Expression Of Vesicular Glutamate Transporter Research Articles

Delayed audiogenic seizure development in a genetic rat model is associated with overactivation of ERK1/2 and disturbances in glutamatergic signaling

Krushinsky-Molodkina (KM) rats genetically prone to audiogenic seizure are characterized by age-dependent expression of audiogenic seizures (AGS). It is known that the critical period of enhanced seizure susceptibility in rodents occurs at 2nd–3rd weeks of postnatal development. However, KM rats do not express AGS at this time-point, but start to demonstrate a stable AGS only after the age of 3 months. We hypothesized that this delay in AGS susceptibility in KM rats is genetically determined and may depend on some alterations in the development of the hippocampal glutamatergic system during the early postnatal period. We analyzed the expression and activity of seizure-related proteins, such as vesicular glutamate transporter 2 (VGLUT2), extracellular signal-regulated kinases 1 and 2 (ERK1/2), synapsin I, and NR2B subunit of the N-methyl-d-aspartate (NMDA) receptor (NR2B) in the hippocampus of KM rats during postnatal development. A significantly higher activity of ERK1/2 in KM rats was observed at 14th, 30th, and 60th days of postnatal development (P14, P30, P60) in comparison with control Wistar rats of the corresponding ages, while in adult (P120) KM rats it was at the same level with Wistar rats. Despite the increased activity of ERK1/2 at P14 and P30, the phosphorylation of synapsin I at Ser62/67 was significantly lower in the hippocampus of KM rats than in Wistar rats of the same ages; however, at P60 and P120, the phosphorylation of synapsin I was enhanced. Our data also revealed the increase of VGLUT2 and NR2B expression at P14, which dramatically decreased at the later stages. Our data indicate that a genetically determined increase in ERK1/2 kinase activity during postnatal ontogenesis in KM rats may be associated with the disturbances in synthesis and activity of the proteins, which are responsible for glutamatergic transmission in the KM rat hippocampus during the seizure susceptibility development.

Cortical Seizures in FoxG1+/- Mice are Accompanied by Akt/S6 Overactivation, Excitation/Inhibition Imbalance and Impaired Synaptic Transmission.

The correct morphofunctional shaping of the cerebral cortex requires a continuous interaction between intrinsic (genes/molecules expressed within the tissue) and extrinsic (e.g., neural activity) factors at all developmental stages. Forkhead Box G1 (FOXG1) is an evolutionarily conserved transcription factor, essential for the cerebral cortex patterning and layering. FOXG1-related disorders, including the congenital form of Rett syndrome, can be caused by deletions, intragenic mutations or duplications. These genetic alterations are associated with a complex phenotypic spectrum, spanning from intellectual disability, microcephaly, to autistic features, and epilepsy. We investigated the functional correlates of dysregulated gene expression by performing electrophysiological assays on FoxG1+/− mice. Local Field Potential (LFP) recordings on freely moving animals detected cortical hyperexcitability. On the other hand, patch-clamp recordings showed a downregulation of spontaneous glutamatergic transmission. These findings were accompanied by overactivation of Akt/S6 signaling. Furthermore, the expression of vesicular glutamate transporter 2 (vGluT2) was increased, whereas the level of the potassium/chloride cotransporter KCC2 was reduced, thus indicating a higher excitation/inhibition ratio. Our findings provide evidence that altered expression of a key gene for cortical development can result in specific alterations in neural circuit function at the macro- and micro-scale, along with dysregulated intracellular signaling and expression of proteins controlling circuit excitability.

Central changes in the Kv10.2 potassium channel in stress-induced hypertension rats.

