RESUMEN
Nav1.7, one of tetrodotoxin-sensitive voltage-gated sodium channels, mainly expressed in the small diameter dorsal root ganglion (DRG) neurons. The expression and accumulation on neuronal membrane of Nav1.7 increased following peripheral tissue inflammation or nerve injury. However, the mechanisms for membrane accumulation of Nav1.7 remained unclear. We report that KIF5b, a highly expressed member of the kinesin-1 family in DRGs, promoted the translocation of Nav1.7 to the plasma membrane in DRG neurons of the rat. Following nociceptive behaviors in rats induced by peripheral spared nerve injury (SNI), synchronously increased KIF5b and Nav1.7 expressions were observed in DRGs. Immunohistochemistry staining demonstrated the co-expressions of KIF5b and Nav1.7 in the same DRG neurons. Immunoprecipitation experiments further confirmed the interactions between KIF5b and Nav1.7. Moreover, intrathecal injections of KIF5b shRNA moderated the SNI-induced both mechanical and thermal hyperalgesia. The rescued analgesic effects also alleviated SNI-induced anxiety-like behaviors. In sum, KIF5b was required for the membrane localizations of Nav1.7, which suggests a novel mechanism for the trafficking of Nav1.7 involved in neuropathic pain.
Asunto(s)
Neuralgia , Traumatismos de los Nervios Periféricos , Ratas , Animales , Ganglios Espinales , Ratas Sprague-Dawley , Neuralgia/metabolismo , Neuronas/metabolismo , HiperalgesiaRESUMEN
It has been proved that endomorphin-2 (EM2) produced obvious analgesic effects in the spinal dorsal horn (SDH), which existed in our human bodies with remarkable affinity and selectivity for the µ-opioid receptor (MOR). Our previous study has demonstrated that EM2 made synapses with the spinoparabrachial projection neurons (PNs) in the SDH and inhibited their activities by reducing presynaptic glutamate release. However, the morphological features of EM2 and the spinoparabrachial PNs in the SDH have not been completely investigated. Here, we examined the morphological features of EM2 and the spinoparabrachial PNs by using triple fluorescence and electron microscopic immunohistochemistry. EM2-immunoreactive (-ir) afferents directly contacted with the spinoparabrachial PNs in lamina I of the SDH. Immunoelectron microscopy (IEM) were used to confirm that these contacts were synaptic connections. It was also observed that EM2-ir axon terminals contacting with spinoparabrachial PNs in lamina I contained MOR, substance P (SP) and vesicular glutamate transporter 2 (VGLUT2). In lamina II, MOR-ir neurons were observed to receive direct contacts from EM2-ir varicosities. The synaptic connections among EM2, MOR, SP, VGLUT2, and the spinoparabrachial PNs were also confirmed by IEM. In sum, our results supply morphological evidences for the analgesic effects of EM2 on the spinoparabrachial PNs in the SDH.
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Post-traumatic stress disorder (PTSD) has become epidemic following severely stressful incidents. Previous studies have shown that brain-derived neurotrophic factor (BDNF) has anxiolytic effects on various anxiety or depression disorders including PTSD. However, the detailed mechanisms of BDNF for treating PTSD were rarely investigated. In the current study, single-prolonged stress (SPS) was used as an animal model recapitulating specific aspects for a PTSD-like phenotype. The effects of BDNF on SPS-induced anxiety-like behaviors were investigated. We showed that the levels of BDNF in the cerebro-spinal fluid (CSF) were significantly reduced after the rats experienced SPS. The SPS-induced reductions of percentages of time spent in the central area to total time in the open field test, were dose-dependently mitigated after BDNF intracerebroventricular (i.c.v.) injections. BDNF i.c.v. administration also dose-dependently increased the preference of the light box in the light-dark box test. Both expressions of tyrosine kinase receptor B (TrkB) protein and mRNA in the prefrontal cortex (PFC) and amygdala were significantly increased after SPS challenges. BDNF i.c.v. administration attenuated these compensatory increases of TrkB. At last, the anxiolytic effects of BDNF on SPS model were also observed by using other two injection methods. These results inspired us to study that different administrations of BDNF were used in patients with PTSD in the future, in-depthly.
