Synaptic reorganization in explanted cultures of rat hippocampus

Due to loss of afferent innervation, synaptic reorganization occurs in organotypic hippocampal slice cultures. With extra- and intracellular recordings, we confirm that the excitatory loop from the dentate gyrus (DG) to CA3 and further to CA1 is preserved. However, hilar stimulation evoked antidromic population spikes in the DG which were followed by a population postsynaptic potential (PPSP); intracellularly, an antidromic spike with a broad shoulder or EPSP/IPSP sequences were induced. Synaptic responses were blocked by glutamate receptor antagonists. Stimulation of CA1 induced a PPSP in DG. Dextranamine stained pyramidal cells of CA1 were shown to project to DG. After removal of area CA3, DG's and mossy fibers' (MF) stimulation still elicited PPSPs and EPSP/IPSP sequences in area CA1 which disappeared when a cut was made through the hippocampal fissure. During bicuculline perfusion, hilar stimulation caused EPSPs in granule cells and spontaneous and evoked repetitive firing appeared even after its isolation from areas CA3 and CA1. Collateral excitatory synaptic coupling between granule cells was confirmed by paired recordings. Besides the preservation of the trisynaptic pathway in this preparation, new functional synaptic contacts appear, presumably due to MF collateral sprouting and formation of pathways between areas CA1 and DG.     

Synaptic loss reflected by secretoneurin-like immunoreactivity in the human hippocampus in Alzheimer's disease

Secretoneurin is a recently described peptide derived by endoproteolytic processing from secretogranin II, previously named chromogranin C. In this study, we have investigated the distribution of secretoneurin-like immunoreactivity in the human hippocampus in controls and in Alzheimer's disease patients, and compared the staining pattern to that of calretinin. Secretoneurin-like immunoreactivity is present throughout the hippocampal formation. At the border of the dentate molecular layer and the granule cell layer, a band of dense secretoneurin immunostaining appeared. In this part, as in the area of the CA2 sector, the high density of secretoneurin-immunoreactivity coincided with calretinin-like immunoreactivity. The mossy fibre system displayed a moderate density of secretoneurin-immunoreactivity. In the entorhinal cortex, a particularly high density of secretoneurin-immunoreactivity was observed. The density of secretoneurin-like immunoreactivity was significantly reduced in the innermost part of the molecular layer and in the outer molecular layer of the dentate gyrus in Alzheimer's disease. For calretinin-like immunoreactivity, a less pronounced decrease was found in the innermost part of the molecular layer. About 40-60% of neuritic plaques were secretoneurin-immunopositive. This study shows that secretoneurin is distinctly distributed in the human hippocampus and that significant changes of secretoneurin-like immunoreactivity occur in Alzheimer's disease, reflecting synaptic loss.     

Synaptic effects of identified interneurons innervating both interneurons and pyramidal cells in the rat hippocampus

GABAergic interneurons sculpt the activity of principal cells and are themselves governed by GABAergic inputs. To determine directly some of the sources and mechanisms of this GABAergic innervation, we have used dual intracellular recordings with biocytin-filled microelectrodes and investigated synaptic interactions between pairs of interneurons in area CA1 of the adult rat hippocampus. Of four synaptically-coupled interneuron-to-interneuron cell pairs, three presynaptic cells were identified as basket cells, preferentially innervating somata and proximal dendrites of pyramidal cells, but one differing from the other two in the laminar distribution of its dendritic and axonal fields. The fourth presynaptic interneuron was located at the border between strata lacunosum moleculare and radiatum, with axon ramifying within stratum radiatum. Action potentials evoked in all four presynaptic interneurons were found to elicit fast hyperpolarizing inhibitory postsynaptic potentials (mean amplitude 0.35 +/- 0.10 mV at a membrane potential of -59 +/- 2.8 mV) in other simultaneously recorded interneurons (n=4). In addition, three of the presynaptic interneurons were also shown to produce similar postsynaptic responses in subsequently recorded pyramidal cells (n=4). Electron microscopic evaluation revealed one of the presynaptic basket cells to form 12 synaptic junctions with the perisomatic domain (seven somatic synapses and five synapses onto proximal dendritic shafts) of the postsynaptic interneuron in addition to innervating the same compartments of randomly-selected local pyramidal cells (50% somatic and 50% proximal dendritic synapses, n=12). In addition, light microscopic analysis also indicated autaptic self-innervation in basket (12 of 12) and bistratified cells (six of six). Electron microscopic investigation of one basket cell confirmed six autaptic junctions made by five of its boutons. Together, these data demonstrate that several distinct types of interneuron have divergent output to both principal cells and local interneurons of the same (basket cells) or different type. The fast synaptic effects, probably mediated by GABA in both postsynaptic interneurons and principal cells are similar. These additional sources of GABA identified here in the input to GABAergic cells could contribute to the differential temporal patterning of distinct GABAergic synaptic networks.     

