RESUMEN

Brain ischemia is one of the leading causes of death and long-term disability worldwide. Cessation of the blood supply to the brain directly stimulates many pathological events, including glutamate overload and neuroinflammation. Glial cell activation occurs shortly after ischemia onset, resulting in the release of proinflammatory cytokines and exacerbation of the detrimental effects of neuroinflammation. Proinflammatory signals influence the infiltration of a wide range of immune cells, including neutrophils, T cells and monocytes/macrophages. In this study, we aimed to verify the potential anti-inflammatory effect of Chicago Sky Blue 6B (CSB6B) in a rat model of focal cerebral ischemia (90-minute middle cerebral artery occlusion). CSB6B was administered 2 h before (pretreatment) or 1.5 h after reperfusion onset (posttreatment). A model of ischemic preconditioning was used as the comparator to pretreatment with CSB6B. The results of indicated that posttreatment with CSB6B had profound anti-inflammatory effects that were associated with reduced neurological deficits and a decreased infarct volume. At 24 h, 3 days and 7 days after brain ischemia, CSB6B administration reduced the protein levels of proinflammatory cytokines, such as Il1ß, Il6, Il18 and TNFα, in the cerebral cortex and the dorsal striatum. Treatment with CSB6B also limited the scope of microglia and astrocyte activation and the infiltration of immune cells. Taken together, this study shows that compounds such as CSB6B might be promising pharmacological tools; however, further studies on the improvements in the drug-like properties of these compounds must be undertaken.

Asunto(s)

Isquemia Encefálica , Fármacos Neuroprotectores , Ratas , Animales , Enfermedades Neuroinflamatorias , Isquemia Encefálica/patología , Citocinas/metabolismo , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/complicaciones , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Modelos Animales de Enfermedad

RESUMEN

Brain aging involves regional alterations of specific cellular subpopulations in the human hippocampus: a network hub for memory consolidation. The present study investigates whether age, sex, education years, and the concentration of neuropathological and inflammatory proteins influence neuronal-type marker expression in the elderly hippocampus. We analyzed the digital images (1 µm/pixel) of postmortem hippocampal sections from 19 non-demented individuals (from 78 to 99 years). This material was obtained from the "Aging Dementia and TBI Study" open database. Brain samples were processed through in situ hybridization (ISH) for the immunodetection of VGLUT1 (glutamatergic transporter) and GAT1 (GABAergic transporter) and mRNAs and Luminex protein quantifications. After image acquisition, we delineated the dentate gyrus, CA 3/2, and CA1 hippocampal subdivisions. Then, we estimated the area fraction in which the ISH markers were expressed. Increased VGLUT1 was observed in multiple hippocampal subfields at late ages. This glutamatergic marker is positively correlated with beta-amyloid and tau proteins and negatively correlated with interleukin-7 levels. Additionally, education years are positively correlated with GAT1 in the hippocampus of elderly women. This GABAergic marker expression is associated with interferon-gamma and brain-derived neurotrophic factor levels. These associations can help to explain how hippocampal sub-regions and neurotransmitter systems undergo distinct physiological changes during normal aging.

Asunto(s)

Hipocampo , Neuronas , Humanos , Femenino , Anciano , Hipocampo/metabolismo , Neuronas/metabolismo , Envejecimiento/metabolismo , Neurotransmisores/metabolismo

RESUMEN

Background: Rapid eye movement (REM) sleep behavior disorder (RBD) predicts cognitive decline in Parkinson's disease (PD) patients without dementia. However, underlying mechanisms remain unknown. Accumulating studies suggest glutamatergic system dysregulation is associated. Objective: To examine the effect of RBD on the rate of cognitive decline in PD patients and investigate whether plasma levels of the neuroexosomal vesicular glutamate transporter-1 (VGLUT-1) and excitatory amino acid transporter-2 (EAAT-2) are altered in PD patients with RBD. Methods: This study included 157 newly diagnosed cognitive normal PD patients and 70 healthy controls (HCs). Based on one-night polysomnography recordings, the PD subjects were divided into PD with and without RBD (PD-RBD and PD-nRBD) groups. All participants received a complete clinical and neuropsychological evaluation at baseline. Plasma levels of neuroexosomal VGLUT-1 and EAAT-2 were measured by ELISA kits. After a 3-year follow-up, we evaluated baseline plasma levels of neuroexosomal glutamate transporters in each group as a predictor of cognitive decline using MoCA score changes over 3 years in regression models. Results: Plasma levels of neuron-derived exosomal EAAT-2 and VGLUT-1 were significantly lower in PD patients than in HCs. Plasma levels of neuroexosomal EAAT-2 were significantly lower in PD-RBD than PD-nRBD group at baseline. At the 3-year follow-up, PD-RBD patients presented greater cognitive decline. Lower baseline blood neuroexosomal EAAT-2 predicted cognitive decline over 3 years in PD-RBD patients (ß = 0.064, P = 0.003). Conclusion: These findings indicate that blood neuroexosomal EAAT-2 is associated with cognitive decline in PD with RBD.

