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Aggregation of the microtubule-associated protein tau into paired helical filaments (PHFs) and neurofibrillary tangles is a defining characteristic of Alzheimer’s Disease. Various plant polyphenols disrupt tau aggregation in vitro but display poor bioavailability and low potency, challenging their therapeutic translation. We previously reported that oral administration of the flavonoid (−)-epicatechin (EC) reduced Amyloid-β (Aβ) plaque pathology in APP/PS1 transgenic mice. Here, we investigated whether EC impacts on tau pathology, independent of actions on Aβ, using rTg4510 mice expressing P301L mutant tau. 4 and 6.5 months old rTg4510 mice received EC (∼18 mg/day) or vehicle (ethanol) via drinking water for 21 days and the levels of total and phosphorylated tau were assessed. At 4 months, tau appeared as two bands of ∼55 kDa, phosphorylated at Ser262 and Ser396 and was unaffected by exposure to EC. At 6.5 months an additional higher molecular weight form of tau was detected at ∼64 kDa which was phosphorylated at Ser262, Ser396 and additionally at the AT8 sites, indicative of the presence of PHFs. EC consumption reduced the levels of the ∼64 kDa tau species and inhibited phosphorylation at Ser262 and AT8 phosphoepitopes. Regulation of the key tau kinase glycogen synthase kinase 3β (GSK3β) by phosphorylation at Ser9 was not altered by exposure to EC in mice or primary neurons. Furthermore, EC did not significantly inhibit GSK3β activity at physiologically-relevant concentrations in a cell free assay. Therefore, a 21-day intervention with EC inhibits or reverses the development of tau pathology in rTg4510 mice independently of direct inhibition of GSK3β.

Rapid eye movement (REM) sleep behaviour disorder (RBD) is a rare parasomnia that may predict the later occurrence of alpha-synucleinopathies. Variants in the gene encoding for the lysosomal enzyme glucocerebrosidase, GBA, strongly increase the risk of RBD. In a GBA1-mouse model recently shown to mimic prodromal stages of α-synucleinopathy, we now demonstrate striking REM and NREM sleep abnormalities accompanied by distinct structural changes in the more widespread sleep neurocircuitry.

Transgenic mice that overproduce beta-amyloid (Aβ) peptides exhibit neurophysiological alterations at the cellular, synaptic and network levels. Recently, increased neuronal activity in nucleus reuniens (Re), has been linked to hyperexcitability within hippocampal-thalamo-cortical networks in the J20 mouse model of amyloidopathy. Here in vitro whole-cell patch clamp recordings were used to compare old pathology-bearing J20 mice and wild-type controls to examine whether alterations to the intrinsic electrophysiological properties of Re neurons could contribute to the amyloidopathy-associated Re hyperactivity. A greater proportion of Re neurons displayed a hyperpolarised membrane potential in J20 mice without changes to the incidence or frequency of spontaneous action potential (AP) generation. Passive membrane properties were independent of transgene expression. Re neurons recorded from J20 mice did not exhibit increased AP generation in response to depolarising current stimuli but did exhibit an increased propensity to rebound burst following hyperpolarising current stimuli. This increase in rebound firing does not appear to result from alterations to T-type calcium channels. Finally, in J20 mice there was an ∼8% reduction in spike width, similar to what we and others have reported in CA1 pyramidal neurons from multiple amyloidopathy mice. We conclude that alterations to the intrinsic properties of Re neurons may contribute to the hyperexcitability observed in hippocampal-thalmo-cortical circuits under pathological Aβ load.Key PointsAlterations in the neurophysiology of hippocampal and cortical neurons has been linked to network hyperexcitability in mouse models of amyloidopathy.The nucleus reuniens (Re) is part of a cognitive network involving the hippocampal formation and prefrontal cortex. Increased cellular activity in Re has been linked to the generation of hippocampal-thalamo-cortical seizure activity in J20 mice.Re neurons display hyperpolarised resting membrane potentials in J20 mice. Passive membrane properties are unaffected by transgene expression. Re neurons recorded from J20 mice did not exhibit increased excitability in response to depolarising current stimuli but did exhibit an increased propensity to rebound burst following hyperpolarising current stimuli. This increased rebound firing was not a result of changes in T-type Ca2+ conductances. Finally we observed a decrease in AP width.These results help us understand how altered Re cellular neurophysiology may contribute to hippocampal-thalamo-cortical hyperexcitability in J20 mice.

Action potential firing in hippocampal pyramidal neurons is regulated by generation of an afterhyperpolarization (AHP). Three phases of AHP are recognized, with the fast AHP regulating action potential firing at the onset of a burst and the medium and slow AHPs supressing action potential firing over hundreds of milliseconds and seconds, respectively. Activation of β-adrenergic receptors suppresses the slow AHP by a protein kinase A-dependent pathway. However, little is known regarding modulation of the medium AHP. Application of the selective β-adrenergic receptor agonist isoproterenol suppressed both the medium and slow AHPs evoked in rat CA1 hippocampal pyramidal neurons recorded from slices maintained in organotypic culture. Suppression of the slow AHP was mimicked by intracellular application of cAMP, with the suppression of the medium AHP by isoproterenol still being evident in cAMP-dialyzed cells. Suppression of both the medium and slow AHPs was antagonized by the β-adrenergic receptor antagonist propranolol. The effect of isoproterenol to suppress the medium AHP was mimicked by two β3-adrenergic receptor agonists, BRL37344 and SR58611A. The medium AHP was mediated by activation of small-conductance calcium-activated K+ channels and deactivation of H channels at the resting membrane potential. Suppression of the medium AHP by isoproterenol was reduced by pretreating cells with the H-channel blocker ZD7288. These data suggest that activation of β3-adrenergic receptors inhibits H channels, which suppresses the medium AHP in CA1 hippocampal neurons by utilizing a pathway that is independent of a rise in intracellular cAMP. This finding highlights a potential new target in modulating H-channel activity and thereby neuronal excitability. NEW & NOTEWORTHY The noradrenergic input into the hippocampus is involved in modulating long-term synaptic plasticity and is implicated in learning and memory. We demonstrate that activation of functional β3-adrenergic receptors suppresses the medium afterhyperpolarization in hippocampal pyramidal neurons. This finding provides an additional mechanism to increase action potential firing frequency, where neuronal excitability is likely to be crucial in cognition and memory.

OBJECTIVES: Functional and structural disconnection of the brain is a prevailing hypothesis to explain cognitive impairment in Alzheimer's Disease (AD). We aim to understand the link between alterations to networks and cognitive impairment using functional connectivity analysis and modelling.METHODS: EEG was recorded from 21 AD patients and 26 controls, mapped into source space using eLORETA, and functional connectivity was calculated using phase locking factor. The mini-mental state exam (MMSE) was used to assess cognitive impairment. A computational model was used to uncover mechanisms of altered functional connectivity.RESULTS: Small-worldness (SW) of functional networks decreased in AD and was positively correlated with MMSE score and the language sub-score. Reduced SW was a result of increased path lengths, predominantly localized to the temporal lobes. Combining observed differences in local oscillation frequency with reduced temporal lobe effective connectivity in the model could account for observed functional network differences.CONCLUSIONS: Temporal lobe disconnection plays a key role in cognitive impairment in AD.SIGNIFICANCE: We combine electrophysiology, neuropsychological scores, and computational modelling to provide novel insight into the relationships between the disconnection hypothesis and cognitive decline in AD.

