[Waiting time to transcatheter aortic valve implantation (TAVI) and mortality during wait period in Algeria]. / Délais d'attente d'implantation de valve aortique transcutanée (TAVI) et mortalité durant la période d'attente en Algérie.

BACKGROUND: Trans Aortic Valve Implantation (TAVI) has become the primary treatment for aortic stenosis in patients over 75 years old. Despite its clinical efficacy, it's adoption in emerging countries remains low due to the high cost of prostheses and limited healthcare funding resources. This leads to prolonged waiting times for the TAVI procedure, which may lead to complications; these data are missing particularly in emerging countries. AIMS: To describe waiting time for TAVI and mortality rate in this waiting period. MATERIALS AND METHODS: This was prospective registry, patients referred for TAVI were prospectively followed; waiting time was calculated from the first visit after referral to TAVI implantation, clinical and, call fellow up was performed every 3 months. We divided patients into two groups: Group 1 (G1) patients still awaiting TAVI (105 patients), and those who underwent TAVI (36 patients). Group 2 (G2) patients who died while awaiting TAVI (16 patients, 10,2 %). RESULTS: Demographic characteristics were similar, with a tendency for older age in G2 (79.5 ± 5.7 years vs. 82.5 ± 7.4 years, p=0,06). G2 exhibited more left ventricular ejection fraction (LVEF) impairment (8.5% vs. 25%, p=0,03) and a higher rate of severe heart failure with dyspnea stages III or IV (2.8% vs. 12.5%, p<0,001). The mean follow-up in G1 was 242.9 ± 137.4 days; the waiting time for TAVI was 231.7 ± 134.1 days, and the average time between the first consultation and death while awaiting TAVI (G2) was 335.1 ± 167.4 days. CONCLUSION: in our series, waiting time is high due to limited Trans aortic heart valve availability, mortality during this wait exceeds 10%. Adverse prognostic factors include impaired LVEF and severe dyspnea stages III or IV.

Intercellular calcium signaling between astrocytes and oligodendrocytes via gap junctions in culture.

To understand further how oligodendrocytes regulate brain function, the mechanism of communication between oligodendrocytes and other cell types needs to be explored. An important mode of communication between various cell types in the nervous system involves gap junctions. Astroglial cells are extensively connected through gap junctions forming the glial syncytium. Although the presence of gap junctions between oligodendrocytes and astrocytes have been well documented, evidence for gap junction-mediated calcium transfer between these two glial populations is still missing. To measure functional coupling between astrocytes and oligodendrocytes and to test whether this coupling is mediated by gap junctions we used laser photostimulation and monitored Ca(2+) propagation in cultures from transgenic animals in which oligodendrocytes express enhanced green fluorescent protein (eGFP). We show that waves of Ca(2+) spread from astrocytes to oligodendrocytes and that these waves are blocked by the broad-spectrum gap junction blocker carbenoxolone, but not the neuron-specific gap junction blocker quinine. We also show that the spread of Ca(2+) waves between astrocytes and oligodendrocytes is bi-directional. Thus, increase of Ca(2+) concentration in astrocytes triggered by surrounding neuronal activity may feed back onto different neuronal populations through oligodendrocytes.

Activity-dependent layer-specific changes in the extracellular chloride concentration and chloride driving force in the rat hippocampus.

