-efficiency channelrhodopsin boosts prefrontal oscillations. Our data causally elucidate the cellular origin of the long-range coupling in the developing brain.

The toxic influence of paraquat on hippocampal neurogenesis in adult mice.

Paraquat, a fast-acting non-selective contact herbicide, is considered an etiological factor related to Parkinson's disease. This study investigated its effects on hippocampal neurogenesis and cognition in adult mice as well as possible mechanisms for the effects. We administered paraquat (1.25 mg/kg, intraperitoneal injection, i.p.) and an equal volume of normal saline for 3 weeks to adult male C57BL/6J mice. The results showed that hippocampus-dependent spatial learning and memory was significantly impaired in paraquat-treated mice. Moreover, paraquat administration inhibited the proliferation of neural progenitor cells, and impaired the survival and altered the fate decision of newly generated cells in the hippocampus. The expression levels of caspase-3 and glial fibrillary acidic protein were significantly higher in paraquat-treated mice than in control mice. Interestingly, paraquat reduced the phosphorylation of Akt, but did not affect the total amount of Akt. In conclusion, our findings suggest that paraquat negatively affected adult hippocampal neurogenesis and cognition function.

The function of sperm-associated antigen 6 in neuronal proliferation and differentiation.

Sperm-associated antigen 6 (SPAG6) is initially found in human testis and is essential for sperm motility and male fertility. Later studies indicate that it also express in the chick Central Nervous System and human embryonic stem cells. However, the function of Spag6 in cortical development is still largely unclear. Using in utero electroporation, we showed that overexpression of Spag6 induced the transfected cells excluded from the proliferation zone of the mouse cortex. Ki67 Co-labeling and BrdU incorporation experiment suggested that overexpression of Spag6 inhibited proliferation of neural progenitor cells. Furthermore, we demonstrated that Spag6-overexpressing cells preferred to differentiated into neurons, which could be labeled by Brn2, rather than GFAP positive astrocytes. Taken together, our data indicate that Spag6 plays an essential role in the process of neuronal proliferation and differentiation.

Reelin and cofilin cooperate during the migration of cortical neurons: a quantitative morphological analysis.

In reeler mutant mice, which are deficient in reelin (Reln), the lamination of the cerebral cortex is disrupted. Reelin signaling induces phosphorylation of LIM kinase 1, which phosphorylates the actin-depolymerizing protein cofilin in migrating neurons. Conditional cofilin mutants show neuronal migration defects. Thus, both reelin and cofilin are indispensable during cortical development. To analyze the effects of cofilin phosphorylation on neuronal migration we used in utero electroporation to transfect E14.5 wild-type cortical neurons with pCAG-EGFP plasmids encoding either a nonphosphorylatable form of cofilin 1 (cofilin(S3A)), a pseudophosphorylated form (cofilin(S3E)) or wild-type cofilin 1 (cofilin(WT)). Wild-type controls and reeler neurons were transfected with pCAG-EGFP. Real-time microscopy and histological analyses revealed that overexpression of cofilin(WT) and both phosphomutants induced migration defects and morphological abnormalities of cortical neurons. Of note, reeler neurons and cofilin(S3A)- and cofilin(S3E)-transfected neurons showed aberrant backward migration towards the ventricular zone. Overexpression of cofilin(S3E), the pseudophosphorylated form, partially rescued the migration defect of reeler neurons, as did overexpression of Limk1. Collectively, the results indicate that reelin and cofilin cooperate in controlling cytoskeletal dynamics during neuronal migration.

Spag6 Negatively Regulates Neuronal Migration During Mouse Brain Development.

