Rat Brain Slices Research Articles

Persistent Firing in Hippocampal CA1 Pyramidal Cells in Young and Aged Rats.

Persistent neuronal firing is often observed in working memory and temporal association tasks both in humans and animals, and is believed to retain necessary information in these tasks. We have reported that hippocampal CA1 pyramidal cells are able to support persistent firing through intrinsic mechanisms in the presence of cholinergic agonists. However, it still remains largely unknown how persistent firing is affected by the development of animals and aging. Using in vitro patch-clamp recordings from CA1 pyramidal cells in rat brain slices, we first show that the cellular excitability of these aged rats was significantly lower than the young rats, responding with fewer spikes to current injection. In addition, we found age-dependent modulations of input resistance, membrane capacitance, and spike width. However, persistent firing in aged (~2 years old) rats was as strong as that in young animals, and the properties of persistent firing were very similar among different age groups. In addition, spike after-hyperpolarization potential (AHP), was not increased by aging and did not correlate with the strength of persistent firing. Lastly, we estimated the depolarization current induced by the cholinergic activation. This current was proportional to the increased membrane capacitance of the aged group and was inversely correlated with the intrinsic excitability of cells. These observations indicate that robust persistent firing can be maintained in aged rats despite reduced excitability, due to the increased amount of cholinergically induced positive current.Significant statement:In an aging society, it is crucial to understand neural mechanisms underlying age-dependent cognitive impairments. In recent years, the importance of intrinsic cellular properties in cognitive functions such as memory has increasingly been recognized. However, research examining age-dependent alteration of intrinsic cellular mechanisms of persistent firing, which is believed to support working memory function, has so far been very scarce. In this study, we demonstrate that the ability to support persistent firing is kept intact in neurons from old rats, despite changes in other properties such as intrinsic excitability. These results identify the ability to support persistent firing as one potential cellular mechanism of rescued cognitive functions under cholinergic enhancement used in Alzheimer's disease and other dementias.

Inhibition of Microglial Activation by Delayed Mild Hypothermia Reduced Preoligodendrocyte Injury in a Neonatal Rat Brain Slice Model.

Periventricular leukomalacia (PVL), characterized by distinctive form of white matter injury, often arises after neonatal cardiac surgery. Proven therapies for PVL are absent. In this study, we designed to quest therapeutic effects of delayed mild hypothermia on PVL and its mechanism in a neonatal rat brain slice model. With the increase of delayed mild hypothermia-treating time, the reduced expression of myelin basic protein and loss of preoligodendrocytes were significantly attenuated after oxygen-glucose deprivation. In addition, the proportion of ionized calcium binding adapter molecule 1 (Iba-1)-positive cells and the expression of Iba-1 were apparently reduced with the increased duration of mild hypothermia treatment. Furthermore, the levels of tumor necrosis factor alpha and interleukin-6 reduced after the mild hypothermia treatment relative to the control. Inhibition of microglial activation with prolonged mild hypothermia may be a potential strategy for white matter protection during cardiopulmonary bypass and hypothermic circulatory arrest.

Anti-glutamatergic Effects of Three Lignan Compounds: Arctigenin, Matairesinol and Trachelogenin - An ex vivo Study on Rat Brain Slices.

Arctigenin is a bioactive dibenzylbutyrolactone-type lignan exhibiting various pharmacological activities. The neuroprotective effects of arctigenin were demonstrated to be mediated via inhibition of AMPA and KA type glutamate receptors in the somatosensory cortex of the rat brain. The aim of this study was to compare the effects of arctigenin with matairesinol and trachelogenin on synaptic activity in ex vivo rat brain slices. Arctigenin, matairesinol and trachelogenin were isolated from Arctium lappa, Centaurea scabiosa and Cirsium arvense, respectively, and applied on brain slices via perfusion medium at the concentration range of 0.5 - 40 µM. The effects of the lignans were examined in the CA1 hippocampus and the somatosensory cortex by recording electrically evoked field potentials. Arctigenin and trachelogenin caused a significant dose-dependent decrease in the amplitude of hippocampal population spikes (POPS) and the slope of excitatory postsynaptic potentials (EPSPs), whereas matairesinol (1 µM and 10 µM) decreased EPSP slope but had no effect on POPS amplitude. Trachelogenin effect (0.5 µM, 10 µM, 20 µM) was comparable to arctigenin (1 µM, 20 µM, 40 µM) (p > 0.05). In the neocortex, arctigenin (10 µM, 20 µM) and trachelogenin (10 µM) significantly decreased the amplitude of evoked potential early component, while matairesinol (1 µM and 10 µM) had no significant effect (p > 0.05). The results suggest that trachelogenin and arctigenin act via inhibition of AMPA and KA receptors in the brain and trachelogenin has a higher potency than arctigenin. Thus, trachelogenin and arctigenin could serve as lead compounds in the development of neuroprotective drugs.

