Flufenamic acid abolishes epileptiform activity in the entorhinal cortex slices by reducing the temporal summation of glutamatergic responses.

Flufenamic acid (FFA) is an anti-inflammatory drug that affects multiple targets and is a widely used research tool in ion channel studies. This pharmacological compound has a low level of selectivity for the transient receptor potential (TRP) channel superfamily, blocking calcium-activated nonselective cation current (ICAN) as well as afterdepolarizations (ADP) induced by it. A number of studies have demonstrated that FFA exerts an anti-epileptic effect in vitro, although the precise mechanism of this effect is not yet identified. The present study used whole-cell patch-clamp recordings and demonstrated that FFA (25 µM) can abolish the generation of seizure-like events (SLE) in entorhinal cortex slices perfused with a 4-aminopyridine-containing solution, depending on the time of application. FFA decreased the temporal summation of synaptic potentials at the onset of SLEs. However, as the epileptiform activity evolved and the SLE onset phase became more abrupt, the blocking effect of FFA diminished. FFA effectively abolished TRP channel-mediated slow ADPs, exerted a weak blockade and slowed the kinetics of GABAa receptor-mediated currents, and did not affect NMDA receptor-mediated evoked currents induced by extracellular stimulation. Although FFA did not directly inhibit NMDA receptor-mediated evoked currents, it decreased the summation of NMDA receptor-mediated potentials in a manner comparable to its effect on the initiation phase of SLE. This suggests that ICAN blockade may be responsible for this effect. Furthermore, our results showed that the selective blocker of melastatin TRP channels (TRPM4) 9-phenanthrol effectively abolished epileptiform activity in a manner analogous to FFA. In contrast, ML-204, the blocker of canonical TRP channels (TRPC), had no discernible effect on this phenomenon. In conclusion, the study demonstrate that FFA abolishes epileptiform activity in the entorhinal cortex by blocking TRPM4 channels and, consequently, decreasing the effectiveness of temporal summation of glutamatergic potentials.

Microglia either promote or restrain TRAIL-mediated excitotoxicity caused by Aß1-42 oligomers.

BACKGROUND: Alzheimer's disease (AD) features progressive neurodegeneration and microglial activation that results in dementia and cognitive decline. The release of soluble amyloid (Aß) oligomers into the extracellular space is an early feature of AD pathology. This can promote excitotoxicity and microglial activation. Microglia can adopt several activation states with various functional outcomes. Protective microglial activation states have been identified in response to Aß plaque pathology in vivo. However, the role of microglia and immune mediators in neurotoxicity induced by soluble Aß oligomers is unclear. Further, there remains a need to identify druggable molecular targets that promote protective microglial states to slow or prevent the progression of AD. METHODS: Hippocampal entorhinal brain slice culture (HEBSC) was employed to study mechanisms of Aß1-42 oligomer-induced neurotoxicity as well as the role of microglia. The roles of glutamate hyperexcitation and immune signaling in Aß-induced neurotoxicity were assessed using MK801 and neutralizing antibodies to the TNF-related apoptosis-inducing ligand (TRAIL) respectively. Microglial activation state was manipulated using Gi-hM4di designer receptor exclusively activated by designer drugs (DREADDs), microglial depletion with the colony-stimulating factor 1 receptor (CSF1R) antagonist PLX3397, and microglial repopulation (PLX3397 withdrawal). Proteomic changes were assessed by LC-MS/MS in microglia isolated from control, repopulated, or Aß-treated HEBSCs. RESULTS: Neurotoxicity induced by soluble Aß1-42 oligomers involves glutamatergic hyperexcitation caused by the proinflammatory mediator and death receptor ligand TRAIL. Microglia were found to have the ability to both promote and restrain Aß-induced toxicity. Induction of microglial Gi-signaling with hM4di to prevent pro-inflammatory activation blunted Aß neurotoxicity, while microglial depletion with CSF1R antagonism worsened neurotoxicity caused by Aß as well as TRAIL. HEBSCs with repopulated microglia, however, showed a near complete resistance to Aß-induced neurotoxicity. Comparison of microglial proteomes revealed that repopulated microglia have a baseline anti-inflammatory and trophic phenotype with a predicted pathway activation that is nearly opposite that of Aß-exposed microglia. mTORC2 and IRF7 were identified as potential targets for intervention. CONCLUSION: Microglia are key mediators of both protection and neurodegeneration in response to Aß. Polarizing microglia toward a protective state could be used as a preventative strategy against Aß-induced neurotoxicity.