Action potential firing in hippocampal pyramidal neurons is regulated by generation of an afterhyperpolarization (AHP). Three phases of AHP are recognized, with the fast AHP regulating action potential firing at the onset of a burst and the medium and slow AHPs supressing action potential firing over hundreds of milliseconds and seconds, respectively. Activation of β-adrenergic receptors suppresses the slow AHP by a protein kinase A-dependent pathway. However, little is known regarding modulation of the medium AHP. Application of the selective β-adrenergic receptor agonist isoproterenol suppressed both the medium and slow AHPs evoked in rat CA1 hippocampal pyramidal neurons recorded from slices maintained in organotypic culture. Suppression of the slow AHP was mimicked by intracellular application of cAMP, with the suppression of the medium AHP by isoproterenol still being evident in cAMP-dialyzed cells. Suppression of both the medium and slow AHPs was antagonized by the β-adrenergic receptor antagonist propranolol. The effect of isoproterenol to suppress the medium AHP was mimicked by two β(3)-adrenergic receptor agonists, BRL37344 and SR58611A. The medium AHP was mediated by activation of small-conductance calcium-activated K(+) channels and deactivation of H channels at the resting membrane potential. Suppression of the medium AHP by isoproterenol was reduced by pretreating cells with the H-channel blocker ZD7288. These data suggest that activation of β(3)-adrenergic receptors inhibits H channels, which suppresses the medium AHP in CA1 hippocampal neurons by utilizing a pathway that is independent of a rise in intracellular cAMP. This finding highlights a potential new target in modulating H-channel activity and thereby neuronal excitability. NEW & NOTEWORTHY The noradrenergic input into the hippocampus is involved in modulating long-term synaptic plasticity and is implicated in learning and memory. We demonstrate that activation of functional β(3)-adrenergic receptors suppresses the medium afterhyperpolarization in hippocampal pyramidal neurons. This finding provides an additional mechanism to increase action potential firing frequency, where neuronal excitability is likely to be crucial in cognition and memory.

Although numerous protocols have been developed for differentiation of neurons from a variety of pluripotent stem cells, most have concentrated on being able to specify effectively appropriate neuronal subtypes and few have been designed to enhance or accelerate functional maturity. Of those that have, most employ time courses of functional maturation that are rather protracted, and none have fully characterized all aspects of neuronal function, from spontaneous action potential generation through to postsynaptic receptor maturation. Here, we describe a simple protocol that employs the sequential addition of just two supplemented media that have been formulated to separate the two key phases of neural differentiation, the neurogenesis and synaptogenesis, each characterized by different signaling requirements. Employing these media, this new protocol synchronized neurogenesis and enhanced the rate of maturation of pluripotent stem cell-derived neural precursors. Neurons differentiated using this protocol exhibited large cell capacitance with relatively hyperpolarized resting membrane potentials; moreover, they exhibited augmented: 1) spontaneous electrical activity; 2) regenerative induced action potential train activity; 3) Na+ current availability, and 4) synaptic currents. This was accomplished by rapid and uniform development of a mature, inhibitory GABAA receptor phenotype that was demonstrated by Ca2+ imaging and the ability of GABAA receptor blockers to evoke seizurogenic network activity in multielectrode array recordings. Furthermore, since this protocol can exploit expanded and frozen prepatterned neural progenitors to deliver mature neurons within 21 days, it is both scalable and transferable to high-throughput platforms for the use in functional screens.

Accumulation of beta-amyloid (Aβ) peptides in the human brain is a canonical pathological hallmark of Alzheimer's disease (AD). Recent work in Aβ-overexpressing transgenic mice indicates that increased brain Aβ levels can be associated with aberrant epileptiform activity. In line with this, such mice can also exhibit altered intrinsic excitability (IE) of cortical and hippocampal neurons: these observations may relate to the increased prevalence of seizures in AD patients. In this study, we examined what changes in IE are produced in hippocampal CA1 pyramidal cells after 2-5 h treatment with an oligomeric preparation of synthetic human Aβ 1-42 peptide. Whole cell current clamp recordings were compared between Aβ-(500 nM) and vehicle-(DMSO 0.05%) treated hippocampal slices obtained from mice. The soluble Aβ treatment did not produce alterations in sub-threshold intrinsic properties, including membrane potential, input resistance, and hyperpolarization activated "sag". Similarly, no changes were noted in the firing profile evoked by 500 ms square current supra-threshold stimuli. However, Aβ 500 nM treatment resulted in the hyperpolarization of the action potential (AP) threshold. In addition, treatment with Aβ at 500 nM depressed the after-hyperpolarization that followed both a single AP or 50 Hz trains of a number of APs between 5 and 25. These data suggest that acute exposure to soluble Aβ oligomers affects IE properties of CA1 pyramidal neurons differently from outcomes seen in transgenic models of amyloidopathy. However, in both chronic and acute models, the IE changes are toward hyperexcitability, reinforcing the idea that amyloidopathy and increased incidence in seizures might be causally related in AD patients.

Chronic cerebral hypoperfusion is a key mechanism associated with white matter disruption in cerebral vascular disease and dementia. In a mouse model relevant to studying cerebral vascular disease, we have previously shown that cerebral hypoperfusion disrupts axon-glial integrity and the distribution of key paranodal and internodal proteins in subcortical myelinated axons. This disruption of myelinated axons is accompanied by increased microglia and cognitive decline. The aim of the present study was to investigate whether hypoperfusion impairs the functional integrity of white matter, its relation with axon-glial integrity and microglial number, and whether by targeting microglia these effects can be improved. We show that in response to increasing durations of hypoperfusion, the conduction velocity of myelinated fibres in the corpus callosum is progressively reduced and that paranodal and internodal axon-glial integrity is disrupted. The number of microglial cells increases in response to hypoperfusion and correlates with disrupted paranodal and internodal integrity and reduced conduction velocities. Further minocycline, a proposed anti-inflammatory and microglia inhibitor, restores white matter function related to a reduction in the number of microglia. The study suggests that microglial activation contributes to the structural and functional alterations of myelinated axons induced by cerebral hypoperfusion and that dampening microglia numbers/proliferation should be further investigated as potential therapeutic benefit in cerebral vascular disease.

Amyloidopathy involves the accumulation of insoluble amyloid β (Aβ) species in the brain's parenchyma and is a key histopathological hallmark of Alzheimer's disease (AD). Work on transgenic mice that overexpress Aβ suggests that elevated Aβ levels in the brain are associated with aberrant epileptiform activity and increased intrinsic excitability (IE) of CA1 hippocampal neurons. In this study we examined if similar changes could be observed in hippocampal CA1 pyramidal neurons from aged PDAPP mice (20-23 month old, Indiana mutation: V717F on APP gene) compared to their age-matched wild-type littermate controls. Whole-cell current clamp recordings revealed that sub-threshold intrinsic properties, such as input resistance, resting membrane potential and hyperpolarization activated "sag" were unaffected, but capacitance was significantly decreased in the transgenic animals. No differences between genotypes were observed in the overall number of action potentials (AP) elicited by 500 ms supra-threshold current stimuli. PDAPP neurons, however, exhibited higher instantaneous firing frequencies after accommodation in response to high intensity current injections. The AP waveform was narrower and shorter in amplitude in PDAPP mice: these changes, according to our in silico model of a CA1/3 pyramidal neuron, depended on the respective increase and reduction of K(+) and Na(+) voltage-gated channels maximal conductances. Finally, the after-hyperpolarization, seen after the first AP evoked by a +300 pA current injection and after 50 Hz AP bursts, was more pronounced in PDAPP mice. These data show that Aβ-overexpression in aged mice altered the capacitance, the neuronal firing and the AP waveform of CA1 pyramidal neurons. Some of these findings are consistent with previous work on younger PDAPP; they also show important differences that can be potentially ascribed to the interaction between amyloidopathy and ageing. Such a change of IE properties over time underlies that the increased incidence of seizure observed in AD patients might rely on different mechanistic pathways during progression of the disease.