The Formation of Morphologically Stable Lipid Nanocarriers for Glioma Therapy

Cerasomes are a promising modification of liposomes with covalent siloxane networks on the surface that provide outstanding morphological stability while maintaining all the useful traits of liposomes. Herein, thin film hydration and ethanol sol injection methods were utilized to produce cerasomes of various composition, which were then evaluated for the purpose of drug delivery. The most promising nanoparticles obtained by the thin film method were studied closely using MTT assay, flow cytometry and fluorescence microscopy on T98G glioblastoma cell line and modified with surfactants to achieve stability and the ability to bypass the blood-brain barrier. An antitumor agent, paclitaxel, was loaded into cerasomes, which increased its potency and demonstrated increased ability to induce apoptosis in T98G glioblastoma cell culture. Cerasomes loaded with fluorescent dye rhodamine B demonstrated significantly increased fluorescence in brain slices of Wistar rats compared to free rhodamine B. Thin film hydration with Tween 80 addition was established as a more reliable and versatile method for cerasome preparation. Cerasomes increased the antitumor action of paclitaxel toward T98G cancer cells by a factor of 36 and were able to deliver rhodamine B over the blood-brain barrier in rats.

γ-Aminobutyric Acid-Ergic Development Contributes to the Enhancement of Electroencephalogram Slow-Delta Oscillations Under Volatile Anesthesia in Neonatal Rats.

General anesthetics (eg, propofol and volatile anesthetics) enhance the slow-delta oscillations of the cortical electroencephalogram (EEG), which partly results from the enhancement of (γ-aminobutyric acid [GABA]) γ-aminobutyric acid-ergic (GABAergic) transmission. There is a GABAergic excitatory-inhibitory shift during postnatal development. Whether general anesthetics can enhance slow-delta oscillations in the immature brain has not yet been unequivocally determined. Perforated patch-clamp recording was used to confirm the reversal potential of GABAergic currents throughout GABAergic development in acute brain slices of neonatal rats. The power density of the electrocorticogram and the minimum alveolar concentrations (MAC) of isoflurane and/or sevoflurane were measured in P4-P21 rats. Then, the effects of bumetanide, an inhibitor of the Na+-K+-2Cl- cotransporter (NKCC1) and K+-Cl- cotransporter (KCC2) knockdown on the potency of volatile anesthetics and the power density of the EEG were determined in vivo. Reversal potential of GABAergic currents were gradually hyperpolarized from P4 to P21 in cortical pyramidal neurons. Bumetanide enhanced the hypnotic effects of volatile anesthetics at P5 (for MACLORR, isoflurane: 0.63% ± 0.07% vs 0.81% ± 0.05%, 95% confidence interval [CI], -0.257 to -0.103, P < .001; sevoflurane: 1.46% ± 0.12% vs 1.66% ± 0.09%, 95% CI, -0.319 to -0.081, P < .001); while knockdown of KCC2 weakened their hypnotic effects at P21 in rats (for MACLORR, isoflurane: 0.58% ± 0.05% to 0.77% ± 0.20%, 95% CI, 0.013-0.357, P = .003; sevoflurane: 1.17% ± 0.04% to 1.33% ± 0.04%, 95% CI, 0.078-0.244, P < .001). For cortical EEG, slow-delta oscillations were the predominant components of the EEG spectrum in neonatal rats. Isoflurane and/or sevoflurane suppressed the power density of slow-delta oscillations rather than enhancement of it until GABAergic maturity. Enhancement of slow-delta oscillations under volatile anesthetics was simulated by preinjection of bumetanide at P5 (isoflurane: slow-delta changed ratio from -0.31 ± 0.22 to 1.57 ± 1.15, 95% CI, 0.67-3.08, P = .007; sevoflurane: slow-delta changed ratio from -0.46 ± 0.25 to 0.95 ± 0.97, 95% CI, 0.38-2.45, P = .014); and suppressed by KCC2-siRNA at P21 (isoflurane: slow-delta changed ratio from 16.13 ± 5.69 to 3.98 ± 2.35, 95% CI, -18.50 to -5.80, P = .002; sevoflurane: slow-delta changed ratio from 0.13 ± 2.82 to 3.23 ± 2.49, 95% CI, 3.02-10.79, P = .003). Enhancement of cortical EEG slow-delta oscillations by volatile anesthetics may require mature GABAergic inhibitory transmission during neonatal development.

