Hippocampal CA1 Pyramidal Neurons Research Articles

Transduction of inflammation from peripheral immune cells to the hippocampus induces neuronal hyperexcitability mediated by Caspase-1 activation

BackgroundRecent studies report infiltration of peripheral blood mononuclear cells (PBMCs) into the central nervous system (CNS) in epileptic disorders, suggestive of a potential contribution of PBMC extravasation to the generation of seizures. Nevertheless, the underlying mechanisms involved in PBMC infiltrates promoting neuronal predisposition to ictogenesis remain unclear. Therefore, we developed an in vitro model mimicking infiltration of activated PBMCs into the brain in order to investigate potential transduction of inflammatory signals from PBMCs to the CNS. MethodsTo establish our model, we first extracted PBMCs from rat spleen, then, immunologically primed PBMCs with lipopolysaccharide (LPS), followed by further activation with nigericin. Thereafter, we co-cultured these activated PBMCs with organotypic cortico-hippocampal brain slice cultures (OCHSCs) derived from the same rat, and compared PBMC-OCHSC co-cultures to OCHSCs exposed to PBMCs in the culture media. We further targeted a potential molecular pathway underlying transduction of peripheral inflammation to OCHSCs by incubating OCHSCs with the Caspase-1 inhibitor VX-765 prior to co-culturing PBMCs with OCHSCs. After 24 h, we analyzed inflammation markers in the cortex and the hippocampus using semiquantitative immunofluorescence. In addition, we analyzed neuronal activity by whole-cell patch-clamp recordings in cortical layer II/III and hippocampal CA1 pyramidal neurons. ResultsIn the cortex, co-culturing immunoreactive PBMCs treated with LPS + nigericin on top of OCHSCs upregulated inflammatory markers and enhanced neuronal excitation. In contrast, no excitability changes were detected after adding primed PBMCs (i.e. treated with LPS only), to OCHSCs. Strikingly, in the hippocampus, both immunoreactive and primed PBMCs elicited similar pro-inflammatory and pro-excitatory effects. However, when immunoreactive and primed PBMCs were cultured in the media separately from OCHSCs, only immunoreactive PBMCs gave rise to neuroinflammation and hyperexcitability in the hippocampus, whereas primed PBMCs failed to produce any significant changes. Finally, VX-765 application to OCHSCs, co-cultured with either immunoreactive or primed PBMCs, prevented neuroinflammation and hippocampal hyperexcitability in OCHSCs. ConclusionsOur study shows a higher susceptibility of the hippocampus to peripheral inflammation as compared to the cortex, mediated via Caspase-1-dependent signaling pathways. Thus, our findings suggest that Caspase-1 inhibition may potentially provide therapeutic benefits during hippocampal neuroinflammation and hyperexcitability secondary to peripheral innate immunity.

Reduced hippocampal inhibition and enhanced autism-epilepsy comorbidity in mice lacking neuropilin 2

The neuropilin receptors and their secreted semaphorin ligands play key roles in brain circuit development by regulating numerous crucial neuronal processes, including the maturation of synapses and migration of GABAergic interneurons. Consistent with its developmental roles, the neuropilin 2 (Nrp2) locus contains polymorphisms in patients with autism spectrum disorder (ASD). Nrp2-deficient mice show autism-like behavioral deficits and propensity to develop seizures. In order to determine the pathophysiology in Nrp2 deficiency, we examined the hippocampal numbers of interneuron subtypes and inhibitory regulation of hippocampal CA1 pyramidal neurons in mice lacking one or both copies of Nrp2. Immunostaining for interneuron subtypes revealed that Nrp2−/− mice have a reduced number of parvalbumin, somatostatin, and neuropeptide Y cells, mainly in CA1. Whole-cell recordings identified reduced firing and hyperpolarized shift in resting membrane potential in CA1 pyramidal neurons from Nrp2+/− and Nrp2−/− mice compared to age-matched wild-type controls indicating decrease in intrinsic excitability. Simultaneously, the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) are reduced in Nrp2-deficient mice. A convulsive dose of kainic acid evoked electrographic and behavioral seizures with significantly shorter latency, longer duration, and higher severity in Nrp2−/− compared to Nrp2+/+ animals. Finally, Nrp2+/− and Nrp2−/− but not Nrp2+/+, mice have impaired cognitive flexibility demonstrated by reward-based reversal learning, a task associated with hippocampal circuit function. Together these data demonstrate a broad reduction in interneuron subtypes and compromised inhibition in CA1 of Nrp2−/− mice, which could contribute to the heightened seizure susceptibility and behavioral deficits consistent with an ASD/epilepsy phenotype.