Ion channels play as a pivotal role in hypertension in the processes of maintenance of vascular tone and sympathetic excitement of hypertension. The Kv10.2 channel (encoded by the Kcnh5 gene) belongs to the EAG voltage-gated superfamily. It is distributed widely in the brain, such as the hippocampus, the cortex, and the olfactory bulb. To date, the expression of Kv10.2 in central nervous system nuclei that regulates cardiovascular function and its inter-relationship with hypertension are still unclear. Here, electric foot-shock stressors with noise were used to establish the stress-induced hypertensive (SIH) rat model. The expression of Kv10.2 in the rostral ventrolateral medulla, the nucleus tractus solitarius, and the paraventricular nucleus (PVN) was examined by immunohistochemical staining and western blots. The following results were obtained: (a) the expression level of Kv10.2 was increased obviously in the paraventricular nucleus of SIH rats, whereas no significant difference was found in the rostral ventrolateral medulla and the nucleus tractus solitarius. (b) Kv10.2 was located in neurons. (c) Vesicular glutamate transporter 1 as a protein mark of glutamate neurons was increased in the paraventricular nucleus of the SIH group. (d) The expression of vesicular glutamate transporter 1 protein in neurons was significantly decreased when the Kcnh5 gene was knocked down by small interfering RNA in vitro. These findings indicate that the changes in Kv10.2 may be related to SIH, which may provide a potential avenue for further investigation of SIH.

The sensory thalamus and visual midbrain in mouse lemurs.

Mouse lemurs are the smallest of extant primates and are thought to resemble early primates in many ways. We provide histological descriptions of the major sensory nuclei of the dorsal thalamus and the superior colliculus (SC) of mouse lemurs (Microcebus murinus). The dorsal lateral geniculate nucleus has the six layers typical of strepsirrhine primates, with matching pairs of magnocellular, parvocellular, and koniocellular layers, one of each pair for each eye. Unlike most primates, magnocellular and parvocellular layers exhibit only small differences in cell size. All layers express vesicular glutamate transporter 2 (VGLUT2), reflecting terminations of retinal inputs, and the expression of VGLUT2 is much less dense in the koniocellular layers. Parvalbumin is densely expressed in all layers, while SMI-32 is densely expressed only in the magnocellular layers. The adjoining pulvinar complex has a posterior nucleus with strong VGLUT2 expression, reflecting terminations from the SC. The SC is laminated with dense expression of VGLUT2 in the upper superficial gray layer, reflecting terminations from the retina. The ventral (MGNv), medial, and dorsal divisions of the medial geniculate complex are only moderately differentiated, although patches of dense VGLUT2 expression are found along the outer border of MGNv. The ventroposterior nucleus has darkly stained cells in Nissl stained sections, and narrow septa separating patchy regions of dense VGLUT2 expression that likely represent different body parts. Overall, these structures resemble those in other strepsirrhine primates, although they are smaller, with the sensory nuclei appearing to occupy proportionately more of the dorsal thalamus than in larger primates.

Thymosin Alpha-1 Inhibits Complete Freund's Adjuvant-Induced Pain and Production of Microglia-Mediated Pro-inflammatory Cytokines in Spinal Cord.

Activation of inflammatory responses regulates the transmission of pain pathways through an integrated network in the peripheral and central nervous systems. The immunopotentiator thymosin alpha-1 (Tα1) has recently been reported to have anti-inflammatory and neuroprotective functions in rodents. However, how Tα1 affects inflammatory pain remains unclear. In the present study, intraperitoneal injection of Tα1 attenuated complete Freund's adjuvant (CFA)-induced pain hypersensitivity, and decreased the up-regulation of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) in inflamed skin and the spinal cord. We found that CFA-induced peripheral inflammation evoked strong microglial activation, but the effect was reversed by Tα1. Notably, Tα1 reversed the CFA-induced up-regulation of vesicular glutamate transporter (VGLUT) and down-regulated the vesicular γ-aminobutyric acid transporter (VGAT) in the spinal cord. Taken together, these results suggest that Tα1 plays a therapeutic role in inflammatory pain and in the modulation of microglia-induced pro-inflammatory cytokine production in addition to mediation of VGLUT and VGAT expression in the spinal cord.