RESUMEN

Neuropathic pain is a debilitating chronic condition provoked by a lesion in the nervous system and it induces functional alterations to the noradrenergic locus coeruleus (LC), affecting distinct dimensions of pain, like sensorial hypersensitivity, pain-induced depression, and anxiety. However, the neurobiological changes induced by nerve damage in the LC remain unclear. Here, we analyzed excitatory and inhibitory inputs to the LC, as well as the possible damage that noradrenergic neurons suffer after the induction of neuropathic pain through chronic constriction injury (CCI). Neuropathic pain was induced in male Sprague-Dawley rats, and the expression of the vesicular glutamate transporter 1 or 2 (VGLUT1 or VGLUT2), vesicular GABA transporter (VGAT), and cleaved caspase-3 (CC3) was analyzed by immunofluorescence 7 (CCI7d) or 28 days after the original lesion (CCI28d). While no significant differences in the density of VGLUT1 puncta were evident, CCI7d induced a significant increase in the perisomatic VGLUT2/VGAT ratio relative to Sham-operated and CCI28d animals. By contrast, when the entire region of LC is evaluated, there was a significant reduction in the density of VGLUT2 puncta in CCI28d animals, without changes in VGLUT2/VGAT ratio relative to the CCI7d animals. Additionally, changes in the noradrenergic soma size, and a lower density of mitochondria and lysosomes were evident in CCI28d animals. Interestingly, enhanced expression of the apoptotic marker CC3 was also evident in the CCI28d rats, mainly co-localizing with glial fibrillary acidic protein but not with any neuronal or noradrenergic marker. Overall, short-term pain appears to lead to an increase of markers of excitatory synapses in the perisomatic region of noradrenergic cells in the LC, an effect that is lost after long-term pain, which appears to activate apoptosis.

RESUMEN

Psychological stress is commonly accepted to be closely associated with masticatory muscle disorder, which is the main symptom of temporomandibular disorder (TMD). Previous studies have confirmed that exposure to stress may cause masticatory muscle hyperactivity. However, the central mechanism underlying this process remains unclear. The mesencephalic trigeminal nucleus (Vme), which resides in the brainstem, is the primary afferent center for masticatory proprioception and plays a key role in oral-motor movements by projecting to the trigeminal motor nucleus (Vmo). Therefore, the present study was designed to examine the role of Vme neurons in masseter overactivity induced by chronic stress. We found that subjecting mice to restraint stress (6 h/day) for 14 days caused significant anxiety-like behavior, obvious masseter overactivity, and markedly enhanced electrophysiological excitability of Vme neurons. By using anterograde tract tracing combined with immunofluorescence staining methods, we observed vesicular glutamate transporter 1 (VGLUT1)-positive glutamatergic projections from the Vme to the Vmo. Moreover, chronic restraint stress (CRS) elevated the expression of VGLUT1 and choline acetyltransferase (ChAT) in Vmo. Furthermore, administration of VGLUT1-targeted short hairpin RNA (shRNA) into the bilateral Vme significantly suppressed the enhanced overexcitability of Vme neurons, downregulated the overexpression of VGLUT1 and ChAT in the Vmo, and attenuated the elevated overactivity of the masseter caused by CRS. Taken together, we showed that CRS can excite neurons in the Vme, enhancing glutamatergic excitatory projections from the Vme to the Vmo and resulting in masseter muscle overactivity. These findings provide us with a novel central mechanism underlying the correlation between psychological factors and TMD.

RESUMEN

Occlusal disharmony has a negative impact on emotion. The mesencephalic trigeminal nucleus (Vme) neurons are the primary afferent nuclei that convey proprioceptive information from proprioceptors and low-threshold mechanoreceptors in the periodontal ligament and jaw muscles in the cranio-oro-facial regions. The dorsomedial part of the principal sensory trigeminal nucleus (Vpdm) and the ventral posteromedial nucleus (VPM) of thalamus have been proven to be crucial relay stations in ascending pathway of proprioception. The VPM sends numerous projections to primary somatosensory areas (SI), which modulate emotion processing. The present study aimed to demonstrate the ascending trigeminal-thalamic-cortex pathway which would mediate malocclusion-induced negative emotion. Unilateral anterior crossbite (UAC) model created by disturbing the dental occlusion was applied. Tract-tracing techniques were used to identify the existence of Vme-Vpdm-VPM pathway and Vpdm-VPM-SI pathway. Chemogenetic and optogenetic methods were taken to modulate the activation of VpdmVGLUT1 neurons and the Vpdm-VPM pathway. Morphological evidence indicated the involvement of the Vme-Vpdm-VPM pathway, Vpdm-VPM-SI pathway and VpdmVGLUT1-VPM pathway in orofacial proprioception in wild-type mice and vesicular glutamate transporter 1 (VGLUT1): tdTomato mice, respectively. Furthermore, chemogenetic inhibition of VpdmVGLUT1 neurons and the Vpdm-VPM pathway alleviated anxiety-like behaviors in a unilateral anterior crossbite (UAC) model, whereas chemogenetic activation induced anxiety-like behaviors in controls and did not aggravate these behaviors in UAC mice. Finally, optogenetic inhibition of the VpdmVGLUT1-VPM pathway in VGLUT1-IRES-Cre mice reversed UAC-induced anxiety comorbidity. In conclusion, these results suggest that the VpdmVGLUT1-VPM neural pathway participates in the modulation of malocclusion-induced anxiety comorbidity. These findings provide new insights into the links between occlusion and emotion and deepen our understanding of the impact of occlusal disharmony on brain dysfunction.