17ß-Estradiol reduces inhibitory synaptic currents in entorhinal cortex neurons through G protein-coupled estrogen receptor-1 activation of extracellular signal-regulated kinase.

Estrogens are believed to modulate cognitive functions in part through the modulation of synaptic transmission in the cortex and hippocampus. Administration of 17ß-estradiol (E2) can rapidly enhance excitatory synaptic transmission in the hippocampus and facilitate excitatory synaptic transmission in rat lateral entorhinal cortex via activation of the G protein-coupled estrogen receptor-1 (GPER1). To assess the mechanisms through which GPER1 activation facilitates synaptic transmission, we assessed the effects of acute 10 nM E2 administration on pharmacologically isolated evoked excitatory and inhibitory synaptic currents in layer II/III entorhinal neurons. Female Long-Evans rats were ovariectomized between postnatal day (PD) 63 and 74 and implanted with a subdermal E2 capsule to maintain continuous low levels of E2. Electrophysiological recordings were obtained between 7 and 20 days after ovariectomy. Application of E2 for 20 min did not significantly affect AMPA or NMDA receptor-mediated excitatory synaptic currents. However, GABA receptor-mediated inhibitory synaptic currents (IPSCs) were markedly reduced by E2 and returned towards baseline levels during the 20-min washout period. The inhibition of GABA-mediated IPSCs was blocked in the presence of the GPER1 receptor antagonist G15. GPER1 can modulate protein kinase A (PKA), but blocking PKA with intracellular KT5720 did not prevent the E2-induced reduction in IPSCs. GPER1 can also stimulate extracellular signal-regulated kinase (ERK), a negative modulator of GABAA receptors, and blocking activation of ERK with PD90859 prevented the E2-induced reduction of IPSCs. E2 can therefore result in a rapid GPER1 and ERK signaling-mediated reduction in GABA-mediated IPSCs. This provides a novel mechanism through which E2 can rapidly modulate synaptic excitability in entorhinal layer II/III neurons and may also contribute to E2 and ERK-dependent alterations in synaptic transmission in other brain areas.

Cholecystokinin B receptor agonists alleviates anterograde amnesia in cholecystokinin-deficient and aged Alzheimer's disease mice.

BACKGROUND: As one major symptom of Alzheimer's disease (AD), anterograde amnesia describes patients with an inability in new memory formation. The crucial role of the entorhinal cortex in forming new memories has been well established, and the neuropeptide cholecystokinin (CCK) is reported to be released from the entorhinal cortex to enable neocortical associated memory and long-term potentiation. Though several studies reveal that the entorhinal cortex and CCK are related to AD, it is less well studied. It is unclear whether CCK is a good biomarker or further a great drug candidate for AD. METHODS: mRNA expressions of CCK and CCK-B receptor (CCKBR) were examined in two mouse models, 3xTg AD and CCK knock-out (CCK-/-) mice. Animals' cognition was investigated with Morris water maze, novel object recognition test and neuroplasticity with in-vitro electrophysiological recording. Drugs were given intraperitoneally to animals to investigate the rescue effects on cognitive deficits, or applied to brain slices directly to explore the influence in inducement of long-term potentiation. RESULTS: Aged 3xTg AD mice exhibited reduced CCK mRNA expression in the entorhinal cortex but reduced CCKBR expression in the neocortex and hippocampus, and impaired cognition and neuroplasticity comparable with CCK-/- mice. Importantly, the animals displayed improved performance and enhanced long-term potentiation after the treatment of CCKBR agonists. CONCLUSIONS: Here we provide more evidence to support the role of CCK in learning and memory and its potential to treat AD. We elaborated on the rescue effect of a promising novel drug, HT-267, on aged 3xTg AD mice. Although the physiological etiology of CCK in AD still needs to be further investigated, this study sheds light on a potential pharmaceutical candidate for AD and dementia.