Evaluation of ciliary-GPCR dynamics using a validated organotypic brain slice culture method.

The primary cilium is a structural organelle present in most mammalian cells. Primary cilia are enriched with a unique protein repertoire distinct from that of the cytosol and the plasma membrane. Such a highly organized microenvironment creates effective machinery for translating extracellular cues into intracellular signals. G protein-coupled receptors (GPCRs) are key receptors in sensing environmental stimuli transmitted via a second messenger into a cellular response. Recent data has demonstrated that a limited number of non-olfactory GPCRs, including melanin-concentrating hormone receptor 1 (MCHR1), are preferentially localized to ciliary membranes of several mammalian cell types, including neuronal cells. Evidence was accumulated to support the functional importance of ciliary-GPCR signaling accompanying ciliary structural changes using cilia-specific cell and molecular biology techniques. Thus, cilia are now considered to function as a unique sensory platform for the integration of GPCR signaling and various cytoplasmic domains. Dissociated neurons expressing ciliary-GPCRs can be a useful tool for examining ciliary dynamics. However, losing preexisting neuronal connectivity may alter neuronal ciliary morphology, such as abnormal elongation. Brain slices prepared under ex vitro conditions are a powerful approach that maintains the cytoarchitecture, enabling researchers to have accurate control over experimental conditions and to study individual cells from subregions of the brain. Here, we present a detailed description of our novel modified method for organotypic culture of rat brain slice and a validated immunostaining protocol to characterize ciliary-GPCR dynamics in coupling with neuropeptides or aminergic activation.

An astrocytic signaling loop for frequency-dependent control of dendritic integration and spatial learning

Dendrites of hippocampal CA1 pyramidal cells amplify clustered glutamatergic input by activation of voltage-gated sodium channels and N-methyl-D-aspartate receptors (NMDARs). NMDAR activity depends on the presence of NMDAR co-agonists such as D-serine, but how co-agonists influence dendritic integration is not well understood. Using combinations of whole-cell patch clamp, iontophoretic glutamate application, two-photon excitation fluorescence microscopy and glutamate uncaging in acute rat and mouse brain slices we found that exogenous D-serine reduced the threshold of dendritic spikes and increased their amplitude. Triggering an astrocytic mechanism controlling endogenous D-serine supply via endocannabinoid receptors (CBRs) also increased dendritic spiking. Unexpectedly, this pathway was activated by pyramidal cell activity primarily in the theta range, which required HCN channels and astrocytic CB1Rs. Therefore, astrocytes close a positive and frequency-dependent feedback loop between pyramidal cell activity and their integration of dendritic input. Its disruption in mice led to an impairment of spatial memory, which demonstrated its behavioral relevance.

Contribution of inwardly rectifying K+ channel 4.1 of supraoptic astrocytes to the regulation of vasopressin neuronal activity by hypotonicity.