Online order control in the psychological refractory period paradigm.

The authors examined the role of online order control in the psychological refractory period (PRP) paradigm. In the first 2 experiments, participants switched between color-letter and letter-color orders so that subtask order was isolated as the only element being switched. The results indicated that order switching impaired the 2 PRP responses and modulated the PRP effect. Importantly, these effects were reduced by advance preparation, demonstrating that order representation was activated before the subtasks themselves. Preparation for subtask order did not reflect preparation for hand order, as shown in Experiment 3. In addition, there was no evidence that subtask order information dissipated between trials. The relevance of the results to theories of the PRP paradigm and task switching is discussed. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Backward response-level crosstalk in the psychological refractory period paradigm.

Bottleneck models of psychological refractory period (PRP) tasks suggest that a Task 1 response should be unaffected by the Task 2 response in the same trial, because selection of the former finishes before selection of the latter begins. Contrary to this conception, the authors found backward response-level crosstalk effects in which Task 2 response force requirements influenced the force-time dynamics of Task 1 responses. Specifically, Task 2 required a hard or soft keypress response. Task 1 responses were harder when the upcoming Task 2 response was to press hard rather than soft, suggesting some activation of Task 2 response parameters before Task 1 processing reached the final ballistic motor output stage. A 3rd experiment using a flankers paradigm showed that this effect did not arise from automatic activation of responses by the stimuli associated with hard and soft responses. This backward response-level crosstalk extends previous findings of backward crosstalk in the PRP paradigm by showing that crosstalk can affect motor output as well as response time and can arise even when the 2 tasks being performed are not semantically related. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The psychological refractory period of stopping.

The author examined whether the act of control of stopping is subject to the psychological refractory period (PRP) and whether stopping causes a PRP for the processing of subsequent stimuli. The task was to execute or to stop a rapid finger tapping. PRP interference was predicted for double-stimulation trials, in which 2 signals to tap or stop were presented in rapid succession. The experiments showed that stopping ongoing action is subject to and produces PRP interference similar to starting. Responses to signals to continue an ongoing action do not produce PRP interference. The results suggest that selection or initiation of new responses, but not mere response choice, constituted the processing bottleneck that caused the PRP in the present task. Further results indicate that the inhibition of not-yet-executed actions, in contrast to action termination, does not suffer PRP interference and that response inhibitions and terminations should be distinguished. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Carbamazepine-induced release of serotonin from rat hippocampus in vitro

PURPOSE: Carbamazepine is one of several antiepileptic drugs (AEDs) that release the inhibitory neurotransmitter serotonin as part of their pharmacodynamic action on brain neurons. We undertook this study to investigate the cellular processes by which carbamazepine (CBZ) releases serotonin from brain tissue. METHODS: Tissue slices were prepared from hippocampi of Sprague-Dawley rats. These hippocampal slices were preincubated in vitro in a buffer so that neurons within the slice would take up tritium-labeled serotonin. Subsequently the slices were superfused with buffer containing CBZ or other chemicals (or both) that increase the overflow of serotonin radioactivity. RESULTS: Carbamazepine produced a concentration-dependent (50, 125, 250, or 500 microM) increase in basal overflow of serotonin radioactivity from superfused rat hippocampal slices in vitro. In contrast, these concentrations did not alter potassium-stimulated release, suggesting that the CBZ-induced release does not depend on depolarization or exocytosis. Blockade of the neuronal membrane serotonin transporter by fluoxetine (1 microM) or citalopram (2 microM) did not alter overflow of serotonin radioactivity produced by 250 microM CBZ. p-chloramphetamine (10 microM) produced a substantial increase in overflow of serotonin radioactivity, and this effect appears to be antagonized by 250 microM CBZ. Uptake of [3H]-labeled serotonin into hippocampal synaptosomes was inhibited by CBZ with a median inhibitory concentration (IC50) of 511+/-33 microM and a Hill coefficient of 0.87+/-0.11, suggesting competitive inhibition of uptake by CBZ. CONCLUSIONS: We conclude that CBZ (a) releases serotonin from hippocampal slices independent of exocytosis and by a mechanism not involving the neuronal membrane serotonin transporter, and (b) at high enough concentration, blocks the neuronal serotonin transporter.     