Histological and Memory Alterations in an Innovative Alzheimer's Disease Animal Model by Vanadium Pentoxide Inhalation.

Background: Previous work from our group has shown that chronic exposure to Vanadium pentoxide (V2O5) causes cytoskeletal alterations suggesting that V2O5 can interact with cytoskeletal proteins through polymerization and tyrosine phosphatases inhibition, causing Alzheimer's disease (AD)-like hippocampal cell death. Objective: This work aims to characterize an innovative AD experimental model through chronic V2O5 inhalation, analyzing the spatial memory alterations and the presence of neurofibrillary tangles (NFTs), amyloid-ß (Aß) senile plaques, cerebral amyloid angiopathy, and dendritic spine loss in AD-related brain structures. Methods: 20 male Wistar rats were divided into control (deionized water) and experimental (0.02 M V2O5 1 h, 3/week for 6 months) groups (n = 10). The T-maze test was used to assess spatial memory once a month. After 6 months, histological alterations of the frontal and entorhinal cortices, CA1, subiculum, and amygdala were analyzed by performing Congo red, Bielschowsky, and Golgi impregnation. Results: Cognitive results in the T-maze showed memory impairment from the third month of V2O5 inhalation. We also noted NFTs, Aß plaque accumulation in the vascular endothelium and pyramidal neurons, dendritic spine, and neuronal loss in all the analyzed structures, CA1 being the most affected. Conclusions: This model characterizes neurodegenerative changes specific to AD. Our model is compatible with Braak AD stage IV, which represents a moment where it is feasible to propose therapies that have a positive impact on stopping neuronal damage.

Light-Driven Sodium Pump as a Potential Tool for the Control of Seizures in Epilepsy.

The marine flavobacterium Krokinobactereikastus light-driven sodium pump (KR2) generates an outward sodium ion current under 530 nm light stimulation, representing a promising optogenetic tool for seizure control. However, the specifics of KR2 application to suppress epileptic activity have not yet been addressed. In the present study, we investigated the possibility of KR2 photostimulation to suppress epileptiform activity in mouse brain slices using the 4-aminopyrindine (4-AP) model. We injected the adeno-associated viral vector (AAV-PHP.eB-hSyn-KR2-YFP) containing the KR2 sodium pump gene enhanced with appropriate trafficking tags. KR2 expression was observed in the lateral entorhinal cortex and CA1 hippocampus. Using whole-cell patch clamp in mouse brain slices, we show that KR2, when stimulated with LED light, induces a substantial hyperpolarization of entorhinal neurons. However, continuous photostimulation of KR2 does not interrupt ictal discharges in mouse entorhinal cortex slices induced by a solution containing 4-AP. KR2-induced hyperpolarization strongly activates neuronal HCN channels. Consequently, turning off photostimulation resulted in HCN channel-mediated rebound depolarization accompanied by a transient increase in spontaneous network activity. Using low-frequency pulsed photostimulation, we induced the generation of short HCN channel-mediated discharges that occurred in response to the light stimulus being turned off; these discharges reliably interrupt ictal activity. Thus, low-frequency pulsed photostimulation of KR2 can be considered as a potential tool for controlling epileptic seizures.

A hippocampus dependent neural circuit loop underlying the generation of auditory mismatch negativity.

