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1.
Croat Med J ; 60(4): 352-360, 2019 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-31483121

RESUMO

AIM: To analyze the effects of glutamatergic agonists and antagonists on the activation of the A1 and A2 noradrenergic neurons localized in caudal ventrolateral medulla and nucleus tractus solitarii, respectively. METHODS: Rats were injected with glutamatergic agonists - kainic acid, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or N-methyl-D-aspartic acid (NMDA), and the brain sections were prepared for immunohistochemistry. Before agonist injections, antagonists - 6-cyano-7-nitroquinoxaline-2,3-dione or dizocilpine were administered. The expression of c-Fos, as the neuronal activation marker, and tyrosine hydroxylase (TH), as the marker of noradrenergic neurons was assessed with dual immunohistochemistry. The percentage of c-Fos-positive noradrenergic neurons relative to all TH-positive neurons in the respective areas of the brain stem was calculated. RESULTS: All three glutamatergic agonists significantly increased the number of the c-Fos-positive noradrenergic neurons in both the A1 and A2 area when compared with control animals. Kainic acid injection activated about 57% of TH-positive neurons in A1 and 40% in A2, AMPA activated 26% in A1 and 38% in A2, and NMDA 77% in A1 and 22% in A2. The injections of appropriate glutamatergic antagonists greatly decreased the number of activated noradrenergic neurons. CONCLUSION: Our results suggest that noradrenergic neurons are regulated and/or activated by glutamatergic system and that these neurons express functional glutamate receptors.


Assuntos
Neurônios Adrenérgicos/efeitos dos fármacos , Tronco Encefálico/efeitos dos fármacos , Fármacos atuantes sobre Aminoácidos Excitatórios/agonistas , Fármacos atuantes sobre Aminoácidos Excitatórios/antagonistas & inibidores , Animais , Feminino , Imuno-Histoquímica , Ácido Caínico/farmacologia , N-Metilaspartato/farmacologia , Proteínas Proto-Oncogênicas c-fos/biossíntese , Ratos , Ratos Sprague-Dawley , Núcleo Solitário/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/biossíntese , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologia
2.
Life Sci ; 232: 116621, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31269415

RESUMO

Kainate (KA) mouse model induced by intraperitoneal injection has been widely used for epilepsy and neurodegeneration studies. KA elicits sustained epileptic activity in mouse brain revealed by recurrent behavioral seizures, deteriorative neurodegeneration and various neurological deficits. However, to date, the vast majority of the studies used male mice only, and few studies on the comparison of brain injury between male and female mice in this model were reported. Epidemiological studies indicate that sex may affect the susceptibility to seizure response and neurodegeneration process. Therefore, this study focused on the effect of sex difference on KA-induced recurrent seizures and mortality, locomotor activity and cognitive impairment, and hippocampal neurodegeneration and reactive gliosis in mice. Our results showed that, compared to females, adult male mice exhibited worse performance in mortality rate, severity of epileptic seizures, and cognitive impairment indicated by novel object recognition task. Unexpectedly, post-KA male and female mice underwent similar decline and recovery of locomotor activity. KA-induced neurodegeneration in the whole hippocampus, particularly in CA1 and CA3 subregions, along with the deteriorative reactive gliosis in astrocytes and microglia, was more severe in males than that in females. These data provided the direct in vivo evidence that indicates the key role of sex difference in studies with KA mouse model, and this could be beneficial for optimizing the design of future studies.


Assuntos
Ácido Caínico/efeitos adversos , Ácido Caínico/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Disfunção Cognitiva/fisiopatologia , Modelos Animais de Doenças , Epilepsia/induzido quimicamente , Feminino , Gliose/induzido quimicamente , Hipocampo/efeitos dos fármacos , Ácido Caínico/farmacologia , Masculino , Camundongos , Microglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Convulsões/induzido quimicamente , Convulsões/fisiopatologia , Fatores Sexuais
3.
Exp Brain Res ; 237(10): 2481-2493, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31321447

RESUMO

Kainic acid (KA)-induced seizures and other experimental models of epilepsy have been proven to be instrumental in identifying novel targets that could be responsible for human icto- and epileptogenesis. We have previously shown that the ablation of pharmacoresistant voltage-gated Ca2+ channels with Cav2.3 as central ion-conducting pore (R-type Ca2+ channel) reduces the sensitivity towards KA-induced epilepsy in mice. In vivo, Cav2.3 channels are thought to be under tight allosteric control by endogenous loosely bound trace metal cations (Zn2+ and Cu2+) that suppress channel gating via a high-affinity trace metal-binding site. Metal dyshomeostasis in the brain, which is a common feature of (KA-induced) seizures, could therefore alter the normal function of Cav2.3 channels and may shift hippocampal and neocortical signaling towards hyperexcitation. To investigate the role of loosely bound metal ions for KA-induced hyperexcitation in vivo, we examined the effects of manipulating brain trace metal homeostasis in mice. To this end, we developed a murine system for intracerebroventricular administration of trace metal ions and/or histidine (His), which can bind Zn2+ and Cu2+ and is involved in their transendothelial transport at the blood-brain barrier. Unexpectedly, our preliminary findings indicate that application of His alone but not in the presence of Zn2+ has substantial beneficial effects on the outcome of KA-induced epilepsy in mice. As such, our results emphasize previous findings on the complex, two-sided role of loosely bound metal ions with regard to neuronal excitation and degeneration under pathophysiological conditions.