The transmembrane distribution of chloride anions (Cl⁻) determines the direction of the Cl⁻ flux through GABA(A) receptors; this establishes whether GABA(A) receptor-mediated responses are hyperpolarizing or depolarizing in neurons. Thus an activity-dependent reduction in the efficacy of inhibitory responses can be the result of an activity-induced reduction of the Cl⁻ driving force. Using Cl(-)-sensitive electrodes, we measured the extracellular Cl⁻ concentration ([Cl⁻](o)) in each layer of the hippocampus under control conditions and after stimulation. In the control condition, [Cl⁻](o) was lower within the CA1 region (112.9 ± 1.3 mM; mean ± SD) than the CA3/dentate gyrus areas (117.7 ± 1.2 mM). Stimulation of CA3 pyramidal cells led to an increase in the [Cl⁻](o). The maximum values were observed in the stratum lacunosum-moleculare (253.4 ± 51.1 mM) and in the hilus (261 ± 43.7 mM), whereas in the granular cell layer, it reached only 159.5 ± 41 mM. The stimulation-induced [Cl⁻](o) increase was followed by a period of decreasing [Cl⁻](o) that fell below the control values. The maximum undershoot (21.6 ± 0.7 mM) was observed in the s. radiatum. Systemic application of the gap junction blocker carbenoxolone significantly decreased the stimulation-induced Cl⁻ extrusion in the dentate gyrus but only slightly modified it in the CA1 area. Carbenoxolone also drastically reduced the Cl⁻ clearance. The time constant of the Cl⁻ clearance was similar between layers (83.4 ± 15.9 ms) but increased after carbenoxolone application (207.1 ± 44.4 ms). Stimulation-induced changes in the [Cl⁻](o) significantly decreased the Cl⁻ driving force and resulted in large fluctuations between layers (Δ = 9.4 mV). The lowest value was observed in the stratum radiatum of the CA1 and the hilar region (7.7 mV), whereas the highest value was calculated for the granule cell layer (16.3 mV). We suggest that a decrease of the extracellular space is mainly responsible for the rapid [Cl⁻](o) increase while the gap junction coupled astrocytic network plays a key role in the activity-dependent redistribution and clearance of Cl⁻ across layers of the hippocampus.

Automated scoring of novel object recognition in rats.

The object recognition task (ORT) has become increasingly popular as a memory test in neuroscience research. Scoring of ORT performance is still mostly done by hand, which can be liable to subjective scoring. To our knowledge, no suited software is available yet since the direction of the nose of the animal cannot be tracked reliably. We have developed a software paradigm that reliably tracks the nose of the rats and have conducted a series of experiments to evaluate the reliability of this newly developed program. We used Wistar rats, which showed good object memory after 1h interval. Subsequently, we used scopolamine (SCOP) to impair the memory performance of the rats. The object exploration was scored by two observers and the automated system. Both observers and the automated system found an impairing drug effect of scopolamine on ORT performance. When using large objects the correlation between the discrimination index d2 of observers was: 0.60 (SCOP) and 0.79 (SAL). However, the correlation between observers and the automated system was quite low: 0.41 (SCOP) and 0.40 (SAL). Reducing the size of the objects increased the reliability between observers and the automated system substantially (0.82-0.87). We conclude that the use of small objects in combination with our program enables reliable automated scoring in the ORT, thus increasing the objectivity and validity of this task.

Transition to seizures in the isolated immature mouse hippocampus: a switch from dominant phasic inhibition to dominant phasic excitation.

The neural dynamics and mechanisms responsible for the transition from the interictal to the ictal state (seizures) are unresolved questions in epilepsy. It has been suggested that a shift from inhibitory to excitatory GABAergic drive can promote seizure generation. In this study, we utilized an experimental model of temporal lobe epilepsy which produces recurrent seizure-like events in the isolated immature mouse hippocampus (P8-16), perfused with low magnesium ACSF, to investigate the cellular dynamics of seizure transition. Whole-cell and perforated patch recordings from CA1 pyramidal cells and from fast- and non-fast-spiking interneurons in the CA1 stratum oriens hippocampal region showed a change in intracellular signal integration during the transition period, starting with dominant phasic inhibitory synaptic input, followed by dominant phasic excitation prior to a seizure. Efflux of bicarbonate ions through the GABA A receptor did not fully account for this excitation and GABAergic excitation via reversed IPSPs was also excluded as the prime mechanism generating the dominant excitation, since somatic and dendritic GABA A responses to externally applied muscimol remained hyperpolarizing throughout the transition period. In addition, abolishing EPSPs in a single neuron by intracellularly injected QX222, revealed that inhibitory synaptic drive was maintained throughout the entire transition period. We suggest that rather than a major shift from inhibitory to excitatory GABAergic drive prior to seizure onset, there is a change in the interaction between afferent synaptic inhibition, and afferent and intrinsic excitatory processes in pyramidal neurons and interneurons, with maintained inhibition and increasing, entrained 'overpowering' excitation during the transition to seizure.

