Depolarizing γ-aminobutyric acid contributes to glutamatergic network rewiring in epilepsy.

OBJECTIVE: Rewiring of excitatory glutamatergic neuronal circuits is a major abnormality in epilepsy. Besides the rewiring of excitatory circuits, an abnormal depolarizing γ-aminobutyric acidergic (GABAergic) drive has been hypothesized to participate in the epileptogenic processes. However, a remaining clinically relevant question is whether early post-status epilepticus (SE) evoked chloride dysregulation is important for the remodeling of aberrant glutamatergic neuronal circuits. METHODS: Osmotic minipumps were used to infuse intracerebrally a specific inhibitor of depolarizing GABAergic transmission as well as a functionally blocking antibody toward the pan-neurotrophin receptor p75 (p75NTR ). The compounds were infused between 2 and 5 days after pilocarpine-induced SE. Immunohistochemistry for NKCC1, KCC2, and ectopic recurrent mossy fiber (rMF) sprouting as well as telemetric electroencephalographic and electrophysiological recordings were performed at day 5 and 2 months post-SE. RESULTS: Blockade of NKCC1 after SE with the specific inhibitor bumetanide restored NKCC1 and KCC2 expression, normalized chloride homeostasis, and significantly reduced the glutamatergic rMF sprouting within the dentate gyrus. This mechanism partially involves p75NTR signaling, as bumetanide application reduced SE-induced p75NTR expression and functional blockade of p75NTR decreased rMF sprouting. The early transient (3 days) post-SE infusion of bumetanide reduced rMF sprouting and recurrent seizures in the chronic epileptic phase. INTERPRETATION: Our findings show that early post-SE abnormal depolarizing GABA and p75NTR signaling fosters a long-lasting rearrangement of glutamatergic network that contributes to the epileptogenic process. This finding defines promising and novel targets to constrain reactive glutamatergic network rewiring in adult epilepsy. Ann Neurol 2017;81:251-265.

Progressive development of synchronous activity in the hippocampal neuronal network is modulated by GluK1 kainate receptors.

Kainate receptors are potent modulators of circuit excitability and have been repeatedly implicated in pathophysiological synchronization of limbic networks. While the role of aberrant GluK2 subunit containing KARs in generation of epileptiform hypersynchronous activity is well described, the contribution of other KAR subtypes, including GluK1 subunit containing KARs remain less well understood. To investigate the contribution of GluK1 KARs in developmental and pathological synchronization of the hippocampal neural network, we used multielectrode array recordings on organotypic hippocampal slices that display first multi-unit activity and later spontaneous population discharges resembling ictal-like epileptiform activity (IEA). Chronic blockage of GluK1 activity using selective antagonist ACET or lentivirally delivered shRNA significantly delayed developmental synchronization of the hippocampal CA3 network and generation of IEA. GluK1 overexpression, on the other hand, had no significant effect on occurrence of IEA, but enhanced the size of the neuron population participating in the population discharges. Correlation analysis indicated that local knockdown of GluK1 locally in the CA3 neurons reduced their functional connectivity, while GluK1 overexpression increased the connectivity to both CA1 and DG. These data suggest that GluK1 KARs regulate functional connectivity between the excitatory neurons, possibly via morphological changes in glutamatergic circuit, affecting synchronization of neuronal populations. The significant effects of GluK1 manipulations on network activity call for further research on GluK1 KAR as potential targets for antiepileptic treatments, particularly during the early postnatal development when GluK1 KARs are strongly expressed in the limbic neural networks.

Mineral, trace element, and toxic metal concentration in hair from dogs with idiopathic epilepsy compared to healthy controls.