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Ansiolíticos , Trastornos por Estrés Postraumático , Animales , Ansiolíticos/farmacología , Ansiedad/tratamiento farmacológico , Ansiedad/metabolismo , Trastornos de Ansiedad/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Modelos Animales de Enfermedad , Hipocampo/metabolismo , RatasRESUMEN
Activating primary afferent TRPV1-positive (TRPV1+) fibers in the spinal dorsal horn triggers exaggerated glutamate release and induces acute pain. However, whether the glutamate postsynaptic responses on dorsal horn neurons are regulated by excessive glutamate is unknown, largely due to intrinsic technical difficulties. In the present study, capsaicin, a specific TRPV1 agonist, was used to activate TRPV1+ fibers in the spinal dorsal horn. Combining three-dimensional (3-D) holographic photostimulation and whole-cell recordings on acute spinal cord slices from adult rodents, we found that postsynaptic glutamate responses were attenuated when activating TRPV1+ fibers with capsaicin. Electron microscopy and Western blot studies found that postsynaptic GluA1 (a subtype of ionotropic glutamate receptors) on the postsynaptic membrane was decreased by acute capsaicin treatment. Therefore, postsynaptic glutamate receptor occupancy and/or downmodulation may underlie this postsynaptic attenuation. Our data thus clarify a scenario in which postsynaptic glutamate responses are largely downregulated upon TRPV1+ activation, and this change may contribute to homeostasis in the dorsal horn circuit when "acute pain" occurs.
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Capsaicina , Ácido Glutámico , Animales , Capsaicina/farmacología , Potenciales Postsinápticos Excitadores , Dolor , Ratas , Ratas Sprague-Dawley , Médula Espinal/metabolismo , Asta Dorsal de la Médula Espinal/metabolismo , Transmisión Sináptica , Canales Catiónicos TRPV/metabolismoRESUMEN
Endomorphin-2 (EM2)-immunoreactive (ir) ï¬bers and terminals in the superï¬cial laminae (lamina I and II) of the spinal dorsal horn (SDH) primarily come from neurons in the ipsilateral dorsal root ganglion (DRG), which are important for nociceptive information transmission and modulation. However, the morphological features of EM2-ir neurons and fibers in the DRG and terminals in the SDH under ultrastructural levels have not been completely revealed. The present study observed the distributions of EM2-ir neurons, fibers and terminals in the DRG and SDH and detected their ultrastructural features using immunoelectron microscopy. EM2-ir neurons in the DRG are primarily small or medium in size and account for 17.2% of all neurons in the DRG. EM2-ir large dense-core granule vesicles (LDCVs) are dispersed in the cytoplasm and fibers. Most of the central processes of DRG neurons were thin myelinated and unmyelinated fibers and contained a few EM2-ir LDCVs. An intensive string of EM2-ir fibers with beads and terminals were observed in the superficial laminae of the SDH, other than EM2-ir neurons. EM2-ir products were also detected sparsely in the fibers and terminals. The average diameter of terminals was 94.41 ± 18.13 nm. EM2-ir terminals formed different types of synapses, most of which were asymmetrical (91%). EM2-ir LDCVs colocalized primarily with spherical small clear vesicles in asymmetrical synapses and flat vesicles in symmetrical synapses. The average length of postsynaptic dense zones (PSDs) measured in the asymmetrical synapses was 317.00 ± 31.67 nm. These results indicate that EM2-containing structures are distributed in the cytoplasm of DRG neurons, the central processes and terminals in the SDH and provide morphological evidence for the antinociceptive effects of EM2 in the SDH.
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Ganglios Espinales , Asta Dorsal de la Médula Espinal , Ratas , Animales , Oligopéptidos , Microscopía Inmunoelectrónica , Médula Espinal/fisiologíaRESUMEN
Empathic pain has attracted the interest of a substantial number of researchers studying the social transfer of pain in the sociological, psychological, and neuroscience fields. However, the neural mechanism of empathic pain remains elusive. Here, we establish a long-term observational pain model in mice and find that glutamatergic projection from the insular cortex (IC) to the basolateral amygdala (BLA) is critical for the formation of observational pain. The selective activation or inhibition of the IC-BLA projection pathway strengthens or weakens the intensity of observational pain, respectively. The synaptic molecules are screened, and the upregulated synaptotagmin-2 and RIM3 are identified as key signals in controlling the increased synaptic glutamate transmission from the IC to the BLA. Together, these results reveal the molecular and synaptic mechanisms of a previously unidentified neural pathway that regulates observational pain in mice.