Astrocytic morphological plasticity and its modulation of adjacent neuronal activity are largely determined by astrocytic volume regulation, in which glial fibrillary acidic protein (GFAP), aquaporin 4 (AQP4), and potassium channels including inwardly rectifying K+ channel 4.1 (Kir4.1) are essential. However, associations of astrocyte-dominant Kir4.1 with other molecules in astrocytic volume regulation and the subsequent influence on neuronal activity remain unclear. Here, we report our study on these issues using primary cultures of rat pups' hypothalamic astrocytes and male adult rat brain slices. In astrocyte culture, hyposmotic challenge (HOC) significantly decreased GFAP monomer expression and astrocytic volume at 1.5 min and increased Kir4.1 expression and inwardly rectifying currents (IRCs) at 10 min. BaCl2 (100 μmol/l) suppressed the HOC-increased IRCs, which was simulated by VU0134992 (2μmol/l), a Kir4.1 blocker. Preincubation of the astrocyte culture with TGN-020 (10μmol/l, a specific AQP4 blocker) made the HOC-increased Kir4.1 currents insignificant. In hypothalamic brain slices, HOC initially decreased and then increased the firing rate of vasopressin (VP) neurons in the supraoptic nucleus. In the presence of BaCl2 or VU0134992, HOC-elicited rebound increase in VP neuronal activity was blocked. GFAP was molecularly associated with Kir4.1, which was increased by HOC at 20 min; this increase was blocked by BaCl2 . These results suggest that HOC-evoked astrocytic retraction or decrease in the volume and length of its processes is associated with increased Kir4.1 activity. Kir4.1 involvement in HOC-elicited astrocytic retraction is associated with AQP4 activity and GFAP plasticity, which together determines the rebound excitation of VP neurons.

Characterization of a Bioactive Peptide T14 in the Human and Rodent Substantia Nigra: Implications for Neurodegenerative Disease.

The substantia nigra is generally considered to show significant cell loss not only in Parkinson's but also in Alzheimer's disease, conditions that share several neuropathological traits. An interesting feature of this nucleus is that the pars compacta dopaminergic neurons contain acetylcholinesterase (AChE). Independent of its enzymatic role, this protein is released from pars reticulata dendrites, with effects that have been observed in vitro, ex vivo and in vivo. The part of the molecule responsible for these actions has been identified as a 14-mer peptide, T14, cleaved from the AChE C-terminus and acting at an allosteric site on alpha-7 nicotinic receptors, with consequences implicated in neurodegeneration. Here, we show that free T14 is co-localized with tyrosine hydroxylase in rodent pars compacta neurons. In brains with Alzheimer's pathology, the T14 immunoreactivity in these neurons increases in density as their number decreases with the progression of the disease. To explore the functional implications of raised T14 levels in the substantia nigra, the effect of exogenous peptide on electrically evoked neuronal activation was tested in rat brain slices using optical imaging with a voltage-sensitive dye (Di-4-ANEPPS). A significant reduction in the activation response was observed; this was blocked by the cyclized variant of T14, NBP14. In contrast, no such effect of the peptide was seen in the striatum, a region lacking the T14 target, alpha-7 receptors. These findings add to the accumulating evidence that T14 is a key signaling molecule in neurodegenerative disorders and that its antagonist NBP14 has therapeutic potential.

TRPV1 channels in nitric oxide-mediated signalling: insight on excitatory transmission in rat CA1 pyramidal neurons.