Expression pattern of voltage-dependent calcium channel subunits in hippocampal inhibitory neurons in mice.

Different subtypes of voltage-dependent calcium channels (VDCCs) generate various types of calcium currents that play important role in neurotransmitter release, membrane excitability, calcium transients and gene expression. Well-established differences in the physiological properties and variable sensitivity of hippocampal GABAergic inhibitory neurons to excitotoxic insults suggest that the calcium homeostasis, thus VDCC subunits expression pattern is likely different in subclasses of inhibitory cells. Using double-immunohistochemistry, here we report that in mice: 1) Cav2.1 and Cav3.1 subunits are expressed in almost all inhibitory neurons; 2) subunits responsible for the L-type calcium current (Cav1.2 and Cav1.3) are infrequently co-localized with calretinin inhibitory cell marker while Cav1.3 subunit, at least in part, tends to compensate for the low expression of Cav1.2 subunit in parvalbumin-, metabotropic glutamate receptor 1alpha- and somatostatin-immunopositive inhibitory neurons; 3) Cav2.2 subunit is expressed in the majority of inhibitory neurons except in calbindin-reactive inhibitory cells; 4) Cav2.3 subunit is expressed in the vast majority of the inhibitory cells except in parvalbumin- and calretinin-immunoreactive neurons where the proportion of expression of this subunit is considerably lower. These data indicate that VDCC subunits are differentially expressed in hippocampal GABAergic interneurons, which could explain the diversity in their electrophysiological properties, the existence of synaptic plasticity in certain inhibitory neurons and their vulnerability to stressful stimuli.

Connexin-47 and connexin-32 in gap junctions of oligodendrocyte somata, myelin sheaths, paranodal loops and Schmidt-Lanterman incisures: implications for ionic homeostasis and potassium siphoning.

The subcellular distributions and co-associations of the gap junction-forming proteins connexin 47 and connexin 32 were investigated in oligodendrocytes of adult mouse and rat CNS. By confocal immunofluorescence light microscopy, abundant connexin 47 was co-localized with astrocytic connexin 43 on oligodendrocyte somata, and along myelinated fibers, whereas connexin 32 without connexin 47 was co-localized with contactin-associated protein (caspr) in paranodes. By thin-section transmission electron microscopy, connexin 47 immunolabeling was on the oligodendrocyte side of gap junctions between oligodendrocyte somata and astrocytes. By freeze-fracture replica immunogold labeling, large gap junctions between oligodendrocyte somata and astrocyte processes contained much more connexin 47 than connexin 32. Along surfaces of internodal myelin, connexin 47 was several times as abundant as connexin 32, and in the smallest gap junctions, often occurred without connexin 32. In contrast, connexin 32 was localized without connexin 47 in newly-described autologous gap junctions in Schmidt-Lanterman incisures and between paranodal loops bordering nodes of Ranvier. Thus, connexin 47 in adult rodent CNS is the most abundant connexin in most heterologous oligodendrocyte-to-astrocyte gap junctions, whereas connexin 32 is the predominant if not sole connexin in autologous ("reflexive") oligodendrocyte gap junctions. These results clarify the locations and connexin compositions of heterologous and autologous oligodendrocyte gap junctions, identify autologous gap junctions at paranodes as potential sites for modulating paranodal electrical properties, and reveal connexin 47-containing and connexin 32-containing gap junctions as conduits for long-distance intracellular and intercellular movement of ions and associated osmotic water. The autologous gap junctions may regulate paranodal electrical properties during saltatory conduction. Acting in series and in parallel, autologous and heterologous oligodendrocyte gap junctions provide essential pathways for intra- and intercellular ionic homeostasis.