Synaptic vesicular localization and exocytosis of L-aspartate in excitatory nerve terminals: a quantitative immunogold analysis in rat hippocampus

To elucidate the role of aspartate as a signal molecule in the brain, its localization and those of related amino acids were examined by light and electron microscopic quantitative immunocytochemistry using antibodies specifically recognizing the aldehyde-fixed amino acids. Rat hippocampal slices were incubated at physiological and depolarizing [K+] before glutaraldehyde fixation. At normal [K+], aspartate-like and glutamate-like immunoreactivities were colocalized in nerve terminals forming asymmetrical synapses on spines in stratum radiatum of CA1 and the inner molecular layer of fascia dentata (i.e., excitatory afferents from CA3 and hilus, respectively). During K+ depolarization there was a loss of aspartate and glutamate from these terminals. Simultaneously the immunoreactivities strongly increased in glial cells. These changes were Ca2+-dependent and tetanus toxin-sensitive and did not comprise taurine-like immunoreactivity. Adding glutamine at CSF concentration prevented the loss of aspartate and glutamate and revealed an enhancement of aspartate in the terminals at moderate depolarization. In hippocampi from animals perfused with glutaraldehyde during insulin-induced hypoglycemia (to combine a strong aspartate signal with good ultrastructure) aspartate was colocalized with glutamate in excitatory terminals in stratum radiatum of CA1. The synaptic vesicle-to-cytoplasmic matrix ratios of immunogold particle density were similar for aspartate and glutamate, significantly higher than those observed for glutamine or taurine. Similar results were obtained in normoglycemic animals, although the nerve terminal contents of aspartate were lower. The results indicate that aspartate can be concentrated in synaptic vesicles and subject to sustained exocytotic release from the same nerve endings that contain and release glutamate.     

Current problems in the measurement of gastrin release. A reproducible measure of physiological gastrin release

The interpretation of gastrin release is confused because of variation in the technique of the radioimmuoassay of gastrin, the lack of a standard stimulus for the release of gastrin and diversity in the method used to express the results. These problems have been analysed (a) by examining the cross-reactivity of three gastrin antisera and using each of the antisera to measure basal gastrin levels in fasting normal subjects, duodenal ulcer and post-vagotomy patients; (b) by determining a satisfactory stimulus for gastrin release in normal subjects; (c) by examining the results to determine the best method of presenting the data. The different a ntisera used were found to give different levels of plasma gastrin in the same sample of plasma. This was not related to the cross reactivity of the antisera. An English breakfast was found to be the most satisfactory stimulus for the release of gastrin. The expression of the results of such a stimulus of gastrin release was affected least by assay variation when the incremental integrated gastrin response was used. It is concluded that the incremental integrated gastrin response to an English breakfast is a satisfactory method for exploring variations in gastrin release.     

Effect of transient cerebral ischaemia on acetylcholine release in the gerbil hippocampus

To clarify the relationship between presynaptic cholinergic dysfunction and postsynaptic cell death in the hippocampus, extracellular levels of acetylcholine (ACh) were assayed and CA1 pyramidal cells were histologically investigated in gerbils which had undergone 2, 5 and 10 min ischaemia. It was found that the KCl- and atropine-induced release of ACh, an index of the functioning cholinergic system at the presynaptic terminals, was significantly lower in the ischaemic groups than in control groups. The hippocampal CA1 pyramidal cell area of the 5 and 10 min ischaemic animals was also significantly decreased, but the 2 min ischaemia caused no cell damage. These findings indicate that the presynaptic terminals of the cholinergic neurone are vulnerable to ischaemic insult and that cholinergic dysfunction precedes postsynaptic CA1 pyramidal cell death in the hippocampus.     

