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To evaluate permanent effects of hippocampal and prefrontal cortex lesion on spatial tasks, lesioned and sham-operated female C57BL/6 mice were exposed to a series of conditioning schemes in IntelliCages housing 8-10 transponder-tagged mice from each treatment group. Sequential testing started at 51-172days after bilateral lesions and lasted for 154 and 218days in two batches of mice, respectively. Spontaneous undisturbed behavioral patterns clearly separated the three groups, hippocampals being characterized by more erratic hyperactivity, and strongly impaired circadian synchronization ability. Hippocampal lesions led to deficits in spatial passive avoidance, as well as in spatial reference and working memory tasks. Impairment was minimal in rewarded preference/reversal schemes, but prominent if behavioral responses required precise circadian timing or included punishment of wrong spatial choices. No differences between sham-operated and prefrontally lesioned subjects in conditioning success were discernible. These results corroborate the view that hippocampal dysfunction spares simple spatial learning tasks but impairs the ability to cope with conflicting task-inherent spatial, temporal or emotional cues. Methodologically, the results show that automated testing and data analysis of socially kept mice is a powerful, efficient and animal-friendly tool for dissecting complex features and behavioral profiles of hippocampal dysfunction characterizing many transgenic or pharmacological mouse models., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

The discovery of adult-born neurons in the hippocampus has triggered a wide range of studies that link the new neurons to various behavioral functions. However, the role of new neurons in behavior is still equivocal. Conflicting results may be due to the difficulty in manipulating neurogenesis without off-target effects as well as the statistical approach used, which fail to account for neurogenesis-independent effects of experimental manipulations on behavior. In this study, we apply a more comprehensive statistical and conceptual approach. Instead of between-group analyses, we consider the within-group relationships between neurogenesis and behavior (ANCOVA and mediation analysis) in a large-scale experiment, in which distinct age- (3 and 5 months) and strain- (DBA and C57) related differences in basal levels of neurogenesis in mice are compared with a large number (∼1,500) of behavioral read outs. The analysis failed to detect any association between anxiety and motor impulsivity with neurogenesis. However, within-group adult hippocampal neurogenesis is associated with the reaction to novelty. Specifically, more neurogenesis is associated with a longer latency to explore and a lower frequency of exploratory actions, overall indicative of a phenotype where animals with more neurogenesis were slower to explore a novel environment. This effect is observed in 5-months-old, but not in 3-months-old mice of both strains. An association between the reaction to novelty and adult neurogenesis can have a major impact on results from previous studies using classical behavioral experiments, in which animals are tested in a--for the animal--novel experimental set-up. The neurogenesis-novelty association found here is also a necessary link in the relation that has been suggested to exist between neurogenesis and psychiatric disorders marked by a failure to cope with novelty., (© 2015 Wiley Periodicals, Inc.)

Recent research indicates that longstanding temporal lobe epilepsy (TLE) is associated with extratemporal, i.e. parietal cortex damage. We investigated egocentric and allocentric memory by use of first-person large-scale virtual reality environments in patients with TLE. We expected that TLE patients with parietal cortex damage were impaired in the egocentric memory task. Twenty-two TLE patients with hippocampal sclerosis (HS) and 22 TLE patients without HS were compared with 42 healthy matched controls on two virtual reality tasks affording to learn a virtual park (allocentric memory) and a virtual maze (egocentric memory). Participants further received a neuropsychological investigation and MRI volumetry at the time of the assessment. When compared with controls, TLE patients with HS had significantly reduced size of the ipsilateral and contralateral somatosensory cortex (postcentral gyrus). When compared with controls or TLE patients without HS, TLE patients with HS were severely impaired learning the virtual maze. Considering all participants, smaller volumes of the left-sided postcentral gyrus were related to worse performance on the virtual maze. It is concluded that the paradigm of egocentric navigation and learning in first-person large-scale virtual environments may be a suitable tool to indicate significant extratemporal damage in individuals with TLE., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Individuals with posttraumatic stress disorder (PTSD) display reduced hippocampus size and impaired cognition. However, studies on individuals with borderline personality disorder (BPD) are rare, and studies on trauma-exposed patients with BPD but without PTSD are lacking., Methods: Twenty-four trauma-exposed women with BPD (10 with PTSD and 14 without) and 25 healthy controls underwent 3-dimensional structural magnetic resonance imaging of the amygdala and hippocampus and a clinical and neuropsychological investigation., Results: Compared with controls, patients with BPD and PTSD displayed significantly reduced amygdala (34%) and hippocampus (12%) size and significantly impaired cognition. Trauma-exposed patients with BPD but without PTSD also showed significantly reduced amygdala (22%) and hippocampus (11%) size but normal cognition. Amygdala and hippocampus size did not differ significantly between patients with and without PTSD., Limitations: The sample sizes of trauma-exposed groups are relatively small. A larger sample size may have revealed statistically significant differences in amygdala size between those with and without PTSD., Conclusion: Our results demonstrate strong amygdala size reduction in trauma-exposed patients with BPD with or without PTSD, much exceeding that reported for trauma-exposed individuals without BPD. Our data suggest that BPD is associated with small amygdala size. Furthermore, evidence is increasing that amygdala and hippocampus size reduction is not only due to PTSD, but also to traumatic exposure.