The selective PDE5 inhibitor, sildenafil, improves object memory in Swiss mice and increases cGMP levels in hippocampal slices.

Previous studies have shown memory enhancing effects of phosphodiesterase type 5 (PDE5) inhibitors in rats. However, differences in nitric oxide (NO)-mediated cyclic GMP (cGMP) signaling in the hippocampus have been described between rats and mice. In the present study we investigated the memory enhancing effects of the PDE5 inhibitor, sildenafil on memory performance in Swiss mice using the object recognition task. Sildenafil (0.3, 1 and 3 mg/kg) was administered orally directly after the first trial. The memory for the objects was retested 24 h later when mice show no memory for the familiar object. Sildenafil improved the object discrimination performance of Swiss mice at a dose of 1 mg/kg. Hippocampal slices of Swiss mice incubated with sildenafil (10 microM) increased cGMP levels in varicosities in the CA3 region of the hippocampus and a number of short, thin fibers. Addition of DEA/NO, an NO donor (10 microM), in the presence of sildenafil (10 microM) strongly increased cGMP immunoreactivity of varicosities in the CA3 region. Double immunostaining of cGMP with the presynaptic marker synaptophysin did not reveal any co-localization of these markers under any circumstance. Taken together, inhibition of PDE5 improves object recognition memory in mice. Furthermore, a postsynaptic role of cGMP could be involved in this respect.

Evaluation of DOI, 8-OH-DPAT, eticlopride and amphetamine on impulsive responding in a reaction time task in rats.

We examined the effects of DOI (2,5-dimethoxy-4-iodoamphetamine), 8-OH-DPAT (8-hydroxy-2-(N,N-dipropylamino)tetralin, eticlopride and amphetamine in a reaction time (RT) task. In this task a trial is initiated after a rat pushes a panel. Rats have to wait (0.5-1.5 s) until a tone is presented before making a response. The number of premature responses, releasing the panel before tone was switched on, was taken as a measure of motor impulsivity. A group of 10 Lewis rats was tested in the RT task after treatment with different doses of drugs which have been shown previously to affect impulsive responding: DOI (0.1, 0.2 mg/kg), 8-OH-DPAT (0.1, 0.3 mg/kg), eticlopride (0.01, 0.03 mg/kg) and D-amphetamine (0.3, 1 mg/kg). A progressive ratio test was used to control for drug effects on food motivation. DOI (0.1 mg/kg) and D-amphetamine (0.3 mg/kg) increased impulsive responding in the RT task. Conversely, 8-OH-DPAT decreased impulsive responding in the RT task. These effects of DOI, D-amphetamine and 8-OH-DPAT on impulsive responding were not associated with changes in food motivation, as assessed by performance in the progressive ratio task. Eticlopride did not affect impulsive responding. The present data suggest that 5-HT2A receptors and dopamine (but not D2 receptors) are associated with motor impulsivity.

Effects of two selective phosphodiesterase type 5 inhibitors, sildenafil and vardenafil, on object recognition memory and hippocampal cyclic GMP levels in the rat.

The present study investigated the effects of two cyclic GMP-specific phosphodiesterase enzyme type 5 inhibitors, sildenafil and vardenafil, on the memory performance in the object recognition task. Both compounds were given per orally (1, 3 and 10 mg/kg sildenafil; 0.1, 0.3, 1 and 3 mg/kg vardenafil) immediately after the exposure to two identical objects. The memory for the objects was tested 24 h later. Vehicle-treated rats spent equal times exploring a new and the familiar object demonstrating that they did not remember the familiar one. However, sildenafil improved the object discrimination performance of the rats with a high discrimination performance at a dose of 3 mg/kg. Rats treated with vardenafil also showed an improved object discrimination performance. Compared with sildenafil, vardenafil appeared to be even more potent in this respect since it already produced a high discrimination performance at a dose of 0.3 mg/kg. The effects of both compounds on cyclic GMP and cyclic AMP accumulation were studied in rat hippocampal slices incubated in vitro. Cyclic GMP levels were increased after incubation with the highest concentration of 100 microM vardenafil (together with 0.1 mM sodium nitroprusside), although no changes in cyclic GMP levels were detected after incubation with different concentrations of sildenafil. Both compounds had no effect on cyclic AMP levels. Additional cyclic GMP immunocytochemistry showed that incubation with vardenafil (in the presence of sodium nitroprusside) resulted in a concentration-dependent staining of cyclic GMP. Staining was predominantly found in neuronal fibres in the hippocampal CA2/CA3 region. It was already detected at a concentration of 0.1 microM vardenafil. Also positive fibres were detected after incubation with sildenafil but at a higher concentration of 10 microM. Taken together, these results suggest that inhibition of phosphodiesterase enzyme type 5 improves object recognition memory. This effect might be explained by increased levels of central cyclic GMP.