RESUMEN

Neurons in the trigeminal mesencephalic nucleus (Vme) have axons that branch peripherally to innervate the orofacial region and project centrally to several motor nuclei in brainstem. The dorsal motor nucleus of vagus nerve (DMV) resides in the brainstem and takes a role in visceral motor function such as pancreatic exocrine secretion. The present study aimed to demonstrate the presence of Vme-DMV circuit, activation of which would elicit a trigeminal neuroendocrine response. A masticatory dysfunctional animal model termed unilateral anterior crossbite (UAC) model created by disturbing the dental occlusion was used. Cholera toxin B subunit (CTb) was injected into the inferior alveolar nerve of rats to help identify the central axon terminals of Vme neurons around the choline acetyltransferase (ChAT) positive motor neurons in the DMV. The level of vesicular glutamate transporter 1 (VGLUT1) expressed in DMV, the level of acetylcholinesterase (AChE) expressed in pancreas, the level of glucagon and insulin expression in islets and serum, and the blood glucose level were detected and compared between UAC and the age matched sham-operation control mice. Data indicated that compared with the controls, there were more CTb/VGLUT1 double labeled axon endings around the ChAT positive neurons in the DMV of UAC groups. Mice in UAC group expressed a higher VGLUT1 protein level in DMV, AChE protein level in pancreas, glucagon and insulin level in islet and serum, and higher postprandial blood glucose level, but lower fasting blood glucose level. All these were reversed at 15-weeks when UAC cessation was performed from 11-weeks (all, P < 0.05). Our findings demonstrated Vme-DMV circuit via which the aberrant occlusion elicited a trigeminal neuroendocrine response such as alteration in the postprandial blood glucose level. Dental occlusion is proposed as a potential therapeutic target for reversing the increased postprandial glucose level.

Asunto(s)

Acetilcolinesterasa , Oclusión Dental , Animales , Ratones , Neuronas Motoras , Ratas , Ratas Sprague-Dawley , Nervio Vago

RESUMEN

Gulf War illness is associated with a combination of exposure to war-related chemical agents and traumatic stress. Currently, there are no effective treatments, and the pathophysiology remains elusive. Neurological problems are among the most commonly reported symptoms. In this study, we investigated the glutamatergic system in the hippocampi of mice exposed to war-related chemical agents and stress. Mice developed Gulf War illness-like symptoms, including mood deficits, cognitive impairments, and fatigue. They exhibited the following pathological changes in hippocampi: elevated extracellular glutamate levels, impaired glutamatergic synapses, astrocyte atrophy, loss of interneurons, and decreased neurogenesis. LDN/OSU-215111 is a small-molecule that can strengthen the structure and function of both the astrocytic processes and the glutamatergic synapses that together form the tripartite synapses. We found that LDN/OSU-215111 effectively prevented the development of mood and cognitive deficits in mice when treatment was implemented immediately following the exposure. Moreover, when symptoms were already present, LDN/OSU-215111 still significantly ameliorated these deficits; impressively, benefits were sustained one month after treatment cessation, indicating disease modification. LDN/OSU-215111 effectively normalized hippocampal pathological changes. Overall, this study provides strong evidence that restoration of tripartite glutamatergic synapses by LDN/OSU-215111 is a potential therapy for Gulf War illness.