Dental malocclusion stimulates neuromuscular circuits associated with temporomandibular disorders.

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.

Glutamatergic Projections to the Cochlear Nucleus are Redistributed in Tinnitus

Tinnitus alters auditory–somatosensory plasticity in the cochlear nucleus (CN). Correspondingly, bimodal auditory–somatosensory stimulation treatment attenuates tinnitus, both in animals and humans (Marks et al., 2018). Therefore, we hypothesized that tinnitus is associated with altered somatosensory innervation of the CN. Here, we studied the expression of vesicular glutamate transporters 1 and 2 (VGLUT1 and VGLUT2) in the CN, which reveals glutamatergic projections from the cochlea as well as somatosensory systems to this brainstem auditory center.Guinea pigs were unilaterally exposed to narrowband noise and behaviorally tested for tinnitus using gap-prepulse inhibition of the acoustic startle. Following physiological and behavioral measures, brain sections were immunohistochemically stained for VGLUT1 or VGLUT2. Puncta density was determined for each region of the ipsilateral and contralateral CN.Tinnitus was associated with an ipsilateral upregulation of VGLUT2 puncta density in the granule cell domain (GCD) and anteroventral CN (AVCN). Furthermore, there was a tinnitus-associated interaural asymmetry for VGLUT1 expression in the AVCN and deep layer of the dorsal CN (DCN3), due to contralateral downregulation of VGLUT1 expression. These tinnitus-related glutamatergic imbalances were reversed upon bimodal stimulation treatment.Tinnitus-associated ipsilateral upregulation of VGLUT2-positive projections likely derives from somatosensory projections to the GCD and AVCN. This upregulation may underlie the neurophysiological hallmarks of tinnitus in the CN. Reversing the increased ipsilateral glutamatergic innervation in the CN is likely a key mechanism in treating tinnitus.

Prox1 mRNA expression in the medial cortex of the turtle (Pseudemys scripta elegans)

The medial cortex of the cerebrum in reptiles is thought to be homologous to the mammalian dentate gyrus, based on cytoarchitectures, fiber connections, and neurochemical profiles. To support this hypothesis, we examined the mRNA expression of vesicular glutamate transporter 1 (vGluT1), a glutamatergic gene marker, and Prox1, a selective gene marker for granule cells of the dentate gyrus, in the turtle medial cortex (zone 2). Reverse transcription-polymerase chain reaction revealed the presence of both mRNAs in the turtle cerebrum. In situ hybridization of zone 2, which is a layer of densely packed neurons in Nissl stains, intensely expressed vGluT1 and Prox1. In zone 3, which is a loosely packed layer, vGluT1 was intensely expressed, whereas Prox1 signals gradated from strong to negative toward zone 4. These findings demonstrate that zone 2 contains glutamatergic neurons and expresses Prox1 mRNA and suggest that zone 2 in the turtle cerebrum is homologous to the mammalian dentate gyrus.

Repeated low level domoic acid exposure increases CA1 VGluT1 levels, but not bouton density, VGluT2 or VGAT levels in the hippocampus of adult mice