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Complejo Nuclear Basolateral , Animales , Complejo Nuclear Basolateral/fisiología , Corteza Cerebral/fisiología , Ácido Glutámico/fisiología , Corteza Insular , Ratones , Dolor , SinapsisRESUMEN
Memory persistence is a fundamental cognitive process for guiding behaviors and is considered to rely mostly on neuronal and synaptic plasticity. Whether and how astrocytes contribute to memory persistence is largely unknown. Here, by using two-photon Ca2+ imaging in head-fixed mice and fiber photometry in freely moving mice, we show that aversive sensory stimulation activates α7-nicotinic acetylcholine receptors (nAChRs) in a subpopulation of astrocytes in the auditory cortex. We demonstrate that fear learning causes the de novo induction of sound-evoked Ca2+ transients in these astrocytes. The astrocytic responsiveness persisted over days along with fear memory and disappeared in animals that underwent extinction of learned freezing behavior. Conditional genetic deletion of α7-nAChRs in astrocytes significantly impaired fear memory persistence. We conclude that learning-acquired, α7-nAChR-dependent astrocytic responsiveness is an integral part of the cellular substrate underlying memory persistence.
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Astrocitos , Miedo , Receptor Nicotínico de Acetilcolina alfa 7 , Animales , Astrocitos/metabolismo , Aprendizaje , Ratones , Transmisión Sináptica , Receptor Nicotínico de Acetilcolina alfa 7/genética , Receptor Nicotínico de Acetilcolina alfa 7/metabolismoRESUMEN
In face of the everlasting battle toward COVID-19 and the rapid evolution of SARS-CoV-2, no specific and effective drugs for treating this disease have been reported until today. Angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV-2, mediates the virus infection by binding to spike protein. Although ACE2 is expressed in the lung, kidney, and intestine, its expressing levels are rather low, especially in the lung. Considering the great infectivity of COVID-19, we speculate that SARS-CoV-2 may depend on other routes to facilitate its infection. Here, we first discover an interaction between host cell receptor CD147 and SARS-CoV-2 spike protein. The loss of CD147 or blocking CD147 in Vero E6 and BEAS-2B cell lines by anti-CD147 antibody, Meplazumab, inhibits SARS-CoV-2 amplification. Expression of human CD147 allows virus entry into non-susceptible BHK-21 cells, which can be neutralized by CD147 extracellular fragment. Viral loads are detectable in the lungs of human CD147 (hCD147) mice infected with SARS-CoV-2, but not in those of virus-infected wild type mice. Interestingly, virions are observed in lymphocytes of lung tissue from a COVID-19 patient. Human T cells with a property of ACE2 natural deficiency can be infected with SARS-CoV-2 pseudovirus in a dose-dependent manner, which is specifically inhibited by Meplazumab. Furthermore, CD147 mediates virus entering host cells by endocytosis. Together, our study reveals a novel virus entry route, CD147-spike protein, which provides an important target for developing specific and effective drug against COVID-19.
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Basigina/genética , COVID-19/genética , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Animales , Basigina/inmunología , COVID-19/inmunología , COVID-19/patología , COVID-19/virología , Interacciones Huésped-Patógeno/inmunología , Humanos , Pulmón/inmunología , Pulmón/patología , Pulmón/virología , Ratones , Pandemias , Unión Proteica/inmunología , Dominios Proteicos/genética , Dominios Proteicos/inmunología , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/genética , Internalización del VirusRESUMEN
As one of the thalamic midline nuclei, the thalamic paraventricular nucleus (PVT) is considered to be an important signal integration site for many descending and ascending pathways that modulate a variety of behaviors, including feeding, emotions, and drug-seeking. A recent study has demonstrated that the PVT is implicated in the acute visceral pain response, but it is unclear whether the PVT plays a critical role in the central processing of chronic pain. Here, we report that the neurons in the posterior portion of the PVT (pPVT) and their downstream pathway are involved in descending nociceptive facilitation regarding the development of neuropathic pain conditions in male rats. Lesions or inhibition of pPVT neurons alleviated mechanical allodynia induced by spared nerve injury (SNI). The excitability of pPVT-central amygdala (CeA) projection neurons was significantly increased in SNI rats. Importantly, selective optogenetic activation of the pPVT-CeA pathway induced obvious mechanical hypersensitivity in naive rats. In addition, we used rabies virus (RV)-based and cell-type-specific retrograde transsynaptic tracing techniques to define a novel neuronal circuit in which glutamatergic neurons in the vlPAG were the target of the pPVT-CeA descending facilitation pathway. Our data suggest that this pPVTGlu+-CeA-vlPAGGlu+ circuit mediates central mechanisms of descending pain facilitation underlying persistent pain conditions.SIGNIFICANCE STATEMENT Studies have shown that the interactions between the posterior portion of the thalamic paraventricular nucleus (pPVT) and central amygdala (CeA) play a critical role in pain-related emotional regulation. However, most reports have associated this circuit with fear and anxiety behaviors. Here, an integrative approach of behavioral tests, electrophysiology, and immunohistochemistry was used to advance the novel concept that the pPVT-CeA pathway activation facilitates neuropathic pain processing. Using rabies virus (RV)-based and cell-type-specific retrograde transsynaptic tracing techniques, we found that glutamatergic neurons in the vlPAG were the target of the pPVT-CeA pathway. Thus, this study indicates the involvement of a pPVTGlu+-CeA-vlPAGGlu+ pathway in a descending facilitatory mechanism underlying neuropathic pain.