Highlights ► Global levels of ubiquitinated proteins increased in the hippocampus of Tg2576 mice. ► No global changes in either SUMO-1 or SUMO-2/3 conjugation in any brain regions analysed. ► SUMO conjugating and deconjugating enzymes, UBC9 and SENP-1, unaltered in Tg2576 mice. ► Total levels of AMPA and kainate receptors were also unaffected in Tg2576 mice. ► Posttranslational modification by ubiquitin may play a role in Alzheimer's disease., Alzheimer's disease (AD) is a major cause of disability in the elderly. The formation of senile plaques and neurofibrillary tangles are the main hallmarks of the disorder, whereas synaptic loss best correlates to the progressive cognitive decline. Interestingly, some of the proteins involved in these pathophysiological processes have been reported to be subject to posttranslational modification by ubiquitin and/or the small ubiquitin-like modifier (SUMO). Here we investigated global changes in protein SUMOylation and ubiquitination in vivo in a model of AD. We used Tg2576 transgenic mice, which overexpress a mutated human amyloid precursor protein (APP) gene implicated in familial AD. As expected, APP protein levels were dramatically increased in the hippocampus, cortex and cerebellum of Tg2576 mice. A significant increase in the global level of ubiquitinated proteins was observed in the hippocampus of Tg2576 mice. Significant or close to significant changes in individual bands of SUMO-1 or SUMO-2/3 conjugation were apparent in all brain regions investigated, although global levels were unaltered between wild-type and transgenic mice. Levels of SUMO-specific conjugating and deconjugating enzymes, UBC9 and SENP-1 were also unaltered in any of the brain regions analysed. Surprisingly, given the well-documented loss of synaptic function, total levels of the excitatory AMPA and kainate receptors were unaffected in the Tg2576 mice. These results suggest that alterations in SUMO substrate conjugation may occur and that global posttranslational modifications by ubiquitin may play an important role in the mechanisms underlying AD.

After-depolarisation is a hallmark of excitability in hippocampal pyramidal cells of CA1 and CA3 regions, because it constitutes the subthreshold relation between inward and outward ionic currents. This relationship determines the nominal response to stimuli and provides the necessary conditions for firing a spike or a burst of action potentials. Nevertheless, after-depolarisation is an inherently transient phenomenon that is not very well understood. We study after-depolarisation using a single-compartment pyramidal-cell model based on recent voltage- and current-clamp experimental data. We systematically investigate CA1 and CA3 behaviour and show that changes to maximal conductances of T-type Ca(2+)-current and muscarinic-sensitive and delayed rectifier K(+)-currents are sufficient to switch the behaviour of the model from a CA3 to a CA1 neuron. We use model analysis to define after-depolarisation and bursting threshold. We also explain the influence of particular ionic currents on this phenomenon. This study ends with a sensitivity analysis that demonstrates the influence of specific currents on excitability. Counter-intuitively, we find that a decrease of Na(+)-current could cause an increase in excitability. Our analysis suggests that a change of high-voltage activated Ca(2+)-current can have a similar effect.

Amyloid beta (Abeta) peptides derived from proteolytic cleavage of amyloid precursor protein (APP) are thought to be a pivotal toxic species in the pathogenesis of Alzheimer's disease (AD). Furthermore, evidence has been accumulating that components of APP processing pathway are involved in non-pathological normal function of the CNS. In this review we aim to cover the extensive body of research aimed at understanding how components of this pathway contribute to neurophysiological function of the CNS in health and disease. We briefly outline changes to clinical neurophysiology seen in AD patients before discussing functional changes in mouse models of AD which range from changes to basal synaptic transmission and synaptic plasticity through to abnormal synchronous network activity. We then describe the various neurophysiological actions that are produced by application of exogenous Abeta in various forms, and finally discuss a number or other neurophysiological aspects of the APP pathway, including functional activities of components of secretase complexes other than Abeta production.

Persistent plastic changes to the intrinsic excitability of neurons have substantial implications for computational processing within the CNS. We have identified and characterized a novel long-lasting form of intrinsic plasticity in hippocampal CA3 pyramidal cells. Although the patterns of action potential firing elicited in this cell population by depolarizing current injections exhibited considerable diversity, practically all cells produced an initial high frequency (>100 Hz) burst of two to five spikes. This burst involved conductances that were responsible for the prominent spike afterdepolarization of CA3 pyramids. Long-lasting changes in the firing behaviour of CA3 cells were produced by conditioning stimuli (CS) consisting of either periods of depolarization in voltage clamp or periods of short (2 or 4 spikes) high frequency (circa 100 Hz) burst firing at 5 or 10 Hz. CS-induced changes included substantial prolongation of the first inter-spike interval and increased spike jitter. Similar CS-induced changes were seen when the test stimulus used to elicit firing resembled a glutamatergic EPSC. In line with this, a long-lasting depression of the ADP was elicited by the same CS that altered firing patterns of CA3 cells. Conditioning-induced changes in both spiking patterns and ADP amplitude were blocked by buffering intracellular Ca2+ with BAPTA. Furthermore, the Kv7 channel blocker XE991, a cognitive enhancer, both enhanced the ADP and completely eliminated its conditioning-induced depression. These findings indicate that a persistent enhancement of Kv7 channels, following a transient increase in cytoplasmic Ca2+, results in a prolonged depression of the ADP in CA3 pyramidal neurones.

The α7 nicotinic acetylcholine receptor (nAChR) has been implicated in Alzheimer's disease and schizophrenia, leading to efforts targeted toward discovering agonists and positive allosteric modulators (PAMs) of this receptor. In a Ca2+ flux fluorometric imaging plate reader assay, SB-206553 (3,5-dihydro-5-methyl - N -3-pyridinylbenzo [1, 2- b :4,5 - b ′]-di pyrrole-1(2 H )-carboxamide), a compound known as a 5-hydroxytryptamine2B/2C receptor antagonist, produced an 8-fold potentiation of the evoked calcium signal in the presence of an EC20 concentration of nicotine and a corresponding EC50 of 1.5 μM for potentiation of EC20 nicotine responses in GH4C1 cells expressing the α7 receptor. SB-206553 was devoid of direct α7 receptor agonist activity and selective against other nicotinic receptors. Confirmation of the PAM activity of SB-206553 on the α7 nAChR was obtained in patch-clamp electrophysiological experiments in GH4C1 cells, where it failed to evoke any detectable currents when applied alone, yet dramatically potentiated the currents evoked by an EC20 (17 μM) and EC100 (124 μM) of acetylcholine (ACh). Native nicotinic receptors in CA1 stratum radiatum interneurons of rat hippocampal slices could also be activated by ACh (200 μM), an effect that was entirely blocked by the α7-selective antagonist methyllycaconitine (MLA). These ACh currents were potentiated by SB-206553, which increased the area of the current response significantly, resulting in a 40-fold enhancement at 100 μM. In behavioral experiments in rats, SB-206553 reversed an MK-801 (dizocilpine maleate)-induced deficit in the prepulse inhibition of acoustic startle response, an effect attenuated in the presence of MLA. This latter observation provides further evidence in support of the potential therapeutic utility of α7 nAChR PAMs in schizophrenia.