Domoic acid (DA) is a neurotoxin produced during harmful algal blooms that accumulates in marine organisms that serve as food resources for humans. While acute DA neurotoxicity can cause seizures and hippocampal lesions, less is known regarding how chronic, subacute DA exposure in adulthood impacts the hippocampus. With more frequent occurrences of harmful algal blooms, it is important to understand the potential impact of repeated, low-level DA exposure on human health. To model repeated, low-dose DA exposure, adult mice received a single low-dose (0.75 ± 0.05 μg/g) of DA or vehicle weekly for 22 consecutive weeks. Quantitative immunohistochemistry was performed to assess the effects of repeated, low-level DA exposure on hippocampal cells and synapses. Vesicular glutamate transporter 1 (VGluT1) immunoreactivity within excitatory boutons in CA1 of DA-exposed mice was increased. Levels of other vesicular transporter proteins (i.e., VGluT2 and the vesicular GABA transporter (VGAT)) within boutons, and corresponding bouton densities, were not significantly altered in CA1, CA3, or dentate gyrus. There were no significant changes in neuron density or glial fibrillary acidic protein (GFAP) immunoreactivity following chronic, low-dose exposure. This suggests that repeated low doses of DA, unlike high doses of DA, do not cause neuronal loss or astrocyte activation in hippocampus in adult mice. Instead, these findings demonstrate that repeated exposure to low levels of DA leads to subtle changes in VGluT1 expression within CA1 excitatory boutons, which may alter glutamatergic transmission in CA1 and disrupt behaviors dependent on spatial memory.

Nucleocytoplasmic export of HDAC5 and SIRT2 downregulation: two epigenetic mechanisms by which antidepressants enhance synaptic plasticity markers.

Antidepressant action has been linked to increased synaptic plasticity in which epigenetic mechanisms such as histone posttranslational acetylation could be involved. Interestingly, the histone deacetylases HDAC5 and SIRT2 are oppositely regulated by stress and antidepressants in mice prefrontal cortex (PFC). Besides, the neuroblastoma SH-SY5Y line is an in vitro neuronal model reliable to study drug effects with clear advantages over animals. We aimed to characterize in vitro the role of HDAC5 and SIRT2 in antidepressant regulation of neuroplasticity. SH-SY5Y cultures were incubated with imipramine, fluoxetine, and reboxetine (10μM, 2 and 24h) as well as the selective HDAC5 (MC3822, 5μM, 24h) or SIRT2 (33i, 5μM, 24h) inhibitors. The regulation of the brain-derived neurotrophic factor (BDNF), the vesicular glutamate transporter 1 (VGLUT1), the acetylated histones 3 (AcH3) and 4 (AcH4), HDAC5, and SIRT2 was studied. Comparatively, the long-term effects of these antidepressants (21days, i.p.) in the mice (C57BL6, 8weeks) PFC were studied. Antidepressants increased both in vitro and in vivo expression of BDNF, VGLUT1, AcH3, and AcH4. Moreover, imipramine and reboxetine increased the phosphorylated form of HDAC5 (P-HDAC5), mediating its cytoplasmic export. Further, SIRT2 was downregulated by all antidepressants. Finally, specific inhibition of HDAC5 and SIRT2 increased neuroplasticity markers. This study supports the validity of the SH-SY5Y model for studying epigenetic changes linked to synaptic plasticity induced by antidepressants as well as the effect of selective HDAC inhibitors. Particularly, nucleocytoplasmic export of HDAC5 and SIRT2 downregulation mediated by antidepressants could enhance synaptic plasticity markers leading to antidepressant action.

C9orf72 Dipeptide Repeats Cause Selective Neurodegeneration and Cell-Autonomous Excitotoxicity in Drosophila Glutamatergic Neurons.