BACKGROUND: Altered trace element status is associated with epilepsy in humans and dogs with idiopathic epilepsy (IE). OBJECTIVES: Compare hair element concentrations in epileptic and healthy dogs. ANIMALS: Sixty-three dogs with IE (53 treated, 10 untreated) and 42 controls. METHODS: Case-control study using ICP-MS to determine hair calcium, magnesium, phosphorus, sodium, potassium, iron, copper, manganese, zinc, selenium, chromium, lead, mercury, cadmium, arsenic, aluminum, and nickel concentration. Groups were compared using nonparametric tests. Results were controlled for diet, sex, age, and hair color using generalized linear mixed models. RESULTS: Compared to healthy controls, dogs with IE had lower hair phosphorus (mean ± SD; IE: 286.19 ± 69.62 µg/g, healthy: 324.52 ± 58.69 µg/g; P = .001), higher hair copper (IE: 10.97 ± 3.51 µg/g, healthy: 8.41 ± 1.27 µg/g; P < .001), zinc (IE: 158.25 ± 19.64 µg/g, healthy: 144.76 ± 32.18 µg/g; P < .001), copper/zinc ratio (IE: 0.07 ± 0.02, healthy: 0.06 ± 0.01; P = .003), selenium (IE: 1.65 ± 0.43 µg/g, healthy: 0.94 ± 0.73 µg/g; P < .001), and arsenic (IE: 0.40 ± 0.78 µg/g, healthy: 0.05 ± 0.08 µg/g; P < .001). When comparing treated and untreated epileptic dogs with healthy dogs, the differences in phosphorus and selenium remained significant for both groups, whereas the differences in copper, zinc, and arsenic were significant only for treated dogs. Potassium bromide treatment was strongly associated with high hair arsenic (P = .000). CONCLUSIONS AND CLINICAL IMPORTANCE: Altered trace element status could be involved in the pathophysiology of IE in dogs. Antiseizure drugs might affect trace element and arsenic metabolism.

Whole blood trace element and toxic metal concentration in dogs with idiopathic epilepsy and healthy dogs: A case-control study.

Background: Idiopathic epilepsy (IE) is the most common neurological disease in dogs. Multiple genes and environmental factors interact to cause clinical signs, although the pathogenesis remains poorly understood. Extensive evidence from recent decades shows that trace elements play a role in epilepsy in humans, and recently it was shown for the first time that also dogs with IE have altered trace element status. On the other hand, toxic metals may cause seizures but research on their role in canine IE is lacking. Therefore, we aimed to investigate trace element and toxic metal concentrations in whole blood from dogs that had been diagnosed with IE and compare them to those of healthy dogs. Materials and methods: Whole blood concentrations of trace elements (selenium, zinc, copper, manganese, iron, and chromium) and toxic metals (arsenic, cadmium, mercury, and lead) were analyzed from 19 dogs that had been diagnosed with IE by board-certified neurologists and 19 healthy control dogs using inductively coupled plasma mass spectrometry. The concentrations in study and control group were compared using the Mann-Whitney U test. Results: Dogs diagnosed with IE had significantly higher blood copper concentration (P = 0.007), higher copper/zinc ratio (P = 0.04), and higher selenium concentration (P < 0.001), as well as lower chromium concentration (P = 0.01) when compared to healthy dogs. Treatment of IE with potassium bromide was associated with a significant elevation in blood arsenic concentration (P = 0.01). Conclusion: In conclusion, the present results support the role of altered trace element status in dogs diagnosed with IE and suggest that copper, selenium, and chromium may be involved in the pathogenesis of canine epilepsy or seizures. The results also suggest that potassium bromide may alter arsenic metabolism in dogs.

Histaminergic neurons protect the developing hippocampus from kainic acid-induced neuronal damage in an organotypic coculture system.