Asunto(s)
Núcleo Amigdalino Central/patología , Núcleos Talámicos de la Línea Media/patología , Vías Nerviosas/patología , Neuralgia/patología , Animales , Conducta Animal , Fenómenos Electrofisiológicos , Hiperalgesia/patología , Procesamiento de Imagen Asistido por Computador , Masculino , Neuralgia/psicología , Neuronas/patología , Nocicepción , Optogenética , Sustancia Gris Periacueductal/patología , Ratas , Ratas Sprague-DawleyRESUMEN
The dorsal medial prefrontal cortex (dmPFC) has been recognized as a key cortical area for nociceptive modulation. However, the underlying neural pathway and the function of specific cell types remain largely unclear. Here, we show that lesions in the dmPFC induced an algesic and anxious state. Using multiple tracing methods including a rabies-based transsynaptic tracing method, we outlined an excitatory descending neural pathway from the dmPFC to the ventrolateral periaqueductal gray (vlPAG). Specific activation of the dmPFC/vlPAG neural pathway by optogenetic manipulation produced analgesic and antianxiety effects in a mouse model of chronic pain. Inhibitory neurons in the dmPFC were specifically activated using a chemogenetic approach, which logically produced an algesic and anxious state under both normal and chronic pain conditions. Antagonists of the GABAA receptor (GABAAR) or mGluR1 were applied to the dmPFC, which produced analgesic and antianxiety effects. In summary, the results of our study suggest that the dmPFC/vlPAG neural pathway might participate in the maintenance of pain thresholds and antianxiety behaviors under normal conditions, while silencing or suppressing the dmPFC/vlPAG pathway might be involved in the initial stages and maintenance of chronic pain and the emergence of anxiety-like behaviors.
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Ansiolíticos/farmacología , Dolor Crónico , Vías Nerviosas , Corteza Prefrontal , Animales , Dolor Crónico/tratamiento farmacológico , Dolor Crónico/genética , Dolor Crónico/metabolismo , Dolor Crónico/fisiopatología , Ratones , Ratones Transgénicos , Vías Nerviosas/metabolismo , Vías Nerviosas/patología , Vías Nerviosas/fisiopatología , Optogenética , Corteza Prefrontal/metabolismo , Corteza Prefrontal/patología , Corteza Prefrontal/fisiopatología , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismoRESUMEN
The central medial nucleus (CM), a prominent cell group of the intralaminar nuclei (ILN) of the thalamus, and the ventrolateral periaqueductal gray matter (vlPAG) are two major components of the medial pain system. Whether vlPAG and CM are input sources of nociceptive information to the basolateral amygdala (BLA) and whether they are involved in neuropathic pain regulation remain unclear. Clarifying the hierarchical organization of these subcortical nuclei (vlPAG, CM, and BLA) can enhance our understanding on the neural circuits for pain regulation. Behavioral test results showed that a CM lesion made by kainic acid (KA) injection could effectively alleviate mechanical hyperalgesia 4, 6, and 8 days after spared nerve injury (SNI) surgery, with the symptoms returning after 10 days. Morphological studies revealed that: (1) the CM received afferents from vlPAG and sent efferents to BLA, indicating that an indirect vlPAG-CM-BLA pathway exists; (2) such CM-BLA projections were primarily excitatory glutamatergic neurons as revealed by fluorescence in situ hybridization; (3) the fibers originated from the CM-formed close contacts with both excitatory and inhibitory neurons in the BLA; and (4) BLA-projecting CM neurons expressed Fos induced by SNI and formed close contacts with fibers from vlPAG, suggesting that the vlPAG-CM-BLA indirect pathway was activated in neuropathic pain conditions. Finally, the vlPAG-CM-BLA indirect pathway was further confirmed using anterograde and monosynaptic virus tracing investigation. In summary, our present results provide behavioral and morphological evidence that the indirect vlPAG-CM-BLA pathway might be a novel pain pathway involved in neuropathic pain regulation.
