IQGAP1 Expression in Spared CA1 Neurons After an Excitotoxic Lesion in the Mouse Hippocampus

Repeated seizures induce permanent alterations in the hippocampal circuits in experimental models with intractable temporal lobe epilepsy. Sprouting and synaptic reorganization induced by seizures has been well-studied in the mossy fiber pathway. However, studies investigating sprouting and synaptic reorganization beyond the mossy fiber pathway are limited. The present study examined the biochemical changes of CA1 pyramidal neurons undergoing morphological changes after excitotoxicity-induced hippocampal CA3 neuronal death. IQ-domain GTPase-activating proteins (IQGAP1), is an effector of Rac1 and Cdc42 and an actin-binding protein, was upregulated in CA1 pyramidal neurons after kainic acid-induced hippocampal CA3 neuronal degeneration. IQGAP1 + cells were colocalized with Nestin, but not in astrocytes or mature neurons. Furthermore, IQGAP1 did not originate from newly divided local precursors or NG2 + cells. IQGAP1 and adenomatous polyposis coli localized in CA1 pyramidal neurons, and Cdc42 activation was followed by IQGAP1 recruitment. These findings suggest that IQGAP1 is upregulated in pre-existed sparing neurons of the CA1 layer undergoing morphological changes after excitoxicity-induced hippocampal CA3 neuronal death. It demonstrates the utility of IQGAP1 as a possible marker for spared pyramidal neurons, which may contribute to structural and functional alternations responsible for the development of epilepsy.     

Temporal interaction between single spikes and complex spike bursts in hippocampal pyramidal cells

Cortical pyramidal cells fire single spikes and complex spike bursts. However, neither the conditions necessary for triggering complex spikes, nor their computational function are well understood. CA1 pyramidal cell burst activity was examined in behaving rats. The fraction of bursts was not reliably higher in place field centers, but rather in places where discharge frequency was 6-7 Hz. Burst probability was lower and bursts were shorter after recent spiking activity than after prolonged periods of silence (100 ms-1 s). Burst initiation probability and burst length were correlated with extracellular spike amplitude and with intracellular action potential rising slope. We suggest that bursts may function as "conditional synchrony detectors," signaling strong afferent synchrony after neuronal silence, and that single spikes triggered by a weak input may suppress bursts evoked by a subsequent strong input.     

Integration and segregation of activity in entorhinal-hippocampal subregions by neocortical slow oscillations

Brain systems communicate by means of neuronal oscillations at multiple temporal and spatial scales. In anesthetized rats, we find that neocortical "slow" oscillation engages neurons in prefrontal, somatosensory, entorhinal, and subicular cortices into synchronous transitions between UP and DOWN states, with a corresponding bimodal distribution of their membrane potential. The membrane potential of hippocampal granule cells and CA3 and CA1 pyramidal cells lacked bimodality, yet it was influenced by the slow oscillation in a region-specific manner. Furthermore, in both anesthetized and naturally sleeping rats, the cortical UP states resulted in increased activity of dentate and most CA1 neurons, as well as the highest probability of ripple events. Yet, the CA3-CA1 network could self-organize into gamma bursts and occasional ripples during the DOWN state. Thus, neo/paleocortical and hippocampal networks periodically reset, self-organize, and temporally coordinate their cell assemblies via the slow oscillation.     

Ionic mechanisms underlying spontaneous CA1 neuronal firing in Ca2+-free solution

Hippocampal CA1 neurons exposed to zero-[Ca(2+)] solutions can generate periodic spontaneous synchronized activity in the absence of synaptic function. Experiments using hippocampal slices showed that, after exposure to zero-[Ca(2+)](0) solution, CA1 pyramidal cells depolarized 5-10 mV and started firing spontaneous action potentials. Spontaneous single neuron activity appeared in singlets or was grouped into bursts of two or three action potentials. A 16-compartment, 23-variable cable model of a CA1 pyramidal neuron was developed to study mechanisms of spontaneous neuronal bursting in a calcium-free extracellular solution. In the model, five active currents (a fast sodium current, a persistent sodium current, an A-type transient potassium current, a delayed rectifier potassium current, and a muscarinic potassium current) are included in the somatic compartment. The model simulates the spontaneous bursting behavior of neurons in calcium-free solutions. The mechanisms underlying several aspects of bursting are studied, including the generation of triplet bursts, spike duration, burst termination, after-depolarization behavior, and the prolonged inactive period between bursts. We show that the small persistent sodium current can play a key role in spontaneous CA1 activity in zero-calcium solutions. In particular, it is necessary for the generation of an after-depolarizing potential and prolongs both individual bursts and the interburst interval.     