We examined memory-related activity within to-be-resected medial temporal lobe (MTL) structures in 12 epilepsy patients with PET before amygdalohippocampectomy and studied the reallocation of memory functions to the contralateral MTL before and after surgery. Learning tasks were designed to activate predominantly the right or left MTL. Those patients who significantly activated to-be-resected ipsilateral MTL structures during the ipsilateral learning task (i.e. the left MTL during verbal learning or the right MTL during nonverbal learning) experienced a postoperative memory decline. Preoperative activation in the contralateral MTL during the ipsilateral learning task positively correlated with the postoperative outcome for ipsilateral memory. There was no significant postoperative reallocation of ipsilateral memory functions to the contralateral MTL.

Previous behavioral studies (Minichiello et al., Neuron 1999;24:401-414) showed that mice deficient for the TrkB receptor in the forebrain were unable to learn a swimming navigation task with an invisible platform and were severely impaired in finding a visible platform in the same setup. Likewise, additional behavioral deficits suggested a malfunction of the hippocampus and proximally connected forebrain structures. In order to discriminate whether the behavioral impairment was caused either by deficits in spatial memory and learning, or alternatively by loss of behavioral flexibility, 8 trkB mutant, 13 wild-type, and 22 heterozygous mice were implanted with transponders and released for 21 days into a large outdoor pen (10 x 10 m). The enclosure contained 2 shelters and 8 computer-controlled feeder boxes, delivering food portions for every mouse only during their first visit. Every third day, mice received food ad libitum inside the shelters. All mice learned to patrol the boxes correctly within a few days. However, significant differences emerged during those days with free food available. Wild-type mice remained inside the shelters, while all homozygous mutants continued to patrol the boxes in their habitual way, the heterozygous mutants showing intermediate scores. These and previous data suggest that one of the natural functions of the mouse hippocampus is to comediate behavioral flexibility, and that TrkB receptors might play an essential role in maintaining the neuronal short-term plasticity necessary for this capacity.

The ability of neurons to display more than a single GABA(A)-receptor subtype per cell requires intricate targeting mechanisms. Analysis by confocal laser scanning microscopy revealed that the alpha2- and alpha5-subunits differed strikingly in their subcellular distribution in hippocampal pyramidal cells and olfactory bulb granule cells, while the distribution of the gamma2-subunit was rather uniform. In mutant mice lacking the alpha5-subunit gene due to a chromosomal deletion, the absence of the alpha5-subunit was accompanied by a corresponding decrease of the gamma2-subunit immunoreactivity. In striking contrast, the subcellular distribution of the alpha2-subunit was unchanged in these mutant mice. These findings indicate that the assembly of distinct GABA(A)-receptor subtypes in the same neuron is regulated independently. Furthermore, the alpha-subunit is a prime candidate for providing domains which direct subcellular targeting.

The time- and voltage-dependent properties of a slowly inactivating K+ current were investigated by using the single-electrode current- and voltage-clamp recording technique in CA3 hippocampal cells of organotypic slice cultures. After a period of prolonged hyperpolarization, the onset of action-potential discharge in response to depolarizing current injection was delayed by several seconds. The conductances underlying this delay were identified in voltage-clamp recordings. A biphasically decaying outward current was evoked when the membrane potential was stepped back to -60 mV after a 30 sec period of hyperpolarization. The fast component was identified as the previously described D-current and was blocked by 100 microM 4-aminopyridine (4-AP). The slow component, which we refer to as IK(slow), appeared to be mediated by K+ ions, because its reversal potential shifted in a Nernstian manner with changes in extracellular K+ concentration. It decayed with a time constant of 7.5 sec and required a hyperpolarizing prepulse below -95 mV for 5.5 sec for 50% recovery from inactivation. IK(slow) was found to be voltage-dependent, with 50% activation occurring at -65 mV and 50% steady-state inactivation occurring at -84 mV. It displayed minimal or no sensitivity to the K(+)-channel blockers 4-AP (0.1-5 mM), Cs+ (1 mM), tetraethylammonium (10-50 mM), Ba2+ (1 mM), dendrotoxin-alpha (5-10 microM), charybdotoxin (0.5-2.5 microM), or glibenclamide (5-10 microM) and was not affected by preventing increases in intracellular Ca2+ concentration with Ca2+ chelators. IK(slow) was reduced by activation of metabotropic glutamatergic and cholinergic receptors. In summary, the biophysical characteristics of IK(slow) suggest a role in determining discharge onset after a period of membrane hyperpolarization, and its modulation by G-protein-coupled receptors reveals an additional function for these receptors in the control of cellular excitability.