Short-Term Epileptiform Activity Potentiates Excitatory Synapses but Does Not Affect Intrinsic Membrane Properties of Pyramidal Neurons in the Rat Hippocampus In Vitro.

Even brief epileptic seizures can lead to activity-dependent structural remodeling of neural circuitry. Animal models show that the functional plasticity of synapses and changes in the intrinsic excitability of neurons can be crucial for epileptogenesis. However, the exact mechanisms underlying epileptogenesis remain unclear. We induced epileptiform activity in rat hippocampal slices for 15 min using a 4-aminopyridine (4-AP) in vitro model and observed hippocampal hyperexcitability for at least 1 h. We tested several possible mechanisms of this hyperexcitability, including changes in intrinsic membrane properties of neurons and presynaptic and postsynaptic alterations. Neither input resistance nor other essential biophysical properties of hippocampal CA1 pyramidal neurons were affected by epileptiform activity. The glutamate release probability also remained unchanged, as the frequency of miniature EPSCs and the paired amplitude ratio of evoked responses did not change after epileptiform activity. However, we found an increase in the AMPA/NMDA ratio, suggesting alterations in the properties of postsynaptic glutamatergic receptors. Thus, the increase in excitability of hippocampal neural networks is realized through postsynaptic mechanisms. In contrast, the intrinsic membrane properties of neurons and the probability of glutamate release from presynaptic terminals are not affected in a 4-AP model.

Sensitivity, specificity and limitation of in vitro hippocampal slice and neuron-based assays for assessment of drug-induced seizure liability

An effective in vitro screening assay to detect seizure liability in preclinical development can contribute to better lead molecule optimization prior to candidate selection, providing higher throughput and overcoming potential brain exposure limitations in animal studies. This study explored effects of 26 positive and 14 negative reference pharmacological agents acting through different mechanisms, including 18 reference agents acting on glutamate signaling pathways, in a brain slice assay (BSA) of adult rat to define the assay's sensitivity, specificity, and limitations. Evoked population spikes (PS) were recorded from CA1 pyramidal neurons of hippocampus (HPC) in the BSA. Endpoints for analysis were PS area and PS number. Most positive references (24/26) elicited a concentration-dependent increase in PS area and/or PS number. The negative references (14/14) had little effect on the PS. Moreover, we studied the effects of 15 reference agents testing positive in the BSA on spontaneous activity in E18 rat HPC neurons monitored with microelectrode arrays (MEA), and compared these effects to the BSA results. From these in vitro studies we conclude that the BSA provides 93% sensitivity and 100% specificity in prediction of drug-induced seizure liability, including detecting seizurogenicity by 3 groups of metabotropic glutamate receptor (mGluR) ligands. The MEA results seemed more variable, both quantitatively and directionally, particularly for endpoints capturing synchronized electrical activity. We discuss these results from the two models, comparing each with published results, and provide potential explanations for differences and future directions.

Micro-injection of propranolol within basolateral amygdala impaired fear and spatial memory and dysregulated evoked responses of CA1 neurons following foot shock stress in rats