Assuntos
Hipocampo/efeitos dos fármacos , Histidina/farmacologia , Íons/metabolismo , Convulsões/tratamento farmacológico , Animais , Modelos Animais de Doenças , Histidina/administração & dosagem , Ácido Caínico/farmacologia , Camundongos Endogâmicos C57BL , Convulsões/induzido quimicamente , Transdução de Sinais/efeitos dos fármacos
4.
Cell Mol Neurobiol ; 39(7): 1039-1049, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31197744

RESUMO

Serotonin (5-HT) has been recognized as a neurotransmitter in the vertebrate retina, restricted mainly to amacrine and bipolar cells. It is involved with synaptic processing and possibly as a mitogenic factor. We confirm that chick retina amacrine and bipolar cells are, respectively, heavily and faintly immunolabeled for 5-HT. Amacrine serotonergic cells also co-express tyrosine hydroxylase (TH), a marker of dopaminergic cells in the retina. Previous reports demonstrated that serotonin transport can be modulated by neurotransmitter receptor activation. As 5-HT is diffusely released as a neuromodulator and co-localized with other transmitters, we evaluated if 5-HT uptake or release is modulated by several mediators in the avian retina. The role of different glutamate receptors on serotonin transport and release in vitro and in vivo was also studied. We show that L-glutamate induces an inhibitory effect on [3H]5-HT uptake and this effect was specific to kainate receptor activation. Kainate-induced decrease in [3H]5-HT uptake was blocked by CNQX, an AMPA/kainate receptor antagonist, but not by MK-801, a NMDA receptor antagonist. [3H]5-HT uptake was not observed in the presence of AMPA, thus suggesting that the decrease in serotonin uptake is mediated by kainate. 5-HT (10-50 µM) had no intrinsic activity in raising intracellular Ca2+, but addition of 10 µM 5-HT decreased Ca2+ shifts induced by KCl in retinal neurons. Moreover, kainate decreased the number of bipolar and amacrine cells labeled to serotonin in chick retina. In conclusion, our data suggest a highly selective effect of kainate receptors in the regulation of serotonin functions in the retinal cells.


Assuntos
Ácido Caínico/farmacologia , Retina/metabolismo , Serotonina/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Embrião de Galinha , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Neurotransmissores/metabolismo , Receptores de Glutamato/metabolismo , Receptores de Ácido Caínico/metabolismo , Retina/citologia , Retina/efeitos dos fármacos , Retina/embriologia , Neurônios Retinianos/efeitos dos fármacos , Neurônios Retinianos/metabolismo , Trítio/metabolismo
5.
J Neurophysiol ; 121(5): 1822-1830, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30892977

RESUMO

Sustained hypoxia (SH) activates chemoreceptors to produce cardiovascular and respiratory responses to bring the arterial partial pressure of O2 back to the physiological range. We evaluated the effect of SH (fraction of inspired O2 = 0.10, 24 h) on glutamatergic synaptic transmission and the interaction neuron-astrocyte in neurons of the nucleus tractus solitarii (NTS). Tractus solitarius (TS) fiber stimulation induced glutamatergic currents in neurons and astrocytes. SH increased α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate (AMPA/kainate) [-183 ± 122 pA (n = 10) vs. -353 ± 101 pA (n = 10)] and N-methyl-d-aspartate (NMDA) current amplitude [61 ± 10 pA (n = 7) vs. 102 ± 37 pA (n = 10)]. To investigate the effects of SH, we used fluoroacetate (FAC), an astrocytic inhibitor, which revealed an excitatory modulation on AMPA/kainate current and an inhibitory modulation of NMDA current in control rats. SH blunted the astrocytic modulation of AMPA [artificial cerebrospinal fluid (aCSF): -353 ± 101 pA vs. aCSF + FAC: -369 ± 76 pA (n = 10)] and NMDA currents [aCSF: 102 ± 37 pA vs. aCSF + FAC: 108 ± 32 pA (n = 10)]. SH increased AMPA current density [control: -6 ± 3.5 pA/pF (n = 6) vs. SH: -20 ± 12 pA/pF (n = 7)], suggesting changes in density, conductance, or affinity of AMPA receptors. SH produced no effect on astrocytic resting membrane potential, input resistance, and AMPA/kainate current. We conclude that SH decreased the neuron-astrocyte interaction at the NTS level, facilitating the glutamatergic transmission, which may contribute to the enhancement of cardiovascular and respiratory responses to baro- and chemoreflexes activation in SH rats. NEW & NOTEWORTHY Using an electrophysiological approach, we have shown that in nucleus tractus solitarii (NTS) from control rats, astrocytes modulate the AMPA and NMDA currents in NTS neurons, changing their excitability. Sustained hypoxia (SH) increased both glutamatergic currents in NTS neurons due to 1) a reduction in the astrocytic modulation and 2) an increase in the density of AMPA receptors. These new findings show the importance of neuron-astrocyte modulation in the excitatory synaptic transmission in NTS of control and SH rats.