Extracting relevant information and transforming it into appropriate behavior, is a fundamental brain function, and requires the coordination between the sensory and cognitive systems, however, the underlying mechanisms of interplay between sensory and cognition systems remain largely unknown. Here, we developed a mouse model for mimicking human auditory mismatch negativity (MMN), a well-characterized translational biomarker for schizophrenia, and an index of early auditory information processing. We found that a subanesthetic dose of ketamine decreased the amplitude of MMN in adult mice. Using pharmacological and chemogenetic approaches, we identified an auditory cortex-entorhinal cortex-hippocampus neural circuit loop that is required for the generation of MMN. In addition, we found that inhibition of dCA1→MEC circuit impaired the auditory related fear discrimination. Moreover, we found that ketamine induced MMN deficiency by inhibition of long-range GABAergic projection from the CA1 region of the dorsal hippocampus to the medial entorhinal cortex. These results provided circuit insights for ketamine effects and early auditory information processing. As the entorhinal cortex is the interface between the neocortex and hippocampus, and the hippocampus is critical for the formation, consolidation, and retrieval of episodic memories and other cognition, our results provide a neural mechanism for the interplay between the sensory and cognition systems.

Ellagic Acid Modulates the Amyloid Precursor Protein Gene via Superoxide Dismutase Regulation in the Entorhinal Cortex in an Experimental Alzheimer's Model.

Patients suffering from Alzheimer's disease (AD) are still increasing worldwide. The development of (AD) is related to oxidative stress and genetic factors. This study investigated the therapeutic effects of ellagic acid (EA) on the entorhinal cortex (ERC), which plays a major role in episodic memory, in the brains of an AD rat model. AD was induced using AlCl3 (50 mg/kg orally for 4 weeks). Rats were divided into four groups: control, AD model, EA (treated with 50 mg/kg EA orally for 4 weeks), and ADEA (AD rats treated with EA after AlCl3 was stopped) groups. All rats were investigated for episodic memory using the novel object recognition test (NORT), antioxidant serum biomarkers, lipid peroxidation, histopathology of the ERC, and quantitative PCR for the superoxide dismutase (SOD) gene. EA therapy in AD rats significantly increased the discrimination index for NORT and the levels of SOD, glutathione, and total antioxidant capacity. Lipid peroxidation products were decreased, and the neurofibrillary tangles and neuritic plaques in the ERC sections were reduced after EA administration. The decrease in ERC thickness in the AD group, caused by caspase-3-mediated apoptosis and neurotoxicity due to amyloid precursor protein, was modulated by the increased SOD mRNA expression. Adjustment of the ERC antioxidant environment and decreased oxidative stress under EA administration enhanced SOD expression, resulting in the modulation of amyloid precursor protein toxicity and caspase-3-mediated apoptosis, thereby restoring episodic memory.

Prenatal sevoflurane exposure causes abnormal development of the entorhinal cortex in rat offspring.

As a gamma-aminobutyric acid type A receptor agonist sevoflurane is a common general anesthetic used in anesthesia and affects the neural development in offspring. We hypothesized that sevoflurane could regulate interneurons via the neuregulin-1-epidermal growth factor receptor-4 (NRG1-ErbB4) pathway in the entorhinal cortex (ECT) of the middle pregnancy. Six female rats in middle pregnancy (14.5 days of pregnancy) were randomly and equally divided into sevoflurane (SeV) and control groups. The rats in the SeV group were exposed to 4% sevoflurane for 3 hours. The expression levels of NRG1 and ErbB4, parvalbumin (PV) and glutamic acid decarboxylase (GAD67), and N-methyl-D-aspartate receptor subunit 2A (NR2A) and subunit 2B (NR2B) in offspring were examined through immunohistochemistry. The pyramidal neurons in the ECT were examined via Golgi staining. The levels of NRG1 and ErbB4 were significantly decreased (P < 0.01) and the levels of PV and GAD67 (interneurons) were found to be decreased in the SeV group (P < 0.01). The level of NR2B was found to be increased while the level of NR2A being decreased in the SeV group (P < 0.01). The development of pyramidal neurons was abnormal in the SeV group (P < 0.05). Conclusively, prenatal sevoflurane exposure could lead to the disturbance of the interneurons by activating the NRG1-ErbB4 pathway and subsequently result in abnormal development of pyramidal neurons in middle pregnancy. Prenatal sevoflurane exposure in middle pregnancy could be potentially harmful to the neural development of rat offspring. This study may reveal a novel pathway in the influence mechanism of sevoflurane on rat offspring.