The central histaminergic neuron system inhibits epileptic seizures, which is suggested to occur mainly through histamine 1 (H1) and histamine 3 (H3) receptors. However, the importance of histaminergic neurons in seizure-induced cell damage is poorly known. In this study, we used an organotypic coculture system and confocal microscopy to examine whether histaminergic neurons, which were verified by immunohistochemistry, have any protective effect on kainic acid (KA)-induced neuronal damage in the developing hippocampus. Fluoro-Jade B, a specific marker for degenerating neurons, indicated that, after the 12 h KA (5 microM) treatment, neuronal damage was significantly attenuated in the hippocampus cultured together with the posterior hypothalamic slice containing histaminergic neurons [HI plus HY (POST)] when compared with the hippocampus cultured alone (HI) or with the anterior hypothalamus devoid of histaminergic neurons. Moreover, alpha-fluoromethylhistidine, an inhibitor of histamine synthesis, eliminated the neuroprotective effect in KA-treated HI plus HY (POST), and extracellularly applied histamine (1 nM to 100 microM) significantly attenuated neuronal damage only at 1 nM concentration in HI. After the 6 h KA treatment, spontaneous electrical activity registered in the CA1 subregion contained significantly less burst activity in HI plus HY (POST) than in HI. Finally, in KA-treated slices, the H3 receptor antagonist thioperamide enhanced the neuroprotective effect of histaminergic neurons, whereas the H1 receptor antagonists triprolidine and mepyramine dose-dependently decreased the neuroprotection in HI plus HY (POST). Our results suggest that histaminergic neurons protect the developing hippocampus from KA-induced neuronal damage, with regulation of neuronal survival being at least partly mediated through H1 and H3 receptors.

Changes in microtubule-associated protein-2 (MAP2) expression during development and after status epilepticus in the immature rat hippocampus.

In this study, we analyzed the spatiotemporal expression patterns of the high-molecular weight (MAP2a and b) and low-molecular weight (MAP2c and d) cytoskeletal microtubule-associated protein-2 (MAP2) isoforms with Western blotting, and the cellular localization of the high-molecular weight MAP2 isoforms with immunocytochemistry in the hippocampi of 1- to 21-day-old rats. Moreover, the temporal profile (from 30 min to 1 week) of MAP2 isoform reactivity to kainic acid-induced status epilepticus was studied in P9 rats. During development, the expression of the high-molecular weight MAP2 isoforms significantly increased, while the low-molecular weight isoforms decreased, the most prominent changes occurring during the second postnatal week. This developmental increase in the high-molecular weight MAP2 expression was also confirmed with immunocytochemistry, which showed increased immunoreactivity, particularly in the molecular layers of the dentate gyrus, and in CA1 and CA3 stratum radiatum. In 9-day-old rats, status epilepticus resulted in a rapid transient increase (about 210%) in the high-molecular weight MAP2 expression, without any effect on the low-molecular weight MAP2. Moreover, disturbed dendritic structure in the CA1 and CA3 stratum radiatum was manifested as formation of varicosities 3h after the kainic acid treatment. The strictly developmentally regulated MAP2 isoform expression suggests different functional roles for these proteins during the postnatal development in the rat hippocampus. Moreover, high-molecular weight MAP2s may play a role in nerve cell survival during cell stress.

Axonal Kainate Receptors Modulate the Strength of Efferent Connectivity by Regulating Presynaptic Differentiation.

Kainate type of glutamate receptors (KARs) are highly expressed during early brain development and may influence refinement of the circuitry, via modulating synaptic transmission and plasticity. KARs are also localized to axons, however, their exact roles in regulating presynaptic processes remain controversial. Here, we have used a microfluidic chamber system allowing specific manipulation of KARs in presynaptic neurons to study their functions in synaptic development and function in vitro. Silencing expression of endogenous KARs resulted in lower density of synaptophysin immunopositive puncta in microfluidically isolated axons. Various recombinant KAR subunits and pharmacological compounds were used to dissect the mechanisms behind this effect. The calcium permeable (Q) variants of the low-affinity (GluK1-3) subunits robustly increased synaptophysin puncta in axons in a manner that was dependent on receptor activity and PKA and PKC dependent signaling. Further, an associated increase in the mean active zone length was observed in electron micrographs. Selective presynaptic expression of these subunits resulted in higher success rate of evoked EPSCs consistent with higher probability of glutamate release. In contrast, the calcium-impermeable (R) variant of GluK1 or the high-affinity subunits (GluK4,5) had no effect on synaptic density or transmission efficacy. These data suggest that calcium permeable axonal KARs promote efferent connectivity by increasing the density of functional presynaptic release sites.

