Female-specific synaptic dysfunction and cognitive impairment in a mouse model of PCDH19 disorder.

Protocadherin-19 (PCDH19) mutations cause early-onset seizures and cognitive impairment. The PCDH19 gene is on the X-chromosome. Unlike most X-linked disorders, PCDH19 mutations affect heterozygous females (PCDH19HET♀ ) but not hemizygous males (PCDH19HEMI♂ ); however, the reason why remains to be elucidated. We demonstrate that PCDH19, a cell-adhesion molecule, is enriched at hippocampal mossy fiber synapses. Pcdh19HET♀ but not Pcdh19HEMI♂ mice show impaired mossy fiber synaptic structure and physiology. Consistently, Pcdh19HET♀ but not Pcdh19HEMI♂ mice exhibit reduced pattern completion and separation abilities, which require mossy fiber synaptic function. Furthermore, PCDH19 appears to interact with N-cadherin at mossy fiber synapses. In Pcdh19HET♀ conditions, mismatch between PCDH19 and N-cadherin diminishes N-cadherin-dependent signaling and impairs mossy fiber synapse development; N-cadherin overexpression rescues Pcdh19HET♀ phenotypes. These results reveal previously unknown molecular and cellular mechanisms underlying the female-specific PCDH19 disorder phenotype.

Maternal Separation-Induced Histone Acetylation Correlates with BDNF-Programmed Synaptic Changes in an Animal Model of PTSD with Sex Differences.

Maternal separation (MS) causes long-lasting epigenetic changes in the brain and increases vulnerability to traumatic events in adulthood. Of interest, there may be sex-specific differences in these epigenetic changes. In this study, the extent of histone acetylation in the hippocampus (HIP) and the expression of BDNF were measured to determine whether BDNF influences risk of PTSD following MS in early life. Rat offspring were separated from their dams (3 h/day or 6 h/day from PND2~PND14). Then, pups were treated with a single prolonged stress (SPS) procedure when they reached adulthood (PND80). In animals stressed with the SPS procedure in adulthood, those that had increased MS intensity in childhood demonstrated more significant changes in performance on tests of anxiety, depression, and contextual fear memory. Reduced levels of total BDNF mRNA and protein were observed after SPS treatment and further declined in groups with greater MS time in childhood. Interestingly, these changes were correlated with decreased H3K9ac levels and increased HDAC2 levels. Additional MS also led to more severe ultrastructural synaptic damage in rats that experienced the SPS procedure, particularly in the CA1 and CA3 region of the HIP, reflecting impaired synaptic plasticity in these regions. Interestingly, male rats in the MS3h-PTSD group showed decreased anxiety, but no similar changes were found in female rats, suggesting a degree of gender specificity in coping with stress after mild MS. In summary, this study suggests that the epigenetic signatures of the BDNF genes can be linked to HIP responses to stress, providing insights that may be relevant for people at risk of stress-related psychopathologies.

Effects of topiramate on the ultrastructure of synaptic endings in the hippocampal CA1 and CA3 sectors in the rat experimental model of febrile seizures: the first electron microscopy report.

The present study aimed at exploring a potentially neuroprotective effect of topiramate (TPM), one of the most commonly used newer-generation, broad-spectrum, antiepileptic drugs against ultrastructural damage of hippocampal synaptic endings in the experimental model of febrile seizures (FS). The study used male young Wistar rats aged 22-30 days, divided into three experimental groups and the control group. Brain maturity in such animals corresponds to that of 1- or 2-year-old children. Hyperthermic stress was evoked by placing animals in a 45°C water bath for four consecutive days. TPM at a dose of 80 mg/kg b.m. was administered with an intragastric tube before and immediately after FS. Specimens (1 mm3) collected from the hippocampal CA1 and CA3 sectors, fixed via transcardial perfusion with a solution of paraformaldehyde and glutaraldehyde, were routinely processed for transmission-electron microscopic analysis. Advanced ultrastructural changes induced by hyperthermic stress were manifested by distinct swelling of hippocampal pre- and post-synaptic axodendritic and axospinal endings, including their vacuolization and disintegration. The axoplasm of the presynaptic boutons contained a markedly decreased number of synaptic vesicles and their abnormal accumulation in the active synaptic region. The synaptic junctions showed a dilated synaptic cleft and a decreased synaptic active zone. TPM used directly after FS was ineffective in the prevention of hyperthermia-induced injury of synaptic endings in hippocampal CA1 and CA3 sectors. However, "prophylactic" administration of TPM, prior to FS induction, demonstrated a neuroprotective effect against synaptic damage in approximately 25% of the synaptic endings in the hippocampal sectors, more frequently located in perivascular zones. It was manifested by smaller oedema of both presynaptic and postsynaptic parts, containing well-preserved mitochondria, increased number and regular distribution of synaptic vesicles within the axoplasm, and increased synaptic active zone. Our current and previous findings suggest that TPM administered "prophylactically", before FS, could exert a favourable effect on some synapses, indirectly, via the vascular factor, i.e. protecting blood-brain barrier components and through better blood supply of the hippocampal CA1 and CA3 sectors, which may have practical implications.