GABA(B) receptors in the median raphe nucleus: distribution and role in the serotonergic control of hippocampal activity.

Previous studies have shown that serotonergic neurons of the median raphe nucleus have a suppressive effect on theta synchronization in the hippocampus. Median raphe lesion, suppression of 5-HT neuronal activity by administration of GABA(A) receptor antagonist or by glutamate blockade or depletion produced long-lasting non-interrupted hippocampal theta in freely behaving rats independent of behavior and in rats anesthetized with urethane. Serotonergic neurons show a characteristic sleep-wake pattern of activity and there is evidence that GABAergic mechanisms play an important role in their regulation. In this study we analyzed the distribution and subcellular localization of GABA(B) receptors in the midbrain raphe complex using combined 5-HT/GABA(B) receptor immunohistochemistry at the light and electron microscopic levels and studied the effects of their pharmacological manipulation on hippocampal electroencephalographic activity in urethane-anesthetized rats. We found that sustained infusion of the GABA(B) receptor agonist baclofen into the median raphe nucleus, using the microdialysis technique, elicited lasting theta activity in the hippocampus. The effect was antagonized by selective GABA(B) receptor antagonists. The predominant localization of GABA(B) receptors in the median, as well as in dorsal raphe was found on serotonergic neurons which strongly indicates that the increase in theta occurrence after baclofen injection resulted from suppression of the serotonergic output originating from the median raphe. On the electron microscopic level, we found GABA(B) receptors located extrasynaptically indicating that these receptors are preferentially activated by strong inputs, i.e. when GABA released from the synaptic terminals is sufficient to spill over from the synaptic cleft. Such conditions might be satisfied during rapid eye movement sleep when GABAergic neurons in the raphe are firing at their highest rate and in rhythmic synchronized bursts. Our data indicate that midbrain raphe GABA(B) mechanisms play an important role in behavioral state control and in hippocampal activity, in particular.

The KCl cotransporter, KCC2, is highly expressed in the vicinity of excitatory synapses in the rat hippocampus.

Immunocytochemical visualization of the neuron-specific K+/Cl- cotransporter, KCC2, at the cellular and subcellular level revealed an area- and layer-specific diffuse labelling, and a discrete staining outlining the somata and dendrites of some interneurons in all areas of the rat hippocampus. KCC2 was highly expressed in parvalbumin-containing interneurons, as well as in subsets of calbindin, calretinin and metabotropic glutamate receptor 1a-immunoreactive interneurons. During the first 2 postnatal weeks, an increase of KCC2 staining was observed in the molecular layer of the dentate gyrus, correlating temporally with the arrival of entorhinal cortical inputs. Subcellular localization demonstrated KCC2 in the plasma membranes. Immunoreactivity in principal cells was responsible for the diffuse staining found in the neuropil. In these cells, KCC2 was detected primarily in dendritic spine heads, at the origin of spines and, at a much lower level on the somata and dendritic shafts. KCC2 expression was considerably higher in the somata and dendrites of interneurons, most notably of parvalbumin-containing cells, as well as in the thorny excrescences of CA3 pyramidal cells and in the spines of spiny hilar and stratum lucidum interneurons. The data indicate that KCC2 is highly expressed in the vicinity of excitatory inputs in the hippocampus, perhaps in close association with extrasynaptic GABAA receptors. A high level of excitation is known to lead to a simultaneous net influx of Na+ and Cl-, as evidenced by dendritic swelling. KCC2 located in the same microenvironment may provide a Cl- extrusion mechanism to deal with both ion and water homeostasis in addition to its role in setting the driving force of Cl- currents involved in fast postsynaptic inhibition.