The arginine-rich dipeptide repeats (DPRs) are highly toxic products from the C9orf72 repeat expansion mutations, which are the most common causes of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, the effects of DPRs in the synaptic regulation and excitotoxicity remain elusive, and how they contribute to the development of FTD is primarily unknown. By expressing DPRs with different toxicity strength in various neuronal populations in a Drosophila model, we unexpectedly found that Glycine-Arginine/Proline-Arginine (GR/PR) with 36 repeats could lead to neurodegenerative phenotypes only when they were expressed in glutamatergic neurons, including motor neurons. We detected increased extracellular glutamate and intracellular calcium levels in GR/PR-expressing larval ventral nerve cord and/or adult brain, accompanied by significant increase of synaptic boutons and active zones in larval neuromuscular junctions. Inhibiting the vesicular glutamate transporter expression or blocking the NMDA receptor in presynaptic glutamatergic motor neurons could effectively rescue the motor deficits and shortened life span caused by poly GR/PR, thus indicating a cell-autonomous excitotoxicity mechanism. Therefore, our results have revealed a novel mode of synaptic regulation by arginine-rich C9 DPRs expressed at more physiologically relevant toxicity levels and provided a mechanism that could contribute to the development of C9-related ALS and FTD.SIGNIFICANCE STATEMENT C9orf72 dipeptide repeats (DPRs) are key toxic species causing ALS/FTD, but their roles in synaptic regulation and excitotoxicity are unclear. Using C9orf72 DPRs with various toxicity strength, we have found that the arginine-rich DPRs cause selective degeneration in Drosophila glutamatergic neurons and revealed an NMDA receptor-dependent cell-autonomous excitotoxicity mechanism. Therefore, this study has advanced our understanding of C9orf72 DPR functions in synaptic regulation and excitotoxicity and provided a new mechanism that could contribute to the development of C9-related ALS and FTD.

Perinatal Exposure to Arsenic in Drinking Water Alters Glutamatergic Neurotransmission in the Striatum of C57BL/6 Mice.

Although exposure to arsenic (As) induces developmental neurotoxicity, there is a lack of data regarding its specific effects on glutamatergic neurotransmission in offspring from dams exposed to As during gestation and lactation. In this study, the body weight, glutamate content, and expression of vesicular glutamate transporter 2 (VGLUT2) and metabotropic glutamate receptors mGluR2 and mGluR3 was examined in the striatum of offspring following treatment of the dams with As (10 or 100mg/L NaAsO2 in drinking water). At postnatal day 21, body weight was decreased significantly, whereas the glutamate content in the striatum of offspring in the 100-mg/L As group were not significantly different from those in the control group. Although mGluR3 expression was not significantly different, VGLUT2 and mGluR2 expression was significantly lower in the striatum of offspring of As-exposed dams. These data indicate that altered glutamatergic neurotransmission may contribute to As-induced developmental neurotoxic effects.

Effects of neonatal MK-801 exposure on recognition memory and excitatory-inhibitory balance of hippocampus in adult female rats

Objective To investigate the long-term effects of repeated neonatal administration of dizocipline maleate (MK-801), the glutamate N-methyl-D-aspartate (NMDA) receptor antagonist, on recognition memory and hippocampal excitatory-inhibitory balance at the synaptic level in adult female rats. Methods Neonatal female Sprague-Dawley (SD) rats were randomly divided into model group and control group. Rats were administrated subcutaneously with MK-801 or normal saline from postnatal day (PND) 5 to PND14 (0.25 mg/kg, twice daily). (1) Object-in-context recognition test was performed on PND73-75. (2) The expression levels of vesicular glutamate transporter 1(VGLUT1) and vesicular GABA transporter(VGAT) in hippocampus were detected by immunohistochemical staining. Results (1) The preference index of model group for new objects was significantly lower than that of the control group (t=-2.762, P=0.012). (2) There was no significant difference in the expression of VGLUT1 in hippocampus of MK-801 mode group(P>0.05). Compared with control group(48.19±2.10), the VGAT level of model group in CA1(39.60±2.19) was lower. Compared with control group(CA1: (0.99±0.05), CA3: (1.28±0.02), the ratio of VGLUT1/VGAT was significantly upregulated in CA1(1.16±0.05)and CA3(1.44±0.03)(P<0.05). Conclusion Early NMDA receptor inhibition produces long-term deleterious effects on associative recognition memory and excitatory-inhibitory balance of hippocampus in female rats. These biochemical abnormalities may contribute to cognitive impairments observed in this study. Key words: MK-801; N-methyl-D-aspartate receptor; Recognition memory; Excitatory-inhibitory imbalance; Rat

The Antiepileptic Ketogenic Diet Alters Hippocampal Transporter Levels and Reduces Adiposity in Aged Rats.