Reducing PRLR expression and JAK2 activity results in an increase in BDNF expression and inhibits the apoptosis of CA3 hippocampal neurons in a chronic mild stress model of depression.

Patients suffering from depression most commonly present with symptoms associated with the autonomic nervous system. Despite the satisfactory results achieved following treatment with vagus nerve stimulation and drug treatment, recurrence is a common occurrence in many patients. As described in numerous studies, prolactin receptor (PRLR) has been identified as an anxiolytic and anti-depressant factor in depression. However, the effect of PRLR on chronic mild stress (CMS)-induced depression remains to be thoroughly demonstrated. Therefore, the present study was conducted on the effect of PRLR gene on brain derived neurotrophic factor (BDNF) expression and hippocampal neuron apoptosis through the establishment of CMS-induced depression mouse models, with aims of providing a new and effective therapeutic option for depression. Microarray-based analysis was initially used to retrieve depression-related expression dataset and PRLR-related signaling pathway. Lentiviral vectors overexpressing PRLR or expressing PRLR-specific shRNA were used to up- or down-regulated the expression of PRLR in mice. Subsequently, the effects of PRLR on hippocampal neurons and pyramidal cells in CA1 and CA3 regions, and ultrastructure in hippocampal region were evaluated. Serum BDNF level and the positive rate of cleaved-Caspase-3 in hippocampal CA3 region were determined. Next, the regulatory mechanism by which PRLR gene silencing influences hippocampal neuron apoptosis via the JAK2-STAT5 signaling pathway was detected. PRLR gene was assumed to participate in the development of depression by regulating the JAK-STAT signaling pathway. Our results found that the mice with CMS-induced depression exhibited locomotion activity and anhedonia. In addition, a decrease in the number of pyramidal cells was observed in the hippocampus while that of apoptotic cells was increased. In addition, serum BDNF level was increased, and the expression of Caspase-3 and Bax in hippocampal neurons and the JAK2-STAT5 signaling pathway was decreased in response to PRLR silencing, along with increased expression of BDNF and Bcl-2. From the aforementioned findings, we concluded that PRLR gene silencing results in the inhibition of hippocampal neuron apoptosis and alleviation of CMS-induced depression by inactivating the JAK2-STAT5 signaling pathway and elevating BDNF expression, providing a new insight for the treatment of depression.

Salidroside mediated stabilization of Bcl -xL prevents mitophagy in CA3 hippocampal neurons during hypoxia.

Chronic hypoxic stress results in deposition of lipofuscin granules in the CA3 region of hippocampal neurons which contributes to neurodegeneration and accelerated neuronal aging. Oxidative stress and mitophagy during hypoxia are crucial to cause aggregation of these lipofuscin granules in hypoxic neurons. Salidroside, a glucoside derivative of ß-Tyrosol, has been reported to protect hypoxic neurons through maintenance of mitochondrial activity. The present study is aimed at investigating the potential of Salidroside in preventing mitophagy during chronic hypoxia and identification of the molecular targets and underlying signaling mechanisms. In-silico analysis for interaction of salidroside with Bcl-xL was carried out using VLife MDS software. The prophylactic efficacy of Salidroside for amelioration of global hypoxia induced neuronal aging was studied in adult male Sprague-Dawley rats exposed to hypobaric hypoxia simulating an altitude of 7600 m for 21 days. Salidroside was supplemented at a daily dose of 25 mg kg-1b.w. p.o. during hypoxic exposure. Ultra-structural and immune-histological studies were conducted to study lipofuscin aggregation and mitophagy. In-silico findings on salidroside mediated stabilization of Bcl-xL were validated by investigating its effect on downstream signaling molecules involved in mitophagy. Administration of Salidroside reduced deposition of lipofuscin in hypoxic CA3 hippocampal neurons and prevented mitophagy. Salidroside stabilizes Bcl-xL in hypoxic neurons resulting in inhibition of PGAM5 phosphatase activity and maintenance of FUNDC1 in phosphorylated state. Salidroside mediated inhibition of pFUNDC1 dephosphorylation prevents FUNDC1-LC3 II interaction which is crucial for mitophagy. The present study demonstrates potential of Salidroside in preventing lipofuscin deposition during chronic hypoxic stress.