Assuntos
Astrócitos/metabolismo , Ácido Glutâmico/farmacologia , Hipóxia/fisiopatologia , Núcleo Solitário/fisiopatologia , Transmissão Sináptica , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/fisiologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Fluoracetatos/farmacologia , Hipóxia/metabolismo , Ácido Caínico/farmacologia , Masculino , Potenciais da Membrana , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Ratos , Ratos Wistar , Núcleo Solitário/metabolismo
6.
Pharm Biol ; 57(1): 238-244, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30922159

RESUMO

CONTEXT: Osthole is a natural coumarin compound most frequently extracted from plants of the Apiaceae family such as Cnidium monnieri (L.) Cusson, Angelica pubescens Maxin.f., and Peucedanum ostruthium (L.). Osthole is considered to have potential therapeutic applications for the treatment of diseases including epilepsy. However, the mechanism of osthole induced-apoptosis in BV-2 microglia cells is not yet clear. OBJECTIVE: To investigate the molecular mechanisms underlying the effect of osthole on PI3K/AKt/mTOR expression in kainic acid (KA)-activated BV-2 microglia cells. MATERIALS AND METHODS: Optimal culture concentration and time of osthole were investigated by MTT assay. The concentration of osthole was tested from 10 to 400 µM and the culture time was tested from 2 to 72 h. Ultrastructure difference among control, KA and osthole group was analyzed under transmission electron microscope. The mRNA expression of PI3K/AKt/mTOR was investigated using reverse transcription (RT)-PCR and the protein expression was investigated using western blotting and immunofluorescence assay. Apoptosis rate of BV-2 cells between each group was measured by flow cytometry. RESULTS: IC50 for cell viability of BV-2 cells by osthole was 157.7 µM. Treated with osthole (140 µM) for 24 h significantly increased the inhibition rate. Pretreatment with osthole inhibited the KA-induced PI3K/AKt/mTOR mRNA and protein expression. The results of flow cytometry analysis showed that the apoptotic rate of osthole group was obviously higher than KA group. CONCLUSIONS: Date showed that osthole may be useful in the treatment of epilepsy and other neurodegenerative diseases that are characterized by over expression of PI3K/Akt/mTOR.


Assuntos
Cumarínicos/farmacologia , Microglia/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ácido Caínico/farmacologia , Camundongos , Microglia/citologia , Microglia/metabolismo , Transdução de Sinais/efeitos dos fármacos
7.
Cell Tissue Res ; 376(3): 309-323, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30788578

RESUMO

One of the major impacts of spinal cord injury (SCI) is the cerebellar neurological malfunction and deformation of its sub-layers. This could be due to the enormous innervation of the spinocerebellar tract from the posterior gray horn in the spinal cord to the ipsilateral cerebellum. Although the neuroprotective role of estradiol in spinal cord (SC) injuries, as well as its ability to delay secondary cell death changes, is well-known, its effect on cerebellar layers is not fully investigated. In this study, a SCI model was achieved by injection of Kainic acid into SC of adult Male Wistar rats in order to assess the effects of SCI on the cerebellum. The animals were classified into SCI group (animals with SCI), estradiol-treated group (animals with SCI and received estradiol), control groups, and sham control group. The microscopical examination 24 h after induction of SCI revealed that KA induced the most characteristics of neurodegeneration including astrocytic propagation and microglial activation. The estradiol was injected intraperitoneally 20 min after induction of SCI, and the samples were collected at 1, 3, 7, 14, and 30 days. Histologically, the estradiol reduced the inflammatory response, enhanced the recovery of molecular, granular, and Purkinje cell layers, and therefore aided in the restoration of layer organization. These findings were also confirmed by immunohistochemical staining and gene expression profiling.