RESUMEN

Exposure to prolonged stress is a major risk-factor for psychiatric disorders such as generalized anxiety and major depressive disorder. Human imaging studies have identified structural and functional abnormalities in the prefrontal cortex of subjects with depression and anxiety disorders, particularly Brodmann's area 25 (BA25). Further, deep brain stimulation of BA25 reduces symptoms of treatment-resistant depression. The rat homolog of BA25 is the infralimbic cortex (IL), which is critical for cognitive appraisal, executive function, and physiological stress reactivity. Previous studies indicate that the IL undergoes stress-induced changes in excitatory/inhibitory balance culminating in reduced activity of glutamate output neurons. However, the regulatory role of IL glutamate output in mood-related behaviors after chronic variable stress (CVS) is unknown. Here, we utilized a lentiviral-packaged small-interfering RNA to reduce translation of vesicular glutamate transporter 1 (vGluT1 siRNA), thereby constraining IL glutamate output. This viral-mediated gene transfer was used in conjunction with a quantitative anatomical analysis of cells expressing the stable immediate-early gene product FosB/ΔFosB, which accumulates in response to repeated neural activation. Through assessment of FosB/ΔFosB-expressing neurons across the frontal lobe in adult male rats, we mapped regions altered by chronic stress and determined the coordinating role of the IL in frontal cortical plasticity. Specifically, CVS-exposed rats had increased density of FosB/ΔFosB-expressing cells in the IL and decreased density in the insula. The latter effect was dependent on IL glutamate output. Next, we examined the interaction of CVS and reduced IL glutamate output in behavioral assays examining coping, anxiety-like behavior, associative learning, and nociception. IL glutamate knockdown decreased immobility during the forced swim test compared to GFP controls, both in rats exposed to CVS as well as rats without previous stress exposure. Further, vGluT1 siRNA prevented CVS-induced avoidance behaviors, while also reducing risk aversion and passive coping. Ultimately, this study identifies the necessity of IL glutamatergic output for regulating frontal cortical neural activity and behavior following chronic stress. These findings also highlight how disruption of excitatory/inhibitory balance within specific frontal cortical cell populations may impact neurobehavioral adaptation and lead to stress-related disorders.

RESUMEN

Changes in perineuronal nets (PNNs) after hearing loss were described in previous studies. The present study aimed to examine how single-sided deafness (SSD) affects the expression of excitatory and inhibitory synaptic transporters and PNNs in the primary auditory cortex (A1). Sprague-Dawley rats (8-week-old females, n = 30) were divided into three groups: (1) the SSD 2-week group (n = 10), (2) the SSD 4-week group (n = 10), and (3) the 4-week control group (n = 10). The expression levels of vesicular glutamate transporter 1 (VGLUT1), VGLUT2, vesicular GABA transporter (VGAT), and genes related to PNNs were measured using quantitative reverse transcription-polymerase chain reaction. The A1 was immunostained for VGLUT1, glutamate acid decarboxylase (GAD) 67, neurocan, aggrecan, brevican, and Wisteria floribunda agglutinin (WFA). The expression levels of VGLUT1, VGLUT2, and VGAT were elevated in the A1 on the ipsilateral side in the SSD groups compared with those in the control groups. Aggrecan expression was elevated in the A1 on the contralateral side in the SSD 2-week group. The SSD groups had elevated expression levels of metalloproteinase (MMP) 9 on the contralateral side. The presynaptic glutamatergic and GABAergic transporters were increased in the A1 on the ipsilateral side after induction of SSD. Changes in the cortical auditory nervous system accompanied changes in the PNNs and their degradation enzymes MMP9 and MMP14.

Asunto(s)

Corteza Auditiva , Sordera , Animales , Corteza Auditiva/metabolismo , Femenino , Metaloproteinasa 9 de la Matriz , Ratas , Ratas Sprague-Dawley , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/genética , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo

RESUMEN

The cerebellar lesions of bovine spongiform encephalopathy (BSE)-infected guinea pigs were characterized as severe atrophy of the cerebellar cortex associated with the loss of granule cells, decrease in the width of the molecular layer, and intense protease-resistant prion protein (PrPSc ) accumulations that are similar to cerebellar lesions in kuru and the VV2 type of sporadic Creutzfeldt-Jakob disease. The aim of this study is to assess the relationships between the distribution and localization of PrPSc and synapses expressing neurotransmitter transporters in order to reveal the pathogenesis of the disease. We used cell-type-specific immunohistochemical makers recognizing glutamatergic and γ-aminobutylic acid (GABA)ergic terminals to identify terminals impaired with PrPSc accumulations. The distribution of PrPSc accumulations and immunoreactivity of synaptic vesicles were studied throughout the neuroanatomical pathways in cerebellar lesions. Time course study demonstrated that PrPSc accumulation showed a tendency to spread from granular layer to molecular layer. The immunoreactivity of vesicular glutamate transporter 1 (VGluT1) was localized in axon terminals of cerebellar granule cells, and decreased in association with the severity of PrPSc accumulations and loss of granule cells. Immunoreactivities of vesicular glutamate transporter 2 (VGluT2) and vesicular GABA transporter (VGAT) that exist in axon terminals of inferior olivary neurons and GABAergic synapses of Purkinje cells, respectively, were preserved well in these lesions. In brainstem, VGluT1 immunoreactivity decreased selectively in pontine nuclei that are a component of the pontocerebellar pathway, although other neurotransmitter immunoreactivities were preserved well. Our findings suggest that the selective loss of VGluT1-immunoreactive synapses subsequent to PrPSc accumulations can contribute to the pathogenesis of cerebellar lesions of BSE-infected guinea pigs.