RESUMEN
Spinal sensory transmission is under descending biphasic modulation, and descending facilitation is believed to contribute to chronic pain. Descending modulation from the brainstem rostral ventromedial medulla (RVM) has been the most studied, whereas little is known about direct corticospinal modulation. Here, we found that stimulation in the anterior cingulate cortex (ACC) potentiated spinal excitatory synaptic transmission and this modulation is independent of the RVM. Peripheral nerve injury enhanced the spinal synaptic transmission and occluded the ACC-spinal cord facilitation. Inhibition of ACC reduced the enhanced spinal synaptic transmission caused by nerve injury. Finally, using optogenetics, we showed that selective activation of ACC-spinal cord projecting neurons caused behavioral pain sensitization, while inhibiting the projection induced analgesic effects. Our results provide strong evidence that ACC stimulation facilitates spinal sensory excitatory transmission by a RVM-independent manner, and that such top-down facilitation may contribute to the process of chronic neuropathic pain.
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Giro del Cíngulo/fisiopatología , Bulbo Raquídeo/fisiopatología , Neuralgia/fisiopatología , Médula Espinal/fisiopatología , Nervio Sural/fisiopatología , Animales , Dolor Crónico , Estimulación Eléctrica , Giro del Cíngulo/diagnóstico por imagen , Giro del Cíngulo/metabolismo , Masculino , Bulbo Raquídeo/diagnóstico por imagen , Bulbo Raquídeo/metabolismo , Microscopía de Fluorescencia por Excitación Multifotónica , Imagen Molecular , Vías Nerviosas , Neuralgia/diagnóstico por imagen , Neuralgia/metabolismo , Neuronas/metabolismo , Neuronas/patología , Optogenética , Dimensión del Dolor , Traumatismos de los Nervios Periféricos , Ratas , Ratas Sprague-Dawley , Médula Espinal/diagnóstico por imagen , Médula Espinal/metabolismo , Nervio Sural/diagnóstico por imagen , Nervio Sural/metabolismo , Transmisión SinápticaRESUMEN
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.
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Neuronas/metabolismo , Núcleos Parabraquiales/metabolismo , Tálamo/metabolismo , Núcleo Espinal del Trigémino/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/genética , Proteína 2 de Transporte Vesicular de Glutamato/genética , Animales , Axones/metabolismo , Biotina/administración & dosificación , Biotina/análogos & derivados , Péptido Relacionado con Gen de Calcitonina/metabolismo , Dendritas/metabolismo , Dextranos/administración & dosificación , Formaldehído , Hibridación Fluorescente in Situ , Inyecciones Subcutáneas , Labio , Masculino , Proteínas Proto-Oncogénicas c-fos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Rodaminas/administración & dosificación , Estilbamidinas/administración & dosificación , Sinapsis/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/metabolismoRESUMEN
BACKGROUND/AIMS: Spinal dorsal horn (SDH) is one of the most important regions for analgesia produced by endomorphin-2 (EM2), which has a higher affinity and specificity for the µ-opioid receptor (MOR) than morphine. Many studies have focused on substantia gelatinosa (SG, lamina II) neurons to elucidate the cellular basis for its antinociceptive effects. However, the complicated types and local circuits of interneurons in the SG make it difficult to understand the real effects of EM2. Therefore, in the present study, we examined the effects of EM2 on projection neurons (PNs) in lamina I. METHODS: Tracing, immunofluoresence, and immunoelectron methods were used to examine the morphological connections between EM2-immunoreactive (-ir) terminals and PNs. By using in vitro whole cell patch clamp recording technique, we investigated the functional effects of EM2 on PNs. RESULTS: EM2-ir afferent terminals directly contacted PNs projecting to the parabrachial nucleus in lamina I. Their synaptic connections were further confirmed by immunoelectron microscopy, most of which were asymmetric synapses. It was found that EM2 had a strong inhibitory effect on the frequency, but not amplitude, of the spontaneous excitatory postsynaptic current (sEPSC) of the spinoparabrachial PNs in lamina I, which could be reversed by MOR antagonist CTOP. However, their spontaneous inhibitory postsynaptic current (sIPSC) and intrinsic properties were not changed after EM2 application. CONCLUSION: Applying EM2 to the SDH could produce analgesia through inhibiting the activities of the spinoparabrachial PNs in lamina I by reducing presynaptic neurotransmitters release from the primary afferent terminals.