Assuntos
Estradiol/uso terapêutico , Células de Purkinje/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Astrócitos/efeitos dos fármacos , Modelos Animais de Doenças , Estradiol/farmacologia , Ácido Caínico/farmacologia , Masculino , Microglia/efeitos dos fármacos , Células de Purkinje/patologia , Células de Purkinje/fisiologia , Ratos , Ratos Wistar , Recuperação de Função Fisiológica/efeitos dos fármacos , Traumatismos da Medula Espinal/induzido quimicamente
8.
eNeuro ; 6(1)2019.
Artigo em Inglês | MEDLINE | ID: mdl-30783614

RESUMO

The biological mechanisms underlying complex forms of learning requiring the understanding of rules based on previous experience are not yet known. Previous studies have raised the intriguing possibility that improvement in complex learning tasks requires the long-term modulation of intrinsic neuronal excitability, induced by reducing the conductance of the slow calcium-dependent potassium current (sIAHP) simultaneously in most neurons in the relevant neuronal networks in several key brain areas. Such sIAHP reduction is expressed in attenuation of the postburst afterhyperpolarization (AHP) potential, and thus in enhanced repetitive action potential firing. Using complex olfactory discrimination (OD) learning as a model for complex learning, we show that brief activation of the GluK2 subtype glutamate receptor results in long-lasting enhancement of neuronal excitability in neurons from controls, but not from trained rats. Such an effect can be obtained by a brief tetanic synaptic stimulation or by direct application of kainate, both of which reduce the postburst AHP in pyramidal neurons. Induction of long-lasting enhancement of neuronal excitability is mediated via a metabotropic process that requires PKC and ERK activation. Intrinsic neuronal excitability cannot be modulated by synaptic activation in neurons from GluK2 knock-out mice. Accordingly, these mice are incapable of learning the complex OD task. Moreover, viral-induced overexpression of Gluk2 in piriform cortex pyramidal neurons results in remarkable enhancement of complex OD learning. Thus, signaling via kainate receptors has a central functional role in higher cognitive abilities.


Assuntos
Aprendizagem por Discriminação/fisiologia , Percepção Olfatória/fisiologia , Córtex Piriforme/fisiologia , Células Piramidais/fisiologia , Receptores de Ácido Caínico/metabolismo , Animais , Agonistas de Aminoácidos Excitatórios/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Ácido Caínico/farmacologia , Masculino , Aprendizagem em Labirinto/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Córtex Piriforme/efeitos dos fármacos , Proteína Quinase C/metabolismo , Células Piramidais/efeitos dos fármacos , Ratos Sprague-Dawley , Receptores de Ácido Caínico/genética , Técnicas de Cultura de Tecidos
9.
Eur J Pharmacol ; 851: 25-35, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30790558

RESUMO

Alzheimer's disease (AD) is characterized by abnormal accumulation of extracellular amyloid beta protein (Aß) plaques and intracellular neurofibrillary tangles, as well as by a state of chronic inflammation in the central nervous system (CNS). Adverse activation of microglia, the brain immune cells, is believed to contribute to AD pathology including excessive neuronal death. Thus, normalizing immune functions of microglia could slow neurodegeneration, and identification of novel compounds capable of modifying microglial functions is an important goal. Since kainic acid (KA) has been shown to modulate microglial morphology and immune functions, we synthesized six new KA analogs (KAAs) and tested their effects on select microglial functions by using three different cell types as microglia models. Four of the KAAs at low micromolar concentrations inhibited secretion of cytotoxins, monocyte chemoattractant protein (MCP)-1, reactive oxygen species and nitric oxide (NO) by immune-stimulated microglia-like cells. We hypothesize that the effects of the novel KAAs on microglia-like cells are not mediated by KA receptors since their biological activity was distinct from that of KA in all assays performed. A structural similarity search identified aldose reductase (AR) as a potential target for the novel KAAs. This hypothesis was supported by use of AR inhibitor zopolrestat, which abolished the inhibitory effects of two KAAs on microglial secretion of NO. Since the newly developed KAAs inhibited pro-inflammatory and cytotoxic functions of microglia, they should be further investigated for their potential beneficial effect on neuroinflammation and neurodegeneration in AD animal models.


Assuntos
Ácido Caínico/análogos & derivados , Ácido Caínico/farmacologia , Microglia/efeitos dos fármacos , Benzotiazóis/farmacologia , Células HL-60 , Humanos , Microglia/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Ftalazinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo
10.
Neuropharmacology ; 149: 149-160, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30763657

RESUMO

Glutamate receptor-mediated excitotoxicity is a common pathogenic process in many neurological conditions including epilepsy. Prolonged seizures induce elevations in extracellular glutamate that contribute to excitotoxic damage, which in turn can trigger chronic neuroinflammatory reactions, leading to secondary damage to the brain. Blocking key inflammatory pathways could prevent such secondary brain injury following the initial excitotoxic insults. Prostaglandin E2 (PGE2) has emerged as an important mediator of neuroinflammation-associated injury, in large part via activating its EP2 receptor subtype. Herein, we investigated the effects of EP2 receptor inhibition on excitotoxicity-associated neuronal inflammation and injury in vivo. Utilizing a bioavailable and brain-permeant compound, TG6-10-1, we found that pharmacological inhibition of EP2 receptor after a one-hour episode of kainate-induced status epilepticus (SE) in mice reduced seizure-promoted functional deficits, cytokine induction, reactive gliosis, blood-brain barrier impairment, and hippocampal damage. Our preclinical findings endorse the feasibility of blocking PGE2/EP2 signaling as an adjunctive strategy to treat prolonged seizures. The promising benefits from EP2 receptor inhibition should also be relevant to other neurological conditions in which excitotoxicity-associated secondary damage to the brain represents a pathogenic event.