Asunto(s)

Cerebelo/patología , Encefalopatía Espongiforme Bovina/patología , Neuronas/patología , Proteínas PrPSc , Animales , Bovinos , Cerebelo/ultraestructura , Femenino , Cobayas , Inmunohistoquímica , Microscopía Electrónica de Transmisión , Neuronas/ultraestructura

RESUMEN

Background@#Depression affects approximately 5% of elderly people and its etiology might be related to chronic stress exposure during neurodevelopmental periods. In this study, we examined the effects of adolescent chronic social stress in aged mice on depressive behaviors and the excitatory-inhibitory (E/I) balance in stress-sensitive regions of the brain.@*Methods@#Sixty-four adolescent, male C57BL/6 mice were randomly assigned to either the 7-week (from post-natal days 29 to 77) social instability stress (stress group, n = 32) or normal housing conditions (control group, n = 32). At 15 months of age, 16 mice were randomly selected from each group for a series of behavioral tests, including two depression-related tasks (the sucrose preference test and the tail suspension test). Three days following the last behavioral test, eight mice were randomly selected from each group for immunohistochemical analyses to measure the cell density of parvalbumin (PV+)- and calretinin (CR+)-positive gamma-aminobutyric-acid (GABA)ergic inhibitory inter-neurons, and the expression levels of vesicular transporters of glutamate-1 (VGluT1) and vesicular GABA transporter (VGAT) in three stress-sensitive regions of the brain (the medial pre-frontal cortex [mPFC], hippocampus, and amygdala).@*Results@#Behaviorally, compared with the control group, adolescent chronic stress increased depression-like behaviors as shown in decreased sucrose preference (54.96 ± 1.97% vs. 43.11 ± 2.85%, t(22) = 3.417, P = 0.003) and reduced latency to immobility in the tail suspension test (92.77 ± 25.08 s vs. 33.14 ± 5.95 s, t(25) = 2.394, P = 0.025), but did not affect anxiety-like behaviors and pre-pulse inhibition. At the neurobiologic level, adolescent stress down-regulated PV+, not CR+, inter-neuron density in the mPFC (F(1, 39) = 19.30, P < 0.001), and hippocampus (F(1, 42) = 5.823, P = 0.020) and altered the CR+, not PV+, inter-neuron density in the amygdala (F(1, 28) = 23.16, P < 0.001). The VGluT1/VGAT ratio was decreased in all three regions (all F > 10.09, all P < 0.004), which suggests stress-induced hypoexcitability in these regions.@*Conclusions@#Chronic stress during adolescence increased depression-like behaviors in aged mice, which may be associated with the E/I imbalance in stress-sensitive brain regions.

RESUMEN

Unilateral anterior crossbite (UAC) has been demonstrated to cause masseter hyperactivity via the periodontal trigeminal mesencephalic nucleus (Vme)-trigeminal motor nucleus circuit. Here, we studied activation of motor neurons of the facial nucleus (VII), hypoglossal nucleus (XII), nucleus ambiguus (Amb), and spinal nucleus of the accessory nerve (SNA) in rats with UAC via their similar connections with Vme. An anterograde tracer, biotinylated dextran amine (BDA), was injected into the Vme to identify the central axon terminals around the motor neurons of VII, XII, Amb, and SNA. The expression of vesicular glutamate transporter 1 (VGLUT1) in neurons of VII, XII, Amb, and SNA, and the expression of acetylcholinesterase (AChE) were measured in the stapedius, lingualis, palatopharyngeal, and sternocleidomastoid muscles. In BDA-treated rats, many BDA-labeled cell bodies in the Vme and terminals in VII, XII, Amb, and SNA were identified. Compared with control rats, rats with UAC showed higher expression of VGLUT1 in these nuclei, and statistically significantly higher expression of AChE in the stapedius, lingualis, and sternocleidomastoid muscles, but not in the palatopharyngeal muscle. These findings suggest that UAC activates orofacial, head, and cervical multimotor behaviors via connections between the Vme and the corresponding motor nuclei.

Asunto(s)

Estimulantes del Sistema Nervioso Central , Maloclusión/complicaciones , Trastornos de la Articulación Temporomandibular/etiología , Trastornos de la Articulación Temporomandibular/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Acetilcolinesterasa/metabolismo , Animales , Colina O-Acetiltransferasa/metabolismo , Modelos Animales de Enfermedad , Núcleo Motor del Nervio Facial/metabolismo , Femenino , Proteínas Ligadas a GPI/metabolismo , Expresión Génica , Bulbo Raquídeo , Neuronas Motoras/metabolismo , Ratas , Ratas Sprague-Dawley , Trastornos de la Articulación Temporomandibular/patología