Task switching and response correspondence in the psychological refractory period paradigm.

Three experiments examined the effects of task switching and response correspondence in a psychological refractory period paradigm. A letter task (vowel-consonant) and a digit task (odd-even) were combined to form 4 possible dual-task pairs in each each: letter-letter, letter-digit, digit-digit, and digit-letter. Foreknowledge of task transition (repeat or switch) and task identity (letter or digit) was varied across experiments: no foreknowledge in Experiment 1, partial foreknowledge (task transition only) in Experiment 2, and full foreknowledge in Experiment 3. For all experiments, the switch cost for Task 2 was additive with stimulus onset asynchrony, and the response-correspondence effect for Task 2 was numerically smaller in the switch condition than in the repeat condition. These outcomes suggest that reconfiguration for Task 2 takes place after the central processing of Task 1 and that the crosstalk correspondence effect is due to response activation by way of stimulus-response associations. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Increased probability of GABA release during withdrawal from morphine

Opioid receptors located on interneurons in the ventral tegmental area (VTA) inhibit GABA(A)-mediated synaptic transmission to dopamine projection neurons. The resulting disinhibition of dopamine cells in the VTA is thought to play a pivotal role in drug abuse; however, little is known about how this GABAA synapse is affected after chronic morphine treatment. The regulation of GABA release during acute withdrawal from morphine was studied in slices from animals treated for 6-7 d with morphine. Slices containing the VTA were prepared and maintained in morphine-free solutions, and GABAA IPSCs were recorded from dopamine cells. The amplitude of evoked IPSCs and the frequency of spontaneous miniature IPSCs measured in slices from morphine-treated guinea pigs were greater than placebo-treated controls. In addition, activation of adenylyl cyclase, with forskolin, and cAMP-dependent protein kinase, with Sp-cAMPS, caused a larger increase in IPSCs in slices from morphine-treated animals. Conversely, the kinase inhibitors staurosporine and Rp-CPT-cAMPS decreased GABA IPSCs to a greater extent after drug treatment. The results indicate that the probability of GABA release was increased during withdrawal from chronic morphine treatment and that this effect resulted from an upregulation of the cAMP-dependent cascade. Increased transmitter release from opioid-sensitive synapses during acute withdrawal may be one adaptive mechanism that results from prolonged morphine treatment.     

Calcium channel antagonist peptides define several components of transmitter release in the hippocampus

The use of subtype-selective voltage-sensitive calcium channel (VSCC) antagonists has established that neurotransmitter release in mammalian brain is mediated by N-like and P-like VSCCs, and that other subtypes also contribute significantly. To determine the roles presynaptic VSCCs play in nervous system function and to evaluate the therapeutic potential of their selective inhibition, it is necessary to define further the contributions of VSCC subtypes to neurotransmitter release. The novel conopeptide, SNX-230 (omega-conopeptide MVIIC), has revealed a new VSCC subtype, the Q-type, in cerebellar granule cells. We have compared the effects of SNX-230 on release of tritiated D-aspartate ([3H]D-Asp; a non-metabolizable analog of glutamate), gamma-aminobutyric acid ([3H]GABA), and norepinephrine ([3H]NE) from rat hippocampal slices to those of the N-type VSCC blocker, SNX-111 (omega-conopeptide MVIIA), and the P-type blocker, omega-agatoxin-IVA (AgaIVA). SNX-230 blocks both [3H]D-Asp and [3H]GABA release completely, whereas AgaIVA blocks them potently but partially and SNX-111 has no effect. These results suggest that glutamate and GABA release are mediated by two VSCC subtypes, a P-type and another, perhaps Q-like. SNX-111 blocks [3H]NE release potently but partially, while SNX-230 blockade is complete, consisting of one very potent phase and one less potent phase. AgaIVA also blocks [3H]NE release potently but partially. These results suggest that at least two VSCC subtypes, an N-type and a novel non-N-type, mediate NE release. Pair-wise combinations of the three ligands indicate that at least three pharmacologically distinct components comprise [3H]NE release in the hippocampus.     