Assuntos
Inflamação/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Receptores de Prostaglandina E Subtipo EP2/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Estado Epiléptico/tratamento farmacológico , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Encéfalo/efeitos dos fármacos , Lesões Encefálicas/tratamento farmacológico , Citocinas/efeitos dos fármacos , Citocinas/metabolismo , Dinoprostona/metabolismo , Modelos Animais de Doenças , Gliose/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Indóis/farmacologia , Ácido Caínico/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Convulsões/fisiopatologia , Transdução de Sinais , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/patologia
11.
Neurosci Lett ; 698: 105-112, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30639396

RESUMO

Motor Neuron Disease disorders, described in domestic animals, are characterized by neuronal degeneration at the spinal cord. Excitotoxicity is a crucial factor for the selective loss of these neurons, being the fundamental processes involved in lesion progression after spinal cord injury, where glutamate is one of the main neurotransmitters involved. Kainic acid (KA) resembles the effects induced by the pathological release of glutamate. Lidocaine administered by different routes exerts some neuroprotective effects in the CNS. The aim of the present work was to determine whether lidocaine simultaneously injected with KA into the spinal cord could prevent the excitotoxic effects of the latter. Sprague-Dawley rats were injected by intraparenchymal route with KA or with KA plus 0.5% lidocaine into the C5 segment. Sham rats were injected with saline. Animals were motor and sensory tested at 0, 1, 2, 3, 7 and 14 post-injection days and then euthanized. Sections of the C5 segment were used for histological and immunohistochemical analysis. No KA-induced motor and sensitive impairments were observed when lidocaine was simultaneously injected with KA. Moreover, neuronal counting was statistically higher when compared with KA-injected animals. Thus, lidocaine could be considered as a neuroprotective drug in diseases and models involving excitotoxicity.


Assuntos
Lidocaína/farmacologia , Neurônios/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , Medula Espinal/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Agonistas de Aminoácidos Excitatórios/farmacologia , Ácido Glutâmico/farmacologia , Ácido Caínico/farmacologia , Masculino , Fármacos Neuroprotetores/farmacologia , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/patologia
12.
Invest Ophthalmol Vis Sci ; 60(1): 183-191, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30640971

RESUMO

Purpose: GCaMP3 is a genetically encoded calcium indicator for monitoring intracellular calcium dynamics. We characterized the expression pattern and functional properties of GCaMP3 in the Thy1-GCaMP3 transgenic mouse retina. Methods: To determine the specificity of GCaMP3 expression, Thy1-GCaMP3 (B6; CBA-Tg(Thy1-GCaMP3)6Gfng/J) retinas were processed for immunohistochemistry with anti-green fluorescent protein (anti-GFP, to enhance GCaMP3 fluorescence), anti-RBPMS (retinal ganglion cell [RGC]-specific marker), and antibodies against amacrine cell markers (ChAT, GABA, GAD67, syntaxin). Calcium imaging was used to characterize functional responses of GCaMP3-expressing (GCaMP+) cells by recording calcium transients evoked by superfusion of kainic acid (KA; 10, 50, or 100 µM). In a subset of animals, optic nerve transection (ONT) was performed 3, 5, or 7 days prior to calcium imaging. Results: GFP immunoreactivity colocalized with RBPMS but not amacrine cell markers in both ONT and non-ONT (control) groups. Calcium transients evoked by KA were reduced after ONT (50 µM KA; ΔF/F0 [SD]; control: 1.00 [0.67], day 3: 0.50 [0.35], day 5: 0.31 [0.28], day 7: 0.35 [0.36]; P < 0.05 versus control). There was also a decrease in the number of GCaMP3+ cells after ONT (cells/mm2 [SD]; control: 2198 [453], day 3: 2224 [643], day 5: 1383 [375], day 7: 913 [178]; P < 0.05). Furthermore, the proportion of GCaMP3+ cells that responded to KA decreased after ONT (50 µM KA, 97%, 54%, 47%, and 58%; control, 3, 5, and 7 days, respectively). Conclusions: Following ONT, functional RGC responses are lost prior to the loss of RGC somata, suggesting that anatomical markers of RGCs may underestimate the extent of RGC dysfunction.