Nitric oxide (NO) is a fascinating signalling molecule implicated in a plethora of biological functions, especially at the synaptic level. Exploring neurotransmission in the hippocampus could be instrumental in the individuation of putative targets for nitric-oxide mediated neuromodulation, especially in terms of the potential repercussions on fundamental processes i.e. synaptic plasticity and excitability-related phenomena. Among these targets, endovanilloid signalling constitutes an object of study since Transient Receptors Vanilloid type 1 (TRPV1) channels possess a NO-sensitive gate modulating its activation. Also, NO has been referred to as a mediator for numerous endocannabinoid effects. Notwithstanding, the linkage between TRPV1 and NO systems in neuromodulation still remains elusive. To this end, we aim at investigating the involvement of TRPV1 in nitric oxide-mediated influence on hippocampal processes. Electrophysiological whole-cell recordings in CA1 pyramidal neurons were applied to evaluate excitatory neurotransmission in rat brain slices. Indeed, miniature excitatory postsynaptic currents (mEPSCs) were analysed upon pharmacological manipulation of TRPV1 and NO signalling pathways. In detail, only the administration of the specific TRPV1 exogenous agonist - capsaicin - reduced the frequency and amplitude of mEPSC similarly to the inhibitor of neuronal nitric oxide synthase (nNOS), 7-nitroindazole (7NI). In contrast, capsazepine, TRPV1 antagonist, does not influence excitatory transmission. The combined TRPV1 activation and nNOS blockade confirm the presence of a putative common mechanism. When we administered the endovanilloid-endocannabinoid ligand, i.e. anandamide, we unveiled a potentiation of neurotransmission that was selectively reverted by 7NI. Our data suggest that nitric oxide influences TRPV1 hippocampal signalling since these channels are not constitutively active, but can be "on-demand" activated to modulate excitation in CA1 pyramidal neurons, and that this effect is linked to nitric oxide production.

Three-dimensional visualization of cerebral blood vessels and neural changes in thick ischemic rat brain slices using tissue clearing

Blood vessels are three-dimensional (3D) in structure and precisely connected. Conventional histological methods are unsuitable for their analysis because of the destruction of functionally important topological 3D vascular structures. Tissue optical clearing techniques enable extensive volume imaging and data analysis without destroying tissue. This study therefore applied a tissue clearing technique to acquire high-resolution 3D images of rat brain vasculature using light-sheet and confocal microscopies. Rats underwent middle cerebral artery occlusion for 45 min followed by 24 h reperfusion with lectin injected directly into the heart for vascular staining. For acquiring 3D images of rat brain vasculature, 3-mm-thick brain slices were reconstructed using tissue clearing and light-sheet microscopy. Subsequently, after 3D rendering, the fitting of blood vessels to a filament model was used for analysis. The results revealed a significant reduction in vessel diameter and density in the ischemic region compared to those in contralesional non-ischemic regions. Immunostaining of 0.5-mm-thick brain slices revealed considerable neuronal loss and increased astrocyte fluorescence intensity in the ipsilateral region. Thus, these methods can provide more accurate data by broadening the scope of the analyzed regions of interest for examining the 3D cerebrovascular system and neuronal changes occurring in various brain disorders.

Functional characterization of cannabidiol effect on the serotonergic neurons of the dorsal raphe nucleus in rat brain slices.

Cannabidiol (CBD), the main non-psychoactive cannabinoid found in the cannabis plant, elicits several pharmacological effects via the 5-HT1A receptor. The dorsal raphe nucleus (DRN) is the main serotonergic cluster in the brain that expresses the 5-HT1A receptor. To date, the effect of CBD on the neuronal activity of DRN 5-HT cells and its interaction with somatodendritic 5-HT1A autoreceptors have not been characterized. Our aim was to study the effect of CBD on the firing activity of DRN 5-HT cells and the 5-HT1A autoreceptor activation by electrophysiological and calcium imaging techniques in male Sprague–Dawley rat brain slices. Perfusion with CBD (30 μM, 10 min) did not significantly change the firing rate of DRN 5-HT cells or the inhibitory effect of 5-HT (50–100 μM, 1 min). However, in the presence of CBD (30 μM, 10 min), the inhibitory effects of 8-OH-DPAT (10 nM) and ipsapirone (100 nM) were reduced by 66% and 53%, respectively. CBD failed to reverse ipsapirone-induced inhibition, whereas perfusion with the 5-HT1A receptor antagonist WAY100635 (30 nM) completely restored by 97.05 ± 14.63% the firing activity of 5-HT cells. Administration of AM251 (1 µM), MDL100907 (30 nM), or picrotoxin (20 μM) did not change the blockade produced by CBD (30 μM) on ipsapirone-induced inhibition. Our study also shows that CBD failed to modify the KCl (15 mM, 4 min)-evoked increase in [Ca2+]i or the inhibitory effect of ipsapirone (1 μM, 4 min) on KCl-evoked [Ca2+]i. In conclusion, CBD does not activate 5-HT1A autoreceptors, but it hindered the inhibitory effect produced by selective 5-HT1A receptor agonists on the firing activity of DRN 5-HT cells through a mechanism that does not involve CB1, 5-HT2A, or GABAA receptors. Our data support a negative allosteric modulation of DRN somatodendritic 5-HT1A receptor by CBD.