Nerve fibre velocity and refractory period distributions in nerve trunks

With the use of a double stimulus technique, nerve fibre velocity range measurements were performed over a single conduction distance in 13 normal subjects and over two conduction distances in another 12 normal subjects. The velocity ranges were found to be dependent upon the conduction distance, owing to unknown refractory period delays. Refractory period values were calculated for the 12 subjects and also direct refractory period distribution measurements were made on 15 normal subjects using a twin stimulus and automatic subtraction technique. Corrections to the velocity range measurements were made upon differing assumptions as to the correlation between refractory period and fibre conduction velocity. It was concluded that a single median value refractory period obtained from the distribution was the best correction to use, based upon the hypothesis that for group A fibres the random scatter of refractory period values is far greater than any variation due to a correlation between refractory period and fibre conduction velocity. It was found important to recognize that calculated values of velocity range are a function not only of the spread of fibre conduction velocities but also of refractory periods.     

Presynaptic modulation of cholecystokinin release by protein kinase C in the rat hippocampus

The role of protein kinase C (PKC) in modulating the release of the octapeptide cholecystokinin (CCK-8) was investigated in rat hippocampal nerve terminals (synaptosomes). The PKC-activating phorbol ester 4beta-phorbol 12,13-dibutyrate (beta-PDBu) dose dependently (5-5,000 nM) increased CCK-8 release in a strictly Ca2+dependent way. This effect was observed only when synaptosomes were stimulated with the K+(A) channel blocker 4-aminopyridine (4-AP; 1 mM) but not with KCl (10-30 mM). The PDBu-induced exocytosis of CCK-8 was completely blocked by the two selective PKC inhibitors chelerythrine and calphostin-C and was not mimicked by alpha-PDBu, an inactive phorbol ester. In addition, an analogue of the endogenous PKC activator diacylglycerol, oleoylacetylglycerol, dose dependently increased CCK-8 exocytosis. Beta-PDBu (50-100 nM) also stimulated the 4-AP-evoked Ca2+-dependent release of the classic transmitter GABA, which co-localizes with CCK-8 in hippocampal interneurons. As a possible physiological trigger for PKC activation, the role of the metabotropic glutamate receptor was investigated. However, the broad receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid did not stimulate, but instead inhibited, both the CCK-8 and the GABA exocytosis. In conclusion, presynaptic PKC may stimulate exocytosis of distinct types of co-localizing neurotransmitters via modulation of presynaptic K+ channels in rat hippocampus.     

Involvement of L-type-like amino acid transporters in S-nitrosocysteine-stimulated noradrenaline release in the rat hippocampus

Nitrogen oxides, such as nitric oxide, have been shown to regulate neuronal functions, including neurotransmitter release. We investigated the effect of S-nitroso-L-cysteine (SNC) on noradrenaline (NA) release in the rat hippocampus in vivo and in vitro. SNC stimulated [3H]NA release from prelabeled hippocampal slices in a dose-dependent manner. SNC stimulated endogenous NA release within 30 min to almost five times the basal level in vivo (microdialysis in freely moving rats). In a Na+-containing Tyrode's buffer, SNC-stimulated [3H]NA release was inhibited 30% by the coaddition of L-leucine. In the Na+-free, choline-containing buffer, SNC-stimulated [3H]NA release, which was similar to that in the Na+-containing buffer, was inhibited markedly by L-leucine, L-alanine, L-methionine, L-phenylalanine, and L-tyrosine. The effects of the other amino acids examined were smaller or very limited. The effect of L-leucine was stronger than that of D-leucine. A specific inhibitor of the L-type amino acid transporter, 2-aminobicyclo[2.2.1]-heptane-2-carboxylate (BCH), inhibited the effects of SNC on [3H]NA release in the Na+-free buffer. Uptake of L-[3H]leucine into the slices in the Na+-free buffer was inhibited by SNC, BCH, and L-phenylalanine, but not by L-lysine. The effect of SNC on cyclic GMP accumulation was not inhibited by L-leucine, although SNC stimulated cyclic GMP accumulation at concentrations up to 25 microM, much less than the concentration that stimulates NA release. These findings suggest that SNC is incorporated into rat hippocampus via the L-type-like amino acid transporter, at least in Na+-free conditions, and that SNC stimulates NA release in vivo and in vitro in a cyclic GMP-independent manner.     