Assuntos
Cálcio/metabolismo , GMP Cíclico/metabolismo , Regulação da Expressão Gênica/fisiologia , Traumatismos do Nervo Óptico , Células Ganglionares da Retina/metabolismo , Antígenos Thy-1/metabolismo , Células Amácrinas/metabolismo , Animais , Biomarcadores/metabolismo , Agonistas de Aminoácidos Excitatórios/farmacologia , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Ácido Caínico/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Microscopia de Fluorescência , Células Ganglionares da Retina/efeitos dos fármacos , Antígenos Thy-1/genética
13.
Brain Pathol ; 29(1): 28-44, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29665128

RESUMO

Kainic acid, an analogue of the excitatory neurotransmitter glutamate, can trigger seizures and neurotoxicity in the hippocampus and other limbic structures in a manner that mirrors the neuropathology of human temporal lobe epilepsy (TLE). However, the underlying mechanisms associated with the neurotoxicity remain unclear. Since amyloid-ß (Aß) peptides, which are critical in the development of Alzheimer's disease, can mediate toxicity by activating glutamatergic NMDA receptors, it is likely that the enhanced glutamatergic transmission that renders hippocampal neurons vulnerable to kainic acid treatment may involve Aß peptides. Thus, we seek to establish what role Aß plays in kainic acid-induced toxicity using in vivo and in vitro paradigms. Our results show that systemic injection of kainic acid to adult rats triggers seizures, gliosis and loss of hippocampal neurons, along with increased levels/processing of amyloid precursor protein (APP), resulting in the enhanced production of Aß-related peptides. The changes in APP levels/processing were evident primarily in activated astrocytes, implying a role for astrocytic Aß in kainic acid-induced toxicity. Accordingly, we showed that treating rat primary cultured astrocytes with kainic acid can lead to increased Aß production/secretion without any compromise in cell viability. Additionally, we revealed that kainic acid reduces neuronal viability more in neuronal/astrocyte co-cultures than in pure neuronal culture, and this is attenuated by precluding Aß production. Collectively, these results indicate that increased production/secretion of Aß-related peptides from activated astrocytes can contribute to neurotoxicity in kainic acid-treated rats. Since kainic acid administration can lead to neuropathological changes resembling TLE, it is likely that APP/Aß peptides derived from astrocytes may have a role in TLE pathogenesis.


Assuntos
Peptídeos beta-Amiloides/fisiologia , Epilepsia do Lobo Temporal/fisiopatologia , Neurônios/efeitos dos fármacos , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Astrócitos/metabolismo , Astrócitos/fisiologia , Encéfalo/patologia , Células Cultivadas , Modelos Animais de Doenças , Hipocampo/patologia , Humanos , Ácido Caínico/farmacologia , Ácido Caínico/toxicidade , Masculino , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Fragmentos de Peptídeos/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos
14.
Brain Res Bull ; 144: 92-100, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30502400

RESUMO

Diminished sensitivity to kainic acid (KA) lesioning action is present in the hippocampus of the lactating dam. Given that KA can alter blood-brain barrier (BBB) permeability, we investigated whether lactation would protect BBB permeability after treatment with this neurotoxin. Adult virgin or lactating (13-15 days pp) female Wistar rats were injected with an i.p. (10 mg/kg b.w.) or i.c.v. (100 ng/1 µl) KA dose. Control groups received a similar volume of vehicle. BBB permeability was assessed by an injection of fluorescent FITC-conjugated dextran tracer (40 kDa, 50 mg/500 µl) or of 4% Evans blue dye (500 µl), given 24 h after KA administration. Extravasation of dye or tracer was detected in virgin but not in lactating rats exposed to KA. In particular, increased extravasation of FITC-dextran was detected in the hippocampal subfields CA1 and CA3 and in the cerebral cortex of virgin rats that received KA, while lactating rats showed increased label for FITC-dextran only in the cerebral cortex. Western blot analysis of tight junction proteins showed a decrease of claudin-5 and occludin but not of zonula occludens-1 in the hippocampus of both virgin and lactating rats. These results allow us to conclude that the BBB of lactating rats is less sensitive to KA lesioning action in comparison to that of virgin rats by mechanisms different than those of tight junction proteins.


Assuntos
Barreira Hematoencefálica/metabolismo , Lactação/metabolismo , Lactação/fisiologia , Animais , Barreira Hematoencefálica/fisiologia , Encéfalo/metabolismo , Claudina-5/metabolismo , Feminino , Hipocampo/metabolismo , Ácido Caínico/farmacologia , Ocludina/metabolismo , Permeabilidade , Ratos , Ratos Wistar
15.
Mol Neurobiol ; 56(7): 5095-5110, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30484111