Rat hippocampal CA3 neuronal injury induced by limb ischemia/reperfusion: A possible restorative effect of alpha lipoic acid.

Limb ischemia reperfusion (I/R) injury is associated with serious local and systemic effects. Reperfusion may augment tissue injury in excess of that produced by ischemia alone. The hippocampus has been reported to be vulnerable to I/R injury. Alpha lipoic acid (ALA) is an endogenous antioxidant with a powerful antioxidative, anti-inflammatory, and antiapoptotic properties. We studied the probable restorative effect of ALA on limb I/R-induced structural damage of rat hippocampus. Forty adult male albino rats were divided equally into four groups: group I (sham); group II (I/R-1 day) has undergone bilateral femoral arteries occlusion (3 h), then reperfusion for 1 day; group III (I/R-7 days) has undergone reperfusion for seven days; group IV (I/R-ALA) has undergone I/R as group III and received an intraperitoneal injection of ALA (100 mg/kg) for 7 days. I/R groups revealed degenerative changes in the pyramidal neuronal perikarya of CA3 field in the form of dark-stained cytoplasm, dilated RER cisternae, mitochondrial alterations, and dense bodies' accumulation. Their dendrites showed disorganized microtubules. Astrogliosis is featured by an increased number and increased immunoreactivity of astrocytes for glial fibrillary acid protein. Morphometric data revealed significant reduction of light neurons, surface area of neurons, and thickness of the CA3 layer. Most blood capillaries exhibited narrow lumen and irregular basal lamina. ALA ameliorated the neuronal damage. Pyramidal neurons revealed preservation of normal structure. Significant increase in the thickness of pyramidal layer in CA3 field and surface area and number of light neurons was observed but astrogliosis persisted. Limb I/R had a deleterious remote effect on the hippocampus aggravated with longer period of reperfusion. This work may encourage the use of ALA in the critical clinical settings with I/R injury.

Ectopic Mossy Fiber Pathfinding in the Hippocampus Caused the Abnormal Neuronal Transmission in the Mouse Models of Psychiatric Disease.

Appropriate axonal pathfinding is a necessary step for the function of neuronal circuits. The mossy fibers (MFs) in the hippocampus of CaMKIIα heterozygous knockout (CaMKIIα-hKO) psychiatric model mice project onto not only the stratum lucidum but also the stratum oriens region in the CA3, which is a projection pattern distinct from that in normal mice. Thus, we examined the electrophysiological properties of the MF-CA3 connection in this mutant mouse on field recordings and found a lower synaptic connection. This study suggested that the phenotype of abnormal MF pathfindings could induce aberrant neuronal functions, which may link to cognition and memory.

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method.

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain  excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Early born neurons are abnormally positioned in the doublecortin knockout hippocampus.