Maturation of cultured hippocampal slices results in increased excitability in granule cells.

The preparation of hippocampal slices results in loss of input neurons to dentate granule cells, which leads to the reorganization of their axons, the mossy fibers, and alters their functional properties in long-term cultures, but its temporal aspects in the immature hippocampus are not known. In this study, we have focused on the early phase of this plastic reorganization process by analyzing granule cell function with field potential and whole cell recordings during the in vitro maturation of hippocampal slices (from 1 to 17 days in vitro, prepared from 6 to 7-day-old rats), and their morphology using extracellular biocytin labelling technique. Acute slices from postnatal 14-22-day-old rats were analyzed to detect any differences in the functional properties of granule cells in these two preparations. In field potential recordings, small synaptically-evoked responses were detected at 2 days in vitro, and their amplitude increased during the culture time. Whole cell voltage clamp recordings revealed intensive spontaneous excitatory postsynaptic currents, and the susceptibility to stimulus-evoked bursting increased with culture time. In acutely prepared slices, neither synaptically-evoked responses in field potential recordings nor any bursting in whole cell recordings were detected. The excitatory activity was under the inhibitory control of gamma-aminobutyric acid type A receptor. Extracellularily applied biocytin labelled dentate granule cells, and revealed sprouting and aberrant targeting of mossy fibers in cultured slices. Our results suggest that reorganization of granule cell axons takes place during the early in vitro maturation of hippocampal slices, and contributes to their increased excitatory activity resembling that in the epileptic hippocampus. Cultured immature hippocampal slices could thus serve as an additional in vitro model to elucidate mechanisms of synaptic plasticity and cellular reactivity in response to external damage in the developing hippocampus.

Subcellular distribution of histamine, GABA and galanin in tuberomamillary neurons in vitro.

Histamine acts as a neurotransmitter in the brain and regulates e.g. sleep, hibernation, vigilance, and release of several other transmitters. All histaminergic neurons are found in the tuberomamillary nucleus (TM), and send axons to almost all parts of the CNS. Despite the obvious importance of these neurons, their development, transmitter storage, and compartmentalization of cotransmitters are poorly known. Histaminergic neurons from fetal rat hypothalamus were studied in primary explant cultures and analyzed by confocal microscopy. Most histaminergic neurons were oval in shape, but round and triangular ones were also found. The average size of the 212 analyzed neurons was 19.2 microm (length), 12.5 microm (width) and 11.7 microm (thickness). The cells possessed two to five microtubule-associated protein (MAP2) positive processes, putative dendrites, and in general one MAP2-negative thin process, a putative axon. Granular histamine-immunoreactivity was found in the cell bodies, axons, and dendrites. In tuberomamillary neurons, most histamine-containing structures displayed immunoreactivity for vesicular monoamine transporter 2 (VMAT2), indicating that the two markers may coexist in the same structures. Lack of VMAT2 in some histamine-immunoreactive structures indicates that another transporter for histamine may exist. In the same neurons, gamma-aminobutyric acid (GABA)-immunoreactivity was found in structures, distinct from those containing histamine, indicating that the two transmitters may be differentially localized, regulated and released. Galanin-immunoreactivity in the cultured tuberomamillary neurons was partially located in the same structures as VMAT2. The results suggest that histamine and GABA, the two principal transmitters of tuberomamillary neurons, are not costored in the same structures in tuberomamillary neurons.

Histamine 1 receptor knock out mice show age-dependent susceptibility to status epilepticus and consequent neuronal damage.