It is well established that activation of group I metabotropic glutamate receptors (mGluRs) produces long-lasting alterations in synaptic efficacy. We now demonstrate that activation of mGluRs can also induce long-term alterations in synchronised network activity that are both induced and expressed in the absence of chemical synaptic transmission. Specifically, in hippocampal slices in which synaptic transmission was eliminated by perfusing with a Ca 2+ -free medium, the selective group I mGluR agonist 3,5-dihydroxyphenylglycine (DHPG) induced a persistent (>3 h) enhancement (>2-fold) of the frequency of synchronised bursting activity. The underlying biochemical mechanism responsible for the induction of this form of plasticity was similar to that for DHPG-induced long-term depression (LTD) in that it required the activation of tyrosine phosphatases. Also, like DHPG-induced LTD, this form of neuronal plasticity could be reversed by application of the mGluR antagonist α-methyl-4-carboxyphenylglycine (MCPG). This unusual form of plasticity, which presumably also occurs when synaptic transmission is intact, could contribute to long-term alterations in synchronised activity in hippocampal neuronal networks.

Background and purpose: M1 muscarinic ACh receptors (mAChRs) represent an attractive drug target for the treatment of cognitive deficits associated with diseases such as Alzheimer's disease and schizophrenia. However, the discovery of subtype-selective mAChR agonists has been hampered by the high degree of conservation of the orthosteric ACh-binding site among mAChR subtypes. The advent of functional screening assays has enabled the identification of agonists such as AC-42 (4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine), which bind to an allosteric site and selectively activate the M1 mAChR subtype. However, studies with this compound have been limited to recombinantly expressed mAChRs. Experimental approach: In this study, we have compared the pharmacological profile of AC-42 and a close structural analogue, 77-LH-28-1 (1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone) at human recombinant, and rat native, mAChRs by calcium mobilization, inositol phosphate accumulation and both in vitro and in vivo electrophysiology. Key results: Calcium mobilization and inositol phosphate accumulation assays revealed that both AC-42 and 77-LH-28-1 display high selectivity to activate the M1 mAChR over other mAChR subtypes. Furthermore, 77-LH-28-1, but not AC-42, acted as an agonist at rat hippocampal M1 receptors, as demonstrated by its ability to increase cell firing and initiate gamma frequency network oscillations. Finally, 77-LH-28-1 stimulated cell firing in the rat hippocampus in vivo following subcutaneous administration. Conclusions and implications: These data suggest that 77-LH-28-1 is a potent, selective, bioavailable and brain-penetrant agonist at the M1 mAChR and therefore that it represents a better tool than AC-42, with which to study the pharmacology of the M1 mAChR. British Journal of Pharmacology (2008) 154, 1104–1115; doi:10.1038/bjp.2008.152; published online 5 May 2008

In the mammalian central nervous system, GABA B receptors mediate slow pre- and postsynaptic inhibition. Using rat hippocampal slices we investigated the role of synaptic GABA B receptors in regulating kainate-induced subthreshold neuronal network oscillations in the gamma frequency range (25-80 Hz). The GABA B receptor agonist baclofen largely eliminated gamma oscillations. The GABA B receptor antagonist CGP55845 reversed this action of baclofen but alone did not alter the power or frequency of ongoing oscillations. To examine the role of synaptically released GABA on network activity, we electrically stimulated stratum radiatum of CA3 whilst recording gamma oscillations from stratum pyramidale. Single stimuli produced a pronounced transient (up to 1 s in duration) inhibition of gamma frequency oscillations. This stimulus-induced shutdown of network activity was enhanced by the GABA uptake inhibitor tiagabine and largely inhibited by CGP55845. Multiple stimuli delivered at frequencies of 1-3 Hz resulted in an activity-dependent fatigue of the inhibition of gamma activity, such that, after a number of stimuli, oscillations could be detected tens of milliseconds after the stimulus. Interestingly, this activity-dependent fatigue of inhibition uncovered a stimulus-dependent temporal entrainment of the gamma oscillations. Furthermore, the amount of repetitive synaptic input that was required to cause this entrainment was dramatically reduced by GABA B receptor antagonism such that it was evident within just a few stimuli. These data suggest that convergent afferent synaptic activity can alter the precise temporal arrangement of neuronal network activity. Furthermore, the flow of such information into a functioning neuronal network is highly regulated by GABA B receptor-mediated synaptic inhibition.

Significance Synaptic transmission between the hippocampus and prefrontal cortex is required for many executive cognitive functions. It is believed that disruption of this communication contributes to symptoms observed in psychiatric disorders including schizophrenia. Hyperdopaminergic tone and hypofunction of NMDA receptor-mediated glutamate transmission are distinctive elements of schizophrenia. Here we demonstrate that activation of low-affinity D2-like dopamine receptors leads to a lasting depression of NMDA receptors at the hippocampal–prefrontal projection of juvenile rats, leading to a marked disruption of synaptic transmission. These data demonstrate a link between dopamine and hypofunction of NMDA receptor-mediated transmission with potential implications for psychiatric disease.

Oscillations in hippocampal local field potentials (LFP) reflect the coordinated, rhythmic activity of constituent interneuronal and principal cell populations. Quantifying changes in oscillatory patterns and power therefore provides a powerful metric through which to infer mechanisms and functions of hippocampal network activity at the mesoscopic level, bridging single-neuron studies to behavioral assays of hippocampal function. Here, complementary protocols that enable mechanistic analyses of oscillation generation in vitro (in slices and a whole hippocampal preparation) and functional analyses of hippocampal circuits in behaving mice are described. Used together, these protocols provide a comprehensive view of hippocampal phenotypes in mouse models, highlighting oscillatory biomarkers of hippocampal function and dysfunction. Curr. Protoc. Mouse Biol. 2:273-294 © 2012 by John Wiley & Sons, Inc. Keywords: theta rhythm; gamma rhythm; electrophysiology; LFP; slice; in vitro; in vivo

Low concentrations of kainate can induce gamma frequency (25–80 Hz) oscillations in hippocampal slices as well as other brain structures in vitro. Little is known, however, about the kainate receptor (KAR) subtypes that underlie this type of rhythmic neuronal network activity. In this study, the role of GLU K5 subunit-containing KARs in kainate-induced hippocampal gamma frequency oscillations was assessed using GLU K5 -selective pharmacological ligands. Activation of GLU K5 -containing subunits using the selective agonists ( RS )-2-amino-3-(3-hydroxy-5- tert -butylisoxazol-4-yl)propanoic acid (ATPA; 0.1–1 μM) or iodowillardiine (0.1–1 μM) failed to induce gamma frequency oscillations in area CA3 of the rat hippocampal slice. Likewise, preincubation with a selective GLU K5 antagonist, ( RS )-3-(2-carboxybenzyl)willardiine (UBP296), did not prevent the appearance of gamma oscillations induced by 150 nM kainate. However, addition of UBP296 (10 μM) to hippocampal slices in which kainate-driven gamma oscillations were pre-established resulted in an approximately 50% reduction in gamma frequency power. These effects occurred in the absence of any effect on AMPA receptor-mediated synaptic transmission. Furthermore, carbachol-induced gamma oscillations were also unaffected by application of UBP296. These results suggest that GLU K5 -containing KARs are not alone sufficient to generate gamma frequency oscillations, but are involved in maintaining neuronal network activity induced by the actions of kainate at other KARs such as GLU K6 .