Rat cortico-striatal sagittal organotypic slice cultures as ex vivo excitotoxic striatal lesion models

Organotypic brain slice cultures are a useful tool to study neurological disease as they provide a 3-dimensional system which more closely recapitulates the in vivo cytoarchitectural complexity than standard 2-dimensional in vitro cell cultures. Building on our previously developed rat brain slice culture protocol, we have extended our findings to develop ex vivo excitotoxic lesion models by treatment of rat sagittal organotypic slices with AMPA or quinolinic acid (QA). We show that treatment of rat sagittal cortico-striatal organotypic slices with 8μM AMPA or 50μM QA causes striatal cell loss with a reduction in neuronal nuclei (NeuN)+ cells and an increase in ethidium homodimer-1 (EthD-1)+ dead cells compared to untreated slices. More specifically, following treatment with QA, we observed a reduction in medium spiny neuron DARPP32 + cells in the striatum and cortex of slices. Treatment of the slices with AMPA does not alter glial fibrillary acidic protein (GFAP) expression, while we observed an acute increase in GFAP expression 1-week post-QA exposure both in the cortex and striatum of slices. This recapitulates the excitotoxic and striatal degeneration observed in rat AMPA and QA lesion models in vivo. Our slice culture platform provides an advance over other systems with the ability to generate acute AMPA- and QA-induced striatal excitotoxicity in sagittal cortico-striatal slices which can be cultured long-term for at least 4 weeks. Our ex vivo organotypic slice culture system provides a long-term cellular platform to model neuronal excitotoxicity, with QA specifically modelling Huntington's disease. This will allow for mechanistic studies of excitotoxicity and neuroprotection, as well as the development and testing of novel therapeutic strategies with reduced cost and ease of manipulation prior to in vivo experimentation.

Pharmacological characterization of the α2A-adrenergic receptor inhibiting rat hippocampal CA3 epileptiform activity: comparison of ligand efficacy and potency

The mechanism underlying the antiepileptic actions of norepinephrine (NE) is unclear with conflicting results. Our objectives are to conclusively delineate the specific adrenergic receptor (AR) involved in attenuating hippocampal CA3 epileptiform activity and assess compounds for lead drug development. We utilized the picrotoxin model of seizure generation in rat brain slices using electrophysiological recordings. Epinephrine (EPI) reduced epileptiform burst frequency in a concentration-dependent manner. To identify the specific receptor involved in this response, the equilibrium dissociation constants were determined for a panel of ligands and compared with established binding values for α1, α2, and other receptor subtypes. Correlation and slope of unity were found for the α2A-AR, but not other receptors. Effects of different chemical classes of α-AR agonists at inhibiting epileptiform activity by potency (pEC50) and relative efficacy (RE) were determined. Compared with NE (pEC50, 6.20; RE, 100%), dexmedetomidine, an imidazoline (pEC50, 8.59; RE, 67.1%), and guanabenz, a guanidine (pEC50, 7.94; RE, 37.9%), exhibited the highest potency (pEC50). In contrast, the catecholamines, EPI (pEC50, 6.95; RE, 120%) and α-methyl-NE (pEC50, 6.38; RE, 116%) were the most efficacious. These findings confirm that CA3 epileptiform activity is mediated solely by α2A-ARs without activation of other receptor systems. These findings suggest a pharmacotherapeutic target for treating epilepsy and highlight the need for selective and efficacious α2A-AR agonists that can cross the blood–brain barrier.

Design and Validation of the First Family of Photo-Activatable Ligands for Melatonin Receptors.