RESUMEN

Previous studies have shown that electroacupuncture (EA) promotes recovery of motor function in Parkinson's disease (PD). However the mechanisms are not completely understood. Clinically, the subthalamic nucleus (STN) is a critical target for deep brain stimulation treatment of PD, and vesicular glutamate transporter 1 (VGluT1) plays an important role in the modulation of glutamate in the STN derived from the cortex. In this study, a 6-hydroxydopamine (6-OHDA)-lesioned rat model of PD was treated with 100 Hz EA for 4 weeks. Immunohistochemical analysis of tyrosine hydroxylase (TH) showed that EA treatment had no effect on TH expression in the ipsilateral striatum or substantia nigra pars compacta, though it alleviated several of the parkinsonian motor symptoms. Compared with the hemi-parkinsonian rats without EA treatment, the 100 Hz EA treatment significantly decreased apomorphine-induced rotation and increased the latency in the Rotarod test. Notably, the EA treatment reversed the 6-OHDA-induced down-regulation of VGluT1 in the STN. The results demonstrated that EA alleviated motor symptoms and up-regulated VGluT1 in the ipsilateral STN of hemi-parkinsonian rats, suggesting that up-regulation of VGluT1 in the STN may be related to the effects of EA on parkinsonian motor symptoms via restoration of function in the cortico-STN pathway.

Asunto(s)

Electroacupuntura/métodos , Enfermedad de Parkinson Secundaria/terapia , Núcleo Subtalámico/metabolismo , Regulación hacia Arriba/fisiología , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Adrenérgicos/toxicidad , Animales , Apomorfina/farmacología , Modelos Animales de Enfermedad , Agonistas de Dopamina/farmacología , Lateralidad Funcional/efectos de los fármacos , Masculino , Haz Prosencefálico Medial/lesiones , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Oxidopamina/toxicidad , Enfermedad de Parkinson Secundaria/inducido químicamente , Enfermedad de Parkinson Secundaria/fisiopatología , Ratas , Ratas Sprague-Dawley , Núcleo Subtalámico/efectos de los fármacos , Núcleo Subtalámico/patología , Tirosina 3-Monooxigenasa/metabolismo , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

Emerging data have identified certain drugs such as scopolamine as rapidly acting antidepressants for major depressive disorder (MDD) that increase glutamate release and induce neurotrophic factors through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) activation in rodent models. However, little research has addressed the direct mechanisms of scopolamine on AMPAR activation or vesicular glutamate transporter 1 (VGLUT1)-mediated glutamate release in the prefrontal cortex (PFC) of mice. Herein, using a chronic unpredictable stress (CUS) paradigm, acute treatment with scopolamine rapidly reversed stress-induced depression-like behaviors in mice. Our results showed that CUS-induced depression-like behaviors, accompanied by a decrease in membrane AMPAR subunit 1 (GluA1), phosphorylated GluA1 Ser845 (pGluA1 Ser845), brain-derived neurotrophic factor (BDNF) and VGF (non-acronymic) and an increase in bicaudal C homolog 1 gene (BICC1) in the PFC of mice, and these biochemical and behavioral abnormalities were ameliorated by acute scopolamine treatments. However, pharmacological block of AMPAR by NBQX infusion into the PFC significantly abolished these effects of scopolamine. In addition, knock down of VGLUT1 by lentiviral-mediated RNA interference in the PFC of mice was sufficient to induce depression-like phenotype, to decrease extracellular glutamate accumulation and to cause similar molecular changes with CUS in mice. Remarkably, VGLUT1 knockdown alleviated the rapid antidepressant-like actions of scopolamine and the effects of scopolamine on membrane GluA1-mediated BDNF, VGF and BICC1 changes. Altogether, our findings suggest that VGLUT1-mediated glutamate release and membrane GluA1 activation may play a critical role in the rapid-acting antidepressant-like effects of scopolamine in mice.

Asunto(s)

Antidepresivos/uso terapéutico , Antagonistas Colinérgicos/uso terapéutico , Depresión/tratamiento farmacológico , Receptores AMPA/metabolismo , Escopolamina/uso terapéutico , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Animales , Antidepresivos/farmacología , Antagonistas Colinérgicos/farmacología , Depresión/etiología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ayuno/efectos adversos , Conducta Alimentaria/efectos de los fármacos , Preferencias Alimentarias/efectos de los fármacos , Ácido Glutámico/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Locomoción/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/metabolismo , Sacarina/administración & dosificación , Escopolamina/farmacología , Conducta Social , Estrés Psicológico/complicaciones , Natación/psicología , Privación de Agua