Human doublecortin (DCX) mutations are associated with severe brain malformations leading to aberrant neuron positioning (heterotopia), intellectual disability and epilepsy. The Dcx protein plays a key role in neuronal migration, and hippocampal pyramidal neurons in Dcx knockout (KO) mice are disorganized. The single CA3 pyramidal cell layer observed in wild type (WT) is present as two abnormal layers in the KO, and CA3 KO pyramidal neurons are more excitable than WT. Dcx KO mice also exhibit spontaneous epileptic activity originating in the hippocampus. It is unknown, however, how hyperexcitability arises and why two CA3 layers are observed.Transcriptome analyses were performed to search for perturbed postnatal gene expression, comparing Dcx KO CA3 pyramidal cell layers with WT. Gene expression changes common to both KO layers indicated mitochondria and Golgi apparatus anomalies, as well as increased cell stress. Intriguingly, gene expression analyses also suggested that the KO layers differ significantly from each other, particularly in terms of maturity. Layer-specific molecular markers and BrdU birthdating to mark the final positions of neurons born at distinct timepoints revealed inverted layering of the CA3 region in Dcx KO animals. Notably, many early-born 'outer boundary' neurons are located in an inner position in the Dcx KO CA3, superficial to other pyramidal neurons. This abnormal positioning likely affects cell morphology and connectivity, influencing network function. Dissecting this Dcx KO phenotype sheds light on coordinated developmental mechanisms of neuronal subpopulations, as well as gene expression patterns contributing to a bi-layered malformation associated with epilepsy.

Regulatory role of the cell adhesion molecule nectin-1 in GABAergic inhibitory synaptic transmission in the CA3 region of mouse hippocampus.

Proper operation of a neural circuit relies on both excitatory and inhibitory synapses. We previously showed that cell adhesion molecules nectin-1 and nectin-3 are localized at puncta adherentia junctions of the hippocampal mossy fiber glutamatergic excitatory synapses and that they do not regulate the excitatory synaptic transmission onto the CA3 pyramidal cells. We studied here the roles of these nectins in the GABAergic inhibitory synaptic transmission onto the CA3 pyramidal cells using nectin-1-deficient and nectin-3-deficient cultured mouse hippocampal slices. In these mutant slices, the amplitudes and frequencies of miniature excitatory postsynaptic currents were indistinguishable from those in the control slices. In the nectin-1-deficient slices, but not in the nectin-3-deficient slices, however, the amplitude of miniature inhibitory postsynaptic currents (mIPSCs) was larger than that in the control slices, although the frequency of the mIPSCs was not different between these two groups of slices. In the dissociated culture of hippocampal neurons from the nectin-1-deficient mice, the amplitude and frequency of mIPSCs were indistinguishable from those in the control neurons. Nectin-1 was not localized at or near the GABAergic inhibitory synapses. These results indicate that nectin-1 regulates the neuronal activities in the CA3 region of the hippocampus by suppressing the GABAergic inhibitory synaptic transmission.

Presynaptic size of associational/commissural CA3 synapses is controlled by fibroblast growth factor 22 in adult mice.

Associational/commissural CA3-CA3 synapses define the recurrent CA3 network that generates the input to CA1 pyramidal neurons. We quantified the fine structure of excitatory synapses in the stratum radiatum of the CA3d area in adult wild type (WT) and fibroblast growth factor 22 knock-out (FGF22KO) mice by using serial 3D electron microscopy. WT excitatory CA3 synapses are rather small yet range 10 fold in size. Spine size, however, was small and uniform and did not correlate with the size of the synaptic junction. To reveal mechanisms that regulate presynaptic structure, we investigated the role of FGF22, a target-derived signal specific for the distal part of area CA3 (CA3d). In adult FGF22KO mice, postsynaptic properties of associational CA3 synapses were unaltered. Presynaptically, the number of synaptic vesicles (SVs), the bouton volume, and the number of vesicles in axonal regions (the super pool) were reduced. This concurrent decrease suggests concerted control by FGF22 of presynaptic size. This hypothesis is supported by the finding that WT presynapses in the proximal part of area CA3 (CA3p) that do not receive FGF22 signaling in WT mice were smaller than presynapses in CA3d in WT but of comparable size in CA3d of FGF22KO mice. Docked SV density was decreased in CA1, CA3d, and CA3p in FGF22KO mice. Because CA1 and CA3p are not directly affected by the loss of FGF22, the smaller docked SV density may be an adaptation to activity changes in the CA3 network. Thus, docked SV density potentially is a long-term regulator for the synaptic release probability and/or the strength of short-term depression in vivo.

Effect of Combined Stress on Morphological Changes and Expression of NO Synthases in Rat Ventral Hippocampus.