Nutritional ketosis is induced by high fat/low carbohydrate dietary regimens, which produce high levels of circulating ketone bodies, shifting metabolism away from glucose utilization. While ketogenic diets (KD) were initially introduced to suppress seizures, they are garnering attention for their potential to treat a myriad of neurodegenerative and metabolic disorders that are associated with advanced age. The feasibility and physiological impact of implementing a long-term KD in old animals, however, has not been systematically examined. In this study, young and aged rats consumed a calorically- and nutritionally-matched KD or control diet for 12 weeks. All KD-fed rats maintained higher levels of BHB and lower levels of glucose relative to controls. However, it took the aged rats longer to reach asymptotic levels of BHB compared to young animals. Moreover, KD-fed rats had significantly less visceral white and brown adipose tissue than controls without a loss of lean mass. Interestingly, the KD led to significant alterations in protein levels of hippocampal transporters for monocarboxylates, glucose, and vesicular glutamate and gamma-aminobutyric acid. Most notably, the age-related decline in vesicular glutamate transporter expression was reversed by the KD. These data demonstrate the feasibility and potential benefits of KDs for treating age-associated neural dysfunction.

Quantitative Analysis of Mouse Dural Afferent Neurons Expressing TRPM8, VGLUT3, and NF200.

To quantify the abundance of dural afferent neurons expressing transient receptor potential channel melastatin 8 (TRPM8), vesicular glutamate transporter 3 (VGLUT3), and neurofilament 200 (NF200) in adult mice. With the increasing use of mice as a model system to study headache mechanisms, it is important to understand the composition of dural afferent neurons in mice. In a previous study, we have measured the abundance of mouse dural afferent neurons that express neuropeptide calcitonin gene-related peptide as well as two TRP channels TRPV1 and TRPA1, respectively. Here, we conducted quantitative analysis of three other dural afferent subpopulations in adult mice. We used the fluorescent tracer Fluoro-Gold to retrogradely label dural afferent neurons in adult mice expressing enhanced green fluorescent protein in discrete subpopulations of trigeminal ganglion (TG) neurons. Mechanoreceptors with myelinated fibers were identified by NF200 immunoreactivity. We also conducted Ca2+ -imaging experiments to test the overlap between TRPM8 and VGLUT3 expression in mouse primary afferent neurons (PANs). The abundance of TRPM8-expressing neurons in dural afferent neurons was significantly lower than that in total TG neurons. The percentages of dural afferent neurons expressing VGLUT3 and NF200 were comparable to those of total TG neurons, respectively. TRPM8 agonist menthol evoked Ca2+ influx in less than 7% VGLUT3-expressing PANs in adult mice. TG neurons expressing TRPM8, VGLUT3, and NF200 all innervate adult mouse dura. TRPM8 and VGLUT3 are expressed in distinct subpopulations of PANs in adult mice. These results provide an anatomical basis to investigate headache mechanisms in mouse models.

Glutamate Deregulation in Ketamine-Induced Psychosis-A Potential Role of PSD95, NMDA Receptor and PMCA Interaction.