RESUMEN
Motor impairment is one of the serious side-effects of morphine, which is an exogenous agonist of the µ-opioid receptor (MOR) as well as a widely used analgesic drug in clinical practice for chronic pain treatment. Endomorphins (EMs, including EM-1 and EM-2), the most effective and specific endogenous agonists of the MOR, exert more potent analgesia in acute and neuropathic pain than other opiates, such as morphine. Although EMs had fewer side-effects comparing to other opiates, motor impairment was still one unwanted reaction which limited its clinical application. In order to prevent and treat the motor impairment, it is critical to reveal the neural mechanisms underlying such locomotion disorder. The purpose of the present study was to reveal the neural mechanisms underlying the effects of EM-2 on the activity of motoneurons in the spinal ventral horn. First, we examine the distribution of EM-2-immunoreactive (IR) primary afferent fibers and their synaptic connections with the motoneurons innervating the skeletal muscles of the lower limb revealed by sciatic nerve retrograde tracing. The results showed that EM-2-IR fibers and terminals were sparsely observed in lamina IX and they formed symmetric synaptic connections with the motoneurons within lamina IX of the spinal ventral horn. Then, whole-cell patch-clamp technique was used to observe the effects of EM-2 on the spontaneous excitatory postsynaptic current (sEPSC) of motoneurons in lamina IX. The results showed that EM-2 could decrease both the frequency and amplitude of the sEPSC of the motoneurons in lamina IX, which was reversed by the MOR antagonist CTOP. These results indicate that EM-2-IR fibers originated from primary afferent fibers form symmetric synaptic connections with motoneurons innervating skeletal muscles of the lower limbs in lamina IX of the spinal ventral horn and EM-2 might exert inhibitory effects on the activities of these motoneurons through both presynaptic and postsynaptic mechanisms.
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Analgésicos Opioides/farmacología , Células del Asta Anterior/efectos de los fármacos , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Oligopéptidos/farmacología , Transmisión Sináptica/efectos de los fármacos , Animales , Células del Asta Anterior/citología , Células del Asta Anterior/metabolismo , Potenciales Postsinápticos Excitadores/fisiología , Masculino , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/inervación , Músculo Esquelético/metabolismo , Antagonistas de Narcóticos/farmacología , Neuronas Aferentes/citología , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/metabolismo , Ratas Sprague-Dawley , Receptores Opioides mu/agonistas , Receptores Opioides mu/antagonistas & inhibidores , Receptores Opioides mu/metabolismo , Nervio Ciático/citología , Nervio Ciático/efectos de los fármacos , Nervio Ciático/metabolismo , Somatostatina/análogos & derivados , Somatostatina/farmacología , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Sinapsis/ultraestructura , Transmisión Sináptica/fisiología , Técnicas de Cultivo de TejidosRESUMEN
Consciousness can be defined by two major attributes: awareness of environment and self, and arousal, which reflects the level of awareness. The return of arousal after general anesthesia presents an experimental tool for probing the neural mechanisms that control consciousness. Here we have identified that systemic or intracerebral injection of the cannabinoid CB1 receptor (CB1R) antagonist AM281 into the dorsomedial nucleus of the hypothalamus (DMH) - but not the adjacent perifornical area (Pef) or the ventrolateral preoptic nucleus of the hypothalamus (VLPO) - accelerates arousal in mice recovering from general anesthesia. Anesthetics selectively activated endocannabinoid (eCB) signaling at DMH glutamatergic but not GABAergic synapses, leading to suppression of both glutamatergic DMH-Pef and GABAergic DMH-VLPO projections. Deletion of CB1R from widespread cerebral cortical or prefrontal cortical (PFC) glutamatergic neurons, including those innervating the DMH, mimicked the arousal-accelerating effects of AM281. In contrast, CB1R deletion from brain GABAergic neurons or hypothalamic glutamatergic neurons did not affect recovery time from anesthesia. Inactivation of PFC-DMH, DMH-VLPO, or DMH-Pef projections blocked AM281-accelerated arousal, whereas activation of these projections mimicked the effects of AM281. We propose that decreased eCB signaling at glutamatergic terminals of the PFC-DMH projection accelerates arousal from general anesthesia through enhancement of the excitatory DMH-Pef projection, the inhibitory DMH-VLPO projection, or both.