1. Gamma-aminobuturic acid-B (GABAB) and adenosine A1 receptors, which are expressed in hippocampal pyramidal cells, are linked to pertussis toxin-sensitive G-proteins known to be coupled negatively to the enzyme adenylyl cyclase. This study investigates the electrophysiological consequences of adenylyl cyclase inhibition in response to stimulation of these receptors. 2. Single-electrode voltage-clamp recordings were obtained from CA3 pyramidal cells in rat hippocampal slice cultures in presence of tetrodotoxin. The calcium-dependent potassium current (IAHP), which is very sensitive to intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP), was used as an electrophysiological indicator of adenylyl cyclase activity. 3. Application of baclofen (10 microM), a selective agonist at GABAB receptors, or adenosine (50 microM) each resulted in a transient decrease followed by a significant enhancement in the amplitude of evoked IAHP. The initial reduction in amplitude of IAHP probably reflects inadequacies in voltage clamp of electronically distant dendritic sites, due to the shunting caused by concomitant activation of potassium conductance by baclofen/adenosine. Comparable increases in membrane conductance in response to the GABAA agonist, muscimol, caused a similar reduction in IAHP. The enhancement of IAHP is consistent with an inhibition of constitutively active adenylyl cyclase. 4. The receptor mediating the responses to adenosine was identified as belonging to the A1 subtype on the basis of its sensitivity to the selective antagonist 8-cyclopentyl-1,3-dipropylxanthine.(ABSTRACT TRUNCATED AT 250 WORDS)

The identification of a large variety of GABAA receptor subunits by molecular cloning suggests the existence of multiple receptor subtypes differing in localization and functional properties. In the present study we analysed immunohistochemically the cellular distribution of GABAA receptors containing the alpha 1 subunit in the rat hippocampus with a subunit-specific antiserum. Prominent staining of numerous interneurons was evident in Ammon's horn and the dentate gyrus, which contrasted with moderate and diffuse immunoreactivity in the dendritic layers of pyramidal and granule cells. Double immunofluorescence staining with antibodies to GABA revealed that a subset of GABAergic neurons in the hippocampus were immunoreactive for the alpha 1 subunit. To determine whether these cells represent distinct subpopulations of interneurons, we analysed the co-localization of the GABAA receptor alpha 1 subunit with selective markers of hippocampal interneurons (selected calcium-binding proteins and neuropeptides). In both Ammon's horn and the dentate gyrus, all parvalbumin-positive neurons and 50% of calretinin-positive neurons were double-labelled, whereas interneurons containing calbindin-D28k were devoid of alpha 1 subunit staining. Similarly, most neurons positive for neuropeptide Y and a subset of somatostatin-positive cells were double-labelled, in contrast to cholecystokinin- and vasoactive intestinal peptide-containing cells, which lacked the alpha 1 subunit staining. These results demonstrate cell-specific expression of GABAA receptors containing the alpha 1 subunit among subsets of hippocampal interneurons, pointing to a pronounced functional specialization of these cells. Furthermore, the prominent expression of GABAA receptors by interneurons suggests that disinhibition may be of major functional relevance in regulating the balance between excitation and inhibition in hippocampal circuits.

Interactions between metabotropic glutamate and N-methyl-D-aspartate (NMDA) receptor-mediated responses were investigated in hippocampal CA3 cells using the single electrode voltage-clamp method. Bath application (2.5-10 microM, 30 s) or iontophoresis of 1-amino-cyclopentyl-trans-1S,3R-dicarboxylate (ACPD), a selective agonist for metabotropic glutamate receptors, resulted in an inward current associated with a decrease in membrane conductance. Following transient bath application of NMDA (5-10 microM, 30-60 s), the ACPD-induced inward current was potentiated for a period of up to 25 min (by 61 +/- 8% with bath application, by 32 +/- 15% with iontophoresis). Transient application of NMDA did not result in a potentiation of ionotropic RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or metabotropic muscarinic responses. ACPD responses were not potentiated following transient AMPA application. Intracellular buffering of calcium with tetrapotassium bis(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) prevented potentiation by NMDA in all cells. Bath application of arachidonic acid did not mimic the NMDA-induced potentiation. These results demonstrate that activation of NMDA receptors can specifically induce a long-lasting potentiation of a metabotropic glutamatergic response in hippocampal CA3 pyramidal cells. The characterization of this interaction may contribute to the elucidation of the physiological significance of metabotropic glutamate receptors.