Adult rats were subjected to 7-day combined stress with stochastic changes of stressors of different modalities (noise, vibration, pulsating bright light) along with mobility restriction and elevated temperature in the chamber during stress exposures (daily 30-min sessions). Circulatory disorders, inhibition of endothelial NO-synthase expression in endothelial cells of the microcirculatory bed, perivascular edema, pronounced degenerative changes, and enhanced expression of inducible NO synthase in CA3 pyramidal neurons in the ventral hippocampus of stressed 12-month-old rats were observed. These findings can attest to the involvement NOdependent mechanisms and different contribution of NO synthase isoforms into the formation of hippocampal neuronal damage.

Voltage Imaging in the Study of Hippocampal Circuit Function and Plasticity.

Synaptic plasticity has the capacity to alter the function of neural circuits, and long-term potentiation (LTP) of synaptic transmission induced by high frequency electrical activity has the capacity to store information in neural circuits. The cellular and molecular mechanisms of LTP have been studied intensively for many years and much progress has been made on this front. By contrast, how synaptic plasticity alters circuit function has received much less attention and remains poorly understood. Voltage imaging provides a powerful general technique for the study of neural circuitry, and studies of synaptic plasticity with voltage imaging are beginning to reveal important aspects of how the function of a neural circuit can change when the strength of its synapses has been modified. The hippocampus has an important role in learning and memory and the plasticity of its synapses has received much attention. Voltage imaging with voltage sensitive dye in the CA1 region of a hippocampal slice has shown that spatial patterns of enhancement following LTP induction can diverge from the spatial patterns elicited by electrical stimulation, suggesting that LTP exhibits a distinct organizational structure. LTP can alter the throughput of electrical activity in the dentate gyrus of a hippocampal slice, to gate transmission on to the CA3 region. The spatial patterns evoked by complex electrical stimulation can be stored within the CA3 region in a hippocampal slice, allowing patterns to be reconstructed with simpler electrical stimulation. Thus, voltage imaging has demonstrated that the CA3 circuit has the capacity for pattern completion. These studies with voltage sensitive dye illustrate a range of interesting and novel questions that can be addressed at the population level. It is hoped that future imaging experiments with single-cell resolution using genetically-encoded voltage sensors will provide a more detailed picture of how synaptic plasticity modifies the information processing capabilities of neural circuits.

Two-Photon Correlation Spectroscopy in Single Dendritic Spines Reveals Fast Actin Filament Reorganization during Activity-Dependent Growth.

Two-photon fluorescence correlation spectroscopy (2P-FCS) within single dendritic spines of living hippocampal pyramidal neurons was used to resolve various subpopulations of mobile F-actin during activity-dependent structural changes such as potentiation induced spine head growth. Two major classes of mobile F-actin were discovered: very dynamic and about a hundred times less dynamic F-actin. Spine head enlargement upon application of Tetraethylammonium (TEA), a protocol previously used for the chemical induction of long-term potentiation (cLTP) strictly correlated to changes in the dynamics and filament numbers in the different actin filament fractions. Our observations suggest that spine enlargement is governed by a mechanism in which longer filaments are first cut into smaller filaments that cooperate with the second, increasingly dynamic shorter actin filament population to quickly reorganize and expand the actin cytoskeleton within the spine head. This process would allow a fast and efficient spine head enlargement using a major fraction of the actin filament population that was already present before spine head growth.

Orchidectomy does not significantly affect spine synapse density in the CA3 hippocampal subfield in St. Kitts vervet monkeys (Chlorocebus aethiops sabaeus).

Gonadal hormones induce significant changes in cognitive function, associated with alterations in the structure of the hippocampus. We have previously shown that androgens increase the number of spine synapses in the CA1 stratum radiatum of the monkey hippocampus. Recent evidence, however, suggests that loss of testicular hormone production may have variable effects on neuroplasticity in different regions of the hippocampus. To test this hypothesis, we examined the effects of orchidectomy in the dentate gyrus and CA3 subfield of the hippocampus in male St. Kitts vervet monkeys (Chlorocebus aethiops sabaeus). Spine synapse density was significantly reduced (39%) after orchidectomy in the dentate gyrus, consistent with previously published reports in CA1 (40%). However, in CA3 orchidectomy induced a much smaller (22%) reduction in synapse density, which did not reach the limits of statistical significance. These results suggest that orchidectomy exerts heterogeneous effects on hippocampal spine synapse density, the CA3 subfield being relatively spared compared to CA1 and the dentate gyrus. This heterogeneity may contribute to the mixed functional responses observed in males following loss of testicular hormone secretions.