The basolateral nucleus of the amygdala (BLA) is responsible for memory retrieval after stress. It regulates hippocampal long-term potentiation (LTP) during stress. Although β-adrenoceptors of the BLA have a critical role in memory, few studies have addressed this question in the BLA, and the results still have been contradictory. The present study was designed to investigate the involvement of β-adrenoceptors of the BLA on hippocampus memory, anxiety, and plasticity in intact and stressed rats. Male Wistar rats were submitted to the electrical foot-shock stress for four consecutive days. Over four consecutive days, animals received bilateral micro-injections of either vehicle or propranolol (4 µg in 1 µl/side) into the BLA (5 min before foot-shock stress). Behavioral (memory, as well as anxiety-like behaviors), electrophysiological, and histological (neural arborization in the hippocampal CA1 pyramidal neurons) studies were performed. Results showed that inhibition of β-adrenoceptors of BLA by propranolol significantly further impaired fear and spatial memory in stressed rats. Similarly, propranolol effectively impaired both memories in the intact animals. Propranolol significantly amplified the slope and amplitude of fEPSP in the CA1 area of the hippocampus only in stressed rats. Foot-shock stress significantly increased the number of dendritic branches in the hippocampus, and propranolol suppressed this effect of stress. It is suggested that β-adrenoceptors in the BLA promote memory and reduce anxiety-like behaviors under tonic and stress conditions. Propranolol dysregulated LTP parameters and reduced dendritic branches, resulting in memory impairment. Probably β-adrenoceptors of BLA regulate evoked responses of CA1 neurons only in stress- and not the tonic condition.

Vitamin B12-folic acid supplementation regulates neuronal immediate early gene expression and improves hippocampal dendritic arborization and memory in old male mice

As the relationship among diet, brain aging and memory is complex, it provides ample opportunity for research in multiple directions including behaviour, epigenetics and neuroplasticity. Nutritional deficiencies together with genetic and environmental factors are the major cause of many age-associated pathologies including memory loss. A compromised vitamin B12-folate status in older people is highly prevalent worldwide. Researchers have established a close association between the adequate level of B12-folate and the maintenance of cognitive brain functions. One of the main reasons for age-associated memory loss is downregulation of neuronal immediate early genes (nIEGs). Therefore, we hypothesize here that vitamin B12-folic acid supplementation in old mice can improve memory by altering the expression status of nIEGs. To check this, 72-week-old male Swiss albino mice were orally administered with 2 μg of vitamin B12 and 22 μg of folic acid/mouse/day for eight weeks. Such supplementation improved recognition memory in old and altered the expression of nIEGs. The expression of nIEGs was further found to be regulated by changes in DNA methylation at their promoter regions and CREB phosphorylation (pCREB). In addition, Golgi-Cox staining showed significant improvement in dendritic length, number of branching points and spine density of hippocampal CA1 pyramidal neurons by B12-folic acid supplementation. Taken together, these findings suggest that vitamin B12-folic acid supplementation regulates nIEGs expression and improves dendritic arborization of hippocampal neurons and memory in old male mice.

Developmental exposure of bisphenol A induces spatial memory deficits by weakening the excitatory neural circuits of CA3-CA1 and EC-CA1 in mice

Bisphenol-A (BPA) is an environmental endocrine disruptor and impairs learning and memory. However, the direct evidence for BPA exposure affecting neural circuits has been limited. In this study, a virus tracing assay has been established to explore the brain's neural circuits. Thy1-Cre mice were used to investigate the effects of BPA on the neural projection of glutamatergic pyramidal neurons in hippocampal CA1 based on Thy1 promoter. These transgenic mice were orally exposed to BPA (0, 0.5 mg/kg/day) from postnatal day (PND) 0 to PND60 and then subjected to behavioral tests. Morris water maze(MWM)and Barnes maze's showed that the spatial memory was seriously impaired in BPA exposed Thy1-Cre mice. Virus tracing assay indicated that CA1 pyramidal neurons mainly received neural inputs from hippocampal CA3, entorhinal cortex (EC), and medial septum (MS). The analysis showed that BPA reduced the number of RV+ neurons in CA3 and EC but not MS. The immunohistochemistry experiment displayed that BPA decreased the percentage of CaMKIIRV+ cells in CA3 and EC. The results demonstrated that the synaptic connection of upstream glutamatergic neurons and CA1 pyramidal cells was weakened by BPA exposure. These point to potentially detrimental effects of BPA exposure on the excitatory neural circuit of CA3-CA1 and EC-CA1 in memory formation. Thus, our findings revealed that the decrease in excitatory neural circuits of CA3-CA1 and EC-CA1 contribute to the BPA-induced spatial memory deficits in Thy1-Cre mice.

ERG Channels Contribute to the Excitability of Pyramidal Neurons in Hippocampal CA1

ERG Channels Contribute to the Excitability of Pyramidal Neurons in Hippocampal CA1

Ethinyl Estadiol/Progestin Oral Contraceptives Depress Spatial Learning and Dysregulate Hippocampal CA3 Microstructure: Implications for Behavioral-Cognitive Effects of Chronic Contraceptive Use?