豚鼠背侧海马锥体细胞自发放电特征

实验采用细胞外玻璃微电极采集豚鼠海马神经元放电信号,并将信号转化为峰峰间期(interspike interval,ISI)以研究麻醉和清醒状态海马锥体细胞自发放电线性和非线性特点。实验建立了豚鼠海马锥体细胞与中间神经元电生理鉴别标准;麻醉和清醒状态下豚鼠海马CA1和CA3区锥体细胞自发放电频率、时程、复杂度等无显著区别;麻醉组豚鼠海马锥体细胞ISI序列的复杂度小于清醒组,锥体细胞分型和ISI变异度等表现不同。实验表明,麻醉和清醒状态下豚鼠海马锥体细胞自发放电呈不同线性和非线性特征。传统和非线性研究手段的结合,可能较全面地反映海马锥体细胞自发放电特性。     

Hippocampal ripple oscillations (200 Hz) in mechanisms of memory consolidation

A current status of knowledge about high-frequency (140-200 Hz) ripple oscillations in the CA1 hippocampal subfield is summarized and considered in the context of two-stage model of the hippocampal memory processing. A large body of evidence suggests highly-selective recruitment of pyramidal cells and interneurons in the generation of the oscillatory pattern after co-operative sharp-wave-related discharge of CA3 pyramidal neurons. We also discuss a role of transmission via gap junctions in the mechanisms of ripple oscillations as well as their adaptive aminergic (histaminergic) modulation. Patterns of neuronal firing in the hippocampus observed during ripple oscillations reproduce space-dependant neuronal activity from the previous waking period. Together with a data about efficacy of high-frequency stimulation for induction of synaptic modification it points out a role for ripples in the formation of long-term memory. Focal ultra fast ripples (up to 500 Hz) have been shown to participate in the development of temporal lobe epilepsy.     

In vivo neuroprotective role of NMDA receptors against kainate-induced excitotoxicity in murine hippocampal pyramidal neurons

Activation of NMDA receptors has been shown to induce either neuronal cell death or neuroprotection against excitotoxicity in cultured cerebellar granule neurons in vitro. We have investigated the effects of pretreatment with NMDA on kainate-induced neuronal cell death in mouse hippocampus in vivo. The systemic administration of kainate (30 mg/kg), but not NMDA (100 mg/kg), induced severe damage in pyramidal neurons of the hippocampal CA1 and CA3 subfields 3-7 days later, without affecting granule neurons in the dentate gyrus. An immunohistochemical study using an anti-single-stranded DNA antibody and TdT-mediated dUTP nick end labeling analysis both revealed that kainate, but not NMDA, induced DNA fragmentation in the CA1 and CA3 pyramidal neurons 1-3 days after administration. Kainate-induced neuronal loss was completely prevented by the systemic administration of NMDA (100 mg/kg) 1 h to 1 day previously. No pyramidal neuron was seen with fragmented DNA in the hippocampus of animals injected with kainate 1 day after NMDA treatment. The neuroprotection mediated by NMDA was prevented by the non-competitive NMDA receptor antagonist MK-801. Taken together these results indicate that in vivo activation of NMDA receptors is capable of protecting against kainate-induced neuronal damage through blockade of DNA fragmentation in murine hippocampus.     