Ketamine causes psychotic episodes and is often used as pharmacological model of psychotic-like behavior in animals. There is increasing evidence that molecular mechanism of its action is more complicated than just N-methyl-D-aspartic acid (NMDA) receptor antagonism and involves interaction with the components of calcium homeostatic machinery, in particular plasma membrane calcium pump (PMCA). Therefore, in this study we aimed to characterize brain region-specific effects of ketamine on PMCA activity, interaction with NMDA receptor through postsynaptic density protein 95 (PSD95) scaffolding proteins and glutamate release from nerve endings. In our study, ketamine induced behavioral changes in healthy male rats consistent with psychotic effects. In the same animals, we were able to demonstrate significant inhibition of plasma membrane calcium ATPase (PMCA) activity in cerebellum, hippocampus and striatum. The expression level and isoform composition of PMCAs were also affected in some of these brain compartments, with possible compensatory effects of PMCA1 substituting for decreased expression of PMCA3. Expression of the PDZ domain-containing scaffold protein PSD95 was induced and its association with PMCA4 was higher in most brain compartments upon ketamine treatment. Moreover, increased PSD95/NMDA receptor direct interaction was also reported, strongly suggesting the formation of multiprotein complexes potentially mediating the effect of ketamine on calcium signaling. We further support this molecular mechanism by showing brain region-specific changes in PSD95/PMCA4 spatial colocalization. We also show that ketamine significantly increases synaptic glutamate release in cortex and striatum (without affecting total tissue glutamate content), inducing the expression of vesicular glutamate transporters and decreasing the expression of membrane glutamate reuptake pump excitatory amino acid transporters 2 (EAAT2). Thus, ketamine-mediated PMCA inhibition, by decreasing total Ca2+ clearing potency, may locally raise cytosolic Ca2+ promoting excessive glutamate release. Regional alterations in glutamate secretion can be further driven by PSD95-mediated spatial recruitment of signaling complexes including glutamate receptors and calcium pumps, representing a novel mechanism of psychogenic action of ketamine.

The effects of unilateral cochlear ablation on the expression of vesicular glutamate transporter 1 in the lower auditory pathway of neonatal rats

ObjectivesUnilateral cochlear damage has profound effects on the central auditory pathways in the brain. MethodsWe examined the effects of unilateral cochlear ablation on VGLUT1 expression in the cochlear nucleus (CN) and the superior olivary complex (SOC) in neonatal rats. ResultsVGLUT1 expression in the CN subdivisions (the AVCN, the PVCN and the DCN-deep layers) and the SOC (the MnTB, the LSO and the MSO) ipsilateral to the ablated side was significantly suppressed by unilateral cochlear ablation. Interestingly, VGLUT1 expression in the PVCN and the DCN-deep layers contralateral to the ablated side was also reduced. ConclusionOur findings indicate that unilateral cochlear ablation affects VGLUT1 expression in the central auditory pathways not only ipsilateral but also contralateral to the ablated side.

Herpes simplex virus-mediated RNA interference targeting vesicular glutamate transporter 3 attenuates tactile allodynia in mice

To construct a replication-deficient herpes simplex virus (HSV-1) for delivering a short hairpin RNA (shRNA) targeting vesicular glutamate transporter 3 (VGLUT3) and observe its effect in alleviating allodynia in mice. The recombinant HSV-1 vector carrying the shRNA targeting Vglut3 (HSV-1-shvglut3) was constructed and inoculated in the sciatic nerve in a mouse model of mechanical allodynia to test its analgesia effect. Mechanical allodynia and heat hypersensitivity of the mice were tested by von Frey filaments and Hargreaves' test, respectively. VGLUT3 expression in the dorsal root ganglion (DRG) was evaluated by immunohistochemistry and Western blotting. Following inoculation in the sciatic nerve, the HSV vector HSV-1-shvglut3 was retrogradely transported to the DRG. Mechanical withdraw thresholds of the mouse models receiving HSV-1-shvglut3 inoculation were reversed to nearly the baseline level, and VGLUT3 expression in the DRG was down-regulated 2 weeks after vector inoculation. The analgesic effect lasted for over 2 weeks in these mice without obvious systematic side effects or changes in heat hypersensitivity threshold. Vglut3 in the DRG is a promising therapeutic target for alleviating mechanical allodynia, and HSV-1 vector-mediated RNA interference is safe and efficient for inducing long-lasting analgesia after peripheral inoculation of the vector.

Age-dependent alterations to paraventricular nucleus insulin-like growth factor 1 receptor as a possible link between sympathoexcitation and inflammation.