Memantine ameliorates cognitive deficit in AD mice via enhancement of entorhinal-CA1 projection.

BACKGROUND: Memantine, a low- to moderate-affinity uncompetitive N-methyl-D-aspartate receptor antagonist, has been shown to improve cognitive functions in animal models of Alzheimer's disease (AD). Here we treated APP/PS1 AD mice with a therapeutic dose of memantine (20 mg/kg/day) and examined its underlying mechanisms in ameliorating cognitive defects. METHODS: Using behavioral, electrophysiological, optogenetic and morphology approaches to explore how memantine delay the pathogenesis of AD. RESULTS: Memantine significantly improved the acquisition in Morris water maze (MWM) in APP/PS1 mice without affecting the speed of swimming. Furthermore, memantine enhanced EC to CA1 synaptic neurotransmission and promoted dendritic spine regeneration of EC neurons that projected to CA1. CONCLUSIONS: Our study reveals the underlying mechanism of memantine in the treatment of AD mice.

2-Pentadecyl-2-oxazoline ameliorates memory impairment and depression-like behaviour in neuropathic mice: possible role of adrenergic alpha2- and H3 histamine autoreceptors.

Neuropathic pain (NP) remains an untreatable disease due to the complex pathophysiology that involves the whole pain neuraxis including the forebrain. Sensory dysfunctions such as allodynia and hyperalgesia are only part of the symptoms associated with neuropathic pain that extend to memory and affectivity deficits. The development of multi-target molecules might be a promising therapeutic strategy against the symptoms associated with NP. 2-pentadecyl-2-oxazoline (PEA-OXA) is a plant-derived agent, which has shown effectiveness against chronic pain and associated neuropsychiatric disorders. The molecular mechanisms by which PEA-OXA exerts its effects are, however, only partially known. In the current study, we show that PEA-OXA, besides being an alpha2 adrenergic receptor antagonist, also acts as a modulator at histamine H3 receptors, and report data on its effects on sensory, affective and cognitive symptoms associated with the spared nerve injury (SNI) model of neuropathic pain in mice. Treatment for 14 days with PEA-OXA after the onset of the symptoms associated with neuropathic pain resulted in the following effects: (i) allodynia was decreased; (ii) affective/cognitive impairment associated with SNI (depression, spatial, and working memories) was counteracted; (iii) long-term potentiation in vivo in the lateral entorhinal cortex-dentate gyrus (perforant pathway, LPP) was ameliorated, (iv) hippocampal glutamate, GABA, histamine, norepinephrine and dopamine level alterations after peripheral nerve injury were reversed, (v) expression level of the TH positive neurons in the Locus Coeruleus were normalized. Thus, a 16-day treatment with PEA-OXA alleviates the sensory, emotional, cognitive, electrophysiological and neurochemical alterations associated with SNI-induced neuropathic pain.

T1AM-TAAR1 signalling protects against OGD-induced synaptic dysfunction in the entorhinal cortex.