Organelle and cellular abnormalities associated with hippocampal heterotopia in neonatal doublecortin knockout mice.

Heterotopic or aberrantly positioned cortical neurons are associated with epilepsy and intellectual disability. Various mouse models exist with forms of heterotopia, but the composition and state of cells developing in heterotopic bands has been little studied. Dcx knockout (KO) mice show hippocampal CA3 pyramidal cell lamination abnormalities, appearing from the age of E17.5, and mice suffer from spontaneous epilepsy. The Dcx KO CA3 region is organized in two distinct pyramidal cell layers, resembling a heterotopic situation, and exhibits hyperexcitability. Here, we characterized the abnormally organized cells in postnatal mouse brains. Electron microscopy confirmed that the Dcx KO CA3 layers at postnatal day (P) 0 are distinct and separated by an intermediate layer devoid of neuronal somata. We found that organization and cytoplasm content of pyramidal neurons in each layer were altered compared to wild type (WT) cells. Less regular nuclei and differences in mitochondria and Golgi apparatuses were identified. Each Dcx KO CA3 layer at P0 contained pyramidal neurons but also other closely apposed cells, displaying different morphologies. Quantitative PCR and immunodetections revealed increased numbers of oligodendrocyte precursor cells (OPCs) and interneurons in close proximity to Dcx KO pyramidal cells. Immunohistochemistry experiments also showed that caspase-3 dependent cell death was increased in the CA1 and CA3 regions of Dcx KO hippocampi at P2. Thus, unsuspected ultrastructural abnormalities and cellular heterogeneity may lead to abnormal neuronal function and survival in this model, which together may contribute to the development of hyperexcitability.

Ghrelin increases hippocampal recombination activating gene 1 expression and spatial memory performance in mice.

Ghrelin, which has been shown to improve memory retention, is an endogenous ligand for the growth hormone secretagogue receptor. Recombination activating gene 1 (Rag1), which plays a critical role in the development and maturation of lymphocytes in the immune system, is also expressed in the central nervous system, particularly in the hippocampus, and has been implicated in memory formation. In the current study, the effects of ghrelin on Rag1 expression in the hippocampus and spatial memory performance in wild-type and Rag1 knockout (KO) mice were examined. The Morris water maze test was used to compare the spatial memory performance of wild-type and Rag1-KO mice. Transmission electron microscopy was used to assess morphological changes in synaptic shape and numbers in hippocampal areas after peripheral administration of ghrelin to wild-type and Rag1-KO mice. In wild-type mice, ghrelin increased synaptic vesicles and postsynaptic membrane deposits in the hippocampal CA3 region, shortened water maze escape latencies, and increased platform spans. In Rag1-KO mice, the escape latencies were significantly increased compared with the group of normal mice. Compared with wild-type mice, Rag1-KO mice showed decreasing platform spans. A tendency toward a shortening of the escape latency times and an increase in the platform spans in Rag1-KO mice was observed after ghrelin injection, but this difference was not statistically significant compared with control mice. After the administration of ghrelin, the expression of Rag1 in the hippocampus was significantly increased compared with control mice. Our results suggest that ghrelin potentiates learning and memory in mice, which may be, at least partly, through the regulation of hippocampal Rag1.

Presynaptic ultrastructural plasticity along CA3→CA1 axons during long-term potentiation in mature hippocampus.