Abstract Combined oral contraceptive pill contains ethinyl estradiol and a synthetic progestin, which prevent ovulation by suppressing the release of the gonadotropins resulting in the inhibition of ovarian follicles’ development. Although advantageous in birth control, the impact on learning and memory is limited necessitating this study on its effect on spatial learning, and hippocampal CA3 microstructure. Thirty two female Wistar rats of average body weight 200 g were equally divided (n = 8) into four groups; 0.002 mg/kg levonorgestrel plus 0.00043 mg/kg ethinyl estradiol (COCP) were administered orally for 21, 42 and 63 days. 24 hours after the last administration the rats underwent Morris water maze test and were sacrificed by transcardial perfusion-fixation. Their hippocampal regions were processed for histological study, and immunolabelled with anti-neuron specific enolase (NSE) and glial fibrillary acidic protein (GFAP). Results showed that the COCP test groups had shorter escape latencies (p ≤ 0.05) in the visible and hidden platform trials. The COCP test groups showed no difference in neuronal population, although some of the hippocampal CA3 pyramidal neurons were either atrophic and/or karyorrhectic, with shrunken and dense nuclei. NSE expression was lower (p ≤ 0.05) in the 21, 42 and 63 days COCP groups, while GFAP expression was lower in the 21 days COCP group, but not different in the 42 and 63 days COCP groups compared with the control. These preliminary results show that COCP influence spatial learning, and may also reduce neuronal metabolic activity, while increasing astrocytic activity in the hippocampal CA3.

Alternative splicing of GluN1 gates glycine site–dependent nonionotropic signaling by NMDAR receptors

N-methyl-D-aspartate (NMDA) receptors (NMDARs), a principal subtype of excitatory neurotransmitter receptor, are composed as tetrameric assemblies of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits. NMDARs can signal nonionotropically through binding of glycine alone to its cognate site on GluN1. A consequence of this signaling by glycine is that NMDARs are primed such that subsequent gating, produced by glycine and glutamate, drives receptor internalization. The GluN1 subunit contains eight alternatively spliced isoforms produced by including or excluding the N1 and the C1, C2, or C2' polypeptide cassettes. Whether GluN1 alternative splicing affects nonionotropic signaling by NMDARs is a major outstanding question. Here, we discovered that glycine priming of recombinant NMDARs critically depends on GluN1 isoforms lacking the N1 cassette; glycine priming is blocked in splice variants containing N1. On the other hand, the C-terminal cassettes-C1, C2, or C2'-each permit glycine signaling. In wild-type mice, we found glycine-induced nonionotropic signaling at synaptic NMDARs in CA1 hippocampal pyramidal neurons. This nonionotropic signaling by glycine to synaptic NMDARs was prevented in mice we engineered, such that GluN1 obligatorily contained N1. We discovered in wild-type mice that, in contrast to pyramidal neurons, synaptic NMDARs in CA1 inhibitory interneurons were resistant to glycine priming. But we recapitulated glycine priming in inhibitory interneurons in mice engineered such that GluN1 obligatorily lacked the N1 cassette. Our findings reveal a previously unsuspected molecular function for alternative splicing of GluN1 in controlling nonionotropic signaling of NMDARs by activating the glycine site.

Rare CASP6N73T variant associated with hippocampal volume exhibits decreased proteolytic activity, synaptic transmission defect, and neurodegeneration