Melatonin is a neurohormone released in a circadian manner with peak levels at night. Melatonin mediates its effects mainly through G protein-coupled MT1 and MT2 receptors. Drugs acting on melatonin receptors are indicated for circadian rhythm- and sleep-related disorders. Tools to study the activation of these receptors with high temporal resolution are lacking. Here, we synthesized a family of light-activatable caged compounds by attaching o-nitrobenzyl (o-NB) or coumarin photocleavable groups to melatonin indolic nitrogen. All caged compounds showed the expected decrease in binding affinity for MT1 and MT2. The o-NB derivative MCS-0382 showed the best uncaging and biological properties, with 250-fold increase in affinity and potency upon illumination. Generation of melatonin from MCS-0382 was further demonstrated by its ability to modulate the excitation of SCN neurons in rat brain slices. MCS-0382 is available to study melatonin effects in a temporally controlled manner in cellular and physiological settings.

Cu-Zn SOD suppresses epilepsy in pilocarpine-treated rats and alters SCN2A/Nrf2/HO-1 expression

Copper-zinc superoxide dismutase (Cu-Zn SOD) is downregulated in epilepsy, however, the role of Cu-Zn SOD in epilepsy remains unclear. Based on the pilocarpine hydrochloride-induced rat model of epilepsy, cortical-striatum brain slices of rats were examined based on field excitatory post-synaptic potentials. Pathological changes were observed by transmission electron microscope. Also using SH-SY5Y cells, flow cytometry and TUNEL staining were applied to investigate cell apoptosis, and ELISA was applied to detect SOD activity. In addition, qRT-PCR and western blot were performed to detect SCN2A/Nrf2/HO-1 gene and protein expression levels, respectively. Cu-Zn SOD over-expression suppressed epilepsy in vivo. In addition, Cu-Zn SOD knockdown notably decreased SOD activity and induced apoptosis in SH-SY5Y cells. Moreover, Cu-Zn SOD silencing decreased the levels of SCN2A, Nrf2 and HO-1. Lastly, Cu-Zn SOD was shown to modulate the NaV1.2/Nrf2/HO-1 axis in rats. In this model, Cu-Zn SOD attenuated epilepsy and was shown to alter the expression level of proteins of the NaV1.2 /Nrf2/HO-1 signalling pathway, indicating that Cu-Zn SOD might be a target for the treatment of epilepsy.

5-HT and α-m-5-HT attenuate excitatory synaptic transmissions onto the lateral amygdala principal neurons via presynaptic 5-HT1B receptors

Accumulating evidence suggests that the serotonergic (5-HT) system in the amygdala has significant effects on affective states. Dysregulation of the 5-HT system in the basolateral amygdaloid complex causes affective disorders. To search for therapeutic targets, subtype specification of 5-HT receptors is crucial. The present study was undertaken to identify the 5-HT receptor subtype responsible for the 5-HT-mediated suppression of excitatory transmission to principal neurons (PNs) in the lateral amygdala (LA). Whole-cell recordings were performed to record excitatory post synaptic currents (EPSCs) in acute rat brain slices. We confirmed that 5-HT and α-m-5-HT, a broad 5-HT2 receptor agonist, attenuated EPSCs in LA PNs. The extent of suppressions by 5-HT and α-m-5-HT remained unchanged in the presence of ritanserin, a broad 5-HT2 receptor antagonist. In the presence of NAS-181, a selective 5-HT1B receptor antagonist, the extent of EPSC suppressions by 5-HT and α-m-5-HT was diminished. CP93129, a selective 5-HT1B receptor agonist, attenuated EPSCs in LA PNs, and this effect was abolished in the presence of NAS-181. Additionally, the paired-pulse ratio of EPSCs was increased by CP93129. Thus, our results indicate that 5-HT and α-m-5-HT attenuate excitatory transmissions to LA PNs via presynaptic 5-HT1B receptors.

The O2-sensitive brain stem, hyperoxic hyperventilation, and CNS oxygen toxicity.