Individual differences in the extent of the infrapyramidal mossy fiber projection (IIP-MF) correlate with performance in tasks sensitive to hippocampal lesions, notably two-way avoidance, radial maze learning, and swimming navigation. Previous studies of swimming navigation suggested that the capacity of reversal learning and measures of directionality might also be related to asymmetries in the distribution of the IIP-MF. In order to verify these findings, the authors crossed the Collins High- and Low-lateralized mice (known to differ in mossy fiber morphology and brain asymmetries) and obtained a F2-generation characterized by strong individual differences in these traits. Twenty-three (13 females, 10 males) mice were tested during 3 days for acquisition of swimming navigation (16 trials) toward a central platform, and during two days (12 trials) for their capacity of reversal learning toward a shifted platform. Morphometry of Timm-stained hippocampi revealed several, partially independent correlations: Larger IIP-MF projections were associated with prolonged crossing over the former platform position during the entire reversal learning; larger IIP-MF projections on the left were correlated with more precise crossing of the former platform position during the first 45 seconds of reversal learning; both extent and asymmetry of IIP-MF correlated positively with overnight improvement of reversal learning; the size of the entire mossy fiber projection (CA4, suprapyramidal and IIP-MF) correlated positively with the time spent in the platform quadrant and measures of initial orientation during acquisition of the task; and the mice showed an ipsilateral turning bias (spin) toward the side with the larger mossy fiber projection. The authors conclude that an intact hippocampus mediates differential processes underlying swimming navigation, and that left and right subfields may have differential functions.

The effects of a novel antagonist of metabotropic glutamate receptors were investigated in CA3 pyramidal cells in hippocampal slice cultures of the rat. Earlier experiments showed that selective activation of metabotropic glutamate receptors with low concentrations of an agonist, 1S, 3R-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD), induced an inward current associated with a decrease in membrane conductance and inhibition of the slow calcium-dependent potassium current. These responses were strongly and reversibly reduced by the antagonist, (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG, 0.5-1 mM). In the presence of antagonists of ionotropic glutamate receptors, stimulation of the afferent mossy fibres evoked postsynaptic responses in CA3 pyramidal cells which paralleled those observed with exogenously applied metabotropic glutamate receptor agonists, i.e. a slow inward current and a reduction of calcium-dependent potassium current. Both responses were greatly reduced by bath-applied MCPG (1 mM). These results show that MCPG acts as an effective antagonist at metabotropic glutamate receptors coupled to potassium conductances in the hippocampus. Furthermore, they confirm that glutamate release from presynaptic terminals can modulate postsynaptic properties by activation of metabotropic glutamate receptors.

Presynaptic receptors for virtually all transmitters have been identified throughout the nervous system. Recent studies in the hippocampus provide new insights into the mechanisms by which the activation of these receptors leads to presynaptic inhibition of transmitter release, and characterize the second messengers involved in coupling presynaptic receptors to their effectors. Presynaptic receptors also provide a tractable route via which the amount of transmitter release may be selectively regulated in therapeutically useful ways.

A depolarization-induced, slowly decaying inward current was examined in slice-cultured CA3 pyramidal cells by voltage-clamp techniques and microfluorometric measurements of cytosolic free Ca2+ concentration ([Ca2+]i). Action potentials elicited by intracellular injection of short-lasting (50-100 ms) depolarizing current pulses were followed by a slowly decaying afterhyperpolarization (AHP). After switching to voltage-clamp mode, short-lasting (50-100 ms) depolarizing voltage jumps from -60 mV to between -30 and 0 mV induced a slowly decaying outward aftercurrent (IAHP) which was depressed by bath application of muscarine (0.5 microM). In the presence of muscarine, the same depolarizations induced a slowly decaying afterdepolarization (ADP) or inward aftercurrent (IADP) in voltage-clamp mode. This current was also induced in the presence of trans(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD, 5 microM), an agonist of metabotropic glutamate receptors, but not in the presence of noradrenalin (5 microM), while both of these agonists depressed IAHP. IADP was depressed by reducing the external Ca2+ concentration from 3.8 to 0.5 mM, by external Co2+ (1 mM) and by external Cd2+ (10-100 microM). Combined voltage-clamp recordings and microfluorometric measurements of [Ca2+]i using the Ca2+ indicator fura-2 revealed that the amplitude of IADP was correlated with the amplitude of depolarization-induced Ca2+ influx. IADP was absent at membrane potentials < -90 mV, and reached maximal amplitudes at approximately -55 mV. Raising the extracellular K+ concentration from 2.7 to 13.5 mM increased the amplitude of IADP and resulted in a positively directed shift of the apparent reversal potential of IADP. When the external Na+ concentration was reduced from 157 to 33 or 18 mM the current reversed at more negative potentials and was reduced to 40 and 21%, respectively, of control amplitude. Lowering the external CI- concentration from 159 to 20 mM did not affect IADP. We conclude that IADP most likely represents a Ca(2+)-activated cation current, rather than a Ca2+ tail current, or an electrogenic Ca2+ extrusion current.