RESUMO

Kainic acid (KA) is an analogue of the excitatory neurotransmitter glutamate that, when injected systemically into adult rats, can trigger seizures and progressive neuronal loss in a manner that mirrors the neuropathology of human mesial temporal lobe epilepsy. However, biomolecular mechanisms responsible for the neuronal loss that occurs as a consequence of this treatment remains elusive. We have recently reported that toxicity induced by KA can partly be mediated by astrocyte-derived amyloid ß (Aß) peptides, which are critical in the development of Alzheimer's disease (AD). Nonetheless, little is known how KA can influence amyloid precursor protein (APP) levels and processing in astrocytes. Thus, in the present study using human U-373 astrocytoma and rat primary astrocytes, we evaluated the role of KA on APP metabolism. Our results revealed that KA treatment increased the levels of APP and its cleaved products (α-/ß-CTFs) in cultured U-373 astrocytoma and primary astrocytes, without altering the cell viability. The cellular and secretory levels of Aß1-40/Aß1-42 were markedly increased in KA-treated astrocytes. We also demonstrated that the steady-state levels of APP-secretases were not altered but the activity of γ-secretase is enhanced in KA-treated U-373 astrocytoma. Furthermore, using selective receptor antagonists, we showed that the effects of KA is mediated by activation of kainate receptors and not NMDA or AMPA receptors. These results suggest that KA can enhance amyloidogenic processing of APP by activating its own receptor leading to increased production/secretion of Aß-related peptides from activated astrocytes which may contribute to the pathogenesis of temporal lobe epilepsy.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Ácido Caínico/farmacologia , Receptores de Ácido Caínico/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Humanos , Ácido Caínico/toxicidade , Receptores de Ácido Caínico/agonistas
16.
Brain Res Bull ; 144: 187-193, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30423353

RESUMO

Temporal lobe epilepsy (TLE) with hippocampal sclerosis is the most common type of drug-resistant epilepsy. Non-human primates are attractive models for studying the pathogenic mechanisms of TLE, with the goal of developing new drugs and interventions. In this study, we developed and tested a Cynomolgus monkey (Macaca fascicularis) model of TLE. A total of 5 Cynomolgus monkeys received3-4 weekly unilateral hippocampal injections of kainic acid (KA) to induce repetitive acute seizures. Animals were monitored via video and electroencephalography (EEG) to assess KA-induced acute seizures and subsequent spontaneous recurrent epileptiform discharges (SREDs). During acute seizures, EEG recording showed bursts of generalized spike discharges arising from the temporal lobe ipsilateral to the KA injection. Three months later, we detected abundant interictal epileptiform discharges (IEDs) during pentobarbital induced anesthesia. Furthermore, two monkeys exhibited synchronized epileptiform discharges accompanied by symptoms mimicking absence seizures. No obvious convulsive symptoms were observed in any monkeys. Overall, our data indicate successful development of a Cynomolgus monkey model of TLE via unilateral hippocampal injection of KA.


Assuntos
Epilepsia do Lobo Temporal/metabolismo , Epilepsia do Lobo Temporal/patologia , Animais , Modelos Animais de Doenças , Eletrodos Implantados , Eletroencefalografia , Epilepsia do Lobo Temporal/induzido quimicamente , Feminino , Hipocampo/patologia , Ácido Caínico/efeitos adversos , Ácido Caínico/farmacologia , Macaca fascicularis , Masculino , Convulsões/patologia , Lobo Temporal/patologia
17.
Mol Neurobiol ; 56(7): 4960-4979, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30421168

RESUMO

During neuronal development, AMPA receptors (AMPARs) and NMDA receptors (NMDARs) are important for neuronal differentiation. Kainate receptors (KARs) are closely related to AMPARs and involved in the regulation of cortical network activity. However, their role for neurite growth and differentiation of cortical neurons is unclear. Here, we used KAR agonists and overexpression of selected KAR subunits and their auxiliary neuropilin and tolloid-like proteins, NETOs, to investigate their influence on dendritic growth and network activity in organotypic cultures of rat visual cortex. Kainate at 500 nM enhanced network activity and promoted development of dendrites in layer II/III pyramidal cells, but not interneurons. GluK2 overexpression promoted dendritic growth in pyramidal cells and interneurons. GluK2 transfectants were highly active and acted as drivers for network activity. GluK1 and NETO1 specifically promoted dendritic growth of interneurons. Our study provides new insights for the roles of KARs and NETOs in the morphological and physiological development of the visual cortex.


Assuntos
Dendritos/fisiologia , Interneurônios/fisiologia , Células Piramidais/fisiologia , Receptores de Ácido Caínico/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Animais , Animais Recém-Nascidos , Dendritos/efeitos dos fármacos , Interneurônios/efeitos dos fármacos , Ácido Caínico/farmacologia , Técnicas de Cultura de Órgãos , Organogênese/efeitos dos fármacos , Organogênese/fisiologia , Subunidades Proteicas/agonistas , Subunidades Proteicas/fisiologia , Células Piramidais/efeitos dos fármacos , Ratos , Ratos Long-Evans , Receptores de Ácido Caínico/agonistas , Córtex Visual/efeitos dos fármacos , Córtex Visual/crescimento & desenvolvimento
18.
Neurochem Res ; 44(3): 600-608, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30097883

RESUMO

Temporal lobe epilepsy (TLE) is an acquired form of focal epilepsy, in which patients not only suffer from unprovoked, devastating seizures, but also from severe comorbidities, such as cognitive dysfunction. Correspondingly, several animal models of TLE exhibit memory dysfunction, especially spatial memory. The Morris water maze test is the most commonly used test for assessing spatial learning and memory in rodents. However, high stress and poor swimming abilities are common confounders and may contribute to misinterpretation. Particularly epileptic mice show altered behaviour during the test as they fail to understand the paradigm context. In the Barnes maze test, a dry-land maze test for spatial learning and memory that uses milder aversive stimuli, these drawbacks have not yet been reported. In the present study, we use this task to evaluate spatial learning and memory in the intrahippocampal kainic acid mouse model of TLE. We demonstrate that the epileptic mice understand the Barnes maze paradigm context, as they learn the location of the escape-chamber by using a serial search strategy but fail to develop the more efficient spatial search strategy. Our data indicate that the Barnes maze may be a better alternative to the Morris water maze for assessing search strategies and impairment of learning and memory in epileptic mice.