Distribution of chloride channel-2-immunoreactive neuronal and astrocytic processes in the hippocampus.

The chloride homeostasis of neurons and non-neuronal cells is maintained in part by a voltage-sensitive inwardly rectifying chloride conductance through the chloride channel-2. This channel is activated by hyperpolarization and extracellular hypotonicity. In the present study, hippocampal sections were immunostained for chloride channel-2, and somata and dendrites of both pyramidal and non-pyramidal cells were found to be immunoreactive. In addition, glial processes in the vicinity of small blood vessels were also immunostained, whereas the neuropil of strata pyramidale and lacunosum-moleculare contained chloride channel-2-positive punctate structures. Electron microscopy and double immunostaining using antibodies against chloride channel-2 and glial fibrillary acidic protein confirmed that the dense network of chloride channel-2-positive processes corresponds to the end feet of astrocytes. The distribution of chloride channel-2-immunoreactive astrocytes was inhomogeneous throughout the hippocampus: strata oriens, pyramidale and lacunosum-moleculare of CA1-CA3 and the outer molecular layer of the dentate gyrus contained the majority of immunoreactive end feet, whereas the other layers showed sparse labeling. Subcellular studies demonstrated that, in addition to astrocytes, chloride channel-2 was localized in the membrane of dendrites, dendritic spines, cell bodies and axon initial segments of neurons, frequently close to, or within active zones of, symmetrical synapses.Thus, chloride channel-2 appears to be involved in transmembrane chloride movements associated with GABAergic synaptic transmission. The specific laminar distribution of chloride channel-2-positive astroglial processes coinciding with that of GABAergic axon terminals suggests that the network of astrocytes may be able to siphon and deliver Cl(-) ions to layers with intense GABAergic transmission, thereby increasing the efficacy of GABA(A) receptor-mediated inhibition.

Localization of the A kinase anchoring protein AKAP79 in the human hippocampus.

The phosphorylation state of the proteins, regulated by phosphatases and kinases, plays an important role in signal transduction and long-term changes in neuronal excitability. In neurons, cAMP-dependent protein kinase (PKA), protein kinase C (PKC) and calcineurin (CN) are attached to a scaffold protein, A kinase anchoring protein (AKAP), thought to anchor these three enzymes to specific sites of action. However, the localization of AKAP, and the predicted sites of linked phosphatase and kinase activities, are still unknown at the fine structural level. In the present study, we investigated the distribution of AKAP79 in the hippocampus from postmortem human brains and lobectomy samples from patients with intractable epilepsy, using preembedding immunoperoxidase and immunogold histochemical methods. AKAP79 was found in the CA1, presubicular and subicular regions, mostly in pyramidal cell dendrites, whereas pyramidal cells in the CA3, CA2 regions and dentate granule cells were negative both in postmortem and in surgical samples. In some epileptic cases, the dentate molecular layer and hilar interneurons also became immunoreactive. At the subcellular level, AKAP79 immunoreactivity was present in postsynaptic profiles near, but not attached to, the postsynaptic density of asymmetrical (presumed excitatory) synapses. We conclude that the spatial selectivity for the action of certain kinases and phosphatases regulating various ligand- and voltage-gated channels may be ensured by the selective presence of their anchoring protein, AKAP79, at the majority of glutamatergic synapses in the CA1, but not in the CA2/CA3 regions, suggesting profound differences in signal transduction and long-term synaptic plasticity between these regions of the human hippocampus.

Construction of a recombinant herpesvirus expressing the jellyfish green fluorescent protein.