Inhibitory processes of hippocampal CA1 pyramidal neurons following kindling-induced epilepsy in the rat

To determine the alterations in cellular function which may contribute to the chronic predisposition of neuronal tissue to epileptiform activity, the membrane properties and inhibitory processes of hippocampal CA1 pyramidal cells were investigated using in vitro slices prepared from commissural-kindled rats. No changes were observed in resting membrane potential, input resistance, spike amplitude, and membrane time constant of "kindled" CA1 pyramidal neurons when compared with controls. There were also no differences between control and kindled preparations in the amplitude of recurrent inhibitory postsynaptic potentials (IPSP) and in the duration of inhibition produced by either alvear (Alv) or stratum radiatum (SR) stimulation. Irrespective of group, repetitive stimulation of the Alv reduced the amplitude of the recurrent IPSP but failed to induce seizurelike activity. On the other hand, repetitive stimulation of SR frequently produced a neuronal burst discharge even though the duration and to some extent the amplitude of orthodromic inhibition was increased. On the basis of these data, it may be suggested that chronic changes in CA1 pyramidal cell membrane properties and transient reductions of inhibitory processes do not underlie the enhanced sensitivity of these neurons to seizure activity associated with kindling.     

Neurophysiological changes associated with selective neuronal damage in hippocampus following transient forebrain ischemia.

Neurophysiological changes of hippocampal neurons were compared before and after transient forebrain ischemia using intracellular recording and staining techniques in vivo. Ischemic depolarization (ID) was used as an indication of severe ischemia. Under halothane anesthesia, approximately 13 min of ID consistently produced severe neuronal damage in the CA1 region of rat hippocampus, while CA3 pyramidal neurons and dentate granule cells remained intact. After such severe ischemia, approximately 60% of the CA1 neurons exhibited a synaptic potentiation. The excitability of these neurons progressively decreased following reperfusion. Approximately 30% of the CA1 neurons showed a synaptic depression following ischemia. The excitability of these neurons transiently decreased following reperfusion. After ischemia of the same severity, both synaptic transmission and excitability of CA3 and granule cells transiently depressed. These data suggest that ischemia-induced synaptic potentiation may be associated with the pathogenesis of neuronal damage following ischemia, and that the synaptic depression may have protective effects on hippocampal neurons after ischemic insult.     

Immunocytochemical Localization of TASK-3 Protein (K2P9.1) in the Rat Brain

Among all K2P channels, TASK-3 shows the most widespread expression in rat brain, regulating neuronal excitability and transmitter release. Using a recently purified and characterized polyclonal monospecific antibody against TASK-3, the entire rat brain was immunocytochemically analyzed for expression of TASK-3 protein. Besides its well-known strong expression in motoneurons and monoaminergic and cholinergic neurons, TASK-3 expression was found in most neurons throughout the brain. However, it was not detected in certain neuronal populations, and neuropil staining was restricted to few areas. Also, it was absent in adult glial cells. In hypothalamic areas, TASK-3 was particularly strongly expressed in the supraoptic and suprachiasmatic nuclei, whereas other hypothalamic nuclei showed lower protein levels. Immunostaining of hippocampal CA1 and CA3 pyramidal neurons showed strongest expression, together with clear staining of CA3 mossy fibers and marked staining also in the dentate gyrus granule cells. In neocortical areas, most neurons expressed TASK-3 with a somatodendritic localization, most obvious in layer V pyramidal neurons. In the cerebellum, TASK-3 protein was found mainly in neurons and neuropil of the granular cell layer, whereas Purkinje cells were only faintly positive. Particularly weak expression was demonstrated in the forebrain. This report provides a comprehensive overview of TASK-3 protein expression in the rat brain.     

Comparison of the Immunoreactivity of Trx2/Prx3 Redox System in the Hippocampal CA1 Region Between the Young and Adult Gerbil Induced by Transient Cerebral Ischemia