Assuntos
Epilepsia do Lobo Temporal/fisiopatologia , Hipocampo/fisiopatologia , Aprendizagem em Labirinto/fisiologia , Aprendizagem Espacial/fisiologia , Animais , Comportamento Animal/fisiologia , Modelos Animais de Doenças , Epilepsia do Lobo Temporal/induzido quimicamente , Ácido Caínico/farmacologia , Camundongos Endogâmicos C57BL , Percepção Espacial/fisiologia , Memória Espacial/fisiologia
19.
Brain Res Bull ; 143: 106-115, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30347263

RESUMO

Effects of kainate or 6-hydroxidopamine (6-OHDA) lesions in the ventromedial prefrontal cortex (vmPFC) on taste-related learning and memory processes were examined. Neurotoxins were applied by iontophoretic method to minimize the extent of lesion and the side effects. Acquisition and retention of conditioned taste avoidance (CTA) was tested to different taste stimuli (0.05 M NaCl, 0.01 M saccharin, 0.01 M citrate and 0.00025 M quinine). In the first experiment, palatability index of taste solutions with these concentrations has been determined as strongly palatable (NaCl, saccharin), weakly palatable (citrate) and weakly unpalatable (quinine) taste stimuli. In two other experiments vmPFC lesions were performed before CTA (acquisition) or after CTA (retrieval). Our results showed that both kainate and 6-OHDA microlesions of vmPFC resulted in deficit of CTA acquisition (to NaCl, saccharin and citrate) and retrieval (to NaCl and saccharin). Deficits were specific to palatable tastants, particularly those that are strongly palatable, and did not occur for unpalatable stimulus. The present data provide evidence for the important role of vmPFC neurons and catecholaminergic innervation of the vmPFC in taste related learning and memory processes.


Assuntos
Ácido Caínico/farmacologia , Oxidopamina/farmacologia , Córtex Pré-Frontal/efeitos dos fármacos , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Condicionamento Clássico/efeitos dos fármacos , Ácido Caínico/metabolismo , Masculino , Memória , Oxidopamina/metabolismo , Ratos , Ratos Wistar , Sacarina , Paladar/fisiologia
20.
Glia ; 66(11): 2397-2413, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30357924

RESUMO

Neurogenesis is sustained throughout life in the mammalian brain, supporting hippocampus-dependent learning and memory. Its permanent alteration by status epilepticus (SE) is associated with learning and cognitive impairments. The mechanisms underlying the initiation of altered neurogenesis after SE are not understood. Glial fibrillary acidic protein-positive radial glia (RG)-like cells proliferate early after SE, but their proliferation dynamics and signaling are largely unclear. We have previously reported a polarized distribution of AMPA receptors (AMPARs) on RG-like cells in vivo and postulated that these may signal their proliferation. Here, we examined the acute effects of kainate on hippocampal precursor cells in vitro and in kainate-induced SE on proliferating and quiescent clones of 5-bromo-2-deoxyuridine prelabeled hippocampal precursors in vivo. In vitro, we found that 5 µM kainate shortened the cell cycle time of RG-like cells via AMPAR activation and accelerated cell cycle re-entry of their progeny. It also shifted their fate choice expanding the population of RG-like cells and reducing the population of downstream amplifying neural progenitors. Kainate enhanced the survival of all precursor cell subtypes. Pharmacologically, kainate's proliferative and survival effects were abolished by AMPAR blockade. Functional AMPAR expression was confirmed on RG-like cells in vitro. In agreement with these observations, kainate/seizures enhanced the proliferation and expansion predominantly of constitutively cycling RG-like cell clones in vivo. Our results identify AMPARs as key potential players in initiating the proliferation of dentate RG-like cells and unravel a possible receptor target for modifying the radial glia-like cell response to SE.


Assuntos
Proliferação de Células/fisiologia , Hipocampo/citologia , Neuroglia/patologia , Receptores de AMPA/metabolismo , Convulsões/patologia , Células-Tronco/patologia , Animais , Animais Recém-Nascidos , Benzodiazepinas/farmacologia , Morte Celular/genética , Células Cultivadas , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Ácido Caínico/farmacologia , Antígeno Ki-67/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Proteínas do Tecido Nervoso/metabolismo , Quinoxalinas/farmacologia , Ratos , Ratos Wistar , Receptores de AMPA/genética
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