Caspase-6 (Casp6) is implicated in Alzheimer disease (AD) cognitive impairment and pathology. Hippocampal atrophy is associated with cognitive impairment in AD. Here, a rare functional exonic missense CASP6 single nucleotide polymorphism (SNP), causing the substitution of asparagine with threonine at amino acid 73 in Casp6 (Casp6N73T), was associated with hippocampal subfield CA1 volume preservation. Compared to wild type Casp6 (Casp6WT), recombinant Casp6N73T altered Casp6 proteolysis of natural substrates Lamin A/C and α-Tubulin, but did not alter cleavage of the Ac-VEID-AFC Casp6 peptide substrate. Casp6N73T-transfected HEK293T cells showed elevated Casp6 mRNA levels similar to Casp6WT-transfected cells, but, in contrast to Casp6WT, did not accumulate active Casp6 subunits nor show increased Casp6 enzymatic activity. Electrophysiological and morphological assessments showed that Casp6N73T recombinant protein caused less neurofunctional damage and neurodegeneration in hippocampal CA1 pyramidal neurons than Casp6WT. Lastly, CASP6 mRNA levels were increased in several AD brain regions confirming the implication of Casp6 in AD. These studies suggest that the rare Casp6N73T variant may protect against hippocampal atrophy due to its altered catalysis of natural protein substrates and intracellular instability thus leading to less Casp6-mediated damage to neuronal structure and function.

Reversible GABAergic dysfunction involved in hippocampal hyperactivity predicts early-stage Alzheimer disease in a mouse model

BackgroundNeuronal hyperactivity related to β-amyloid (Aβ) is considered an early warning sign of Alzheimer disease (AD). Although increasing evidence supports this opinion, the underlying mechanisms are still unknown.MethodsHere, we recorded whole-cell synaptic currents and membrane potentials using patch clamping of acute hippocampal slices from human amyloid precursor protein (APP)/presenilin-1 transgenic (5XFAD) mice and their wild-type littermates. Biochemical methods, electron microscopic imaging, behavioral tests, and intraventricular drug delivery applied with osmotic pumps were used in this study.ResultsWe confirmed hyperactivity of hippocampal CA1 pyramidal neurons in 5XFAD mice using whole-cell electrophysiological recording at 2.5 months old, when local Aβ-positive plaques had not developed and only mild cognitive dysfunction occurred. We further discovered attenuated inhibitory postsynaptic currents and unchanged excitatory postsynaptic currents in CA1 pyramidal neurons, in which the intrinsic excitability was unchanged. Moreover, the density of both γ-aminobutyric acid A (GABAA) receptor subunits, α1 and γ2, was reduced in synapses of the hippocampus in transgenic mice. Intriguingly, early intervention with the GABAA receptor agonist gaboxadol reversed the hippocampal hyperactivity and modestly ameliorated cognitive performance in 5XFAD mice under our experimental conditions.ConclusionsInhibitory postsynaptic disruption critically contributes to abnormalities in the hippocampal network and cognition in 5XFAD mice and possibly in AD. Therefore, strengthening the GABAergic system could be a promising therapy for AD in the early stages.

Enkephalin release from VIP interneurons in the hippocampal CA2/3a region mediates heterosynaptic plasticity and social memory.

The hippocampus contains a diverse array of inhibitory interneurons that gate information flow through local cortico-hippocampal circuits to regulate memory storage. Although most studies of interneurons have focused on their role in fast synaptic inhibition mediated by GABA release, different classes of interneurons express unique sets of neuropeptides, many of which have been shown to exert powerful effects on neuronal function and memory when applied pharmacologically. However, relatively little is known about whether and how release of endogenous neuropeptides from inhibitory cells contributes to their behavioral role in regulating memory formation. Here we report that vasoactive intestinal peptide (VIP)-expressing interneurons participate in social memory storage by enhancing information transfer from hippocampal CA3 pyramidal neurons to CA2 pyramidal neurons. Notably, this action depends on release of the neuropeptide enkephalin from VIP neurons, causing long-term depression of feedforward inhibition onto CA2 pyramidal cells. Moreover, VIP neuron activity in the CA2 region is increased selectively during exploration of a novel conspecific. Our findings, thus, enhance our appreciation of how GABAergic neurons can regulate synaptic plasticity and mnemonic behavior by demonstrating that such actions can be mediated by release of a specific neuropeptide, rather than through classic fast inhibitory transmission.