In the present study, we compared the immunoreactivities and levels of Trx/prx redox system, thioredoxin 2 (Trx2), thioredoxin reductase 2 (TrxR2) and peroxiredoxin 3 (Prx3), as well as neuronal death in the hippocampal CA1 region between the adult and young gerbil after 5 min of transient cerebral ischemia. At 4 days post-ischemia, pyramidal neurons (about 90%) in the adult stratum pyramidale of the CA1 region showed "delayed neuronal death (DND)"; however, at this time point, few pyramidal neurons showed DND in the young stratum pyramidale. At 7 days post-ischemia, about 56% of pyramidal neurons showed DND in the young stratum pyramidale. The immunoreactivities of all the antioxidants in the young sham-group were similar to those in the adult sham-group. At 4 days post-ischemia, the immunoreactivity of TrxR2, not Trx2 and Prx3 in the adult ischemia-group was dramatically decreased in CA1 pyramidal neurons. At this time point, the immunoreactivities of all the antioxidants in the young ischemia-group were apparently increased compared to the adult ischemia-group. From 7 days pots-ischemia, non-pyramidal cells showed the immunoreactivities of all the antioxidants in the ischemic CA1 region; however, in the young ischemia-groups, the immunoreactivities were much lower than those in the adult ischemia-groups. In brief, our results showed that the immunoreactivities of Trx2, TrxR2 and Prx3 were dramatically increased in CA1 pyramidal neurons of the young ischemia-groups at 4 days post-ischemia compared to those in the adult ischemia-groups induced by transient cerebral ischemia.     

Network properties of memory trace formation in the hippocampus.

Based on the experimental evidence from his laboratory and the relevant literature the Author outlines a formal model of memory trace formation. During exploratory (theta) behaviors the neocortical information is transmitted to the hippocampus via the fast-firing granule cells which may induce a weak and transient heterosynaptic potentiation in a subgroup of CA3 pyramidal cells. The weakly potentiated CA3 neurons will then initiate population bursts upon the termination of exploratory activity (sharp wave state). It is assumed that recurrent excitation during the population burst is strongest on those cells which initiated the population event. It is suggested that the strong excitatory drive brought about by the sharp wave-concurrent population bursts during consummatory behaviors, immobility, and slow wave sleep may be sufficient for the induction of long-term synaptic modification in the initiator neurons of the CA3 region and in their targets in CA1. In this two-stage model both exploratory (theta) and sharp wave states of the hippocampus are essential and any interference that might modify the structure of the population bursts (e.g., epileptic spikes) are detrimental to memory trace formation.     

Mapping of rat hippocampal neurons with NeuN after ischemia/reperfusion and Ginkgo biloba extract (EGb 761) pretreatment

1. The neuroprotective effect of Ginkgo biloba extract (EGb 761) against transient forebrain ischemia following 7 days of reperfusion was studied in male Wistar rats after four-vessel occlusion for 20 min.2. NeuN, a neuronal specific nuclear protein was used for immunohistochemical detection of surviving pyramidal neurons in the hippocampus, as well as counterstaining with hematoxylin in the same sections for detection of neurons that underwent delayed neuronal death and for glial nuclei staining. GFAP immunohistochemistry was used for detection of astrocytes in the studied area of CA1 region.3. In the group of rats pretreated 7 days with Ginkgo biloba extract (EGb 761), following 20 min of ischemia and 7 days of reperfusion without EGb 761, increased number of NeuN immunoreactive cells were counted in the most vulnerable CA1 pyramidal layer of hippocampus. On the other hand, the group of rats with 7 days of EGb 761 pretreatment following 20 min of ischemia and 7 days of reperfusion with EGb 761 showed decreased number of surviving NeuN immunoreactive CA1 pyramidal cells in comparison with the first above-mentioned experimental group.4. Increased number of reactive astrocytes immunolabeled for GFAP (Glial fibrilary acidic protein) was observed in both experimental groups in the stratum oriens and stratum lacunosum and moleculare.5. Twenty minutes of ischemia is lethal for most population of CA1 pyramidal cell layer. Our results showed that prophylactic oral administration of Ginkgo biloba extract (EGb 761) in the dose 40 mg/kg/day during the 7 days protects the most vulnerable CA1 pyramidal cells against 20 min of ischemia.     