RESUMEN

Glutamate is concentrated into synaptic vesicles (SV) by the vesicular glutamate transporters (VGLUT) 1 and 2. VGLUTs also harbor a Na+/Pi-transport activity when residing at the plasma membrane. Here we aimed to identify whether the diurnal switches of VGLUT1 parallels interactions with or modification of endocytic proteins. VGLUT1 and dynamin bind to SH3 domains of either endophilin (Enph) or intersectin 1 (ITSN1) harboring one or five SH3 domains A-E, respectively. We followed diurnal variations by pull down experiments using SH3 fusion protein and brains from mice entrained in a strict 24-h light-dark cycle (12-h light Zeitgeber (ZT) 0, 6; 12-h dark ZT 12 and 18). In pull downs with EnphSH3 interaction with VGLUT1 is high during the resting light and reduced during the active dark period while dynamin binding does not vary. This diurnal light/dark pattern depends on a functional period 2 gene and changes when animals are kept in complete darkness. Pull downs using ITSN1SH3 A reveal diurnally varying binding of VGLUT1 with slightly reduced VGLUT1/dynamin ratios at the beginning of the light (ZT 0) or the dark (ZT 12) period. Phosphorylation increases binding of VGLUT1 but not of dynamin to EnphSH3. In contrast binding of dynamin to ITSN1SH3 A decreases under phosphorylating conditions with no changes in VGLUT1 binding. Phosphorylation of dynamin at Ser 774 is high at ZT 6 and ZT 18 when more VGLUT1 is at the plasma membrane but low at ZT 0 and ZT 12 the diurnal peaks of VGLUT1 endocytosis. In conclusion the diurnally varying endocytosis of VGLUT1 involves differential interactions with the SH3 domains of Enph and ITSN1 and correlates with the de-phosphorylation of dynamin1.

Asunto(s)

Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Encéfalo/metabolismo , Ritmo Circadiano/fisiología , Dinaminas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Animales , Membrana Celular/metabolismo , Endocitosis/fisiología , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Fosforilación , Fotoperiodo , Unión Proteica , Vesículas Sinápticas/metabolismo

RESUMEN

Previous studies have shown that electroacupuncture (EA) promotes recovery of motor function in Parkinson's disease (PD). However the mechanisms are not completely understood. Clinically, the subthalamic nucleus (STN) is a critical target for deep brain stimulation treatment of PD, and vesicular glutamate transporter 1 (VGluT1) plays an important role in the modulation of glutamate in the STN derived from the cortex. In this study, a 6-hydroxydopamine (6-OHDA)-lesioned rat model of PD was treated with 100 Hz EA for 4 weeks. Immunohistochemical analysis of tyrosine hydroxylase (TH) showed that EA treatment had no effect on TH expression in the ipsilateral striatum or substantia nigra pars compacta, though it alleviated several of the parkinsonian motor symptoms. Compared with the hemi-parkinsonian rats without EA treatment, the 100 Hz EA treatment significantly decreased apomorphine-induced rotation and increased the latency in the Rotarod test. Notably, the EA treatment reversed the 6-OHDA-induced down-regulation of VGluT1 in the STN. The results demonstrated that EA alleviated motor symptoms and up-regulated VGluT1 in the ipsilateral STN of hemi-parkinsonian rats, suggesting that up-regulation of VGluT1 in the STN may be related to the effects of EA on parkinsonian motor symptoms via restoration of function in the cortico-STN pathway.

Asunto(s)

Animales , Masculino , Ratas , Adrenérgicos , Toxicidad , Apomorfina , Farmacología , Modelos Animales de Enfermedad , Agonistas de Dopamina , Farmacología , Electroacupuntura , Métodos , Lateralidad Funcional , Haz Prosencefálico Medial , Heridas y Lesiones , Actividad Motora , Fisiología , Neuronas , Metabolismo , Oxidopamina , Toxicidad , Enfermedad de Parkinson Secundaria , Terapéutica , Ratas Sprague-Dawley , Núcleo Subtalámico , Metabolismo , Patología , Tirosina 3-Monooxigenasa , Metabolismo , Regulación hacia Arriba , Fisiología , Proteína 1 de Transporte Vesicular de Glutamato , Metabolismo

RESUMEN

The intrinsic cardiac nervous system modulates cardiac function by acting as an integration site for regulating autonomic efferent cardiac output. This intrinsic system is proposed to be composed of a short cardio-cardiac feedback control loop within the cardiac innervation hierarchy. For example, electrophysiological studies have postulated the presence of sensory neurons in intrinsic cardiac ganglia (ICG) for regional cardiac control. There is still a knowledge gap, however, about the anatomical location and neurochemical phenotype of sensory neurons inside ICG. In the present study, rat ICG neurons were characterized neurochemically with immunohistochemistry using glutamatergic markers: vesicular glutamate transporters 1 and 2 (VGLUT1; VGLUT2), and glutaminase (GLS), the enzyme essential for glutamate production. Glutamatergic neurons (VGLUT1/VGLUT2/GLS) in the ICG that have axons to the ventricles were identified by retrograde tracing of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) injected in the ventricular wall. Co-labeling of VGLUT1, VGLUT2, and GLS with the vesicular acetylcholine transporter (VAChT) was used to evaluate the relationship between post-ganglionic autonomic neurons and glutamatergic neurons. Sequential labeling of VGLUT1 and VGLUT2 in adjacent tissue sections was used to evaluate the co-localization of VGLUT1 and VGLUT2 in ICG neurons. Our studies yielded the following results: (1) ICG contain glutamatergic neurons with GLS for glutamate production and VGLUT1 and 2 for transport of glutamate into synaptic vesicles; (2) atrial ICG contain neurons that project to ventricle walls and these neurons are glutamatergic; (3) many glutamatergic ICG neurons also were cholinergic, expressing VAChT; (4) VGLUT1 and VGLUT2 co-localization occurred in ICG neurons with variation of their protein expression level. Investigation of both glutamatergic and cholinergic ICG neurons could help in better understanding the function of the intrinsic cardiac nervous system.

