RGS14 is a natural suppressor of both synaptic plasticity in CA2 neurons and hippocampal-based learning and memory

Learning and memory have been closely linked to strengthening of synaptic connections between neurons (i.e., synaptic plasticity) within the dentate gyrus (DG)–CA3–CA1 trisynaptic circuit of the hippocampus. Conspicuously absent from this circuit is area CA2, an intervening hippocampal region that is poorly understood. Schaffer collateral synapses on CA2 neurons are distinct from those on other hippocampal neurons in that they exhibit a perplexing lack of synaptic long-term potentiation (LTP). Here we demonstrate that the signaling protein RGS14 is highly enriched in CA2 pyramidal neurons and plays a role in suppression of both synaptic plasticity at these synapses and hippocampal-based learning and memory. RGS14 is a scaffolding protein that integrates G protein and H-Ras/ERK/MAP kinase signaling pathways, thereby making it well positioned to suppress plasticity in CA2 neurons. Supporting this idea, deletion of exons 2–7 of the RGS14 gene yields mice that lack RGS14 (RGS14-KO) and now express robust LTP at glutamatergic synapses in CA2 neurons with no impact on synaptic plasticity in CA1 neurons. Treatment of RGS14-deficient CA2 neurons with a specific MEK inhibitor blocked this LTP, suggesting a role for ERK/MAP kinase signaling pathways in this process. When tested behaviorally, RGS14-KO mice exhibited marked enhancement in spatial learning and in object recognition memory compared with their wild-type littermates, but showed no differences in their performance on tests of nonhippocampal-dependent behaviors. These results demonstrate that RGS14 is a key regulator of signaling pathways linking synaptic plasticity in CA2 pyramidal neurons to hippocampal-based learning and memory but distinct from the canonical DG–CA3–CA1 circuit.     

Interlamellar CA1 network in the hippocampus

To understand the cellular basis of learning and memory, the neurophysiology of the hippocampus has been largely examined in thin transverse slice preparations. However, the synaptic architecture along the longitudinal septo-temporal axis perpendicular to the transverse projections in CA1 is largely unknown, despite its potential significance for understanding the information processing carried out by the hippocampus. Here, using a battery of powerful techniques, including 3D digital holography and focal glutamate uncaging, voltage-sensitive dye, two-photon imaging, electrophysiology, and immunohistochemistry, we show that CA1 pyramidal neurons are connected to one another in an associational and well-organized fashion along the longitudinal axis of the hippocampus. Such CA1 longitudinal connections mediate reliable signal transfer among the pyramidal cells and express significant synaptic plasticity. These results illustrate a need to reconceptualize hippocampal CA1 network function to include not only processing in the transverse plane, but also operations made possible by the longitudinal network. Our data will thus provide an essential basis for future computational modeling studies on information processing operations carried out in the full 3D hippocampal network that underlies its complex cognitive functions.The hippocampus is widely used to study functional connectivity of the brain with the hope that principles that operate within its relatively simple architecture may be extended to more complex cortical structures. Its manageable number of cell types also provides an attractive opportunity to examine fundamental issues in neuroscience such as the relationship between network circuitry and function. For example, considerable effort has been devoted toward elucidating the circuitry supporting episodic memory—a property closely linked to the hippocampus. CA3 pyramidal neurons form extensive recurrent connections with each other (1). Such connections are able to learn to associate components of an input pattern with each other (2), which, in turn, has greatly influenced thinking on the mechanisms of memory formation and recall (3). Under appropriate conditions, computer simulations reveal that recurrent neural networks have the capacity to learn temporal sequences and to carry out pattern completion (4, 5). Interestingly, although they are quite near to the CA3 region, CA1 pyramidal neurons reportedly form remarkably few associational connections (6, 7). This distinctive difference in network architecture might suggest that, although area CA1 could serve to decode the output of CA3, it would not possess the intrinsic ability for autoassociational computations. This idea would imply that the ability of CA1 to carry out independent information processing operations may be more limited than that of CA3. However, even after removal of all input from area CA3, CA1 pyramidal neurons still have the capacity to transform location-modulated signals from the entorhinal cortex into accurate spatial firing patterns (8). In addition, deficits in temporal sequence learning are more severe after selective lesions to CA1 than to CA3 (9). Finally, CA1 is more closely linked to memory of temporal order of visual objects and especially over long intervals (10). Thus, area CA1 appears to have a greater ability for intrinsic information processing than would be expected based on current understanding of its circuitry. Intrinsic processing could represent autoassociational computations through direct excitatory synaptic contacts among the CA1 pyramidal cells, but as noted, there is little evidence for such connectivity within CA1. This puzzle led us to reexamine the apparent sparseness of associational synaptic connections between CA1 pyramidal neurons using experimental techniques that were not previously available for this investigation.The “trisynaptic circuits” (dentate gyrus: CA3–CA1) oriented transversely to the hippocampal long axis, the basis of the “lamellar hypothesis” (11), has greatly influenced thinking about the structure-function relationships of this structure. This hypothesis suggests that the hippocampus is organized as a stack of parallel, trisynaptic circuits. Although this view has been challenged by the observation of fibers running across lamellae, especially in dentate gyrus and CA3 area (12, 13), the hypothesis supported an explosion in the use of the transverse slice for electrophysiological studies of the hippocampus. However, axons oriented along the longitudinal axis are unavoidably severed in the preparation of the transverse slice, meaning that these studies are heavily weighted in favor of conclusions based on fibers traveling within the transverse plane. We used a whole hippocampus preparation, as well as longitudinal and transverse slice preparations, to obtain a more accurate picture of synaptic connections among CA1 pyramidal neurons in three dimensions. Remarkably, we found prominent associational connectivity along the longitudinal axis. Furthermore, synapses of the longitudinal network possess the capacity for synaptic plasticity that includes a novel memory mechanism we recently described called dendritic hold and read (DHR) (14). These findings may help to explain the intrinsic ability of area CA1 to process information transfer and provide novel data that will lead to more realistic models of hippocampal function in three dimensions.     

Age-related changes of NGF, BDNF, parvalbumin and neuronal nitric oxide synthase immunoreactivity in the mouse hippocampal CA1 sector

We investigated the age-related alterations in nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), parvalbumin and neuronal nitric oxide synthase (nNOS) immunoreactivity of the mouse hippocampal CA1 sector. NGF and BDNF immunoreactivity was unchanged in the hippocampal CA1 pyramidal neurons from 2 to 50-59 weeks of birth. In contrast, a significant increase in the NGF and BDNF immunoreactivity was observed in glial cells of the hippocampal CA1 sector from 40-42 to 50-59 weeks of birth. On the other hand, the number of parvalbumin- and nNOS-positive interneurons was unchanged in the hippocampal CA1 sector during aging processes, except for a significant decrease of nNOS-positive interneurons 2 weeks of birth. Our results indicate that NGF and BDNF immunoreactivity was unaltered in the hippocampal CA1 pyramidal neurons during aging processes. In contrast, a significant increase in the NGF and BDNF immunoreactivity was observed in glial cells of the hippocampal CA1 sector during aging processes. The present study also shows that the number of parvalbumin- and nNOS-positive interneurons was unchanged in the hippocampal CA1 sector during aging processes, except for a significant decrease of nNOS-positive interneurons 2 weeks of birth. These results demonstrate that the expression of glial NGF and BDNF may play a key role for helping survival and maintenance of pyramidal neurons and neuronal functions in the hippocampal CA1 sector during aging processes. Furthermore, our findings suggest that parvalbumin- and nNOS-positive interneurons in the hippocampal CA1 sector are resistant to aging processes. Moreover, our findings suggest that nitric oxide synthesized by the nNOS may play some role for neuronal growth during postnatal development.     

Differential responses of hippocampal subfields to cortical up-down states

The connectivity of the hippocampal trisynaptic circuit, formed by the dentate gyrus, the CA3 and the CA1 region, is well characterized anatomically and functionally in vitro. The functional connectivity of this circuit in vivo remains to be understood. Toward this goal, we investigated the influence of the spontaneous, synchronized oscillations in the neocortical local field potential, reflecting up-down states (UDS) of cortical neurons, on the hippocampus. We simultaneously measured the extracellular local field potential in association cortex and the membrane potential of identified hippocampal excitatory neurons in anesthetized mice. Dentate gyrus granule cells showed clear UDS modulation that was phase locked to cortical UDS with a short delay. In contrast, CA3 pyramidal neurons showed mixed UDS modulation, such that some cells were depolarized during the cortical up state and others were hyperpolarized. CA1 pyramidal neurons, located farther downstream, showed consistent UDS modulation, such that when the cortical and dentate gyrus neurons were depolarized, the CA1 pyramidal cells were hyperpolarized. These results demonstrate the differential functional connectivity between neocortex and hippocampal subfields during UDS oscillations.     

3’-甲氧基葛根素抗大鼠脑缺血再灌注损伤作用

目的探讨3’-甲氧基葛根素（3＇-MOP）在大鼠脑缺血再灌损伤中的神经保护作用。方法24只大鼠随机分为脑缺血再灌组（IR组）、IR＋3’-MOP组和对照组。HE染色观察海马CA1区神经元的组织形态学变化;TUNEL染色检测海马CA1区神经元阳性凋亡细胞数。结果IR＋3＇-MOP组海马CA1区神经元的损伤减轻,细胞存活数显著多于IR组,而凋亡细胞数显著少于IR组。结论3＇-MOP可经抑制凋亡的发生,减轻海马CA1区神经元缺血再灌损伤。     

Cyclooxygenase-2 inhibition prevents delayed death of CA1 hippocampal neurons following global ischemia

The inducible isoform of the enzyme cyclooxygenase-2 (COX2) is an immediate early gene induced by synaptic activity in the brain. COX2 activity is an important mediator of inflammation, but it is not known whether COX2 activity is pathogenic in brain. To study the role of COX2 activity in ischemic injury in brain, expression of COX2 mRNA and protein and the effect of treatment with a COX2 inhibitor on neuronal survival in a rat model of global ischemia were determined. Expression of both COX2 mRNA and protein was increased after ischemia in CA1 hippocampal neurons before their death. There was increased survival of CA1 neurons in rats treated with the COX2-selective inhibitor SC58125 {1-[(4-methylsulfonyl) phenyl]-3-trifluoro-methyl-5-[(4-fluoro)phenyl] pyrazole} before or after global ischemia compared with vehicle controls. Furthermore, hippocampal prostaglandin E₂ concentrations 24 h after global ischemia were decreased in drug-treated animals compared with vehicle-treated controls. These results suggest that COX2 activity contributes to CA1 neuronal death after global ischemia.     

The PIDDosome mediates delayed death of hippocampal CA1 neurons after transient global cerebral ischemia in rats

A brief period of global brain ischemia, such as that induced by cardiac arrest or cardiopulmonary bypass surgery, causes cell death in vulnerable hippocampal CA1 pyramidal neurons days after reperfusion. Although numerous factors have been suggested to account for this phenomenon, the mechanisms underlying it are poorly understood. We describe a cell death signal called the PIDDosome, a protein complex of p53-induced protein with a death domain (PIDD), receptor-interacting protein-associated ICH-1/CED-3 homologous protein with a death domain (RAIDD), and procaspase-2. We induced 5 min of transient global cerebral ischemia (tGCI) using bilateral common carotid artery occlusion with hypotension. Western blot analysis showed that expression of twice-cleaved fragment of PIDD (PIDD-CC) increased in the cytosolic fraction of the hippocampal CA1 subregion and preceded procaspase-2 activation after tGCI. Caspase-2 cleaved Bid in brain homogenates. Co-immunoprecipitation and immunofluorescent studies demonstrated that PIDD-CC, RAIDD, and procaspase-2 were co-localized and bound directly, which indicates the formation of the PIDD death domain complex. Furthermore, we tested inhibition of PIDD expression by using small interfering RNA (siRNA) treatment that was initiated 48 h before tGCI. Administration of siRNA against PIDD decreased not only expression of PIDD-CC, but also activation of procaspase-2 and Bid, resulting in a decrease in histological neuronal damage and DNA fragmentation in the hippocampal CA1 subregion after tGCI. These results imply that PIDD plays an important role in procaspase-2 activation and delayed CA1 neuronal death after tGCI. We propose that PIDD is a hypothetical molecular target for therapy against neuronal death after tGCI.     

Gamma oscillations dynamically couple hippocampal CA3 and CA1 regions during memory task performance

The hippocampal formation is believed to be critical for the encoding, consolidation, and retrieval of episodic memories. Yet, how these processes are supported by the anatomically diverse hippocampal networks is still unknown. To examine this issue, we tested rats in a hippocampus-dependent delayed spatial alternation task on a modified T maze while simultaneously recording local field potentials from dendritic and somatic layers of the dentate gyrus, CA3, and CA1 regions by using high-density, 96-site silicon probes. Both the power and coherence of gamma oscillations exhibited layer-specific changes during task performance. Peak increases in the gamma power and coherence were found in the CA3-CA1 interface on the maze segment approaching the T junction, independent of motor aspects of task performance. These results show that hippocampal networks can be dynamically coupled by gamma oscillations according to specific behavioral demands. Based on these findings, we propose that gamma oscillations may serve as a physiological mechanism by which CA3 output can coordinate CA1 activity to support retrieval of hippocampus-dependent memories.     

Photostimulation using caged glutamate reveals functional circuitry in living brain slices.

An approach for high-spatial-resolution mapping of functional circuitry in living mammalian brain slices has been developed. The locations of neurons making functional synaptic connections to a single neuron are revealed by photostimulation of highly restricted areas of the slice (50-100 microns in diameter) while maintaining a whole-cell recording of the neuron of interest. Photostimulation is achieved by bathing brain slices in a molecularly caged form of the neurotransmitter glutamate [L-glutamic acid alpha-(4,5-dimethoxy-2-nitrobenzyl) ester], which is then converted to the active form by brief pulses (< 1 ms in duration) of ultraviolet irradiation. Direct activation of receptors on recorded neurons in rat hippocampus and ferret visual cortex demonstrates that photostimulation is reliable and reproducible and can be repeated at the same site at least 30 times without obvious decrement in neuronal responsiveness. Photostimulation of presynaptic neurons at sites distant to the recorded neuron evoked synaptic responses in hippocampal and cortical cells at distances of up to several millimeters from the recorded neuron. Stimulation of 25-100 distinct presynaptic sites while recording from a single postsynaptic neuron was easily achieved. Caged glutamate-based photostimulation eliminates artifacts and limitations inherent in conventional stimulation methods, including stimulation of axons of passage, desensitization, and poor temporal resolution of "puffer" pipettes, and current artifacts of iontophoretic application. This approach allows detailed physiological investigation and manipulation of the complex intrinsic circuitry of the mammalian brain.     

Enhancement of CA3 hippocampal network activity by activation of group II metabotropic glutamate receptors

Impaired function or expression of group II metabotropic glutamate receptors (mGluRIIs) is observed in brain disorders such as schizophrenia. This class of receptor is thought to modulate activity of neuronal circuits primarily by inhibiting neurotransmitter release. Here, we characterize a postsynaptic excitatory response mediated by somato-dendritic mGluRIIs in hippocampal CA3 pyramidal cells and in stratum oriens interneurons. The specific mGluRII agonists DCG-IV or LCCG-1 induced an inward current blocked by the mGluRII antagonist LY341495. Experiments with transgenic mice revealed a significant reduction of the inward current in mGluR3(-/-) but not in mGluR2(-/-) mice. The excitatory response was associated with periods of synchronized activity at theta frequency. Furthermore, cholinergically induced network oscillations exhibited decreased frequency when mGluRIIs were blocked. Thus, our data indicate that hippocampal responses are modulated not only by presynaptic mGluRIIs that reduce glutamate release but also by postsynaptic mGluRIIs that depolarize neurons and enhance CA3 network activity.     

Remodeling of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor subunit composition in hippocampal neurons after global ischemia

Transient global ischemia induces selective delayed cell death, primarily of principal neurons in the hippocampal CA1. However, the molecular mechanisms underlying ischemia-induced cell death are as yet unclear. The present study shows that global ischemia triggers a pronounced and cell-specific reduction in GluR2 [the subunit that limits Ca(2+) permeability of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors] in vulnerable CA1 neurons, as evidenced by immunofluorescence of brain sections and Western blot analysis of microdissected hippocampal subfields. At 72 h after ischemia (a time before cell death), virtually all CA1 pyramidal neurons exhibited greatly reduced GluR2 immunolabeling throughout their somata and dendritic processes. GluR2 immunolabeling was unchanged in pyramidal cells of the CA3 and granule cells of the dentate gyrus, regions resistant to ischemia-induced damage. Immunolabeling of the AMPA receptor subunit GluR1 was unchanged in CA1, CA3, and dentate gyrus. Western analysis indicated that GluR2 subunit abundance was markedly reduced in CA1 at 60 and 72 h after the ischemic insult; GluR1 abundance was unchanged in all subfields at all times examined. These findings, together with the previous observation of enhanced AMPA-elicited Ca(2+) influx in postischemic CA1 neurons, show that functional GluR2-lacking, Ca(2+)-permeable AMPA receptors are expressed in vulnerable neurons before cell death. Thus, the present study provides an important link in the postulated causal chain between global ischemia and delayed death of CA1 pyramidal neurons.     

Active summation of excitatory postsynaptic potentials in hippocampal CA3 pyramidal neurons

The manner in which the thousands of synaptic inputs received by a pyramidal neuron are summed is critical both to our understanding of the computations that may be performed by single neurons and of the codes used by neurons to transmit information. Recent work on pyramidal cell dendrites has shown that subthreshold synaptic inputs are modulated by voltage-dependent channels, raising the possibility that summation of synaptic responses is influenced by the active properties of dendrites. Here, we use somatic and dendritic whole-cell recordings to show that pyramidal cells in hippocampal area CA3 sum distal and proximal excitatory postsynaptic potentials sublinearly and actively, that the degree of nonlinearity depends on the magnitude and timing of the excitatory postsynaptic potentials, and that blockade of transient potassium channels linearizes summation. Nonlinear summation of synaptic inputs could have important implications for the computations performed by single neurons and also for the role of the mossy fiber and perforant path inputs to hippocampal area CA3.     

Melatonin inhibits outward delayed rectifier potassium currents in hippocampal CA1 pyramidal neuron via intracellular indole-related domains

In the present study, we investigated the effect of melatonin on the outward delayed rectifier potassium currents (IK) in CA1 pyramidal neurons of rat hippocampal slices using patch-clamp technique in whole-cell configuration. In a concentration-dependent manner, melatonin caused a reduction of IK with a half-maximal inhibitory concentration (IC50) of 3.75 mm. The inhibitory effect had rapid onset and was readily reversible. Melatonin shifted steady-state inactivation of IK in hyperpolarizing direction but did not alter its steady-state activation. Neither luzindole, an MT1/MT2 receptor antagonist, nor prazosin, an MT3 receptor antagonist, blocked melatonin-induced current reduction. The results indicate that melatonin-induced IK inhibition was not via activation of its own membrane receptors. 5-Hydroxytryptamine (5-HT), a melatonin precursor and an agonist of serotonin receptors, when it was given in pipette internal solution but not bath solution, produced a similar inhibitory effect to that of melatonin. Moreover, indole, a major component of melatonin, reversibly and dose dependently inhibited IK with an IC50 of 3.44 mm. Present results suggest that melatonin inhibits IK in hippocampal CA1 pyramidal neurons probably through its interaction with the intracellular indole-related domains of potassium channels.     

Electrophysiological evidence for a hyperpolarizing, galanin (1-15)-selective receptor on hippocampal CA3 pyramidal neurons

The effects of the 29-amino acid neuropeptide galanin [GAL (1-29)], GAL(1-15), GAL(1-16), and the GAL subtype 2 receptor agonist D-tryptophan(2)-GAL(1-29) were studied in the dorsal hippocampus in vitro with intracellular recording techniques. GAL(1-15) induced, in the presence of tetrodotoxin, a dose-dependent hyperpolarization in hippocampal CA3 neurons. Most of the GAL(1-15)-sensitive neurons did not respond to GAL(1-29), GAL(1-16), or D-tryptophan(2)-GAL(1-29). These results indicate the presence of a distinct, yet-to-be cloned GAL(1-15)-selective receptor on CA3 neurons in the dorsal hippocampus.     

Characterization of the CA1 pyramidal neurons in rat model of hepatic cirrhosis: insights into their electrophysiological properties

Although the key contributors of altering neurological function in hepatic encephalopathy are relatively well known, the electrophysiological mechanism of CA1 damage, a key vulnerable area during hyperammonemia, have not yet been defined. Therefore, here we focus on the electrophysiological mechanisms of cognitive impairments following bile duct ligation (BDL). We performed patch-clamp recordings from the CA1 pyramidal neurons in hippocampus of male Wistar rats, which underwent sham or BDL surgery. A striking electrophysiological change of hippocampal neurons in experimental model of BDL was observed in the present study. Spontaneous firing frequency and rate of action potential (AP) rebound was decreased and afterhyperpolarization amplitude (AHP) was increased significantly in hippocampal cells of BDL animals compared to sham group. Together, the results suggest that altered intrinsic properties of the hippocampal neurons may contribute to the cognitive abnormalities during hepatic encephalopathy (HE), highlighting the electrophysiological mechanisms for providing new treatments against HE.

     

Ultrastructural morphometric analysis of lipofuscin in pyramidal cells of the human Ammon's horn

Lipofuscin is a waste product of autolysosomal metabolism. The amount of lipofuscin in the cytoplasm depends on cell type, cell function and age. In most studies, either the fluorescent or the stained component of lipofuscin was investigated. Quantitative morphological investigations of the lipofuscin composition separated into the vacuolar and granular component were missing. For the hippocampal pyramidal cells we have determined the lipofuscin quantity and, separately, the vacuolar and granular component at the ultrastructural level. The hippocampal subfields CA 1, CA 2, CA 3 and CA 4 were observed at the ages 20, 40, 60 and 80 years (+/-3 years). Quantitative determinations of the vacuolar and granular component of neuronal lipofuscin in pyramidal cells were performed with a semi-automatic image analysis system. In CA 1 pyramidal cells the lipofuscin content was significantly lower than in the other sectors, which did not differ significantly in their lipofuscin content. The amount of the granular component in relation to the vacuolar component in CA 1 was larger than in the other sectors. With advancing age the lipofuscin content per cell increased. The vacuolar component of all hippocampal subfields experienced a larger increase than the granular component. Consequently the relation of the vacuolar and granular component changed; the relative amount of the vacuolar component increased, while that of the granular component decreased with age. The differences between sector CA 1 and the other hippocampal subfields were discussed with reference to differences of metabolic and functional activity of the neurons. Cytoprotective factors like Calbindin D28k were discussed for CA 1.     

CA1 neurons in the human hippocampus are critical for autobiographical memory, mental time travel, and autonoetic consciousness

Autobiographical memories in our lives are critically dependent on temporal lobe structures. However, the contribution of CA1 neurons in the human hippocampus to the retrieval of episodic autobiographical memory remains elusive. In patients with a rare acute transient global amnesia, highly focal lesions confined to the CA1 field of the hippocampus can be detected on MRI. We studied the effect of these lesions on autobiographical memory using a detailed autobiographical interview including the remember/know procedure. In 14 of 16 patients, focal lesions in the CA1 sector of the hippocampal cornu ammonis were detected. Autobiographical memory was significantly affected over all time periods, including memory for remote periods. Impairment of episodic memory and autonoetic consciousness exhibited a strong temporal gradient extending 30 to 40 y into the past. These results highlight the distinct and critical role of human hippocampal CA1 neurons in autobiographical memory retrieval and for re-experiencing detailed episodic memories.     

Operative GABAergic inhibition in hippocampal CA1 pyramidal neurons in experimental epilepsy

Patch–clamp recordings of CA1 interneurons and pyramidal cells were performed in hippocampal slices from kainate- or pilocarpine-treated rat models of temporal lobe epilepsy. We report that γ-aminobutyric acid (GABA)ergic inhibition in pyramidal neurons is still functional in temporal lobe epilepsy because: (i) the frequency of spontaneous GABAergic currents is similar to that of control and (ii) focal electrical stimulation of interneurons evokes a hyperpolarization that prevents the generation of action potentials. In paired recordings of interneurons and pyramidal cells, synchronous interictal activities were recorded. Furthermore, large network-driven GABAergic inhibitory postsynaptic currents were present in pyramidal cells during interictal discharges. The duration of these interictal discharges was increased by the GABA type A antagonist bicuculline. We conclude that GABAergic inhibition is still present and functional in these experimental models and that the principal defect of inhibition does not lie in a complete disconnection of GABAergic interneurons from their glutamatergic inputs.