Activity dynamics and behavioral correlates of CA3 and CA1 hippocampal pyramidal neurons

The CA3 and CA1 pyramidal neurons are the major principal cell types of the hippocampus proper. The strongly recurrent collateral system of CA3 cells and the largely parallel-organized CA1 neurons suggest that these regions perform distinct computations. However, a comprehensive comparison between CA1 and CA3 pyramidal cells in terms of firing properties, network dynamics, and behavioral correlations is sparse in the intact animal. We performed large-scale recordings in the dorsal hippocampus of rats to quantify the similarities and differences between CA1 (n > 3,600) and CA3 (n > 2,200) pyramidal cells during sleep and exploration in multiple environments. CA1 and CA3 neurons differed significantly in firing rates, spike burst propensity, spike entrainment by the theta rhythm, and other aspects of spiking dynamics in a brain state-dependent manner. A smaller proportion of CA3 than CA1 cells displayed prominent place fields, but place fields of CA3 neurons were more compact, more stable, and carried more spatial information per spike than those of CA1 pyramidal cells. Several other features of the two cell types were specific to the testing environment. CA3 neurons showed less pronounced phase precession and a weaker position versus spike-phase relationship than CA1 cells. Our findings suggest that these distinct activity dynamics of CA1 and CA3 pyramidal cells support their distinct computational roles.     

High expression of GLT-1 in hippocampal CA3 and dentate gyrus subfields contributes to their inherent resistance to ischemia in rats

It is well known that neurons in the CA3 and dentate gyrus (DG) subfields of the hippocampus are resistant to short period of ischemia which is usually lethal to pyramidal neurons in hippocampal CA1 subfield. The present study was undertaken to clarify whether the inherent higher resistance of neurons in CA3 and DG to ischemia is associated with glial glutamate transporter-1 (GLT-1) in rats. Western blot analysis and immunohistochemistry assay showed that the basal expressions of GLT-1 in both CA3 and DG were much higher than that in CA1 subfield. Mild global brain ischemia for 8 min induced delayed death of almost all CA1 pyramidal neurons and marked GLT-1 down-regulation in the CA1 subfield, but it was not lethal to the neurons in either CA3 or DG and induced GLT-1 up-regulation and astrocyte activation showed normal soma and aplenty slender processes in the both areas. When the global brain ischemia was prolonged to 25 min, neuronal death was clearly observed in CA3 and DG accompanied with down-regulation of GLT-1 expression and abnormal astrocytes represented with hypertrophic somas, but shortened processes. After down-regulating of GLT-1 expression and function by its antisense oligodeoxynucleotides or inhibiting GLT-1 function by dihydrokainate, an inhibitor of GLT-1, the mild global brain ischemia for 8 min, which usually was not lethal to CA3 and DG neurons, induced the neuronal death in CA3 and DG subfields. Taken together, the higher expression of GLT-1 in the CA3 and DG contributes to their inherent resistance to ischemia.     

Expression and changes of endogenous insulin-like growth factor-1 in neurons and glia in the gerbil hippocampus and dentate gyrus after ischemic insult

In the present study, we focused upon expression and changes of endogenous insulin-like growth factor-1 (IGF-1) in the hippocampus of the Mongolian gerbil after ischemic insult. In sham-operated animals, IGF-1 immunoreactivity was absent from the hippocampus. IGF-1-immunoreactive (IR) neurons were detected at 12 h and 1 day after ischemic insult. In the hippocampal CA1 area, the IGF-IR neurons were non-pyramidal cells (GABAergic neurons). In the hippocampal CA2/3 areas, the IGF-1-IR neurons were pyramidal and non-pyramidal cells, and in the dentate gyrus the IGF-1-IR neurons were hilar neurons. Four days after ischemia-reperfusion, IGF-1 immunoreactivity disappeared from neurons, and significantly increased in astrocytes and microglia. These results suggest that the induction of IGF-1 in the CA1 area during the early stage (12-24 h after ischemic insult) is associated with the relative vulnerabilities of pyramidal glutamatergic neurons and non-pyramidal GABAergic neurons. The later increase (4 days after ischemic insult) of IGF-1 expression and protein content was found to promote the activities of astrocytes and microglia. These increases of IGF-1 in astrocytes and in microglia are associated with mechanisms that compensate for the effects of delayed neuronal death.     