Accumulation of beta‐amyloid (Aβ) peptides in the human brain is a canonical pathological hallmark of Alzheimer's disease (AD). Recent work in Aβ‐overexpressing transgenic mice indicates that increased brain Aβ levels can be associated with aberrant epileptiform activity. In line with this, such mice can also exhibit altered intrinsic excitability (IE) of cortical and hippocampal neurons: these observations may relate to the increased prevalence of seizures in AD patients. In this study, we examined what changes in IE are produced in hippocampal CA1 pyramidal cells after 2–5 h treatment with an oligomeric preparation of synthetic human Aβ 1–42 peptide. Whole cell current clamp recordings were compared between Aβ‐(500 nM) and vehicle‐(DMSO 0.05%) treated hippocampal slices obtained from mice. The soluble Aβ treatment did not produce alterations in sub‐threshold intrinsic properties, including membrane potential, input resistance, and hyperpolarization activated “sag”. Similarly, no changes were noted in the firing profile evoked by 500 ms square current supra‐threshold stimuli. However, Aβ 500 nM treatment resulted in the hyperpolarization of the action potential (AP) threshold. In addition, treatment with Aβ at 500 nM depressed the after‐hyperpolarization that followed both a single AP or 50 Hz trains of a number of APs between 5 and 25. These data suggest that acute exposure to soluble Aβ oligomers affects IE properties of CA1 pyramidal neurons differently from outcomes seen in transgenic models of amyloidopathy. However, in both chronic and acute models, the IE changes are toward hyperexcitability, reinforcing the idea that amyloidopathy and increased incidence in seizures might be causally related in AD patients. © 2014 The Authors Hippocampus Published by Wiley Periodicals, Inc.

5-HT(4) receptors are widely distributed in both peripheral and central nervous systems where they couple, via a G-protein, to the activation of adenylate cyclase. In the brain, the highest 5-HT(4) receptor densities are found in the limbic system, including the hippocampus and frontal cortex. It has been suggested that activation of these receptors may be of therapeutic benefit in diseases that produce cognitive deficits such as Alzheimer's disease (AD). Previous electrophysiological studies have shown that the 5-HT(4) agonist, Zacopride, can increase population spike amplitude recorded in region CA1 of rat hippocampal slices in a cyclic AMP (cAMP)/cAMP-dependent protein kinase A-dependent manner. We report here that the 5-HT(4) agonist, Prucalopride, and the 5-HT(4) partial agonist, SL65.0155, produce a similar effect in rat hippocampal slices and that the specific 5-HT(4) antagonist, GR113808, blocks these effects. To investigate the potential use of 5-HT(4) agonists in the treatment of AD, Prucalopride was applied to hippocampal slices from a transgenic mouse line that overexpresses the Abeta peptide. Despite the deficit in synaptic transmission present in these mice, the percentage increase of the CA1 population spike induced by Prucalopride was the same as that observed in wild-type mice. These data support 5-HT(4) receptors as a target for cognitive enhancement and suggest that a partial agonist would be sufficient to produce benefits, while reducing potential peripheral side effects. In addition, we show that 5-HT(4) receptors remain functional in the presence of excess Abeta peptide and may therefore be a useful target in AD.

Several psychiatric diseases, including schizophrenia, are thought to have a developmental aetiology, but to date no clear link has been made between psychiatric disease and a specific developmental process. LPA1 is a Gi-coupled seven transmembrane receptor with high affinity for lysophosphatidic acid. Although LPA1 is expressed in several peripheral tissues, in the nervous system it shows relatively restricted temporal expression to neuroepithelia during CNS development and to myelinating glia in the adult. We report the detailed neurological and behavioural analysis of mice homozygous for a targeted deletion at the lpa1 locus. Our observations reveal a marked deficit in prepulse inhibition, widespread changes in the levels and turnover of the neurotransmitter 5-HT, a brain region-specific alteration in levels of amino acids, and a craniofacial dysmorphism in these mice. We suggest that the loss of LPA1 receptor generates defects resembling those found in psychiatric disease.

The suprachiasmatic nuclei (SCN) of the hypothalamus house the main circadian pacemaker in mammals. Vasoactive intestinal polypeptide (VIP) is the most abundant neuropeptide in the SCN and has been shown to phase-shift the electrical activity rhythm of SCN cells in vitro. However, the effects of VIP on the cellular activity of rat SCN neurones are unknown. In this study, we examined the acute effects of VIP on the extracellularly recorded spontaneous firing rate of SCN neurones in an in-vitro hypothalamic slice preparation. Furthermore, with the use of receptor-selective agonists and antagonists, we determined which receptors might mediate the effects of VIP in the SCN. Approximately 50% of cells responded to VIP; the main type of response was suppression in firing rate, although a few cells were activated. Suppression responses to VIP were mimicked by the VPAC(2) receptor agonist Ro 25-1553 and blocked by the selective VPAC(2) receptor antagonist PG 99-465. The PAC(1) receptor agonist maxadilan evoked responses from 40% of SCN cells, and activations to this agonist were not altered by PG 99-465. Responses to VIP were not blocked by antagonists to ionotropic glutamate receptors, but the duration of suppression was modulated by the GABA(A) receptor antagonist bicuculline. Our data indicate that VIP alters the electrical activity of rat SCN neurones in vitro, via both VPAC(2) and PAC(1) receptors.

analysis of the Tg2576 dentate gyrus proteome and transcriptome that is induced in response to cognitive-enhancing treatment with RSG that led us to discover that the PPARg and ERK/CREB/CBP signaling axes converge to enhance cognition. We found that these key integrators of cognitive performance in the Tg2576 model were central nodes within the GWAS LOAD loci network. Conclusions: These findings suggest the intriguing possibility that these GWAS LOAD loci may represent a functionally interrelated network with the PPARg and ERK/CREB/ CBP signaling axes. The molecular determinants of the convergence of these pathways may represent new targets for therapeutic intervention in cognitive enhancement in AD.

The disrupted in schizophrenia 1 (DISC1) gene is found at the breakpoint of an inherited chromosomal translocation, and segregates with major mental illnesses. Its potential role in central nervous system (CNS) malfunction has triggered intensive investigation of the biological roles played by DISC1, with the hope that this may shed new light on the pathobiology of psychiatric disease. Such work has ranged from investigations of animal behavior to detailed molecular-level analysis of the assemblies that DISC1 forms with other proteins. Here, we discuss the evidence for a role of DISC1 in synaptic function in the mammalian CNS.

BACE1 is the rate-limiting enzyme that cleaves amyloid precursor protein (APP) to produce the amyloid β peptides that accumulate in Alzheimer's disease (AD). BACE1, which is elevated in AD patients and APP transgenic mice, also cleaves the β2-subunit of voltage-gated sodium channels (Navβ2). Although increased BACE1 levels are associated with Navβ2 cleavage in AD patients, whether Navβ2 cleavage occurs in APP mice had not yet been examined. Such a finding would be of interest because of its potential impact on neuronal activity: previous studies demonstrated that BACE1-overexpressing mice exhibit excessive cleavage of Navβ2 and reduced sodium current density, but the phenotype associated with loss of function mutations in either Navβ-subunits or pore-forming α-subunits is epilepsy. Because mounting evidence suggests that epileptiform activity may play an important role in the development of AD-related cognitive deficits, we examined whether enhanced cleavage of Navβ2 occurs in APP transgenic mice, and whether it is associated with aberrant neuronal activity and cognitive deficits. We found increased levels of BACE1 expression and Navβ2 cleavage fragments in cortical lysates from APP transgenic mice, as well as associated alterations in Nav1.1α expression and localization. Both pyramidal neurons and inhibitory interneurons exhibited evidence of increased Navβ2 cleavage. Moreover, the magnitude of alterations in sodium channel subunits was associated with aberrant EEG activity and impairments in the Morris water maze. Together, these results suggest that altered processing of voltage-gated sodium channels may contribute to aberrant neuronal activity and cognitive deficits in AD.