Central nervous system oxygen toxicity (CNS-OT) is a complex disorder that presents, initially, as a sequence of cardio-respiratory abnormalities and nonconvulsive signs and symptoms (S/Sx) of brain stem origin that culminate in generalized seizures, loss of consciousness, and postictal cardiogenic pulmonary edema. The risk of CNS-OT and its antecedent “early toxic indications” are what limits the use of hyperbaric oxygen (HBO2) in hyperbaric and undersea medicine. The purpose of this review is to illustrate, based on animal research, how the temporal pattern of abnormal brain stem responses that precedes an “oxtox hit” provides researchers a window into the early neurological events underlying seizure genesis. Specifically, we focus on the phenomenon of hyperoxic hyperventilation, and the medullary neurons presumed to contribute in large part to this paradoxical respiratory response; neurons in the caudal Solitary complex (cSC) of the dorsomedial medulla, including putative CO2 chemoreceptor neurons. The electrophysiological and redox properties of O2-/CO2-sensitive cSC neurons identified in rat brain slice experiments are summarized. Additionally, evidence is summarized that supports the working hypothesis that seizure genesis originates in subcortical areas and involves cardio-respiratory centers and cranial nerve nuclei in the hind brain (brainstem and cerebellum) based on, respectively, the complex temporal pattern of abnormal cardio-respiratory responses and various nonconvulsive S/Sx that precede seizures during exposure to HBO2.

Modulation of seizure-like events by the small conductance and ATP-sensitive potassium ion channels

Potassium ion channels are extensively involved in the regulation of epileptic seizures. The small conductance calcium-sensitive potassium channels (SK channels) and ATP-sensitive potassium (KATP) channels are activated by calcium ion entry and decrease ATP levels, respectively. These channels can underlie the post-burst afterhyperpolarization and be upregulated during seizures, providing negative feedback during epileptic activity. Using the whole-cell patch-clamp method in rat brain slices, we investigated the effect of SK- and KATP-affecting drugs on seizure-like events (SLEs) in the 4-aminopyridine model of epileptic seizures in vitro. We demonstrate that SK and KATP channels contribute to sustaining the high-frequency firing of the principal neurons in the deep layers of the entorhinal cortex during injections of depolarizing current and epileptiform discharges. Neither the pharmacological blockade nor the activation of these channels was able to prevent the epileptiform activity in brain slices. However, the blockade of KATP channels increases the SLE duration, suggesting that these channels may contribute to the termination of SLEs. Thus, KATP channels can be considered a promising target for pharmacological interventions for the treatment of epilepsy.

Mediation of Sinusoidal Network Oscillations in the Locus Coeruleus of Newborn Rat Slices by Pharmacologically Distinct AMPA and KA Receptors.

Brain control by locus coeruleus (LC) neurons involves afferent glutamate (Glu) inputs. In newborns, LC Glu receptors and responses may be sparse due to immaturity of the brain circuits providing such input. However, we reported, using newborn rat brain slices, that Glu and its ionotropic receptor (iGluR) agonist NMDA transform spontaneous local field potential (LFP) rhythm. Here, we studied whether α-amino-3-hydroxy-5-methyl-4-isoxazole propionic-acid (AMPA) and kainate (KA) iGluR subtypes also transform the LFP pattern. AMPA (0.25–0.5 µM) and KA (0.5–2.5 µM) merged ~0.2 s-lasting bell-shaped LFP events occurring at ~1 Hz into ~40% shorter and ~4-fold faster spindle-shaped and more regular sinusoidal oscillations. The AMPA/KA effects were associated with a 3.1/4.3-fold accelerated phase-locked single neuron spiking due to 4.0/4.2 mV depolarization while spike jitter decreased to 64/42% of the control, respectively. Raising extracellular K+ from 3 to 9 mM increased the LFP rate 1.4-fold or elicited slower multipeak events. A blockade of Cl−-mediated inhibition with gabazine (5 μM) plus strychnine (10 μM) affected neither the control rhythm nor AMPA/KA oscillations. GYKI-53655 (25 μM) blocked AMPA (but not KA) oscillations whereas UBP-302 (25 μM) blocked KA (but not AMPA) oscillations. Our findings revealed that AMPA and KA evoke a similar novel neural network discharge pattern transformation type by acting on pharmacologically distinct AMPAR and KA receptors. This shows that already the neonatal LC can generate oscillatory network behaviors that may be important, for example, for responses to opioids.