Abnormalities in thyroid hormones (TH) availability and/or metabolism have been hypothesized to contribute to Alzheimer's disease (AD) and to be a risk factor for stroke. Recently, 3-iodothyronamine (T1AM), an endogenous amine putatively derived from TH metabolism, gained interest for its ability to promote learning and memory in the mouse. Moreover, T1AM has been demonstrated to rescue the ß-Amyloid dependent LTP impairment in the entorhinal cortex (EC), a brain area crucially involved in learning and memory and early affected during AD. In the present work, we have investigated the effect of T1AM on ischemia-induced EC synaptic dysfunction. In EC brain slices exposed to oxygen-glucose deprivation (OGD), we demonstrated that the acute perfusion of T1AM (5 µM) was capable of preventing ischemia-induced synaptic depression and that this protective effect was mediated by the trace amine-associated receptor 1 (TAAR1). Moreover, we demonstrated that activation of the BDNF-TrkB signalling is required for T1AM action during ischemia. The protective effect of T1AM was more evident when using EC slices from transgenic mutant human APP (mhAPP mice) that are more vulnerable to the effect of OGD. Our results confirm that the TH derivative T1AM can rescue synaptic function after transient ischemia, an effect that was also observed in a Aß-enriched environment.

The anticonvulsant zonisamide positively modulates recombinant and native glycine receptors at clinically relevant concentrations.

GABAA and glycine receptors mediate fast synaptic inhibitory neurotransmission. Despite studies showing that activation of cerebral glycine receptors could be a potential strategy in the treatment of epilepsy, few studies have assessed the effects of existing anticonvulsant therapies on recombinant or native glycine receptors. We, therefore, evaluated the actions of a series of anticonvulsants at recombinant human homo-oligomeric glycine receptor α1, α2 and α3 subtypes expressed in Xenopus oocytes using two-electrode voltage-clamp methods, and then assessed the most effective drug at native glycine receptors from entorhinal cortex neurons using whole-cell voltage-clamp recordings. Ganaxolone, tiagabine and zonisamide positively modulated glycine induced currents at recombinant homomeric glycine receptors. Of these, zonisamide was the most efficacious and exhibited an EC50 value ranging between 450 and 560 µM at α1, α2 and α3 subtypes. These values were not significantly different indicating a non-selective modulation of glycine receptors. Using a therapeutic concentration of zonisamide (100 µM), the potency of glycine was significantly shifted from 106 to 56 µM at α1, 185 to 112 µM at α2, and 245 to 91 µM at α3 receptors. Furthermore, zonisamide (100 µM) potentiated exogenous homomeric and heteromeric glycine mediated currents from layer II pyramidal cells of the lateral or medial entorhinal cortex. As therapeutic concentrations of zonisamide positively modulate recombinant and native glycine receptors, we propose that the anticonvulsant effects of zonisamide may, at least in part, be mediated via this action.

D-serine mitigates cell loss associated with temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) is the most common type of drug-resistant epilepsy in adults, with an unknown etiology. A hallmark of TLE is the characteristic loss of layer 3 neurons in the medial entorhinal area (MEA) that underlies seizure development. One approach to intervention is preventing loss of these neurons through better understanding of underlying pathophysiological mechanisms. Here, we show that both neurons and glia together give rise to the pathology that is mitigated by the amino acid D-serine whose levels are potentially diminished under epileptic conditions. Focal administration of D-serine to the MEA attenuates neuronal loss in this region thereby preventing epileptogenesis in an animal model of TLE. Additionally, treatment with D-serine reduces astrocyte counts in the MEA, alters their reactive status, and attenuates proliferation and/or infiltration of microglia to the region thereby curtailing the deleterious consequences of neuroinflammation. Given the paucity of compounds that reduce hyperexcitability and neuron loss, have anti-inflammatory properties, and are well tolerated by the brain, D-serine, an endogenous amino acid, offers new hope as a therapeutic agent for refractory TLE.

Intramuscular Injection of BOTOX® Boosts Learning and Memory in Adult Mice in Association with Enriched Circulation of Platelets and Enhanced Density of Pyramidal Neurons in the Hippocampus.