Sperm-associated antigen 6 (Spag6) is a Chlamydomonas reinhardtii PF16 homologous gene detected in the human testis and is crucial for sperm motility. Neuronal migration is a dynamic process requiring coordinated cytoskeletal remodeling, and Spag6 is co-localized with microtubules in Chinese hamster ovary cells and COS-1 cells. However, the role of Spag6 in neuronal migration remains unclear. Here, we demonstrated that Spag6 was continuously expressed in the developing cerebral cortex. Using in utero electroporation (IUE), we found that overexpression of Spag6 delayed the rate of neuronal migration, rather than affecting the ultimate fate of cortical neurons. Furthermore, overexpression of Spag6 caused a significant decrease in neurite number and length of cortical neurons. Our results indicated that Spag6 controlled neuronal migration as well as neurite branching and elongation.

The mouse radial spoke protein 3 is a nucleocytoplasmic shuttling protein that promotes neurogenesis.

Radial spoke protein 3 (RSP3) was first identified in Chlamydomonas as a component of radial spoke, which is important for flagellar motility. The mammalian homolog of the Chlamydomonas RSP3 protein is found to be a mammalian protein kinase A-anchoring protein that binds ERK1/2. Here we show that mouse RSP3 is a nucleocytoplasmic shuttling protein. The full-length RSP3-EGFP fusion protein is mainly located in the cytoplasm of Chinese hamster ovary cells. However, by using deletion mutants of RSP3, we identified two nuclear localization signals and a nuclear export signal in RSP3. Moreover, using in utero electroporation, we found that overexpression of RSP3 in the developing cerebral cortex promotes neurogenesis. The layer II/III of the neocortex was much thicker in the RSP3-transfected region than that of the untransfected region in the neocortex. We also show that RSP3 is specifically located in the primary cilia of the radial glial cells, where it acts as a signaling mediator that regulates neurogenesis. Thus, our results suggest that RSP3 is a nucleocytoplasmic shuttling protein and plays an essential role in neurogenesis.

Morphological changes of radial glial cells during mouse embryonic development.

During brain development, the radial glial cell acts as a scaffold to support radial migration of postmitotic neurons. However, the morphological changes of radial glial cells during embryo development are poorly understood. We used in utero electroporation and immunohistochemistry to study the dynamics of radial glial cells accompanied by cortical development in mice from embryonic day 14 to postnatal day 0. We found that different segments of radial glial cells changed by the growth of different layers of cortex, such as marginal zone, cortical plate, intermediate zone and ventricular zone. Moreover, the length, angle and number of branches of the radial glial cell changed significantly at the late stage of neurogenesis. All these changes were consistent with the distinct phases of locomotion. Thus, we speculated that morphological changes of the radial glial cell were associated with the neuronal migration and dendritic development.

Exposure to swainsonine impairs adult neurogenesis and spatial learning and memory.

Swainsonine (SW) is an indolizidine triol plant alkaloid isolated from the species Astragalus, colloquially termed locoweed. Ingestion induces severe neurological symptoms of livestock and wildlife, including ataxia, trembling, exaggerated fright reactions. Toxicity to the central and peripheral nervous system is caused by inhibition of lysosomal a-mannosidase (AMA) and accumulation of intracellular oligosaccharide. However, the effects of SW on adult neurogenesis and cognition have remained unclear. Therefore, the present study was conducted to examine the effects of SW on adult neurogenesis and learning as well as memory performance in adult mice. SW (10µg/mL in drinking water) was administered orally to mice for 4 weeks. Our results showed that SW reduced proliferation and survival of neural progenitor cells (NPCs) in culture, and in the hippocampus of adult mice. In addition, exposure to SW led to down-regulation of doublecortin (DCX) and synaptophysin (SYP) in the hippocampus. However, caspase 3 and glial fibrillary acidic protein (GFAP) levels were significantly increased in SW-treated mice. Finally, SW-treated mice exhibited deficits in hippocampus-dependent spatial learning and memory. Our findings suggest that SW affects adult neurogenesis and cognitive function.

The SH2 domain is crucial for function of Fyn in neuronal migration and cortical lamination.