The effects of astaxanthin treatment on a rat model of Alzheimer’s disease

Alzheimer's disease (AD), a progressive neurodegenerative disorder characterized by memory loss and dementia, could be a consequence of the abnormalities of cortical milieu, such as oxidative stress, inflammation, and/or accompanied with the aggregation of β-amyloid. The majority of AD patients are sporadic, late-onset AD, which predominantly occurs over 65 years of age. Our results revealed that the ferrous amyloid buthionine (FAB)-infused sporadic AD-like model showed deficits in spatial learning and memory and with apparent loss of choline acetyltransferase (ChAT) expression in medial septal (MS) nucleus. In hippocampal CA1 region, the loss of pyramidal neurons was accompanied with cholinergic fiber loss and neuroinflammatory responses including glial reaction and enhanced expression of inducible nitric oxide synthase (iNOS). Surviving hippocampal CA1 pyramidal neurons showed the reduction of dendritic spines as well. Astaxanthin (ATX), a potent antioxidant, reported to improve the outcome of oxidative-stress-related diseases. The ATX treatment in FAB-infused rats decreased neuroinflammation and restored the ChAT + fibers in hippocampal CA1 region and the ChAT expression in MS nucleus. It also partly recovered the spine loss on hippocampal CA1 pyramidal neurons and ameliorated the behavioral deficits in AD-like rats. From these data, we believed that the ATX can be a potential option for slowing the progression of Alzheimer's disease.

Translating Ketamine’s Rapid Neuroplastic Effects From Animals to Humans

Studies in animals have demonstrated rapid restoration of stress-induced deficits of neuroplasticity as one of Ketamine’s mechanisms of action. A result from a rat model demonstrated immediate increases of dendritic spines in hippocampal CA1 pyramidal neurons after ketamine. Here, we aimed to detect ketamine’s rapid alterations of hippocampal plasticity in humans.

Increased Excitatory Synaptic Transmission Associated with Adult Seizure Vulnerability Induced by Early-Life Inflammation in Mice.

Early-life inflammatory stress increases seizure susceptibility later in life. However, possible sex- and age-specific differences and the associated mechanisms are largely unknown. C57BL/6 mice were bred in house, and female and male pups were injected with lipopolysaccharide (LPS; 100 μg/kg, i.p.) or vehicle control (saline solution) at postnatal day 14 (P14). Seizure threshold was assessed in response to pentylenetetrazol (1% solution, i.v.) in adolescence (∼P40) and adulthood (∼P60). We found that adult, but not adolescent, mice treated with LPS displayed ∼34% lower seizure threshold compared with controls. Females and males showed similar increased seizure susceptibility, suggesting that altered brain excitability was age dependent, but not sex dependent. Whole-cell recordings revealed no differences in excitatory synaptic activity onto CA1 pyramidal neurons from control or neonatally inflamed adolescent mice of either sex. However, adult mice of both sexes previously exposed to LPS displayed spontaneous EPSC frequency approximately twice that of controls, but amplitude was unchanged. Although these changes were not associated with alterations in dendritic spines or in the NMDA/AMPA receptor ratio, they were linked to an increased glutamate release probability from Schaffer collateral, but not temporoammonic pathway. This glutamate increase was associated with reduced activity of presynaptic GABAB receptors and was independent of the endocannabinoid-mediated suppression of excitation. Our new findings demonstrate that early-life inflammation leads to long-term increased hippocampal excitability in adult female and male mice associated with changes in glutamatergic synaptic transmission. These alterations may contribute to enhanced vulnerability of the brain to subsequent pathologic challenges such as epileptic seizures.SIGNIFICANCE STATEMENT Adult physiology has been shown to be affected by early-life inflammation. Our data reveal that early-life inflammation increases excitatory synaptic transmission onto hippocampal CA1 pyramidal neurons in an age-dependent manner through disrupted presynaptic GABAB receptor activity on Schaffer collaterals. This hyperexcitability was seen only in adult, and not in adolescent, animals of either sex. The data suggest a maturation process, independent of sex, in the priming action of early-life inflammation and highlight the importance of studying mature brains to reveal cellular changes associated with early-life interventions.

Cation leak underlies neuronal excitability in an HCN1 developmental and epileptic encephalopathy.