Here we report the insertion of a synthetic version of the cDNA encoding the jellyfish (Aequorea victoria) green fluorescent protein (gfph ) into the genome of pseudorabies (Aujeszky's disease) virus (PrV). A putative latency promoter (PLAT) located at the inverted repeat region of the PrV genome was chosen as the target site for the insertion. Recombinant viral DNA designated as vLAT-gfp was generated as a result of homologous recombination between the transfected viral DNA and a plasmid containing the GFP-expression cassette flanked by viral sequences homologous to the target region. Plaques containing recombinant virus were selected visually using a fluorescent microscope. We demonstrated a GFP-expression in infected neurons of rat brain which showed normal morphology at early stage of viral infection by monitoring fluorescent light emission.

Presynaptically located CB1 cannabinoid receptors regulate GABA release from axon terminals of specific hippocampal interneurons.

To understand the functional significance and mechanisms of action in the CNS of endogenous and exogenous cannabinoids, it is crucial to identify the neural elements that serve as the structural substrate of these actions. We used a recently developed antibody against the CB1 cannabinoid receptor to study this question in hippocampal networks. Interneurons with features typical of basket cells showed a selective, intense staining for CB1 in all hippocampal subfields and layers. Most of them (85.6%) contained cholecystokinin (CCK), which corresponded to 96.9% of all CCK-positive interneurons, whereas only 4.6% of the parvalbumin (PV)-containing basket cells expressed CB1. Accordingly, electron microscopy revealed that CB1-immunoreactive axon terminals of CCK-containing basket cells surrounded the somata and proximal dendrites of pyramidal neurons, whereas PV-positive basket cell terminals in similar locations were negative for CB1. The synthetic cannabinoid agonist WIN 55,212-2 (0.01-3 microM) reduced dose-dependently the electrical field stimulation-induced [3H]GABA release from superfused hippocampal slices, with an EC50 value of 0. 041 microM. Inhibition of GABA release by WIN 55,212-2 was not mediated by inhibition of glutamatergic transmission because the WIN 55,212-2 effect was not reduced by the glutamate blockers AP5 and CNQX. In contrast, the CB1 cannabinoid receptor antagonist SR 141716A (1 microM) prevented this effect, whereas by itself it did not change the outflow of [3H]GABA. These results suggest that cannabinoid-mediated modulation of hippocampal interneuron networks operate largely via presynaptic receptors on CCK-immunoreactive basket cell terminals. Reduction of GABA release from these terminals is the likely mechanism by which both endogenous and exogenous CB1 ligands interfere with hippocampal network oscillations and associated cognitive functions.

GABAergic cells are the major postsynaptic targets of mossy fibers in the rat hippocampus.

Dentate granule cells communicate with their postsynaptic targets by three distinct terminal types. These include the large mossy terminals, filopodial extensions of the mossy terminals, and smaller en passant synaptic varicosities. We examined the postsynaptic targets of mossy fibers by combining in vivo intracellular labeling of granule cells, immunocytochemistry, and electron microscopy. Single granule cells formed large, complex "mossy" synapses on 11-15 CA3 pyramidal cells and 7-12 hilar mossy cells. In contrast, GABAergic interneurons, identified with immunostaining for substance P-receptor, parvalbumin, and mGluR1a-receptor, were selectively innervated by very thin (filopodial) extensions of the mossy terminals and by small en passant boutons in both the hilar and CA3 regions. These terminals formed single, often perforated, asymmetric synapses on the cell bodies, dendrites, and spines of GABAergic interneurons. The number of filopodial extensions and small terminals was 10 times larger than the number of mossy terminals. These findings show that in contrast to cortical pyramidal neurons, (1) granule cells developed distinct types of terminals to affect interneurons and pyramidal cells and (2) they innervated more inhibitory than excitatory cells. These findings may explain the physiological observations that increased activity of granule cells suppresses the overall excitability of the CA3 recurrent system and may form the structural basis of the target-dependent regulation of glutamate release in the mossy fiber system.

The absence of a major Ca2+ signaling pathway in GABAergic neurons of the hippocampus.