Voltage-sensitive calcium channels (VSCC) are vital to the normal physiology of many cell types, including neurones, skeletal, cardiac and smooth muscle cells, heart pacemaker tissue and endocrine cells. Whole-cell recording is a functional electrophysiological assay that allows real-time measurement of macroscopic VSCC activity at the level of single cells. Using this technique, it is possible to probe the molecular physiology, pharmacology, and biophysics of VSCC proteins with a level of precision rarely surpassed in cell biological studies. With best practice voltage-dependent gating behaviors of VSCCs can be interrogated with temporal resolution

Cognitive decline occurs during normal aging and is likely to be reflected in the neurophysiological properties of neural circuits with key roles in cognition, for example those of the limbic system. To identify candidate neurophysiological changes we used patch clamp methods to compare the intrinsic excitability properties of hippocampal CA1 pyramidal neurons of mature adult (8-10 month) and aged (22-24 month) mice. Resting potential, input resistance, and the "sag" observed on injection of hyperpolarizing current were not age-dependent. In contrast, the patterns of spike firing observed with depolarizing current injections demonstrated the presence of an age-related hypoexcitability. Action potential waveform analysis revealed that spike thresholds were approximately 3 mV more depolarized in aged animals. In line with this, voltage clamp recordings of Na(+) currents from nucleated macropatches exhibited an approximate 3 mV depolarizing shift in the voltage-dependence of activation gating. Inactivation curves, in contrast, were not different. These data indicate alterations in Na(+) channel activation gating contribute to neuronal hypoexcitability in aging, and therefore may be a factor in age-related cognitive decline.

Burst firing is an important property of hippocampal pyramidal neurons. Group I metabotropic glutamate receptors (mGluRs) produce a multitude of effects on both the synaptic and intrinsic properties of neurons. We investigated whether brief activation of these receptors results in persistent modifications to the intrinsic excitability of rat hippocampal CA3 pyramidal cells (CA3-PCs). In whole-cell current-clamp recordings, current stimuli consisting of filtered, pseudo-random noise produced action potential firing with a mean frequency of ∼1.5-2 Hz. Analysis of spike intervals revealed that this firing included a substantial component (∼20%) of high-frequency (∼100 Hz) bursting activity. Activation of group I mGluRs with (S)-3,5-dihydroxyphenylglycine [(S)-DHPG] selectively eliminated the high-frequency bursts, an effect that persisted30 min after (S)-DHPG washout. The fast after-depolarizing potential (ADP) of CA3-PCs is known to be important for generating high-frequency action potential bursting. This ADP was persistently depressed following a short application of (S)-DHPG. This effect was blocked by the mGluR1 antagonist, (S)-(+)-α-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385). In contrast, the depression was resistant to the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate, N-methyl-D-aspartate (NMDA) and γ-aminobutyric acid (GABA)(A) antagonists. Unlike other manipulations that generate persistent depression of the ADP in CA3-PCs, DHPG-mediated ADP depression was insensitive to the Kv7 channel inhibitor 10,10-bis(4-Pyridinylmethyl)-9(10H)-anthracenone dihydrochloride (XE991) and strong intracellular Ca(2+) buffering by 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Synaptic activation of mGluRs in the associational-commissural pathway also resulted in persistent depression of the ADP in postsynaptic CA3-PCs, which was blocked by LY367385. These data represent the first evidence that synaptic activation of mGluR1 can modulate the intrinsic excitability properties of hippocampal neurons.

Channelrhodopsins are light-activated channels originally isolated from algae that are being used increasingly as tools to non-invasively stimulate neurones. Despite their widespread use some aspects of their biophysical properties have not been fully characterised. Here we report detailed investigation of the gating kinetics and voltage-dependence of ChR2 transiently expressed in HEK-293 cells. Currents were elicited using light pulses of defined duration and intensity generated by a blue LED. Datasets were gathered both at room temperature (RT, ~22 °C) and 37 °C. Current responses to light rose rapidly to a peak and then desensitized to a steady state plateau. When illumination was terminated currents rapidly deactivated. Recovery from desensitization at −85 mV was slow with half-times of 1.4 and 3.1 s at 37 °C and ~22 °C, respectively. At both temperatures, the reversal potential of ChR2 responses was a few mV positive to 0 mV. Both the peak and plateau phases of ChR2 responses exhibited strong inward rectification with only small outward currents at positive membrane potentials. The rates of ChR2 activation, deactivation and desensitization were ~2 times faster at 37 °C than at ~22 °C. Both the activation and deactivation kinetics of ChR2 were significantly slowed by depolarization at both temperatures. Additionally, the degree of steady state desensitization was greater at more depolarized potentials. The macroscopic desensitization kinetics were not voltage-dependent, but recovery from desensitization was slowed by depolarization. These gating behaviour data provide an important basis for more detailed analysis of the properties and limitations of ChR2 use in more complex systems.

Aβ peptides derived from the cleavage of amyloid precursor protein are widely believed to play an important role in the pathophysiology of Alzheimer's disease. A common way to study the impact of these molecules on CNS function is to compare the physiology of transgenic mice that overproduce Aβ with non-transgenic animals. In the hippocampus, this approach has been frequently applied to the investigation of synaptic transmission and plasticity in the perforant and Schaffer collateral commissural pathways, the first and third components of the classical hippocampal trisynaptic circuit, respectively. Similar studies however have not been carried out on the remaining component of the trisynaptic circuit, the mossy fibre pathway. Using transverse hippocampal slices prepared from ~2 year old animals we have compared mossy fibre synaptic function in wild-type mice and their Tg2576 littermates which age-dependently overproduce Aβ. Input-output curves were not altered in slices from Tg2576 mice, but these animals exhibited a significant loss of the prominent frequency-facilitation expressed by the mossy fibre pathway. In addition to this change in short term synaptic plasticity, high frequency stimulation-induced, NMDA-receptor-independent LTP was absent in slices from the transgenic mice. These data represent the first description of functional deficits in the mossy fibre pathway of Aβ-overproducing transgenic mice.

Glutamate is released from synaptic vesicles following formation of a fusion pore, connecting the vesicle interior with the synaptic cleft. Release is proposed to result from either full fusion of the vesicle with the terminal membrane or by ‘kiss-and-run’, where release occurs through the fusion pore. ‘Kiss-and-run’ seems implausible as passive diffusion of glutamate through the pore is too slow to account for the rapidity of release. Vesicular accumulation of glutamate is driven by a proton gradient, resulting in the co-release of protons during exocytosis. We tested whether the proton gradient between the vesicle and cleft contributes to glutamate exocytosis. Collapse of the gradient reduced hippocampal glutamatergic transmission, an effect that was not associated with presynaptic changes in excitability, transmitter release probability, or postsynaptic sensitivity. These data indicate that approximately half of glutamate release utilizes the proton gradient between vesicle and cleft, suggesting a significant proportion of release by ‘kiss-and-run’.

Prolonged Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels is crucial in activating the Ca2+-sensitive transcription factor NFAT, which is responsible for directing T cell proliferation and cytokine gene expression. To establish whether targeting CRAC might counteract intestinal inflammation, we evaluated the in vitro effect of a selective CRAC inhibitor on T cell cytokine production and T-bet expression by lamina propria mononuclear cells (LPMC) and biopsy specimens from inflammatory bowel disease (IBD) patients. The inhibitory activity of the CRAC blocker was investigated through patch-clamp experiments on rat basophilic leukemia cells and fluorometric imaging plate reader intracellular Ca2+ assays using thapsigargin-stimulated Jurkat T cells and its detailed selectivity profile defined using a range of in vitro radioligand binding and functional assays. Anti-CD3/CD28-stimulated LPMC and biopsy specimens from 51 patients with IBD were cultured with a range of CRAC inhibitor concentrations (0.01–10 μM). IFN-γ, IL-2, IL-8, and IL-17 were analyzed by ELISA. T-bet was determined by immunoblotting. We found that the CRAC blocker concentration-dependently inhibited CRAC current in rat basophilic leukemia cells and thapsigargin-induced Ca2+ influx in Jurkat T cells. A concentration-dependent reduction in T-bet expression and production of IFN-γ, IL-2, IL-17, but not IL-8, was observed in IBD LPMC and biopsy specimens treated with the CRAC inhibitor. In conclusion, we provide evidence that the suppression of CRAC channel function may dampen the increased T cell response in the inflamed gut, thus suggesting a promising role for CRAC inhibitor drugs in the therapeutic management of patients with IBD.