Pathogenic variants in HCN1 are associated with developmental and epileptic encephalopathies. The recurrent de novo HCN1 M305L pathogenic variant is associated with severe developmental impairment and drug-resistant epilepsy. We engineered the homologue Hcn1 M294L heterozygous knock-in (Hcn1M294L) mouse to explore the disease mechanism underlying an HCN1 developmental and epileptic encephalopathy. The Hcn1M294L mouse recapitulated the phenotypic features of patients with the HCN1 M305L variant, including spontaneous seizures and a learning deficit. Active epileptiform spiking on the electrocorticogram and morphological markers typical of rodent seizure models were observed in the Hcn1M294L mouse. Lamotrigine exacerbated seizures and increased spiking, whereas sodium valproate reduced spiking, mirroring drug responses reported in a patient with this variant. Functional analysis in Xenopus laevis oocytes and layer V somatosensory cortical pyramidal neurons in ex vivo tissue revealed a loss of voltage dependence for the disease variant resulting in a constitutively open channel that allowed for cation 'leak' at depolarized membrane potentials. Consequently, Hcn1M294L layer V somatosensory cortical pyramidal neurons were significantly depolarized at rest. These neurons adapted through a depolarizing shift in action potential threshold. Despite this compensation, layer V somatosensory cortical pyramidal neurons fired action potentials more readily from rest. A similar depolarized resting potential and left-shift in rheobase was observed for CA1 hippocampal pyramidal neurons. The Hcn1M294L mouse provides insight into the pathological mechanisms underlying hyperexcitability in HCN1 developmental and epileptic encephalopathy, as well as being a preclinical model with strong construct and face validity, on which potential treatments can be tested.

Структурные основы изменения межнейронной коммуникации нейронов поля СА3 гиппокампа белых крыс после тяжелой черепно-мозговой травмы

Цель - изучение пирамидных нейронов поля СА3 гиппокампа белых крыс в динамике после тяжелой черепно-мозговой травмы (ТЧМТ). Методы. ТЧМТ моделировали под наркозом с помощью свободно падающего груза массой 200-250 г с высоты 50 см на теменно-затылочную область. Гиппокамп изучали в контроле (n=5), через 1, 3, 5, 7 и 14 сут после ТЧМТ (n=25). Общую оценку состояния нейронов поля СА3 проводили на препаратах окрашенных гематоксилином-эозином, численную плотность нейронов - при окраске по Нисслю, цитоскелет нейронов изучали с помощью реакции иммунотипирования нейрон-специфического структурного белка (МАР-2), синаптические терминали - иммунотипирования синаптофизина (p38). Для визуализации MAP-2 нейронов и р38 синаптических терминалей использовали мультимерный набор NovolinkTM (DAB) Polymer Detection System (Leica Biosystems Newcastle Ltd, Великобритания). Морфометрический анализ проводили на цветных растровых и бинарных изображениях с использованием плагинов программы ImageJ 1.52s. Определяли относительную площадь зон отека-набухания, численную плотность пирамидных нейронов, количество дистрофически и некробиотически измененных нейронов, общую и относительную площадь синаптических терминалей. Результаты. Через 1 сут после ТЧМТ нарастали явления отека-набухания, увеличивалось количество дистрофически и некробиотически измененных нейронов, уменьшалась общая и относительная площадь терминалей. В течение 14 сут общая плотность нейронов уменьшилась на 31%. Параллельно активировались механизмы нейро- и синаптической пластичности, в результате чего восстанавливался цитоскелет поврежденных нейронов и увеличивалось количество межнейронных синапсов (в 1,32 раза выше контроля). Заключение. Восстановление структур межнейронной коммуникации происходило на фоне уменьшения общей численной плотности пирамидных нейронов. Выявленные изменения рассматриваются как основа перманентной компенсаторно-восстановительной реорганизации межнейронных отношений гиппокампа на фоне вторичной ишемии головного мозга. Aim. To study changes in hippocampal CA3 pyramidal neurons of white rats after severe traumatic brain injury (STBI). Methods. STBI was modeled with a free-falling weight (200-250 g) impact. The hippocampus was studied in control rats (n=5) 1, 3, 5, 7, and 14 days after STBI (n=25). The CA3 field neurons were examined on preparations stained with hematoxylin-eosin and the number of neurons was determined with Nissl staining. The neuronal cytoskeleton was studied by immunotyping of the neuron-specific structural protein MAP-2, and synaptic terminals were studied by immunotyping of synaptophysin (p38). Neuronal MAP-2 and p38 were visualized with a multimeric Novolink™ (DAB) Polymer Detection Systems kit (Leica Biosystems Newcastle Ltd, Great Britain). Morphometric analysis was performed on color raster and binary images using ImageJ 1.52s plugins to determine the relative area of edema and swelling zones, number density of pyramidal neurons, content of dystrophic and necrobiotically altered neurons, and total and relative areas of synaptic terminals. Results. On the next day after STBI, manifestations of edema and swelling and the content of dystrophic and necrobiotically altered neurons were increased whereas the total and relative areas of terminals were decreased. In 14 days, the total density of neurons decreased by 31%, which was in parallel with activation of mechanisms for neuro- and synaptic plasticity. As a result, the cytoskeleton of damaged neurons recovered, and the content of interneuronal synapses increased 1.32 times compared to the control. Conclusion. The structural recovery of interneuronal communication was associated with a decrease in the total number density of pyramidal neurons. These changes were regarded as a base for permanent compensatory and restorative reorganization of hippocampal interneuronal relations in secondary cerebral ischemia.