Neurons in the developing brain form the cortical plate (CP) in an inside-out manner, in which the late-born neurons are located more superficially than the early-born neurons. Fyn, a member of the Src family kinases, plays an important role in neuronal migration by binding to many substrates. However, the role of the Src-homology 2 (SH2) domain in function of Fyn in neuronal migration remains poorly understood. Here, we demonstrate that the SH2 domain is essential for the action of Fyn in neuronal migration and cortical lamination. A point mutation in the Fyn SH2 domain (FynR176A) impaired neuronal migration and their final location in the cerebral cortex, by inducing neuronal aggregation and branching. Thus, we provide the first evidence of the Fyn SH2 domain contributing to neuronal migration and neuronal morphogenesis.

The aspartic acid of Fyn at 390 is critical for neuronal migration during corticogenesis.

The mammalian cerebral cortex develops through the coordinated migration of postmitotic neurons. Fyn, a member of the Src tyrosine kinase family (SFKs), is involved in the neuronal migration and the absence of Fyn leads to abnormal migration. However, the molecular mechanism whereby Fyn acts on migrating neurons has remained unclear. Here, we employed two Fyn mutants (Fyn259T and FynD390A) to investigate the function of Fyn kinase domain in neuronal migration. Using in utero electroporation, we co-transfected the migrating neurons in embryonic cortex with these mutants combined with plasmid expressing GFP. Interestingly, although both of them impaired neuronal migration, FynD390A, rather than Fyn259T, induced remarkable morphology change. Our work provides in vivo and in vitro evidence that the aspartic acid of Fyn at 390 is indispensable for the radial migration, and it is required for precise cooperation with focal adhesion kinase.

Subcellular localization and function of mouse radial spoke protein 3 in mammalian cells and central nervous system.

Radial spoke protein 3 (RSP3) was first identified in Chlamydomonas as a component of the radial spoke. The mammalian homologue of the Chlamydomonas RSP3 gene is mainly expressed in testis and developing central nervous system (CNS). However, the subcellular localization and function of mammalian RSP3 in the developing brain and mammalian cells remain poorly understood. Here we show that the mouse RSP3 accumulates at the perinuclear region of Chinese hamster ovary (CHO) and 293T cells. Detailed analysis shows that the mouse RSP3 is not co-localized with the endoplasmic reticulum or Golgi apparatus markers in CHO cells. Using in utero electroporation, we found that over-expression of mammalian RSP3 increases the percentage of neurons reaching the upper cortical plate. In vivo analysis shows that the mouse RSP3 mainly accumulates in the proximal cytoplasmic dilation of the leading process of the migrating cortical neurons. Furthermore, we find that the mammalian RSP3 concentrates in the ependymal cilia as a component of the cilia. Thus, our data provide the first evidence for the subcellular localization and function of mammalian RSP3 in mammalian cells and developing CNS.

Effective suppression of acrylamide neurotoxicity by lithium in mouse.

The primary objective of this investigation was to assess the neuroprotective efficacy of lithium in an acrylamide (ACR)-induced neuropathy model in mice. In this study, Kunming male mice were administered ACR (25 mg/kg bw, i.p. once a day) with or without lithium (25 mg/kg bw, i.p. once a day) for 2 weeks. All ACR-administered mice exhibited severe symptoms of neuropathy. We found that treatment with lithium effectively alleviated behavioral deficits in animals elicited by acrylamide. Interestingly, the reduction of hippocampal neurogenesis resulting from ACR injection was promoted by administration of lithium. Further, lithium treatment significantly offset ACR-induced depletion in p-GSK-3ß (Ser9) levels in hippocampus. Collectively our findings suggest the propensity of lithium to attenuate ACR-induced neuropathy. Further studies are necessary to understand the precise molecular mechanism by which the lithium attenuates neuropathy. Nevertheless, our data clearly demonstrate the beneficial effects of lithium on ACR-induced neuropathy in mice and suggest its possible therapeutic application as an adjuvant in the management of other forms of neuropathy in humans.