Regional and cellular distribution of neural visinin-like protein immunoreactivities (VILIP-1 and VILIP-3) in human brain

Neural visinin-like proteins (VILIPs) are members of the neuronal subfamily of intracellular EF-hand calcium sensor proteins termed the NCS family, which are thought to play important roles in cellular signal transduction. While numerous studies suggest a wide but uneven distribution of these proteins in rat and chicken brain, their location in, and possible significance for, the human brain, remains to be established. We used specific polyclonal antisera to map the human brain for VILIP-1 and VILIP-3 immunoreactivities. VILIP-1 was detected in cortical pyramidal cells and interneurons, septal, subthalamic and hippocampal neurons (subfields CA1 and CA4 pyramidal cells and especially hilar interneurons) as well as in cerebellar Golgi, basket, granule, stellate and dentate nucleus neurons. Purkinje cells were free of immunoreaction. VILIP-3 was more restricted in its distribution. It was identified in cerebellar Purkinje cells and a subpopulation of granule neurons. Further, neurons belonging to different nuclei of the brain stem and multiple subcortical nerve cells stained for visinin-like protein 3. A weak immunoreaction appeared in cortical and hippocampal neurons. Intracellularly the immunoreactivity appeared in the perikarya, dendrites and some axons. Sometimes, immunostaining was found in the neuropil. Glia did not express visinin-like proteins. Our findings support, from a neuroanatomical viewpoint, the idea that these calcium sensor proteins may be of relevance for neuronal signalling in the human CNS.     

Glutamate release from astrocytes as a non-synaptic mechanism for neuronal synchronization in the hippocampus.

Synchronization of activity of anatomically distributed groups of neurons represents a fundamental event in the processing of information in the brain. While this phenomenon is believed to result from dynamic interactions within the neuronal circuitry, how exactly populations of neurons become synchronized remains largely to be clarified. We propose that astrocytes are directly involved in the generation of neuronal synchrony in the hippocampus. By using a combination of experimental approaches in hippocampal slice preparations, including patch-clamp recordings and confocal microscopy calcium imaging, we studied the effect on CA1 pyramidal neurons of glutamate released from astrocytes upon various stimuli that trigger Ca2+ elevations in these glial cells, including Schaffer collateral stimulation. We found that astrocytic glutamate evokes synchronous, slow inward currents (SICs) and Ca2+ elevations in CA1 pyramidal neurons by acting preferentially, if not exclusively, on extrasynaptic NMDA receptors. Due to desensitization, AMPA receptors were not activated by astrocytic glutamate unless cyclothiazide was present. In the virtual absence of extracellular Mg2+, glutamate released from astrocytes was found to evoke, in paired recordings, highly synchronous SICs from two CA1 pyramidal neurons and, in Ca2+ imaging experiments, Ca2+ elevations that occurred synchronously in domains composed of 2-12 CA1 neurons. In the presence of extracellular Mg2+ (1 mM), synchronous SICs in two neurons as well as synchronous Ca2+ elevations in neuronal domains were still observed, although with a reduced frequency. Our results reveal a functional link between astrocytic glutamate and extrasynaptic NMDA receptors that contributes to the overall dynamics of neuronal synchrony. Our observations also raise a series of questions on possible roles of this astrocyte-to-neuron signaling in pathological changes in the hippocampus such as excitotoxic neuronal damage or the generation of epileptiform activity.     

Scale-Free Topology of the CA3 Hippocampal Network: A Novel Method to Analyze Functional Neuronal Assemblies

Cognitive mapping functions of the hippocampus critically depend on the recurrent network of the CA3 pyramidal cells. However, it is still not known in detail how network activity patterns emerge, or how they encode information. By using functional multineuron calcium imaging, we simultaneously recorded the activity of >100 neurons in the CA3 region of hippocampal slice cultures. We utilized a novel computational method to analyze the multichannel spike trains and to depict functional neuronal assemblies. By means of event synchronization and the correlation matrix analysis method, we found that: 1), the average functional neuronal cluster consists of 23 neurons, and neurons could be part of multiple assemblies; 2), the clustering strength, size, and mean distance among cells in neuronal assemblies follow a power-law-like distribution; 3), the clustering strength and size of neuronal assemblies are not correlated with the total number of neurons and their physical distance; and 4), the clustering distance of neuronal assemblies is weakly correlated with the total number of neurons and their physical distance. These findings suggest that the functional organization of the spontaneously firing CA3 hippocampal network is a scale-free structure in slice culture.