The central histaminergic neuron system is an important regulator of activity stages such as arousal and sleep. In several epilepsy models, histamine has been shown to modulate epileptic activity and histamine 1 (H1) receptors seem to play a key role in this process. However, little is known about the H1 receptor-mediated seizure regulation during the early postnatal development, and therefore we examined differences in severity of kainic acid (KA)-induced status epilepticus (SE) and consequent neuronal damage in H1 receptor knock out (KO) and wild type (WT) mice at postnatal days 14, 21, and 60 (P14, P21, and P60). Our results show that in P14 H1 receptor KO mice, SE severity and neuronal damage were comparable to those of WT mice, whereas P21 KO mice had significantly decreased survival, more severe seizures, and enhanced neuronal damage in various brain regions, which were observed only in males. In P60 mice, SE severity did not differ between the genotypes, but in KO group, neuronal damage was significantly increased. Our results suggest that H1 receptors could contribute to regulation of seizures and neuronal damage age-dependently thus making the histaminergic system as a challenging target for novel drug design in epilepsy.

Kainic acid-induced neurodegeneration and activation of inflammatory processes in organotypic hippocampal slice cultures: treatment with cyclooxygenase-2 inhibitor does not prevent neuronal death.

In the postnatal rodent hippocampus status epilepticus (SE) leads to age- and region-specific excitotoxic neuronal damage, the precise mechanisms of which are still incompletely known. Recent studies suggest that the activation of inflammatory responses together with glial cell reactivity highly contribute to excitotoxic neuronal damage. However, pharmacological tools to attenuate their activation in the postnatal brain are still poorly elucidated. In this study, we investigated the role of inflammatory mediators in kainic acid (KA)-induced neuronal damage in organotypic hippocampal slice cultures (OHCs). A specific cyclooxygenase-2 (COX-2) inhibitor N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS-398) was used to study whether or not it could ameliorate neuronal death. Our results show that KA treatment (24 h) resulted in a dose-dependent degeneration of CA3a/b pyramidal neurons. Furthermore, COX-2 immunoreactivity was pronouncedly enhanced particularly in CA3c pyramidal neurons, microglial and astrocyte morphology changed from a resting to active appearance, the expression of the microglial specific protein, Iba1, increased, and prostaglandin E2 (PGE2) production increased. These indicated the activation of inflammatory processes. However, the expression of neither proinflammatory cytokines, i.e. tumour necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß), nor the anti-inflammatory cytokine IL-10 mRNA was significantly altered by KA treatment as studied by real-time PCR. Despite activation of an array of inflammatory processes, neuronal damage could not be rescued either with the combined pre- and co-treatment with a specific COX-2 inhibitor, NS-398. Our results suggest that KA induces activation of a repertoire of inflammatory processes in immature OHCs, and that the timing of anti-inflammatory treatment to achieve neuroprotection is a challenge due to developmental properties and the complexity of inflammatory processes activated by noxious stimuli. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

Involvement of histamine 1 receptor in seizure susceptibility and neuroprotection in immature mice.

The central histaminergic neuronal system is a powerful modulator of brain activity, and its functional disturbance is related to e.g. epilepsy. We have recently shown in the slice culture system that histaminergic neurons attenuate kainic acid (KA)-induced epileptiform activity and neuronal damage in the hippocampus through histamine 1 (H1) receptors. We now further examined the role of H1 receptors in the regulation of KA-induced seizures and neuronal damage in immature 9-day-old H1 receptor knock out (KO) mice. In the H1 receptor KO mice, behavioral seizures were significantly more severe and duration of seizures was significantly longer when compared to the wild type (WT) mice at the KA dose of 2mg/kg. Moreover, neuronal damage correlated with seizure severity, and it was significantly increased in the thalamus and retrosplenial granular cortex (RGC) of the KO mice. The H1 receptor antagonist triprolidine treatment supported these findings by showing significantly increased seizures severity and neuronal damage in the septum, thalamus, CA3 region of the hippocampus, and RGC in the KA-treated WT mice. Our present novel findings suggest that H1 receptors play a pivotal role in the regulation of seizure intensity and duration as well as seizure-induced neuronal damage in the immature P9 mice.

The calpain inhibitor MDL-28170 and the AMPA/KA receptor antagonist CNQX inhibit neurofilament degradation and enhance neuronal survival in kainic acid-treated hippocampal slice cultures.