Metabotropic glutamate receptors (mGluRs) are involved in many forms of neuronal plasticity. In the hippocampus, they have well-defined roles in long-lasting forms of both synaptic and intrinsic plasticity. Here, we describe a novel form of long-lasting intrinsic plasticity that we call (S)-3,5-dihydroxyphenylglycine (DHPG)-mediated long-term depression of excitability (DHPG-LDE), and which is generated following transient pharmacological activation of group I mGluRs. In extracellular recordings from hippocampal slices, DHPG-LDE was expressed as a long-lasting depression of antidromic compound action potentials (cAPs) in CA1 or CA3 cells following a 4-min exposure to the group I mGluR agonist (S)-DHPG. A similar phenomenon was also seen for orthodromic fibre volleys evoked in CA3 axons. In single-cell recordings from CA1 pyramids, DHPG-LDE was manifest as persistent failures in antidromic action potential generation. DHPG-LDE was blocked by (S)-(+)-a-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385), an antagonist of mGluR1, but not 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP), an mGluR5 inhibitor. Although insensitive to antagonists of alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate/kainate and gamma-aminobutyric acid(A) receptors, DHPG-LDE was blocked by antagonists of N-methyl-D-aspartate (NMDA) receptors. Similarly, in single-cell recordings, DHPG-mediated antidromic spike failures were eliminated by NMDA receptor antagonism. Long after (S)-DHPG washout, DHPG-LDE was reversed by mGluR1 antagonism. A 4-min application of (S)-DHPG also produced an NMDA receptor-dependent persistent depolarization of CA1 pyramidal cells. This depolarization was not solely responsible for DHPG-LDE, because a similar level of depolarization elicited by raising extracellular K(+) increased the amplitude of the cAP. DHPG-LDE did not involve HCN channels or protein synthesis, but was eliminated by blockers of protein kinase C or tyrosine phosphatases.

Persistent plastic changes to the intrinsic excitability of neurons have substantial implications for computational processing within the CNS. We have identified and characterized a novel long-lasting form of intrinsic plasticity in hippocampal CA3 pyramidal cells. Although the patterns of action potential firing elicited in this cell population by depolarizing current injections exhibited considerable diversity, practically all cells produced an initial high frequency (100 Hz) burst of two to five spikes. This burst involved conductances that were responsible for the prominent spike afterdepolarization of CA3 pyramids. Long-lasting changes in the firing behaviour of CA3 cells were produced by conditioning stimuli (CS) consisting of either periods of depolarization in voltage clamp or periods of short (2 or 4 spikes) high frequency (circa 100 Hz) burst firing at 5 or 10 Hz. CS-induced changes included substantial prolongation of the first inter-spike interval and increased spike jitter. Similar CS-induced changes were seen when the test stimulus used to elicit firing resembled a glutamatergic EPSC. In line with this, a long-lasting depression of the ADP was elicited by the same CS that altered firing patterns of CA3 cells. Conditioning-induced changes in both spiking patterns and ADP amplitude were blocked by buffering intracellular Ca(2+) with BAPTA. Furthermore, the Kv7 channel blocker XE991, a cognitive enhancer, both enhanced the ADP and completely eliminated its conditioning-induced depression. These findings indicate that a persistent enhancement of Kv7 channels, following a transient increase in cytoplasmic Ca(2+), results in a prolonged depression of the ADP in CA3 pyramidal neurones.

In the mammalian central nervous system, GABA(B) receptors mediate slow pre- and postsynaptic inhibition. Using rat hippocampal slices we investigated the role of synaptic GABA(B) receptors in regulating kainate-induced subthreshold neuronal network oscillations in the gamma frequency range (25-80 Hz). The GABA(B) receptor agonist baclofen largely eliminated gamma oscillations. The GABA(B) receptor antagonist CGP55845 reversed this action of baclofen but alone did not alter the power or frequency of ongoing oscillations. To examine the role of synaptically released GABA on network activity, we electrically stimulated stratum radiatum of CA3 whilst recording gamma oscillations from stratum pyramidale. Single stimuli produced a pronounced transient (up to 1 s in duration) inhibition of gamma frequency oscillations. This stimulus-induced shutdown of network activity was enhanced by the GABA uptake inhibitor tiagabine and largely inhibited by CGP55845. Multiple stimuli delivered at frequencies of 1-3 Hz resulted in an activity-dependent fatigue of the inhibition of gamma activity, such that, after a number of stimuli, oscillations could be detected tens of milliseconds after the stimulus. Interestingly, this activity-dependent fatigue of inhibition uncovered a stimulus-dependent temporal entrainment of the gamma oscillations. Furthermore, the amount of repetitive synaptic input that was required to cause this entrainment was dramatically reduced by GABA(B) receptor antagonism such that it was evident within just a few stimuli. These data suggest that convergent afferent synaptic activity can alter the precise temporal arrangement of neuronal network activity. Furthermore, the flow of such information into a functioning neuronal network is highly regulated by GABA(B) receptor-mediated synaptic inhibition.

We report the detailed expression profile of TRPM2 mRNA within the human central nervous system (CNS) and demonstrate increased TRPM2 mRNA expression at 1 and 4 weeks following ischemic injury in the rat transient middle cerebral artery occlusion (tMCAO) stroke model. Microglial cells play a key role in pathology produced following ischemic injury in the CNS and possess TRPM2, which may contribute to stroke-related pathological responses. We show that TRPM2 mRNA is present in the human C13 microglial cell line and is reduced by antisense treatment. Activation of C13 cells by interleukin-1beta leads to a fivefold increase of TRPM2 mRNA demonstrating transcriptional regulation. To confirm mRNA distribution correlated with functional expression, we combined electrophysiology, Ca2+ imaging, and antisense approaches. C13 microglia exhibited, when stimulated with hydrogen peroxide (H2O2), increased [Ca2+]i, which was reduced by antisense treatment. Moreover, patch-clamp recordings from C13 demonstrated that increased intracellular adenosine diphosphoribose (ADPR) or extracellular H2O2 induced an inward current, consistent with activation of TRPM2. In addition we confirm the functional expression of a TRPM2-like conductance in primary microglial cultures derived from rats. Activation of TRPM2 in microglia during ischemic brain injury may mediate key aspects of microglial pathophysiological responses.

Using rat hippocampal slices, extracellularly recorded antidromic compound action potentials (cAP) were produced in CA1 pyramidal cell populations by electrical stimulation of the alveus at 0.5 Hz. These responses were additionally examined across a range of stimulus frequencies between 0.5 and 100 Hz. Anticonvulsant drugs in clinical use were applied via perfusion of the recording chamber. Three anticonvulsants produced a concentration-dependent inhibition of the cAP evoked at low frequency (0.5 Hz). The following IC(50) values were observed: lamotrigine, 210 microM (interpolated); carbamazepine, 210 microM (interpolated); phenytoin, 400 microM (extrapolated). The extent of inhibition produced was increased when trains of 30 cAPs were evoked at frequenciesor 30 Hz. This frequency dependence was quantified by measuring a response integral for a range of compound concentrations. Three other compounds valproate (5 mM), topiramate (500 microM) and levetiracetam (500 microM) produced no clear effect at any stimulus frequency tested. Using this simple neurophysiological assay it has been possible to compare the use-dependent inhibition of hippocampal action potentials by a range of anticonvulsants, providing a useful adjunct to patch clamp studies of such molecules at Na(+) channels. There is no clear correlation between the activity in this model and the clinical efficacy of these drugs in different forms of epilepsy.