We have developed an in vitro model of chronic epilepsy in order to study the consequences of prolonged periods of epileptic activity. After applying the convulsants bicuculline and/or picrotoxin to mature rat hippocampal slice cultures for 3 days, large numbers of swollen and vacuolated cells were observed throughout all hippocampal subfields. The number of dendritic spines of pyramidal cells was massively reduced. These changes were similar to those observed previously in post-mortem studies of hippocampal tissue from human epilepsy patients. Intracellular recordings from CA3 pyramidal cells revealed that spontaneous synaptic activity was greatly reduced in treated cultures. gamma-Aminobutyric acid-mediated inhibition was apparently not affected by sustained convulsant activity, although synaptic excitation was markedly depressed. Acute re-application of bicuculline to treated cultures elicited, upon stimulation of the mossy fibre tract, a typical interictal burst lasting several hundred milliseconds, with a wave form similar to those occurring in untreated cultures, but of a shorter duration. In contrast, ictal bursts (lasting tens of seconds), which always occur spontaneously in control cultures during initial perfusion of bicuculline, were not observed in treated cultures. These pathological changes were reversible when treated cultures were returned to normal medium for 1 week. The surviving cells had a healthy morphology and a normal complement of dendritic spines. Spontaneous synaptic activity was normal, and ictal bursts occurred spontaneously upon perfusion of bicuculline. The findings suggest that the morphological and functional changes are a consequence, rather than a direct cause of epilepsy.

The determination of the physiological role of glutamatergic metabotropic actions has been hampered by the lack of potent and specific antagonists. It has recently been reported that 4C3HPG (RS-4-carboxy-3-hydroxyphenylglycine) can antagonize metabotropic responses in the central nervous system. The effects of 4C3HPG on metabotropic responses evoked by trans-ACPD were investigated in CA3 pyramidal cells in hippocampal slice cultures. Our results show that in hippocampus 4C3HPG fails to antagonize responses mediated by metabotropic glutamate receptors.

1. Depolarization-induced elevations of intracellular calcium concentration ([Ca2+]i) were examined in slice-cultured hippocampal pyramidal and nonpyramidal cells of the CA3 region by combined intracellular and multisite fura-2 recording techniques. 2. In pyramidal cells, spiking activity induced by depolarizing current pulses (200-800 ms) induced transient elevations of somatic as well as of proximal dendritic [Ca2+]i. The calcium signals from the proximal dendrites were larger in amplitude and decayed much faster than those from the soma. Depolarization of presumed interneurons induced comparable somatic and dendritic calcium transients, which decayed faster than those observed in pyramidal cell somata. 3. The calcium transients of pyramidal cells, but not those of nonpyramidal cells, were associated with a slow afterhyperpolarization (sAHP), whose time course was correlated with that of the somatic calcium signal. We conclude that the lack of a sAHP in non-pyramidal cells cannot be explained by the absence of an efficient rise in [Ca2+]i but rather by the absence of the potassium conductance underlying the sAHP in pyramidal cells.

This study examined the release of endogenous amino acids from acute hippocampal slices, upon stimulation of the Schaffer collateral-commissural fibres. One-minute samples of superfusate were collected via a cannula placed over the CA1 stratum radiatum, and were analysed by reversed-phase high performance liquid chromatography. Evoked potentials were recorded to ascertain stimulation efficacy. Four minutes of continuous 50 Hz stimulation produced a tetrodotoxin-sensitive release of aspartate and glycine in the second minute of stimulation, as well as a tetrodotoxin-sensitive release of cysteine sulphinic acid, during stimulation and of homocysteic acid, following stimulation. Such 50 Hz stimulation also produced a tetrodotoxin-insensitive decrease in methionine levels, but no significant changes in any of the other 15 amino acids measured. Four minutes of continuous 1 Hz stimulation produced no changes in the levels of any of the amino acids measured, but four 600-ms trains of 100 Hz stimulation, which, unlike the 1 Hz stimulation, produced long-term potentiation, resulted in significant increases in levels of cysteine sulphinic acid and homocysteic acid, but not of any of the other amino acids measured. These results suggest that aspartate, glycine, homocysteic acid, and cysteine sulphinic acid play a role in synaptic transmission in the Schaffer collateral-commissural fibres, and that cysteine sulphinic acid and homocysteic acid may be released specifically by high-frequency stimulation.