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Endocannabinoides/fisiología , Hipotálamo/fisiología , Receptor Cannabinoide CB1/metabolismo , Transmisión Sináptica , Anestesia General , Animales , Nivel de Alerta , Neuronas GABAérgicas/fisiología , Hipotálamo/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Morfolinas/farmacología , Red Nerviosa/efectos de los fármacos , Red Nerviosa/fisiología , Pirazoles/farmacología , Ratas Sprague-Dawley , Receptor Cannabinoide CB1/antagonistas & inhibidoresRESUMEN
Opiates are commonly used analgesics that often cause clinical respiratory depression. However, their underlying mechanisms remain unclear. Endomorphin-2 (EM2) is a novel, endogenous tetrapeptide opioid with very high affinity and selectivity for the µ-opioid receptor (MOR). The pre-Bötzinger complex (pre-BötC) is considered the center of respiratory rhythm generation, and the synaptic connections in this region are essential for respiratory rhythm. The present study identified EM2-like immunoreactive (LI) axonal terminals in the pre-BötC of adult rats. Some EM2-LI axonal terminals made principally symmetric synapses with neurokinin 1 receptor (NK1R)-LI or MOR-LI neuronal dendritic processes in the pre-BötC. Unilateral microinjection of EM2 into the pre-BötC decreased breathing frequency and amplitude. A prior microinjection of the selective MOR antagonist ß-funaltrexamine (ß-FNA) into the pre-BötC prevented the effects of EM2. The present results suggest that EM2-LI axonal terminals modulate NK1R-expressing neurons in the pre-BötC and that EM2 plays a role in respiratory depression through MORs in the pre-BötC.
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Tronco Encefálico/metabolismo , Oligopéptidos/metabolismo , Receptores Opioides mu/metabolismo , Mecánica Respiratoria/fisiología , Animales , Tronco Encefálico/química , Tronco Encefálico/efectos de los fármacos , Masculino , Naltrexona/análogos & derivados , Naltrexona/farmacología , Antagonistas de Narcóticos/farmacología , Oligopéptidos/análisis , Oligopéptidos/farmacología , Unión Proteica/fisiología , Ratas , Ratas Sprague-Dawley , Receptores Opioides mu/análisis , Receptores Opioides mu/antagonistas & inhibidores , Mecánica Respiratoria/efectos de los fármacosRESUMEN
5-hydroxytryptamine (5-HT) transmission in the medial prefrontal cortex (mPFC) enhances or suppresses signal outflow to influence emotion-/cognition-based function performances and, putatively, the autonomic responses. The top-down cortical modulation of autonomic activities may be mediated in part through projections from mPFC to brain stem dorsal vagal complex (DVC). The abundant and heterogeneous densities of 5-HT fibers across laminae in mPFC suggest serotonergic innervation of mPFC-DVC projection neurons whereby endogenous 5-HT acts to regulate autonomic activities. The present study investigated the physical relationship between 5-HT fibers and the autonomic-related mPFC neurons by examining and quantitatively characterizing the 5-HT contacts upon retrogradely labeled mPFC-DVC projection neurons in pre- and infra-limbic cortices (PrL/IL) with light and electron microscopies combined with immunocytochemistry for 5-HT and presynaptic vesicle marker synaptophysin (Syn). 5-HT varicosities were observed, under confocal microscope, to form close appositions to or, at ultrastructural level, to form asymmetric axodendritic synapses and direct contacts upon the target neurons. About 16% of the entire 5-HTergic varicosities in lamina V of PrL/IL coexpressed Syn and about 24% of the peri-somatic 5-HTergic swellings demonstrated Syn-immunoreactivity (ir), suggesting a low frequency of putative synapses estimated at optical level. Ultrastructurally, examination of thirty-seven serially cut thin 5-HT boutons closely apposed to the labeled dendritic profiles demonstrated that only three contacts presented with identifiable asymmetric, synaptic membrane specializations. These data provide the first and direct morphological evidence supporting that endogenous 5-HT may be released mainly via direct contacts bearing no identifiable synaptic specializations as well as synapses, targeting autonomic-related mPFC neurons for autonomic regulation.