The cytoskeleton controls the architecture and survival of the central nervous system neurons by maintaining the stability of axons, dendrites and cellular architecture, and any disturbance in this genuine structure could compromise cell survival. The developmentally regulated intracellular intermediate filament protein neurofilament (NF), composed of the light (NF-L), medium (NF-M) and high (NF-H) molecular weight isoforms, is expressed abundantly in nerve cells but its significance in nerve cell survival in stress situations in the brain is unknown. We have used Western blotting, immunocytochemistry, and Fluoro-Jade B and thionine stainings to clarify the effect of kainic acid (KA) treatment on NF protein stability, and its importance for neuronal survival in hippocampal slice cultures. The contribution of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/KA glutamate receptor subtypes, calpain proteases and L-type Ca2+-channels to these processes were also assessed. Our results indicated that KA-induced degradation of NF was a fast process, similarly affecting all three NF proteins. It was effectively inhibited by the AMPA/KA receptor antagonist CNQX and the calpain inhibitor MDL-28170, whereas the Ca2+-channel blocker nifedipine and the NMDA receptor antagonist MK-801 had no significant effect. Moreover, KA-induced neuronal damage was effectively decreased in cultures treated with CNQX and MDL-28170. Our results suggest that the stability of NF proteins is an important factor contributing to neuronal survival after excitotoxic injury, and that both AMPA/KA receptor antagonists and calpain inhibitors might serve as neuroprotectants against this type of insult in the immature hippocampus.

Histamine-immunoreactive neurons in the mouse and rat suprachiasmatic nucleus.

Among the well-established roles of the neurotransmitter histamine (HA) is that as a regulator of the sleep-wake cycle, which early gained HA a reputation as a 'waking substance'. The tuberomammillary nucleus (TMN) of the posterior hypothalamus, which contains the sole source of neuronal HA in the brain, is reciprocally connected to the suprachiasmatic nucleus (SCN) which, in turn, is best known as the pacemaker of circadian rhythms in mammals. We report HA-immunoreactive (-ir) neurons in the mouse and rat SCN that neither display immunoreactivity (-iry) for the HA-synthesizing enzyme histidine decarboxylase (HDC) nor contain HDC mRNA. Further, HA-iry was absent in the SCN of HDC knockout mice, but present in appropriate control animals, indicating that the observed HA-iry is HDC dependent. Experiments with hypothalamic slice cultures and i.c.v. injection of HA suggest that HA in the SCN neurons originates in the TMN and is transported from the TMN along histaminergic fibres known to innervate the SCN. These results could indicate the existence of a hitherto unknown uptake mechanism for HA into neurons. Through HA uptake and, putatively, re-release of the captured HA, these neurons could participate in the HA-mediated effects on the circadian system in concert with direct histaminergic inputs from the TMN to the SCN. The innervation of the SCN by several neurotransmitter systems could provide a way for other systems to affect the HA-containing neuronal cell bodies in the SCN.

Resistance of neurofilaments to degradation, and lack of neuronal death and mossy fiber sprouting after kainic acid-induced status epilepticus in the developing rat hippocampus.

Neurofilament (NF) proteins, the major constituent of intermediate filaments in neurons, have an important role in cellular stability and plasticity. We have now studied the short-term (hours) and long-term (up to 1 week) effects of kainic acid (KA)-induced status epilepticus (SE) on the reactivity of NF proteins, and mossy fiber (MF) sprouting and neuronal death up to 4 weeks in 9-day-old rats. In Western blotting, the expression of the phosphorylation-independent epitopes of NF-L, NF-M, and NF-H rapidly but transiently increased after the treatment, whereas the phosphorylated NF-M remained elevated for 7 days. However, the treatment did not change the immunoreactivity of NF proteins, and no neuronal death or mossy fiber sprouting was detected at any time point. Our findings indicate seizure-induced reactivity of NF proteins but their resistance to degradation, which could be of importance in neuronal survival and may also prevent MF sprouting in the developing hippocampus.