Divalent metals such as cobalt are frequently used by neurophysiologists to prevent synaptic transmission, because they are thought to selectively block presynaptic calcium conductance. Recording intracellularly from hippocampal CA3 pyramidal cells we show that Co2+ (2 mM) is not specific in this action but also diminishes postsynaptic responses mediated by agonists acting at ionotropic and metabotropic glutamatergic receptors, as well as GABAA, GABAB, adenosine, and cholinergic receptors. These findings indicate that a more selective substance should be employed for experiments where neurotransmitter release must be blocked.

1. Intracellular recording techniques were used to study synaptic potentials in CA3 pyramidal cells elicited with mossy fibre stimulation in partially disinhibited hippocampal slice cultures. Two experimental protocols were used: (1) high concentrations (20-40 microM) of the A-type gamma-aminobutyric acid (GABAA) receptor antagonist bicuculline plus low concentrations (2-4 microM) of the glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), or (2) low concentrations (1-2.5 microM) of bicuculline alone. 2. Under the first condition, stimulation of mossy fibre afferents evoked epileptic bursts alternating with a response consisting of an excitatory postsynaptic potential (EPSP) followed by an unusually large and long-lasting hyperpolarizing potential with a maximal amplitude in the range of -30 mV from the resting membrane potential. 3. This paroxysmal inhibitory potential (PIP) had a reversal potential near that of potassium. The amplitude of the PIP was not dependent on action potentials superimposed on the preceding EPSP, and was present in cells recorded with microelectrodes containing the Ca2+ chelator EGTA. These data suggest that the PIP is not a Ca(2+)-activated K+ potential. 4. The PIP was prolonged by the GABA-uptake blocker nipecotic acid, was reduced by hyperpolarizing interneurons with the opioid agonist FK 33-824, and was abolished by the GABAB-receptor antagonist CGP 35 348. These data indicate that the PIP is mediated by the activation of GABAB receptors following GABA release from interneurons. 5. The NMDA-receptor antagonist D-2-amino-5-phosphonovalerate (D-APV) strongly reduced the amplitude of the PIP, but had no effect on the GABAB receptor-mediated inhibitory postsynaptic potential (IPSP) under control conditions. 6. Under the first condition, regular stimulation elicited a cyclical pattern of evoked responses. There was either an alternation between an epileptic burst and a PIP or, at shorter interstimulus intervals, a sequence of gradually increasing PIPs followed by an epileptic burst, which then reset the cycle. 7. Under the second condition, in low concentrations of bicuculline alone, the early GABAA-mediated IPSP was little affected, but the late GABAB-mediated IPSP was greatly enhanced. These enhanced late IPSPs were comparable in amplitude and duration to the PIPs seen under the first conditions, could exhibit cyclical behaviour, and were reduced by D-APV. 8. Application of CGP 35 348 abolished the late IPSP under control conditions, but had no effect on hippocampal excitability. In contrast, CGP 35 348 blocked the PIP elicited in low bicuculline, and consequently led to intense epileptic discharge.(ABSTRACT TRUNCATED AT 400 WORDS)

The extent of the infrapyramidal mossy fiber projection in CA3 (IIP-MF) at the midseptotemporal level correlates negatively with two-way avoidance learning and positively with performance in the radial maze, both tasks known to be sensitive to hippocampal lesions. If hippocampal structural variations are causing behavioral variations, one must predict positive correlations between the extent of the IIP-MF and performance in swimming navigation. Thus, the authors studied learning and reversal learning of swimming navigation in mice in which the size of the IIP-MF had been randomized by means of systematic crosses and in 2 mouse strains known for differential infrapyramidal projections (C57BL/6 and DBA/2). In 19 random-bred mice (9 male, 10 female), the extent of the IIP-MF showed negative correlations with swimming time after platform reversal (day 4: r = -0.50, P < .03; day 5 r = -0.73, P < .001), but none during acquisition of the task. In addition, statistical analysis suggested an influence of asymmetrically distributed mossy fiber projections during reversal learning. The strain comparison between 18 DBA/2 and 16 C57BL/6 male mice confirmed these results: no strain difference during days 1-3, and a significantly faster swimming time in the strain C57BL/6 (with large IIP-MF) at day 5 (second day of reversal), associated with significantly more crossings of the former platform location during the early phases of reversal learning. This latter measure was also negatively correlated with asymmetry of the IIP-MF in both strains. Finally, variations of the IIP-MF were correlated partially with adjustment of swimming speed that appeared to depend on size and asymmetry of CA4 as well. Thus, natural variations in the size of the IIP-MF distribution, and, perhaps, of CA4, appear to linearly influence processes directly involved in complex spatial learning.