HIV-1 Tat and Morphine Differentially Disrupt Pyramidal Cell Structure and Function and Spatial Learning in Hippocampal Area CA1: Continuous versus Interrupted Morphine Exposure.

About half the people infected with human immunodeficiency virus (HIV) have neurocognitive deficits that often include memory impairment and hippocampal deficits, which can be exacerbated by opioid abuse. To explore the effects of opioids and HIV on hippocampal CA1 pyramidal neuron structure and function, we induced HIV-1 transactivator of transcription (Tat) expression in transgenic mice for 14 d and co-administered time-release morphine or vehicle subcutaneous implants during the final 5 d (days 9–14) to establish steady-state morphine levels. Morphine was withheld from some ex vivo slices during recordings to begin to assess the initial pharmacokinetic consequences of opioid withdrawal. Tat expression reduced hippocampal CA1 pyramidal neuronal excitability at lower stimulating currents. Pyramidal cell firing rates were unaffected by continuous morphine exposure. Behaviorally, exposure to Tat or high dosages of morphine impaired spatial memory Exposure to Tat and steady-state levels of morphine appeared to have largely independent effects on pyramidal neuron structure and function, a response that is distinct from other vulnerable brain regions such as the striatum. By contrast, acutely withholding morphine (from morphine-tolerant ex vivo slices) revealed unique and selective neuroadaptive shifts in CA1 pyramidal neuronal excitability and dendritic plasticity, including some interactions with Tat. Collectively, the results show that opioid-HIV interactions in hippocampal area CA1 are more nuanced than previously assumed, and appear to vary depending on the outcome assessed and on the pharmacokinetics of morphine exposure.

Repeated victorious and defeat experiences induce similar apical dendritic spine remodeling in CA1 hippocampus of rats

In this study, apical dendritic spine density of neurons in hippocampal, amygdalar and prefrontal cortical areas was compared in rats that were repeatedly winning or losing social conflicts. Territorial male wild-type Groningen (WTG) rats were allowed multiple daily attacks (>20 times) on intruder males in the resident-intruder paradigm. Frequent winning experiences are known to facilitate uncontrolled aggressive behavior reflected in aggressive attacks on anesthetized males which was also observed in the winners in this study. Both winners and losers were socially housed during the experiments; winners with females to stimulate territorial behavior, and losers with two other losing male rats. Twenty-four hours after the last social encounter, brains from experienced residential winners and repeatedly defeated intruder rats were collected and neuronal morphology in selected brain regions was studied via Golgi-Cox staining. Results indicate that spine density in the apical dendrites of the hippocampal CA1 reduced similarly in both winners and losers. In addition, winners showed increased spine densities at the proximal segments (20−30 μm) of the basolateral amygdala neurons and losers tended to show a decreased spine density at the more proximal segments of the infralimbic region of prefrontal cortex neurons. No effect of winning and losing was observed in the medial amygdala. The atrophic effect of repeated defeats in hippocampal and prefrontal regions was anticipated despite the fact that social housing of the repeatedly losing intruder males may have played a protective role. The reduction of hippocampal spine density in the winners seems surprising but supports previous findings in hierarchical dominant males in rat colonies. The dominants showed even greater shrinkage of the apical dendritic arbors of hippocampal CA3 pyramidal neurons compared to the stressed subordinates.