In area CA1 of the mature hippocampus, synaptogenesis occurs within 30 minutes after the induction of long-term potentiation (LTP); however, by 2 hours many small dendritic spines are lost, and those remaining have larger synapses. Little is known, however, about associated changes in presynaptic vesicles and axonal boutons. Axons in CA1 stratum radiatum were evaluated with 3D reconstructions from serial section electron microscopy at 30 minutes and 2 hours after induction of LTP by theta-burst stimulation (TBS). The frequency of axonal boutons with a single postsynaptic partner was decreased by 33% at 2 hours, corresponding perfectly to the 33% loss specifically of small dendritic spines (head diameters <0.45 µm). Docked vesicles were reduced at 30 minutes and then returned to control levels by 2 hours following induction of LTP. By 2 hours there were fewer small synaptic vesicles overall in the presynaptic vesicle pool. Clathrin-mediated endocytosis was used as a marker of local activity, and axonal boutons containing clathrin-coated pits showed a more pronounced decrease in presynaptic vesicles at both 30 minutes and 2 hours after induction of LTP relative to control values. Putative transport packets, identified as a cluster of less than 10 axonal vesicles occurring between synaptic boutons, were stable at 30 minutes but markedly reduced by 2 hours after the induction of LTP. APV blocked these effects, suggesting that the loss of axonal boutons and presynaptic vesicles was dependent on N-methyl-D-aspartic acid (NMDA) receptor activation during LTP. These findings show that specific presynaptic ultrastructural changes complement postsynaptic ultrastructural plasticity during LTP.

[Electrophysiological and morphohistochemical study of adrenalectomy on the hippocampal neurons].

Chronic insufficiency of adrenal hormones is a pathology leading to brain dysfunction. In electrophysiological studies, by extracellular recording of spike activity of single hippocampal neurons (HN) caused by high-frequency stimulation of the entorhinal cortex (EC) in rats with unilateral removal of adrenal (adrenalectomy - AE), we analyzed mechanisms of adaptation of neural networks to chronic hormonal deprivation. The balance of excitatory and inhibitory responses, recorded in HN of intact rats was submitted to characteristic changes in dynamics of development of neurodegeneration: the dominating in norm inhibitory responses were decreased at all AE terms (from 42 % to 25 % by the 18th week). On the contrary, the minimal in norm percent of excitatory responses sharply increased (from 17 % to 60 %) at the 25-27th day after AE, which indicates a possible increase of cholinergic neurotransmission. There was recorded a high level of the mean frequency of peristimulus spiking from the 25-27th day to the 18th week after AE, which indicates the high glutamate level or pronounced activation of NMDA receptors. On the whole, the ratio of excitatory/inhibitory HN responses indicates discoordinated activity of neuronal chains of EC-HN under conditions of AE. Histochemical analysis revealed an increased sensitivity to AE in neurons of the CA1 fields. After disturbance of neuronal structure by the 5th day, 25-27 days after AE, there was observed proliferation cell elements in the CA1 field; as a result, by the 10th week, there is observed the complete filling of "devastated" areas of hippocampus and a sharp rise of phosphatase activity in nuclei of dentate gyrus. 18 weeks after AE, the majority of the CA1 field neurons are submitted to chromatolysis, and the phosphatase activity falls. The presented data make certain contribution to understanding of mechanisms of control of cognitive brain functions and plasticity in interconnection with the hormonal factor.

Effects of maternal mild zinc deficiency and zinc supplementation in offspring on spatial memory and hippocampal neuronal ultrastructural changes.

OBJECTIVE: Knowledge about the hippocampal morphologic mechanisms of learning and memory for maternal mild zinc deficiency during pregnancy/lactation followed by zinc supplementation of pups after weaning is limited. This study examined the effects of zinc deficiency and zinc supplementation on cognition and hippocampal neurons. METHODS: One-day pregnant rats were randomly divided into four groups (n = 12): control (CO), pair-fed (PF), zinc-deprived (ZD), and oral zinc-supplemented (OZS). The CO and PF groups were fed a control diet (zinc 25 µg/g diet), and the others were fed a mildly zinc-deficient diet (zinc 2 µg/g diet) during pregnancy and lactation. After weaning (day 21), offspring in the OZS group were switched to a control diet. After 35 d, the behavioral function of the offspring was tested with the Morris water maze test. The ultrastructure of the hippocampal CA3 area was observed under a transmission electron microscope. RESULTS: Compared with the CO and PF groups, rats in the ZD group spent more time finding the latent platform and swam longer distances (P < 0.05). The time used finding the platform and the swimming distance in the OZS group were similar to those in the CO and PF groups (P > 0.05). In addition, apoptotic neuronal changes in the hippocampus were observed in the ZD group, whereas the reversal of neuronal morphologic changes was observed in the OZS group. CONCLUSION: The changes in hippocampal neuron morphology were consistent with the changes in the learning and memory ability of mildly zinc-deficient and zinc-supplemented offspring.