Modifications to neural circuits of the paraventricular hypothalamic nucleus (PVN) have been implicated in sympathoexcitation and systemic cardiovascular dysfunction. However, to date, the role of insulin-like growth factor 1 receptor (IGF-1R) expression on PVN pathophysiology is unknown. Using confocal immunofluorescence quantification and electrophysiological recordings from acute PVN slices, we investigated the mechanism through which age-dependent IGF-1R depletion contributes to the progression of inflammation and sympathoexcitation in the PVN of spontaneously hypertensive rats (SHR). Four and twenty weeks old SHR and Wistar Kyoto (WKY) rats were used for this study. Our data showed that angiotensin I/II and pro-inflammatory high mobility box group protein 1 (HMGB1) exhibited increased expression in the PVN of SHR versus WKY at 4weeks (p<0.01), and were even more highly expressed with age in SHR (p<0.001). This correlated with a significant decrease in IGF-1R expression, with age, in the PVN of SHR when compared with WKY (p<0.001) and were accompanied by related changes in astrocytes and microglia. In subsequent analyses, we found an age-dependent change in the expression of proteins associated with IGF-1R signaling pathways involved in inflammatory responses and synaptic function in the PVN. MAPK/ErK was more highly expressed in the PVN of SHR by the fourth week (p<0.001; vs. WKY), while expression of neuronal nitric oxide synthase (p<0.001) and calcium-calmodulin-dependent kinase II alpha (CamKIIα; p<0.001) were significantly decreased by the 4th and 20th week, respectively. Age-dependent changes in MAPK/ErK expression in the PVN correlated with an increase in the expression of vesicular glutamate transporter (p<0.001 vs. WKY), while decreased levels of CamKIIα was associated with a decreased expression of tyrosine hydroxylase (p<0.001) by the 20th week. In addition, reduced labeling for ϒ-aminobutyric acid in the PVN of SHR (p<0.001) correlated with a decrease in neuronal nitric oxide synthase labeling (p<0.001) when compared with the WKY by the 20th week. Electrophysiological recordings from neurons in acute slice preparations of the PVN of 4weeks old SHR revealed spontaneous post-synaptic currents of higher frequency when compared with neurons from WKY PNV slices of the same age (p<0.001; n=14 cells). This also correlated with an increase in PSD-95 in the PVN of SHR when compared with the WKY (p<0.001). Overall, we found an age-dependent reduction of IGF-1R, and related altered expression of associated downstream signaling molecules that may represent a link between the concurrent progression of synaptic dysfunction and inflammation in the PVN of SHR.

Reduced thermal sensitivity and increased opioidergic tone in the TASTPM mouse model of Alzheimer's disease.

Individuals with Alzheimer's disease (AD) are in susceptible patient groups in which pain is an important clinical issue that is often underdiagnosed. However, it is unclear whether decreased pain complaints in patients with AD result from elevated pain tolerance or an impaired ability to communicate sensations. Here, we explored if AD-related pathology is present in key regions of the pain pathway and assessed whether nociceptive thresholds to acute noxious stimulation are altered in the double-mutant APPswe × PS1.M146V (TASTPM) transgenic mouse model of AD. TASTPM mice exhibited an age-dependant cognitive deficit at the age of 6 months, but not at 4 months, a deficit that was accompanied by amyloid plaques in the cortex, hippocampus, and thalamus. In the spinal cord, β-amyloid (APP/Aβ) immunoreactivity was observed in dorsal and ventral horn neurons, and the expression of vesicular glutamate transporter 2 (VGLUT2) was significantly reduced, while the expression of the inhibitory peptides enkephalins was increased in TASTPM dorsal horn, consistent with an increased inhibitory tone. TASTPM mice displayed reduced sensitivity to acute noxious heat, which was reversed by naloxone, an opioid antagonist. This study suggests that increased inhibition and decreased excitation in the spinal cord may be responsible for the reduced thermal sensitivity associated with AD-related pathology.