Reflex-inhibiting stimuli and the refractory period of the acoustic startle reflex in the rat.

Previous studies show that if acoustic startle stimuli are presented in pairs, then the reaction to the 2nd stimulus (S2) is reduced, with the size of the refractory decrement determined by the interstimulus interval and the relative intensity of the 1st stimulus (S1). If a neutral stimulus (p) is presented just prior to S1, then the reaction to S1 is similarly inhibited, revealing the phenomenon of prestimulus inhibition. In 2 experiments with male albino Holtzman rats (N = 24) it was found that suppression of the reflex to S2 by S1 was unaffected by prestimulus inhibition of S1 (i.e., reflex amplitudes associated with S2 were identical in pS1-S2 series and S1-S2 series). In contrast, a reduction in the intensity of S1 relative to S2 did reduce the effect of S1 on S2. These data indicate that prestimulus inhibition of the reflex to S1 does not result because the preliminary stimulus attenuates the sensory impact of S1. The inhibitory process may be presumed to have a central locus. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Synaptic target selectivity and input of GABAergic basket and bistratified interneurons in the CA1 area of the rat hippocampus

To assess the position of interneurons in the hippocampal network, fast spiking cells were recorded intracellularly in vitro and filled with biocytin. Sixteen non-principal cells were selected on the basis of 1) cell bodies located in the pyramidal layer and in the middle of the slice, 2) extensive labeling of their axons, and 3) a branching pattern of the axon indicating that they were not axo-axonic cells. Examination of their efferent synapses (n = 400) demonstrated that the cells made synapses on cell bodies, dendritic shafts, spines, and axon initial segments (AIS). Statistical analysis of the distribution of different postsynaptic elements, together with published data (n = 288) for 12 similar cells, showed that the interneurons were heterogeneous with regard to the frequency of synapses given to different parts of pyramidal cells. When the cells were grouped according to whether they had less or more than 40% somatic synaptic targets, each population appeared homogeneous. The population (n = 19) innervating a high proportion of somata (53 +/- 10%, SD) corresponds to basket cells. They also form synapses with proximal dendrites (44 +/- 12%) and rarely with AISs and spines. One well-filled basket cell had 8,859 boutons within the slice, covering an area of 0.331 mm2 of pyramidal layer tangentially and containing 7,150 pyramidal cells, 933 (13%) of which were calculated to be innervated, assuming that each pyramidal cell received nine to ten synapses. It was extrapolated that the intact axon probably had about 10,800 boutons innervating 1,140 pyramids. The proportion of innervated pyramidal cells decreased from 28% in the middle to 4% at the edge of the axonal field. The other group of neurons, the bistratified cells (n = 9), showed a preference for dendritic shafts (79 +/- 8%) and spines (17 +/- 8%) as synaptic targets, rarely terminating on somata (4 +/- 8%). Their axonal field was significantly larger (1,250 +/- 180 microns) in the medio-lateral direction than that of basket cells (760 +/- 130 microns). The axon terminals of bistratified cells were smaller than those of basket cells. Furthermore, in constrast to bistratified cells, basket cells had a significant proportion of dendrites in stratum lacunosum-moleculare suggesting a direct entorhinal input. The results define two distinct types of GABAergic neuron innervating pyramidal cells in a spatially segregated manner and predict different functions for the two inputs. The perisomatic termination of basket cells is suited for the synchronization of a subset of pyramidal cells that they select from the population within their axonal field, whereas the termination of bistratified cells in conjunction with Schaffer collateral/commissural terminals may govern the timing of CA3 input and/or voltage-dependent conductances in the dendrites.