Hyperpolarization-activated cyclic nucleotide gated (HCN) ion channels regulate membrane potential, neurotransmitter release, and patterning of synchronized neuronal activity. Currently, there is an intense debate as to whether or not these ion channels play a pro- or anticonvulsant role in vivo. To gain an insight into this question, we have examined how inhibitors of the response mediated by HCN channels (referred to as Ih) affect epileptiform activity induced in adult hippocampal slices. The archetypal Ih blocker ZD-7288 produced a concentration-dependent inhibition of both nonsynaptic- (low Ca2+/elevated K+ aCSF) and synaptic- (low Mg2+ aCSF, elevated K+ aCSF or convulsant application (bicuculline or pentylenetetrazol)) based epileptiform activities. The IC50 value for ZD-7288 induced inhibition of epileptiform activity was similar across all forms of epileptiform response and was below concentrations producing nonspecific inhibition of glutamatergic synaptic transmission. Furthermore, capsazepine, which exhibits similar potency to ZD-7288 at inhibiting Ih, failed to inhibit glutamatergic synaptic transmission per se but produced a significant inhibition of bicuculline-induced epileptiform activity. These data suggest that broad spectrum inhibition of Ih reduces neuronal hyperexcitability in the hippocampus. Synapse 59:308–316, 2006. © 2006 Wiley-Liss, Inc.

Synchronous neuronal firing can be induced in hippocampal slices in the absence of synaptic transmission by lowering extracellular Ca2+ and raising extracellular K+. However, the ionic mechanisms underlying this nonsynaptic synchronous firing are not well understood. In this study we have investigated the role of KCNQ /Kv7 channels in regulating this form of nonsynaptic bursting activity. Incubation of rat hippocampal slices in reduced (2+]o and increased (6.3 mM) [K+]o, blocked synaptic transmission, increased neuronal firing, and led to the development of spontaneous periodic nonsynaptic epileptiform activity. This activity was recorded extracellularly as large (4.7 ± 1.9 mV) depolarizing envelopes with superimposed high-frequency synchronous population spikes. These intraburst population spikes initially occurred at a high frequency (about 120 Hz), which decayed throughout the burst stabilizing in the gamma-frequency band (30–80 Hz). Further increasing [K+]o resulted in an increase in the interburst frequency without altering the intraburst population spike frequency. Application of retigabine (10 μM), a Kv7 channel modulator, completely abolished the bursts, in an XE-991–sensitive manner. Furthermore, application of the Kv7 channel blockers, linopirdine (10 μM) or XE-991 (10 μM) alone, abolished the gamma frequency, but not the higher-frequency population spike firing observed during low Ca2+/high K+ bursts. These data suggest that Kv7 channels are likely to play a role in the regulation of synchronous population firing activity.

The role of calcium-activated potassium channels in the regulation of neuronal hyperexcitability, as in epilepsy, is unclear. To examine this issue, we have used the acute hippocampal slice model of epileptiform activity to investigate the effects of an enhancer of SK channel activity, 1-ethyl-benzimidazolinone (EBIO). That EBIO is an SK channel modulator was confirmed by its potentiation of hSK1, hSK2, hSK3 and hIK currents (EC(50) values in the range of 130-870 microM) and its apamin (1 microM) sensitive reduction of the number of action potentials fired in CA3 pyramidal neurons in response to a depolarizing current step. In addition, while EBIO did not significantly affect electrically evoked glutamatergic synaptic transmission, it did inhibit epileptiform activity (IC(50) values in the range of 150-325 microM) induced by (1) modifying the extracellular ionic environment by removing extracellular Mg(2+) or elevating extracellular K(+) from 3.0 to 8.5 mM and (2) disinhibiting the slice using 3 mM pentylenetetrazol or combined application of 10 microM gabazine and 10 microM CGP55845. Furthermore, its inhibitory effect in the full disinhibition model of epileptiform activity (10 microM gabazine + 10 microM CGP55845) was occluded by the SK channel blocker apamin (300 nM-1 microM) which in its own right increased the duration and reduced the frequency of individual epileptiform bursts. In conclusion, compounds that enhance the activation of small conductance Ca(2+) -activated K(+) channels are effective inhibitors of epileptiform activity in vitro.

Gabapentin (Neurontin) has been successfully used in the treatment of both epilepsy and neuropathic pain. Despite its widespread clinical use, its mechanism of action is very poorly understood. Indeed, the only protein it is known to interact with is the alpha2delta subunit of the voltage-gated Ca(2+) channel complex. In a recent article, gabapentin was reported to inhibit synaptic transmission in the spinal cord through an inhibitory effect on presynaptic P/Q-type Ca(2+) channels in both glutamatergic primary afferents and glycinergic interneuones. To examine if such inhibition of P/Q-channel-mediated synaptic transmission by gabapentin generalised to other synaptic pathways, we tested the actions of gabapentin of P/Q-type Ca(2+) channel-mediated synaptic responses in the CA1 subfield of the hippocampus. We found that gabapentin was completely inactive on such synaptic responses even at 10 times the maximally effective concentration used in the spinal cord. A small ( approximately 10%) but consistent depression of control synaptic responses was elicited by 10 microM gabapentin. No greater response was observed at a 10 times higher concentration. From these data we conclude that gabapentin is not a generic inhibitor of presynaptic P/Q-type channels and its actions at the spinal level must represent a feature of the P/Q-type channel not present in the hippocampus. Given the known interactions of this compound, the best candidate for this is the presence, subtype, or state of the alpha2delta subunit.

GABAergic synaptic transmission plays an important role in the patterning of epileptiform activity. We have previously shown that global loss of GABA(B) receptor function due to transgenic deletion of the GABA(B1) receptor subunit exacerbates epileptiform activity induced by pharmacological manipulations in hippocampal slices. Here we show that a similar hyperexcitable phenotype is observed in hippocampal slices prepared from a transgenic mouse expressing a GABA(B2) receptor subunit lacking its C terminal tail (the DeltaGB2-Ct mouse); a molecular manipulation that also produces complete loss of GABA(B) receptor function. Thus, epileptiform bursts that are sensitive to NMDA receptor antagonists (induced by either the GABA(A) receptor antagonist bicuculline (10muM) or removal of extracellular Mg(2+)) were significantly longer in duration in DeltaGB2-Ct slices relative to WT slices. We now extend these observations to demonstrate that a stimulus train induced bursting (STIB) protocol also evokes significantly longer bicuculline sensitive bursts of activity in DeltaGB2-Ct slices compared to WT. Furthermore, synchronous GABA(A) receptor-mediated potentials recorded in the presence of the potassium channel blocker 4-aminopyridine (4-AP, 100muM) and the ionotropic glutamate receptor antagonists NBQX (20muM) and D-AP5 (50muM) were significantly prolonged in duration in DeltaGB2-Ct versus WT slices. These data suggest that the loss of GABA(B) receptor function in DeltaGB2-Ct hippocampal slices promotes depolarising GABA(A) receptor-mediated events, which in turn, leads to the generation of ictal-like events, which may contribute to the epilepsy phenotype observed in vivo.