Rhythmic gamma frequency light flickering ameliorates stress-related behaviors and cognitive deficits by modulating neuroinflammatory response through IL-12-Mediated cytokines production in chronic stress-induced mice.

Chronic stress enhances the risk for psychiatric disorders and induces depression and cognitive impairment. Gamma oscillations are essential for neurocircuit function, emotion, and cognition. However, the influence of gamma entrainment by sensory stimuli on specific aspects of chronic stress-induced responses remains unclear. Mice were subjected to corticosterone (CORT) administration and chronic restraint stress (CRS) for weeks, followed by rhythmic gamma frequency light flickering exposure. Local field potentials (LFPs) were recorded from the V1, CA1, and PFC regions to verify the light flicker on gamma oscillations. Behavioral tests were used to examine stress-related and memory-related behaviors. Golgi staining was performed to observe changes in spine morphology. Synaptosomes were isolated to determine the expression of synapse-related proteins through immunoblotting. RNA sequencing (RNA-seq) was applied to explore specific changes in the transcriptome. Immunofluorescence staining, real-time quantitative polymerase chain reaction (qPCR), and ELISA were used to evaluate microglial activation and cytokine levels. In this study, we demonstrated that rhythmic 40 Hz LF attenuated stress-related behavior and cognitive impairments by ameliorating the microstructural alterations in spine morphology and increasing the expression of GluN2A and GluA1 in chronically stressed mice. Transcriptome analysis revealed that significantly downregulated genes in LF-exposed CRS mice were enriched in neuroimmune-related signaling pathways. Rhythmic 40 Hz LF exposure significantly decreased the number of Iba1-positive microglia in the PFC and hippocampus, and the expression levels of the M1 markers of microglia iNOS and CD68 were reduced significantly in CRS mice. In addition, 40 Hz LF exposure suppressed the secretion of cytokines IL-12, which could regulate the production of IFN-γ and IL-10 in stressed mice. Our results demonstrate that exposure to rhythmic 40 Hz LF induces the neuroimmune response and downregulation of neuroinflammation with attenuated stress-related behaviors and cognitive function in CRS-induced mice. Our findings highlight the importance of sensory-evoked gamma entrainment as a potential therapeutic strategy for stress-related disorders treatment. Abbreviations: CORT, Chronic corticosterone treatment; CRS, Chronic restraint stress; IACUC, Institutional Animal Care and Use Committee; LF, light flickers; FST, Forced swim test; NSFT, Novelty-suppressed feeding test; SPT, Sucrose preference test; NSFT, Novelty-suppressed feeding; qPCR, Quantitative real-time polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PVDF, polyvinylidene fluoride; PBS, phosphate-buffered saline; PBS-T, phosphate-buffered saline plus 0.1% Tween 20; PVDF, polyvinylidene fluoride; GFAP, Glial fibrillary acidic protein; DAPI, 4',6-Diamid- ino-2-phenylindole; Iba1, Ionized calcium-binding adaptor molecule 1; iNOS, Inducible nitric oxide synthase; IL-10, Interleukin-10; IL6, Interleukin 6; IL-1ß, Interleukin 1ß; IL-12, Interleukin 12; TNF-α, Tumor necrosis factor alpha; IFN-γ, Interferon-gamma; TLR6 and 9, Toll-like Receptor 6 and 9.

VGLUT1 or VGLUT2 mRNA-positive neurons in spinal trigeminal nucleus provide collateral projections to both the thalamus and the parabrachial nucleus in rats.

The trigemino-thalamic (T-T) and trigemino-parabrachial (T-P) pathways are strongly implicated in the sensory-discriminative and affective/emotional aspects of orofacial pain, respectively. These T-T and T-P projection fibers originate from the spinal trigeminal nucleus (Vsp). We previously determined that many vesicular glutamate transporter (VGLUT1 and/or VGLUT2) mRNA-positive neurons were distributed in the Vsp of the adult rat, and most of these neurons sent their axons to the thalamus or cerebellum. However, whether VGLUT1 or VGLUT2 mRNA-positive projection neurons exist that send their axons to both the thalamus and the parabrachial nucleus (PBN) has not been reported. Thus, in the present study, dual retrograde tract tracing was used in combination with fluorescence in situ hybridization (FISH) for VGLUT1 or VGLUT2 mRNA to identify the existence of VGLUT1 or VGLUT2 mRNA neurons that send collateral projections to both the thalamus and the PBN. Neurons in the Vsp that send collateral projections to both the thalamus and the PBN were mainly VGLUT2 mRNA-positive, with a proportion of 90.3%, 93.0% and 85.4% in the oral (Vo), interpolar (Vi) and caudal (Vc) subnucleus of the Vsp, respectively. Moreover, approximately 34.0% of the collateral projection neurons in the Vc showed Fos immunopositivity after injection of formalin into the lip, and parts of calcitonin gene-related peptide (CGRP)-immunopositive axonal varicosities were in direct contact with the Vc collateral projection neurons. These results indicate that most collateral projection neurons in the Vsp, particularly in the Vc, which express mainly VGLUT2, may relay orofacial nociceptive information directly to the thalamus and PBN via axon collaterals.

Melatonin Suppresses Neuropathic Pain via MT2-Dependent and -Independent Pathways in Dorsal Root Ganglia Neurons of Mice.

Melatonin (Mel) and its receptors (MT1 and MT2) have a well-documented efficacy in treating different pain conditions. However, the anti-nociceptive effects of Mel and Mel receptors in neuropathic pain (NP) are poorly understood. To elucidate this process, pain behaviors were measured in a dorsal root ganglia (DRG)-friendly sciatic nerve cuffing model. We detected up-regulation of MT2 expression in the DRGs of cuff-implanted mice and its activation by the agonist 8-M-PDOT (8MP). Also, Mel attenuated the mechanical and thermal allodynia induced by cuff implantation. Immunohistochemical analysis demonstrated the expression of MT2 in the DRG neurons, while MT1 was expressed in the satellite cells. In cultured primary neurons, microarray analysis and gene knockdown experiments demonstrated that MT2 activation by 8MP or Mel suppressed calcium signaling pathways via MAPK1, which were blocked by RAR-related orphan receptor alpha (RORα) activation with a high dose of Mel. Furthermore, expression of nitric oxide synthase 1 (NOS1) was down-regulated upon Mel treatment regardless of MT2 or RORα. Application of Mel or 8MP in cuff-implanted models inhibited the activation of peptidergic neurons and neuro-inflammation in the DRGs by down-regulating c-fos, calcitonin gene-related peptide [CGRP], and tumor necrosis factor-1α [TNF-1α] and interleukin-1ß [IL-1ß]. Addition of the MT2 antagonist luzindole blocked the effects of 8MP but not those of Mel. In conclusion, only MT2 was expressed in the DRG neurons and up-regulated upon cuff implantation. The analgesic effects of Mel in cuff-implanted mice were closely associated with both MT2-dependent (MAPK-calcium channels) and MT2-independent (NOS1) pathways in the DRG.