BOTOX® is a therapeutic form of botulinum neurotoxin. It acts by blocking the release of acetylcholine (ACh) from the synaptic vesicles at the neuromuscular junctions, thereby inhibiting the muscle contraction. Notably, many neurological diseases have been characterized by movement disorders in association with abnormal levels of ACh. Thus, blockade of aberrant release of ACh appears to be a potential therapeutic strategy to mitigate many neurological deficits. BOTOX® has widely been used to manage a number of clinical complications like neuromuscular disorders, migraine and neuropathic pain. While the beneficial effects of BOTOX® against movement disorders have extensively been studied, its possible role in the outcome of cognitive function remains to be determined. Therefore, we investigated the effect of BOTOX® on learning and memory in experimental adult mice using behavioural paradigms such as open field task, Morris water maze and novel object recognition test in correlation with haematological parameters and histological assessments of the brain. Results revealed that a mild dose of BOTOX® treatment via an intramuscular route in adult animals improves learning and memory in association with increased number of circulating platelets and enhanced structural plasticity in the hippocampus. In the future, this minimally invasive treatment could be implemented to ameliorate different forms of dementia resulting from abnormal ageing and various neurocognitive disorders including Alzheimer's disease (AD).

Acute application of Centella asiatica extract enhanced AMPAR-mediated postsynaptic currents in rat entorhinal cortex.

Centella asiatica is notable for its wide range of biological activities beneficial to human health, particularly its cognitive enhancement and neuroprotective effects. The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors are ionotropic glutamate receptors mediating fast excitatory neurotransmission essential in long-term potentiation widely thought to be the cellular mechanism of learning and memory. The method of whole-cell patch-clamp was used to study the effect of the acute application of Centella asiatica extract on the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated spontaneous excitatory postsynaptic currents in the entorhinal cortex of rat brain slices. The respective low dose of test compounds significantly increased the amplitude of spontaneous excitatory postsynaptic currents while having no significant effects on the frequency. The findings suggested that Centella asiatica extract increased the response of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors at the postsynaptic level, revealing the potential role of Centella asiatica in modulating the glutamatergic responses in the entorhinal cortex of rat brain slices to produce cognitive enhancement effects.

Activity- and pH-dependent adenosine shifts at the end of a focal seizure in the entorhinal cortex.

Adenosine (ADO) is an endogenous modulator of neuronal excitability, with anticonvulsant and neuroprotective effects. It has been proposed that the activity-dependent release of ADO promoted by the extracellular acidification occurring during seizures contributes to seizure termination. To verify this hypothesis, we recorded field potentials, pH and ADO changes measured with enzymatic biosensors during acute focal seizures in the medial entorhinal cortex (mEC) of the isolated guinea-pig brain maintained in vitro. The effect of ADO on seizure-like events (SLEs) induced by GABAa receptor antagonism with bicuculline methiodide (BMI; 50 µM) was assessed by arterial applications of 1 mM ADO. ADO either reduced or prevented epileptiform activity. The A1 receptor antagonist DPCPX (100-500 µM) prolonged BMI-induced seizures and was able to precipitate SLEs in the absence of proconvulsant. Simultaneous recordings of brain activity, extracellular ADO and pH shifts demonstrated that ADO decreases at the onset and progressively rises toward the end of SLEs induced by either BMI or 4-aminopyridine (4AP; 50 µM), reaching maximal values 1-5 min after SLE termination. ADO changes were preceded by a SLE-dependent extracellular acid shift. Both pH acidification and ADO changes were abolished by 22 mM HEPES in the arterial perfusate. In these conditions, SLE duration was prolonged. Our data confirm that ADO plays a role in regulating brain excitability. Its increase depends on seizure-induced acid pH shift and it is maximal after the end of the SLE. These findings strongly suggest that ADO contributes to termination of focal seizures and to the establishment of the postictal depression.

Cyclosomatostatin-induced catalepsy in aged rats: Specific change of brain c-Fos protein expression in the lateral entorhinal cortex.