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Vías Autónomas/metabolismo , Tronco Encefálico/metabolismo , Neuronas/metabolismo , Corteza Prefrontal/metabolismo , Serotonina/metabolismo , Animales , Vías Autónomas/citología , Tronco Encefálico/citología , Inmunohistoquímica , Masculino , Microscopía Fluorescente , Microscopía Inmunoelectrónica , Técnicas de Trazados de Vías Neuroanatómicas , Neuronas/citología , Corteza Prefrontal/citología , Ratas Sprague-Dawley , Sinaptofisina/metabolismoRESUMEN
INTRODUCTION: We employed a nanosilver-collagen scaffold and tested its effects on inhibiting bacteria and facilitating nerve regeneration. METHODS: Based on our previous research, we prepared bionic scaffolds with different concentrations of nanosilver and examined their internal structures by scanning electron microscopy and energy dispersive spectroscopy. We implanted these scaffolds or autologous nerve grafts into rats to repair a 10-mm injury of the sciatic nerve. RESULTS: The 2 mg/ml group showed a >10 mm bacterial inhibition zone in all 3 types of bacterial culture dishes. At day 60 postsurgery, the 2 mg/ml group also showed the highest amplitude of evoked potential (AMP) and nerve conduction velocity (NCV). The regenerating nerves in the 2 mg/ml group were denser and more mature, and with thicker and well-arrayed myelin sheath. CONCLUSIONS: These results demonstrate that nanosilver scaffolds (2 mg/ml group) were effective in inhibiting bacteria both in vitro and in vivo, and reduced the contamination-caused immune responses, which in turn promoted nerve regeneration and functional recovery.
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Nanopartículas , Nervio Ciático/microbiología , Nervio Ciático/cirugía , Plata/farmacología , Ingeniería de Tejidos , Andamios del Tejido , Animales , Escherichia coli/efectos de los fármacos , Recuento de Leucocitos , Microscopía Electrónica de Transmisión , Modelos Animales , Músculo Esquelético/patología , Regeneración Nerviosa , Tamaño de los Órganos , Pseudomonas aeruginosa/efectos de los fármacos , Ratas Sprague-Dawley , Nervio Ciático/lesiones , Nervio Ciático/ultraestructura , Staphylococcus aureus/efectos de los fármacosRESUMEN
AIMS: The present study aims to test whether astrocytes contribute to glucocorticoid-mediated diabetic mechanical allodynia. METHODS: Streptozotocin (STZ)-induced diabetic rats were used in our study. The intrathecal operation was performed 21 days after the onset of diabetes. Diabetic mechanical allodynia was present 28 d after the onset of diabetes, and the mechanical threshold was tested using von Frey filaments. Immunohistochemistry, including immunofluorescent histochemical staining, was performed to observe the morphology of the spinal dorsal horn (SDH). Western blot analysis was employed as a semi-quantitative assay of the expression levels of GFAP and NDRG2 associated with diabetic mechanical allodynia. RESULTS: Diabetic rats displayed mechanical allodynia and activated astrocytes in the SDH 28 days after the onset of diabetes. This allodynia was attenuated by intrathecal administration of the astrocyte-specific inhibitor l-α-aminoadipate. In parallel, intrathecal injection of RU486, a glucocorticoid receptor antagonist, inhibited the activation of astrocytes in the SDH, alleviating the diabetes-induced mechanical allodynia. Furthermore, we found that dorsal horn astrocytes express abundant N-myc downstream-regulated gene 2 (NDRG2), which contributes to astrocyte reactivity. NDRG2 was over-expressed in activated astrocytes in diabetic rats with mechanical allodynia. Intrathecal injection of RU486 prevented the over-expression of NDRG2, which reversed the astrocyte reactivity and diabetic tactile allodynia. CONCLUSIONS: These results suggest that glucocorticoid-mediated over-expression of NDRG2 may contribute to the activation of dorsal horn astrocytes, which play a crucial role in diabetic mechanical allodynia. Thus, inhibiting glucocorticoid receptors and/or astrocyte reactivity in the SDH may be a therapeutic strategy for treating diabetic tactile allodynia.