The Ca2+/calmodulin-dependent protein phosphatase 2B or calcineurin (CN) participates in several Ca2+-dependent signal transduction cascades and, thus, contributes to the short and long term regulation of neuronal excitability. By using a specific antibody to CN, we demonstrate its absence from hippocampal interneurons and illustrate a physiological consequence of such CN deficiency. Consistent with the lack of CN in interneurons as detected by immunocytochemistry, the CN inhibitors FK-506 or okadaic acid significantly prolonged N-methyl-D-aspartate channel openings recorded in the cell-attached mode in hippocampal principal cells but not those recorded in interneurons. Interneurons were also devoid of Ca2+/calmodulin-dependent protein kinase IIalpha, yet many of their nuclei contained the cyclic AMP-responsive element binding protein. On the basis of the CN and Ca2+/calmodulin-dependent protein kinase IIalpha deficiency of interneurons, entirely different biochemical mechanisms are expected to govern Ca2+-dependent neuronal plasticity in interneurons versus principal cells.

Dendritic properties of hippocampal CA1 pyramidal neurons in the rat: intracellular staining in vivo and in vitro.

Dendritic morphology and passive cable properties determine many aspects of synaptic integration in complex neurons, together with voltage-dependent membrane conductances. We investigated dendritic properties of CA1 pyramidal neurons intracellularly labeled during in vivo and in vitro physiologic recordings, by using similar intracellular staining and three-dimensional reconstruction techniques. Total dendritic length of the in vivo neurons was similar to that of the in vitro cells. After correction for shrinkage, cell extent in three-dimensional representation was not different between the two groups. Both in vivo and in vitro neurons demonstrated a variable degree of symmetry, with some neurons showing more cylindrical symmetry around the main apical axis, whereas other neurons were more elliptical, with the variation likely due to preparation and preservation conditions. Branch order analysis revealed no difference in the number of branch orders or dendritic complexity. Passive conduction of dendritic signals to the soma in these neurons shows considerable attenuation, particularly with higher frequency signals (such as synaptic potentials compared with steady-state signals), despite a relatively short electrotonic length. Essential aspects of morphometric appearance and complex dendritic integration critical to CA1 pyramidal cell functioning are preserved across neurons defined from the two different hippocampal preparations used in this study.

Interneurons in the hippocampal dentate gyrus: an in vivo intracellular study.

Interneurons in the dentate area were characterized physiologically and filled with biocytin in urethane-anaesthetized rats. On the basis of axonal targets the following groups could be distinguished. (i) Large multipolar interneurons with spiny dendrites in the deep hilar region densely innervated the outer molecular layer and contacted both granule cells and parvalbumin-positive neurons (hilar interneuron with perforant pathway-associated axon terminals; HIPP cells). (ii) A pyramidal-shaped neuron with a cell body located in the subgranular layer innervated mostly the inner molecular layer and the granule cell layer (hilar interneuron with commissural-associational pathway-associated axon terminals; HICAP cell). It contacted both granule cells and interneurons. Axon collaterals of HIPP and HICAP neurons covered virtually the entire septo-temporal extent of the dorsal dentate gyrus. (iii) Calbindin-immunoreactive neurons with horizontal dendrites in stratum oriens of the CA3c region gave rise to a rich axon arbor in strata oriens, pyramidale and radiatum and innervated almost the entire extent of the dorsal hippocampus, with some collaterals entering the subicular area (putative trilaminar cell). (iv) Hilar basket cells innervated mostly the granule cell layer and to some extent the inner molecular layer and the CA3c pyramidal layer. HIPP and trilaminar interneurons could be antidromically activated by stimulation of the fimbria. Only the HICAP cells could be monosynaptically discharged by the perforant path input. All interneurons examined showed phase-locked activity to the extracellularly recorded theta/gamma oscillations or to irregular dentate electroencephalogram spikes. These observations indicate that the interconnected interneuronal system plays a critical role in coordinating population of the dentate gyrus and Ammon's hom.