Glutamate (GLU) mediates its 'fast' excitatory transmitter action in the brain by directly gating cation-selective ion channels ('ionotropic' receptors). However, GLU can also activate another type of receptor, coupled to phospholipase C ('metabotropic' receptor). In hippocampal cells, stimulation of this metabotropic receptor by GLU, or by a racemic mixture of (1S-3R and 1R-3S) 1-aminocyclopentyl-1,3-dicarboxylate (ACPD), induces a slower excitation mediated by inhibition of K+ currents. We have assessed whether this slow form of metabotropic receptor excitation can contribute to the effects of synaptically released GLU in hippocampal slice cultures, by recording the responses of CA3 pyramidal cells to afferent mossy fibre stimulation. When the fast ionotropic response was blocked pharmacologically, mossy fibre stimulation produced a slow depolarizing postsynaptic potential associated with a decrease in membrane conductance, a depression of the slow after-hyperpolarization following a train of action potentials, and reduced accommodation during the action potential train. Under voltage-clamp, mossy fibre stimulation produced a slow voltage-dependent inward current which resembled that produced by application of exogenous ACPD or quisqualate (QUIS), and which was occluded by these metabotropic agonists. We therefore suggest that synaptically released GLU can induce two types of postsynaptic responses: a fast excitation through activation of ionotropic receptors and a slower excitation associated with inhibition of K+ conductances through activation of metabotropic receptors. This is analogous to the dual action of acetylcholine on ionotropic (nicotinic) and metabotropic (muscarinic) receptors.

In co-cultures prepared from the septum and the hippocampus, cholinergic fibers originating in the septal slices grew into the neighboring hippocampal tissue and established functional cholinergic connections with pyramidal cells. To get further insight into the mechanisms governing cholinergic fiber growth, we have added TTX to the growth medium (2 x 10(-7) M) to block propagated electrical activity. Under these conditions, considerably fewer cholinergic cells appeared to survive. A few cholinergic fibers still invaded hippocampal target tissue, but their number was markedly reduced compared with control cultures. Simultaneous application of NGF together with TTX, however, not only increased enzyme levels and enhanced survival of cholinergic neurons, but also led to hippocampal ingrowth in virtually all septo-hippocampal co-cultures. These data, therefore, suggest, that in the absence of spiking activity, cholinergic fibers are capable of growing into a co-cultured target tissue. To test the specificity of growth of septal cholinergic fibers, we have co-cultured septal slices with slices of various brain areas which in situ lack a major cholinergic innervation, in particular the cerebellum. In the vast majority of such co-cultures, cholinergic fibers remained restricted within the septal slices, without innervating cerebellar tissue. This failure might in part be related to the lack of trophic factors released by the target tissue. We have, therefore, grown septo-cerebellar cultures in the presence and absence of NGF. Following application of 100 ng/ml NGF during the entire growth of the cultures, numerous AChE-positive fibers originating in the septal slices invaded the co-cultured cerebellar slices.(ABSTRACT TRUNCATED AT 250 WORDS)

To investigate the relation between perforant path and the pattern of glutamate-like immunoreactivity in its target regions in the rat hippocampal formation, unilateral lesions of various size and location were placed to interrupt certain contingents of these afferent fibers. Postembedding immunohistochemistry at the level of light microscopy yielded the same pattern of immunoreactivity in the hippocampal formation contralateral to the lesion as in untreated animals. On the ipsilateral side, however, extensive transections of the perforant path led to a drastic loss of glutamate-immunoreactive terminal-like elements in the outer part of the dentate molecular layer. More restricted lesions induced a loss of punctate glutamate-like immunoreactivity in narrower bands within this zone. The width and the location of the affected bands appeared to depend on the extent of the transections and their topographical relation to the perforant path fiber system. These results and those obtained using a postembedding immunogold method at the level of electron microscopy strongly indicate that perforant path terminals in the dentate molecular layer of the rat contain high levels of glutamate and, thus, provide further support for an already well-documented role of this excitatory amino acid as neurotransmitter in this system.

Excitatory amino acids mediate fast synaptic transmission in the central nervous system through the activation of at least three distinct ionotropic receptors: N-methyl-D-aspartate (NMDA), the alpha-amino-3-hydroxy-5-methyl-isoxasole-4-propionate (AMPA)/quisqualate (QUIS) and the kainate subtypes (for reviews, see refs 1, 2). They also activate the additional QUIS 'metabotropic' receptor (sensitive to trans-1-amino-cyclopentyl-1,3-dicarboxylate, ACPD) linked to inositol phospholipid metabolism. We have used hippocampal slice cultures to study the electrophysiological consequences of the metabotropic response. We find that activation of an ACPD-sensitive QUIS receptor produces a 'slow' excitation of CA3 pyramidal cells, resulting from depression of a Ca2(+)-dependent K+ current and a voltage-gated K+ current. Combined voltage-clamp and microfluorometric recordings show that, although these receptors can trigger an increase in intracellular Ca2+ concentration, suppression of K+ currents is independent of changes in intracellular Ca2+. These effects closely resemble those induced by activating muscarinic acetylcholine receptors in the same neurons and suggest that excitatory amino acids not only act as fast ionotropic transmitters but also as slow neuromodulatory transmitters.