Asunto(s)

Ganglios Autónomos/citología , Ganglios Autónomos/metabolismo , Ácido Glutámico/metabolismo , Ventrículos Cardíacos/inervación , Neuronas/citología , Neuronas/metabolismo , Análisis de Varianza , Animales , Dermoscopía , Femenino , Glutaminasa/metabolismo , Ventrículos Cardíacos/metabolismo , Inmunohistoquímica , Masculino , Técnicas de Trazados de Vías Neuroanatómicas , Ratas Sprague-Dawley , Proteínas de Transporte Vesicular de Acetilcolina/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo

RESUMEN

Morphologically mixed chemical/electrical synapses at axon terminals, with the electrical component formed by gap junctions, is common in the CNS of lower vertebrates. In mammalian CNS, evidence for morphologically mixed synapses has been obtained in only a few locations. Here, we used immunofluorescence approaches to examine the localization of the neuronally expressed gap junction forming protein connexin36 (Cx36) in relation to the axon terminal marker vesicular glutamate transporter-1 (vglut1) in the spinal cord and the trigeminal motor nucleus (Mo5) of rat and mouse. In adult rodents, immunolabeling for Cx36 appeared exclusively as Cx36-puncta, and was widely distributed at all rostro-caudal levels in most spinal cord laminae and in the Mo5. A high proportion of Cx36-puncta was co-localized with vglut1, forming morphologically mixed synapses on motoneurons, in intermediate spinal cord lamina, and in regions of medial lamina VII, where vglut1-containing terminals associated with Cx36 converged on neurons adjacent to the central canal. Unilateral transection of lumbar dorsal roots reduced immunolabeling of both vglut1 and Cx36 in intermediate laminae and lamina IX. Further, vglut1-terminals displaying Cx36-puncta were contacted by terminals labeled for glutamic acid decarboxylase65, which is known to be contained in presynaptic terminals on large-diameter primary afferents. Developmentally, mixed synapses begin to emerge in the spinal cord only after the second to third postnatal week and thereafter increase to adult levels. Our findings demonstrate that axon terminals of primary afferent origin form morphologically mixed synapses containing Cx36 in broadly distributed areas of adult rodent spinal cord and Mo5.

Asunto(s)

Conexinas/metabolismo , Neuronas Motoras/metabolismo , Médula Espinal/metabolismo , Sinapsis/metabolismo , Animales , Sinapsis Eléctricas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Sprague-Dawley , Núcleos del Trigémino/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Aferentes Viscerales/metabolismo , Proteína delta-6 de Union Comunicante

RESUMEN

BACKGROUND: Images in biomedical imaging research are often affected by non-specific background noise. This poses a serious problem when the noise overlaps with specific signals to be quantified, e.g. for their number and intensity. A simple and effective means of removing background noise is to prepare a filtered image that closely reflects background noise and to subtract it from the original unfiltered image. This approach is in common use, but its effectiveness in identifying and quantifying synaptic puncta has not been characterized in detail. NEW ANALYSIS: We report on our assessment of the effectiveness of isolating punctate signals from diffusely distributed background noise using one variant of this approach, "Difference of Gaussian(s) (DoG)" which is based on a Gaussian filter. RESULTS: We evaluated immunocytochemically stained, cultured mouse hippocampal neurons as an example, and provided the rationale for choosing specific parameter values for individual steps in detecting glutamatergic nerve terminals. The intensity and width of the detected puncta were proportional to those obtained by manual fitting of two-dimensional Gaussian functions to the local information in the original image. COMPARISON WITH EXISTING METHODS: DoG was compared with the rolling-ball method, using biological data and numerical simulations. Both methods removed background noise, but differed slightly with respect to their efficiency in discriminating neighboring peaks, as well as their susceptibility to high-frequency noise and variability in object size. CONCLUSIONS: DoG will be useful in detecting punctate signals, once its characteristics are examined quantitatively by experimenters.

Asunto(s)

Técnica del Anticuerpo Fluorescente , Interpretación de Imagen Asistida por Computador , Neuronas/citología , Distribución Normal , Sinapsis/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Hipocampo/citología , Ratones , Microscopía Confocal , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo