Lacunosum Moleculare Research Articles

Differential distribution of KCC2 along the axo-somato-dendritic axis of CA1 pyramidal cells

Event Abstract Back to Event Differential distribution of KCC2 along the axo-somato-dendritic axis of CA1 pyramidal cells Rita Baldi1*, Csaba Varga1 and Gabor Tamas1 1 Hungarian Academy of Sciences, University of Szeged, Hungary The neuron-specific K-Cl cotransporter (KCC2) plays a crucial role in adjusting intracellular chloride concentrations. Thus, the relative abundance of KCC2 along neuronal plasma membrane compartments can influence the reversal potential of chloride and GABAA receptor mediated synapses as shown for perisomatic and axo-axonic GABAergic synapses. We reported the lack of KCC2 in the axon compared to the soma in the cerebral cortex and extended the quantification of KCC2 to the dendrites of CA1 pyramidal cells with high resolution preembedding immunolocalization. Our results show that KCC2 has an uneven distribution profile along the axo-somato-dendritic axis of CA1 pyramidal neurons. Confirming our results on neocortical pyramidal cells, the axon initial segment contained ~ 5 % of somatic KCC2 cocentrations on CA1 pyramids. Compared to the proximal apical dendrites, the concentration of KCC2 decreased by ~ 37 % in the distal part of stratum radiatum (p<0.0001). Interestingly, the density of gold particles indicating KCC2 concentration was higher in lacunosum moleculare than in the distal part of stratum radiatum, but still ~ 22 % lower than in the proximal dendritic membranes (p<0.03). Basal dendrites showed KCC2 concentrations similar to proximal dendrites of str. radiatum and str. lacunosum moleculare and thus basal dendrites contained more KCC2 than dendrites in the distal str. radiatum. In conclusion, KCC2 is present in the soma as well as in the basal and apical dendritic shafts and spines of CA1 pyramidal cells. Moreover, the density of KCC2 seems to correlate with the relative abundance of GABAergic synapses innervating different segments of the apical dendrites in CA1 pyramidal cells. Conference: 12th Meeting of the Hungarian Neuroscience Society, Budapest, Hungary, 22 Jan - 24 Jan, 2009. Presentation Type: Poster Presentation Topic: Research on the cerebral cortex and related structures Citation: Baldi R, Varga C and Tamas G (2009). Differential distribution of KCC2 along the axo-somato-dendritic axis of CA1 pyramidal cells. Front. Syst. Neurosci. Conference Abstract: 12th Meeting of the Hungarian Neuroscience Society. doi: 10.3389/conf.neuro.01.2009.04.187 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 06 Mar 2009; Published Online: 06 Mar 2009. * Correspondence: Rita Baldi, Hungarian Academy of Sciences, University of Szeged, Szeged, Hungary, baldi.rita@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Rita Baldi Csaba Varga Gabor Tamas Google Rita Baldi Csaba Varga Gabor Tamas Google Scholar Rita Baldi Csaba Varga Gabor Tamas PubMed Rita Baldi Csaba Varga Gabor Tamas Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Bidirectional Hebbian Plasticity at Hippocampal Mossy Fiber Synapses on CA3 Interneurons

Hippocampal area CA3 is critically involved in the formation of nonoverlapping neuronal subpopulations ("pattern separation") to store memory representations as distinct events. Efficient pattern separation relies on the strong and sparse excitatory input from the mossy fibers (MFs) to pyramidal cells and feedforward inhibitory interneurons. However, MF synapses on CA3 pyramidal cells undergo long-term potentiation (LTP), which, if unopposed, will degrade pattern separation because MF activation will now recruit additional CA3 pyramidal cells. Here, we demonstrate MF LTP in stratum lacunosum-moleculare (L-M) interneurons induced by the same stimulation protocol that induces MF LTP in pyramidal cells. This LTP was NMDA receptor (NMDAR) independent and occurred at MF Ca(2+)-impermeable AMPA receptor synapses. LTP was prevented by with voltage clamping the postsynaptic cell soma during high-frequency stimulation (HFS), intracellular injections of the Ca(2+) chelator BAPTA (20 mm), or bath applications of the L-type Ca(2+) channel blocker nimodipine (10 microm). We propose that MF LTP in L-M interneurons preserves the sparsity of pyramidal cell activation, thus allowing CA3 to maintain its role in pattern separation. In the presence of the mGluR1alpha antagonist LY367385 [(S)-(+)-a-amino-4-carboxy-2-methylbenzeneacetic acid] (100 microm), the same HFS that induces MF LTP in naive slices triggered NMDAR-independent MF LTD. This LTD, like LTP, required activation of the L-type Ca(2+) channel and also was induced after blockade of IP(3) receptors with heparin (4 mg/ml) or the selective depletion of receptor-gated Ca(2+) stores with ryanodine (10 or 100 microm). We conclude that L-M interneurons are endowed with Ca(2+) signaling cascades suitable for controlling the polarity of MF long-term plasticity induced by joint presynaptic and postsynaptic activities.

Cuprizone treatment induces demyelination and astrocytosis in the mouse hippocampus

Memory impairment is outstanding within the spectrum of cognitive deficits in multiple sclerosis (MS) patients. Demyelination has been reported in the hippocampus formation of MS patients. The degree of hippocampus lesions in MS strongly correlates with progression of cognitive dysfunction. Because no appropriate animal model for the study of hippocampus demyelination has been established, we used the cuprizone mouse model to investigated demyelination in young adult and aged mice. The myelin status was analyzed by classical histological staining, immunocytochemistry for proteolipoprotein, and electron microscopy. Oligodendrocyte, astroglial, and microglia markers were studied. Cuprizone intoxication induced an almost complete demyelination of distinct hippocampus subregions to a similar extent in young adult and aged male mice. Demyelination was pronounced in a subset of white and gray matter areas, i.e., the stratum lacunosum moleculare containing the perforant path, medial alveus, stratum pyramidale in the cornu ammonis 2/3 region, and hilus region. Besides demyelination, affected areas displayed hypertrophic and hyperplastic astrocytosis. No significant effect on microglia invasion was detected at any investigated time point (0, 3, 5, and 7 weeks). We conclude that cuprizone-induced demyelination provides an adequate animal model to investigate appropriate therapy strategies for the prevention of hippocampus demyelination.

Developmental Sensitivity of Hippocampal Interneurons to Ethanol: Involvement of the Hyperpolarization-Activated Current, Ih

Ethanol (EtOH) has powerful effects on GABA(A) receptor-mediated neurotransmission, and we have previously shown that EtOH-induced enhancement of GABA(A) receptor-mediated synaptic transmission in the hippocampus is developmentally regulated. Because synaptic inhibition is determined in part by the firing properties of interneurons, we have investigated the mechanisms whereby EtOH influences the spontaneous firing characteristics and hyperpolarization-activated cation current (Ih) of hippocampal interneurons located in the near to the border of stratum lacunosum moleculare and s. radiatum of adolescent and adult rats. EtOH did not affect current injection-induced action potentials of interneurons that do not exhibit spontaneous firing. However, in neurons that fire spontaneously, EtOH enhanced the frequency of spontaneous action potentials (sAPs) in a concentration-dependent manner, an effect that was more pronounced in interneurons from adolescent rats, compared with adult rats. EtOH also modulated the afterhyperpolarization (AHP) that follows sAPs by shortening the tau(slow) decay time constant, and this effect was more pronounced in slices from adolescent rats. EtOH increased Ih amplitudes, accelerated Ih activation kinetics, and increased the maximal Ih conductance in interneurons from animals in both age groups. These effects were also more pronounced in interneurons from adolescents and persisted in the presence of glutamatergic and GABAergic blockers. However, EtOH failed to affect sAP firing in the presence of ZD7288 or cesium chloride. These results suggest that Ih may be of mechanistic significance in the effect of EtOH on interneuron spontaneous firing.

GABAB Receptor Modulation of Feedforward Inhibition through Hippocampal Neurogliaform Cells

Feedforward inhibition of neurons is a fundamental component of information flow control in the brain. We studied the roles played by neurogliaform cells (NGFCs) of stratum lacunosum moleculare of the hippocampus in providing feedforward inhibition to CA1 pyramidal cells. We recorded from synaptically coupled pairs of anatomically identified NGFCs and CA1 pyramidal cells and found that, strikingly, a single presynaptic action potential evoked a biphasic unitary IPSC (uIPSC), consisting of two distinct components mediated by GABA(A) and GABA(B) receptors. A GABA(B) receptor-mediated unitary response has not previously been observed in hippocampal excitatory neurons. The decay of the GABA(A) receptor-mediated response was slow (time constant = 50 ms), and was tightly regulated by presynaptic GABA(B) receptors. Surprisingly, the GABA(B) receptor ligands baclofen and (2S)-3-{[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl}(phenylmethyl)phosphinic acid (CGP55845), while affecting the NGFC-mediated uIPSCs, had no effect on action potential-evoked presynaptic Ca2+ signals monitored in individual axonal boutons of NGFCs with two-photon microscopy. In contrast, baclofen clearly depressed presynaptic Ca2+ transients in non-NGF interneurons. Changes in extracellular Ca2+ concentration that mimicked the effects of baclofen or CGP55845 on uIPSCs significantly altered presynaptic Ca2+ transients. Electrophysiological data suggest that GABA(B) receptors expressed by NGFCs contribute to the dynamic control of the excitatory input to CA1 pyramidal neurons from the temporoammonic path. The NGFC-CA1 pyramidal cell connection therefore provides a unique and subtle mechanism to shape the integration time domain for signals arriving via a major excitatory input to CA1 pyramidal cells.

Coincidence detection of convergent perforant path and mossy fibre inputs by CA3 interneurons

We performed whole-cell recordings from CA3 s. radiatum (R) and s. lacunosum-moleculare (L-M) interneurons in hippocampal slices to examine the temporal aspects of summation of converging perforant path (PP) and mossy fibre (MF) inputs. PP EPSPs were evoked from the s. lacunosum-moleculare in area CA1. MF EPSPs were evoked from the medial extent of the suprapyramidal blade of the dentate gyrus. Summation was strongly supralinear when examining PP EPSP with MF EPSP in a heterosynaptic pair at the 10 ms ISI, and linear to sublinear at longer ISIs. This pattern of nonlinearities suggests that R and L-M interneurons act as coincidence detectors for input from PP and MF. Summation at all ISIs was linear in voltage clamp mode demonstrating that nonlinearities were generated by postsynaptic voltage-dependent conductances. Supralinearity was not detected when the first EPSP in the pair was replaced by a simulated EPSP injected into the soma, suggesting that the conductances underlying the EPSP boosting were located in distal dendrites. Supralinearity was selectively eliminated with either Ni2+ (30 microm), mibefradil (10 microm) or nimodipine (15 microm), but was unaffected by QX-314. This pharmacological profile indicates that supralinearity is due to recruitment of dendritic T-type Ca2+channels by the first subthreshold EPSP in the pair. Results with the hyperpolarization-activated (Ih) channel blocker ZD 7288 (50 microm) revealed that Ih restricted the time course of supralinearity for coincidently summed EPSPs, and promoted linear to sublinear summation for asynchronous EPSPs. We conclude that coincidence detection results from the counterbalanced activation of T-type Ca2+ channels and inactivation of Ih.

Dendritic and axonal localization of cytotoxic T-lymphocyte antigen-2 alpha protein in mouse brain

Cytotoxic T-lymphocyte antigen-2 alpha (CTLA-2α) is a novel cysteine proteinase inhibitor protein originally discovered and expressed in mouse activated T-cells and mast cells. Expressed recombinant CTLA-2α is shown to exhibit selective inhibition of cathepsin L-like cysteine proteinases. We have recently reported the expression pattern of CTLA-2α mRNA in mouse brain by in situ hybridization, demonstrating that it is mainly enriched within neuronal populations. In this study we present the distribution profile of the protein by immunohistochemical analysis. Results showed that CTLA-2α protein is preferentially localized in dendritic and axonal compartments. In telencephalon, strong labeling was detected in dendrites in the cerebral cortices, stratum radiatum and stratum lacunosum moleculare and within axonal fibers of stratum lucidum where mossy fibers emanating from all parts of the granule cell layer of dentate gyrus terminate at pyramidal neurons and interneurons. In diencephalon, moderate staining was found in all thalamic nuclei but was strong in medial habenular nucleus and the hypothalamic nuclei including suprachiasmatic nucleus, optic chiasm, arcuate nucleus and median eminence. In mesencephalon, strong immunoreactivity was detected in superior colliculus, inferior colliculus and paramedian raphe nucleus. In the rhombencephalon, the pontine nucleus and transverse fibers of the pons revealed strong staining but were moderate in vestibular nuclei. Strong immunoreactivity was also observed in the internal white matter, granule cell layer and Purkinje cell layer within cerebellum. On Western blot analysis, a band of 14 kDa for CTLA-2α from protein extracts of the cerebrum, cerebellum, pons and medulla was detected. The distribution pattern and functional considerations of CTLA-2α in the brain are discussed.

Discordant localization of WFA reactivity and brevican/ADAMTS-derived fragment in rodent brain

BackgroundProteoglycan (PG) in the extracellular matrix (ECM) of the central nervous system (CNS) may act as a barrier for neurite elongation in a growth tract, and regulate other characteristics collectively defined as structural neural plasticity. Proteolytic cleavage of PGs appears to alter the environment to one favoring plasticity and growth. Brevican belongs to the lectican family of aggregating, chondroitin sulfate (CS)-bearing PGs, and it modulates neurite outgrowth and synaptogenesis. Several ADAMTSs (a disintegrin and metalloproteinase with thrombospondin motifs) are glutamyl-endopeptidases that proteolytically cleave brevican. The purpose of this study was to localize regions of adult CNS that contain a proteolytic-derived fragment of brevican which bears the ADAMTS-cleaved neoepitope sequence. These regions were compared to areas of Wisteria floribunda agglutin (WFA) reactivity, a common reagent used to detect "perineuronal nets" (PNNs) of intact matrix and a marker which is thought to label regions of relative neural stability.ResultsWFA reactivity was found primarily as PNNs, whereas brevican and the ADAMTS-cleaved fragment of brevican were more broadly distributed in neuropil, and in particular regions localized to PNNs. One example is hippocampus where the ADAMTS-cleaved brevican fragment is found surrounding pyramidal neurons, in neuropil of stratum oriens/radiatum and the lacunosum moleculare. The fragment was less abundant in the molecular layer of the dentate gyrus. Mostly PNNs of scattered interneurons along the pyramidal layer were identified by WFA. In lateral thalamus, the reticular thalamic nucleus stained abundantly with WFA whereas ventral posterior nuclei were markedly immunopositive for ADAMTS-cleaved brevican. Using Western blotting techniques, no common species were reactive for brevican and WFA.ConclusionIn general, a marked discordance was observed in the regional localization between WFA and brevican or the ADAMTS-derived N-terminal fragment of brevican. Functionally, this difference may correspond to regions with varied prevalence for neural stability/plasticity.

Specific inhibitory synapses shift the balance from feedforward to feedback inhibition of hippocampal CA1 pyramidal cells

Feedforward and feedback inhibition are two fundamental modes of operation widespread in the nervous system. We have functionally identified synaptic connections between rat CA1 hippocampal interneurons of the stratum oriens (SO) and interneurons of the stratum lacunosum moleculare (SLM), which can act as feedback and feedforward interneurons, respectively. The unitary inhibitory postsynaptic currents (uIPSCs) detected with K-gluconate-based patch solution at -50 mV had an amplitude of 20.0 +/- 2.0 pA, rise time 2.2 +/- 0.2 ms, decay time 25 +/- 2.2 ms, jitter 1.4 +/- 0.2 ms (average +/- SEM, n = 39), and were abolished by the gamma-aminobutyric acid (GABA)(A) receptor antagonist 2-(3-carboxypropyl)-3-amino-6-methoxyphenyl-pyridazinium bromide (SR 95531). Post hoc anatomical characterization revealed that all but one of the identified presynaptic neurons were oriens-lacunosum moleculare (O-LM) cells, whereas the postsynaptic neurons were highly heterogeneous, including neurogliaform (n = 4), basket (n = 4), Schaffer collateral-associated (n = 10) and perforant path-associated (n = 9) cells. We investigated the short-term plasticity expressed at these synapses, and found that stimulation at 10-40 Hz resulted in short-term depression of uIPSCs. This short-term plasticity was determined by presynaptic factors and was not target-cell specific. We found that the feedforward inhibition elicited by the direct cortical input including the perforant path onto CA1 pyramidal cells was modulated through the inhibitory synapses we have characterized. Our data show that the inhibitory synapses between interneurons of the SO and SLM shift the balance between feedback and feedforward inhibition onto CA1 pyramidal neurons.

Convergence of entorhinal and CA3 inputs onto pyramidal neurons and interneurons in hippocampal area CA1—An anatomical study in the rat

The entorhinal cortex (EC) conveys information to hippocampal field CA1 either directly by way of projections from principal neurons in layer III, or indirectly by axons from layer II via the dentate gyrus, CA3, and Schaffer collaterals. These two pathways differentially influence activity in CA1, yet conclusive evidence is lacking whether and to what extent they converge onto single CA1 neurons. Presently we studied such convergence. Different neuroanatomical tracers injected into layer III of EC and into CA3, respectively, tagged simultaneously the direct entorhino-hippocampal fibers and the indirect innervation of CA1 neurons by Schaffer collaterals. In slices of fixed brains we intracellularly filled CA1 pyramidal cells and interneurons in stratum lacunosum-moleculare (LM) and stratum radiatum (SR). Sections of these slices were scanned in a confocal laser scanning microscope. 3D-reconstruction was used to determine whether boutons of the labeled input fibers were in contact with the intracellularly filled neurons. We analyzed 12 pyramidal neurons and 21 interneurons. Perforant path innervation to pyramidal neurons in our material was observed to be denser than that from CA3. All pyramidal neurons and 17 of the interneurons received contacts of both perforant pathway and Schaffer input on their dendrites and cell bodies. Four interneurons, which were completely embedded in LM, received only labeled perforant pathway input. Thus, we found convergence of both projection systems on single CA1 pyramidal and interneurons with dendrites that access the layers where perforant pathway fibers and Schaffer collaterals end.

Activity-dependent Regulation of h Channel Distribution in Hippocampal CA1 Pyramidal Neurons

The hyperpolarization-activated cation current, I(h), plays an important role in regulating intrinsic neuronal excitability in the brain. In hippocampal pyramidal neurons, I(h) is mediated by h channels comprised primarily of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel subunits, HCN1 and HCN2. Pyramidal neuron h channels within hippocampal area CA1 are remarkably enriched in distal apical dendrites, and this unique distribution pattern is critical for regulating dendritic excitability. We utilized biochemical and immunohistochemical approaches in organotypic slice cultures to explore factors that control h channel localization in dendrites. We found that distal dendritic enrichment of HCN1 is first detectable at postnatal day 13, reaching maximal enrichment by the 3rd postnatal week. Interestingly we found that an intact entorhinal cortex, which projects to distal dendrites of CA1 but not area CA3, is critical for the establishment and maintenance of distal dendritic enrichment of HCN1. Moreover blockade of excitatory neurotransmission using tetrodotoxin, 6-cyano-7-nitroquinoxaline-2,3-dione, or 2-aminophosphonovalerate redistributed HCN1 evenly throughout the dendrite without significant changes in protein expression levels. Inhibition of calcium/calmodulin-dependent protein kinase II activity, but not p38 MAPK, also redistributed HCN1 in CA1 pyramidal neurons. We conclude that activation of ionotropic glutamate receptors by excitatory temporoammonic pathway projections from the entorhinal cortex establishes and maintains the distribution pattern of HCN1 in CA1 pyramidal neuron dendrites by activating calcium/calmodulin-dependent protein kinase II-mediated downstream signals.

GABAergic projections from the hippocampus to the retrosplenial cortex in the rat

The retrosplenial cortex (RS) in rats has been implicated in a wide range of behaviors, including spatial navigation and memory. Relevant to this, the RS is closely interconnected with the hippocampus by multiple direct and indirect routes. Here, by injecting the retrograde tracer cholera toxin subunit B conjugated with Alexa488 (CTB-Alexa488) in the granular retrosplenial cortex (GRS), we demonstrate a moderately dense non-pyramidal projection from CA1. Neurons are in several layers, but mainly (about 65%) at the border of the stratum radiatum (SR) and stratum lacunosum moleculare (SLM). In particular, by double-labeling with GAD67 or gamma-aminobutyric acid (GABA), we establish that these neurons are GABAergic. Further immunocytochemical screening for calcium-binding proteins, somatostatin (SS) or cholecystokinin (CCK) failed to identify additional neurochemical subgroups; but a small subset (about 14%) is positive for the m2 muscarinic acetylcholine receptor (M2R). Terminations target layer 1 of the GRS, as shown by biotinylated dextran amine (BDA) injections into CA1 and confirmed by a very superficial injection of CTB-Alexa488 in GRS. The superficial injection shows that there is a sparse GABAergic projection from the subiculum to layer 1 of the GRS, in addition to the dense excitatory connections to layer 3. The role of these dual inhibitory-excitatory pathways - within the subiculum, and in parallel from CA1 and the subiculum - remains to be determined, but may be related to synchronized oscillatory activity in the hippocampal complex and GRS, or to the generation of rhythmic activity within the GRS.

The CNS glycoprotein Shadoo has PrPC-like protective properties and displays reduced levels in prion infections

The cellular prion protein, PrPC, is neuroprotective in a number of settings and in particular prevents cerebellar degeneration mediated by CNS-expressed Doppel or internally deleted PrP (‘ΔPrP'). This paradigm has facilitated mapping of activity determinants in PrPC and implicated a cryptic PrPC-like protein, ‘π'. Shadoo (Sho) is a hypothetical GPI-anchored protein encoded by the Sprn gene, exhibiting homology and domain organization similar to the N-terminus of PrP. Here we demonstrate Sprn expression and Sho protein in the adult CNS. Sho expression overlaps PrPC, but is low in cerebellar granular neurons (CGNs) containing PrPC and high in PrPC-deficient dendritic processes. In Prnp0/0 CGNs, Sho transgenes were PrPC-like in their ability to counteract neurotoxic effects of either Doppel or ΔPrP. Additionally, prion-infected mice exhibit a dramatic reduction in endogenous Sho protein. Sho is a candidate for π, and since it engenders a PrPC-like neuroprotective activity, compromised neuroprotective activity resulting from reduced levels may exacerbate damage in prion infections. Sho may prove useful in deciphering several unresolved facets of prion biology.

Kv4.3-mediated A-type K+ currents underlie rhythmic activity in hippocampal interneurons.

Hippocampal-dependent learning and memory processes are associated with theta frequency rhythmic activity. Interneuron and pyramidal cell network interactions underlie this activity, but contributions of interneuron voltage-gated membrane conductances remain unclear. We show that interneurons at the CA1 lacunosum-moleculare (LM) and radiatum (RAD) junction (LM/RAD) display voltage-dependent subthreshold membrane potential oscillations (MPOs) generated by voltage-gated tetrodotoxin-sensitive Na+ and 4-aminopyridine (4-AP)-sensitive K+ currents. They also exhibit prominent 4-AP-sensitive A-type K+ currents, with gating properties showing activation at subthreshold membrane potentials. We found that LM/RAD cells are part of specific interneuron subpopulations expressing the K+ channel subunit Kv4.3 and their transfection with Kv4.3 small interfering RNA selectively impaired A-type K+ currents and MPOs. Thus, our findings reveal a novel function of Kv4.3-mediated A-type K+ currents in the generation of intrinsic MPOs in specific subpopulations of interneurons that may participate in hippocampal theta-related rhythmic activity.

Propagation of postsynaptic currents and potentials via gap junctions in GABAergic networks of the rat hippocampus

The integration of synaptic signalling in the mammalian hippocampus underlies higher cognitive functions such as learning and memory. We have studied the gap junction-mediated cell-to-cell and network propagation of GABA(A) receptor-mediated events in stratum lacunosum moleculare interneurons of the rat hippocampus. Propagated events were identified both in voltage- and current-clamp configurations. After blockade of ionotropic excitatory synaptic transmission, voltage-clamp recordings with chloride-loaded electrodes (predicted GABA(A) receptor reversal potential: 0 mV) at -15 mV revealed the unexpected presence of spontaneous events of opposite polarities. Inward events were larger and kinetically faster when compared to outward currents. Both types of events were blocked by gabazine, but only outward currents were significantly affected by the gap junction blocker carbenoxolone, indicating that outward events originated in electrically coupled neurons. These results were in agreement with computational modelling showing that propagated events were modulated in size and shape by their relative distance to the gap junction site. Paired recordings from electrically coupled interneurons performed with high- and low-chloride pipettes (predicted GABA(A) receptor reversal potentials: 0 mV and -80 mV, respectively) directly demonstrated that depolarizing postsynaptic events could propagate to the cell recorded with the low-chloride solution. Cell-to-cell propagation was abolished by carbenoxolone, and was not observed in uncoupled pairs. Application of 4-aminopyridine on slices resulted in spontaneous network activation of interneurons, which was driven by excitatory GABA(A) receptor-mediated input. Population activity was greatly depressed by carbenoxolone, suggesting that propagation of depolarizing synaptic GABAergic potentials may be a critical determinant of interneuronal synchronous bursting in the hippocampus.

Modulation of excitability in CA1 pyramidal neurons via the interplay of entorhinal cortex and CA3 inputs

Hippocampal CA1 pyramidal neurons receive extrahippocampal and intrahippocampal inputs. The Schaffer collateral (SC) pathway, projecting to the stratum radiatum of the CA1 field, serves as the primary excitatory input to CA1 cells. The temporoammonic pathway (TA), converging at stratum lacunosum moleculare (SLM), appears to be inhibitory. The relative temporal occurrence of the stimuli conveyed by the two pathways seems to modulate the excitability of CA1 pyramidal neurons. Specifically, the excitatory effect of SC inputs is greatly attenuated when preceded by TA stimulation within a specific time window. This phenomenon is referred to as the spike-blocking effect. Dvorak-Carbone and Schuman found that spike-blocking efficacy temporally coincide with the GABAb signalling pathway, while spike blocking was almost abolished in the presence of GABAb antagonist. In this work, we study the contribution of the GABAb receptor on the aforementioned phenomenon using a refined version of a previously published multicompartmental model of a CA1 pyramidal cell. We also investigate to what extend spike blocking is affected by the spatial arrangement of coincident synaptic inputs in the dendrites of the model neuron. We find that in addition to a temporal regulation, the arrangement of synaptic contacts provides a location-dependent modulation of excitability by the EC input. When synapses are scattered throughout the layered-model cell spike blocking is enhanced whereas spike blocking is reduced with clustered synaptic arrangement. We conclude that spike blocking may act as a mechanism for discriminating weak from strong synaptic inputs.

Difference in time course of modulation of synaptic transmission by group II versus group III metabotropic glutamate receptors in region CA1 of the hippocampus

We investigated the time course of modulation of synaptic transmission by group II and group III metabotropic glutamate receptors in region CA1 of the hippocampus. In the presence of 50 microM picrotoxin, pressure pulse application of 1 mM glutamate resulted in a fast onset of suppression of synaptic transmission in stratum lacunosum moleculare and a slower onset of suppression in stratum radiatum, with both effects returning to baseline over the course of several minutes. Application of 50 microM of the group II agonist (2R,4R)-APDC in stratum lacunosum moleculare resulted in the same fast onset of suppression while having no effect in stratum radiatum. Pressure pulse application of 100 microM DL-AP4 in stratum lacunosum moleculare and stratum radiatum resulted in a much slower onset of suppression of synaptic transmission than (2R,4R)-APDC. Suppression by (2R,4R)-APDC was accompanied by a rapid enhancement of paired pulse facilitation, indicative of a presynaptic mechanism. This demonstrates that activation of group II mGluRs in the hippocampus causes a fast onset of suppression in stratum lacunosum moleculare, while activation of group III mGluRs causes a slower onset of suppression. The difference in time course for group II vs. group III mGluRs suggests a different functional role, with group II playing a potential role in making synapses act as low pass filters.

Electroconvulsive seizure increases adult hippocampal angiogenesis in rats

Electroconvulsive seizure has a proven therapeutic application in the treatment of severe depression and treatment-resistant depression. Despite the efficacy of electroconvulsive seizure as a non-chemical antidepressant treatment, the mechanism of action is unclear. Elevation in hippocampal trophic factor expression and concomitant cellular proliferation are thought to play a role in its action. We examined whether the reported induction of angiogenic factors and endothelial cell proliferation leads to an increase in vascular density. Two hippocampal regions, the dentate gyrus and the stratum lacunosum moleculare (SLM), were examined employing a combination of vascular density quantification, angiogenic gene expression analysis and immunohistochemistry. A 6% increase in vascular density was observed in the dentate gyrus but this did not achieve statistical significance. The SLM of the hippocampus exhibited a robust 20-30% increase in vascular density and was accompanied by an increase in expression of inhibitor of differentiation-3. There was also an induction of the angiogenesis markers alphaVbeta3 integrin and Del1. Increases in the vascular density of the SLM could be in response to enhanced metabolic activity in this region. This is supported by the induction of glutamine synthetase and the glutamate transporter GLT1.

Structural insights from high-resolution diffusion tensor imaging and tractography of the isolated rat hippocampus

The hippocampus is a critical structure for learning and memory formation injured by diverse neuropathologies such as epilepsy or Alzheimer's disease. Recently, clinical investigations have attempted to use diffusion tensor MRI as a more specific surrogate marker for hippocampal damage. To first better understand the tissue architecture of healthy hippocampal regions, this study characterized 10 rat hippocampi with diffusion tensor imaging (DTI) at 50-μm in-plane image resolution using a 14.1-T magnet. Chemical fixation of the dissected and straightened rat hippocampus provided a simple, effective way to reduce partial volume effects when segmenting hippocampal regions and improved mean signal-to-noise per unit time (e.g. 50.6 ± 4.4 at b = 1250 s/mm 2 in 27 min). Contrary to previous reports that water diffusion is homogeneous throughout the nervous system, statistically different mean diffusivities were observed (e.g. 0.238 ± 0.054 and 0.318 ± 0.084 μm 2/ms for the molecular and granule cell layers respectively) (ANOVA, P < 0.05). Different hippocampal subregions had lower fractional anisotropy than uniformly fibrous structures like corpus callosum because of their complex architecture. DTI-derived color fiber orientation maps and tractography demonstrated most components of the trisynaptic intrahippocampal pathway (e.g. orientations in stratum lacunosum–moleculare were dominated by perforant and Schaffer fibers) and also permitted some assessment of connectivity in the rat hippocampus.

The Impact of Astrocytic Gap Junctional Coupling on Potassium Buffering in the Hippocampus

Astrocytic gap junctions have been suggested to contribute to spatial buffering of potassium in the brain. Direct evidence has been difficult to gather because of the lack of astrocyte-specific gap junction blockers. We obtained mice with coupling-deficient astrocytes by crossing conditional connexin43-deficient mice with connexin30(-/-) mice. Similar to wild-type astrocytes, genetically uncoupled hippocampal astrocytes displayed negative resting membrane potentials, time- and voltage-independent whole-cell currents, and typical astrocyte morphologies. Astrocyte densities were also unchanged. Using potassium-selective microelectrodes, we assessed changes in potassium buffering in hippocampal slices of mice with coupling-deficient astrocytes. We demonstrate that astrocytic gap junctions accelerate potassium clearance, limit potassium accumulation during synchronized neuronal firing, and aid in radial potassium relocation in the stratum lacunosum moleculare. Furthermore, slices of mice with coupling-deficient astrocytes displayed a reduced threshold for the generation of epileptiform events. However, it was evident that radial relocation of potassium in the stratum radiatum was not dependent on gap junctional coupling. We suggest that the perpendicular array of individual astrocytes in the stratum radiatum makes these cells ideally suited for spatial buffering of potassium released by pyramidal cells, independent of gap junctions. In general, a surprisingly large capacity for K+ clearance was conserved in mice with coupling-deficient astrocytes, indicating that gap junction-dependent processes only partially account for K+ buffering in the hippocampus.