Aging represents the largest risk factor for developing Parkinson's disease (PD); another salient feature of this disorder is a decreased brain levels of somatostatin. Recently, in aged Wistar rats, we simulated the central somatostatinergic deficiency by intracerebroventricular injections of a somatostatin antagonist, cyclosomatostatin (cSST). The treated animals displayed catalepsy, a state that resembles the extrapyramidal signs of Parkinson's disease; young animals were insensitive to cSST. The neuroanatomical substrates responsible for the increased cataleptogenic activity of cSST in aged animals, are currently unknown. To study this issue, we assessed the cSST effect on brain c-Fos-protein expression in aged and young rats; thirty three brain regions were examined. cSST was employed at the dose cataleptogenic for aged animals and non-cataleptogenic for young ones. c-Fos expression patterns in the 'cataleptic' and 'non-cataleptic' animals were very similar, with the only distinction being a decrease in the c-Fos expression in the aged lateral entorhinal cortex (LEntCx). This decrease was not observed when the cSST-induced cataleptic response was inhibited by administration of diphenhydramine and nicotine. Thus, the development of catalepsy in the aged Wistar rats appeared to be associated with a hypoactivation of the LEntCx; possibly, there exists a mechanistic link between the LEntCx hypoactivation and increased susceptibility of aged rats to catalepsy. Apparently, these findings may provide novel insight into the link between mechanisms of parkinsonian motor disorders and aging.

KCC2 antagonism and gabaergic synchronization in the entorhinal cortex in the absence of ionotropic glutamatergic receptor signalling.

γ-Aminobutyric acid (GABA), which is released by interneurons, plays an active role in generating interictal epileptiform spikes during blockade of ionotropic glutamatergic signalling, but it remains unclear whether and how the K+-Cl- cotransporter 2 (KCC2) influences these paroxysmal events. Therefore, we employed tetrode recordings in the in vitro rat entorhinal cortex (EC) to analyze the effects of the KCC2 antagonist VU0463271 on 4-aminopyridine (4AP)-induced interictal spikes that were pharmacologically isolated by applying ionotropic glutamatergic receptor antagonists. After the addition of VU0463271, these interictal spikes continued to occur at similar rates as in control (i.e., during application of 4AP with ionotropic glutamatergic receptor antagonists) but were smaller and shorter. Despite the absence of ionotropic glutamatergic receptor signalling, both interneurons and principal cells increased their firing during interictal spikes. Moreover, we found that KCC2 antagonism increased interneuron firing but decreased principal cell firing during the interictal spike rising phase; in contrast, during the falling phase, interneuron firing decreased in the presence of VU0463271 while no change was observed in principal cell firing. Overall, our results show that KCC2 antagonism enhances interneuron excitability at the onset of interictal spikes generated by the EC neuronal networks during blockade of ionotropic glutamatergic transmission but disrupts later neuronal recruitment.

Cholinergic Switch between Two Types of Slow Waves in Cerebral Cortex.

Sleep slow waves are known to participate in memory consolidation, yet slow waves occurring under anesthesia present no positive effects on memory. Here, we shed light onto this paradox, based on a combination of extracellular recordings in vivo, in vitro, and computational models. We find two types of slow waves, based on analyzing the temporal patterns of successive slow-wave events. The first type is consistently observed in natural slow-wave sleep, while the second is shown to be ubiquitous under anesthesia. Network models of spiking neurons predict that the two slow wave types emerge due to a different gain on inhibitory versus excitatory cells and that different levels of spike-frequency adaptation in excitatory cells can account for dynamical distinctions between the two types. This prediction was tested in vitro by varying adaptation strength using an agonist of acetylcholine receptors, which demonstrated a neuromodulatory switch between the two types of slow waves. Finally, we show that the first type of slow-wave dynamics is more sensitive to external stimuli, which can explain how slow waves in sleep and anesthesia differentially affect memory consolidation, as well as provide a link between slow-wave dynamics and memory diseases.