The 24-hr sleep-wake distribution and power spectra of the electroencephalogram were determined in rabbits that had been implanted with cortical and hippocampal electrodes. A diurnal preference for sleep was observed. The spectral power density in nonrapid eye movement sleep (NREM sleep) of the cortex showed a decreasing trend in most frequencies within the 12-hr light period. In the 12-hr dim period no clear trend was present. Most hippocampal EEG frequencies decreased in NREM sleep in the first two hours of the light period, and thereafter stayed on a constant level. Sleep deprivation elicited the following changes: a prolonged increase of NREM sleep and a short increase of REM sleep; in the cortex, an increase of slow-wave activity (SWA; power density in the 0.25-2.0 Hz frequency band) in NREM sleep, which declined in the course of recovery; an enhancement of slow-wave (1.25-3 Hz) and theta (6.25-7 Hz) activity in REM sleep. The hippocampus showed an increase in NREM sleep power density in almost all frequencies. In REM sleep the hippocampus exhibited an increase in power density in the 6.25-7 Hz and 12.25-13 Hz bands, whereas in the 7.25-8 Hz band the values were below baseline. The results show that SWA in NREM sleep and theta activity in REM sleep are enhanced by sleep deprivation, as has been observed in other mammalian species. The EEG changes in the hippocampus resembled those in the cortex.

Combined intracellular and microfluorometric recording techniques were used to evaluate whether the inhibition by cholinergic or adrenergic transmitters of the Ca2(+)-activated potassium current (IAHP) in hippocampal CA3 pyramidal cells was mediated by an alteration of depolarization-induced change in cytosolic free Ca2+ concentration [(Ca2+]i). Low concentrations of isoproterenol (1-10 microM) and muscarine (0.25-1 microM) reversibly abolished IAHP without affecting concomitant Ca2+ transients or the steady-state [Ca2+]i. Only after application of higher concentrations of muscarine, [Ca2+]i increased; in the presence of potassium channel blockers, muscarine depressed Ca2+ currents and concomitant Ca2+ transients. These observations provide direct evidence that the inhibition of IAHP by isoproterenol and muscarine are not mediated by an alteration of Ca2+ dynamics.

The dynamics of cytosolic free Ca2+ ([Ca2+]i) of single voltage-clamped CA3 pyramidal cells in hippocampal slice cultures is reviewed. [Ca2+]i amounts to about 30 nM at resting membrane potential and increases slowly when the membrane potential is clamped at more positive values (up to 500 nM at -30 mV). Short lasting depolarizations (40-100 ms) induce a transient rise in [Ca2+]i which activates a slow aftercurrent (IAHP). The muscarinic or beta-adrenergic depression of IAHP is not accompanied by any change in the dynamics of Ca2+ and appears, therefore, to result primarily from an inhibition of the K(+)-current itself or of the ability of Ca2+ to activate the current. At higher concentrations than those required to inhibit IAHP, muscarine produces a pronounced inward current and this is accompanied by a rise in resting [Ca2+]i concentration.

Although there is good evidence favoring L-glutamate as a major excitatory amino acid transmitter, relatively little is known about the distribution of nerve terminals using this substance. A method visualizing glutamate-like immunoreactivity at the light microscopic level by means of a monoclonal antibody, mAb 2D7, is described. --The antigen used for immunization was a glutaraldehyde-linked glutamate-BSA conjugate, and hybridomas were differentially screened by ELISA for production of antibodies recognizing glutamate- but not aspartate-BSA. The crossreactivity of 'anti-glutamate' mAb 2D7 as estimated in absorption tests was low even with conjugates closely related to glutamate-BSA.--Semithin sections from rapidly perfusion-fixed, plastic-embedded rat brain tissues were etched and stained by a combination of the peroxidase-antiperoxidase method and silver enhancement of the diaminobenzidine reaction product. Only this amongst several other immunohistochemical methods tried produced labeling patterns which showed terminal-like elements in brain regions such as olfactory bulb, hippocampus and cerebellum, and which were mostly consistent with already available information on systems using glutamate as neurotransmitter. Particularly striking was the staining of elements reminiscent of mossy fiber terminals in hippocampus and cerebellum as well as of cerebellar parallel fiber terminals.

1. Single-electrode voltage-clamp recordings were made from CA3 pyramidal cells in organotypic hippocampal slice cultures for measurement of membrane currents underlying both the gamma-aminobutyric acid (GABA)-mediated, Cl- -dependent inhibitory postsynaptic potential (IPSC), evoked in response to stimulation of the mossy fiber pathway, and responses to iontophoretically applied GABA. Their reversal potentials are presumed to equal the equilibrium potential for Cl- (37). Mechanisms underlying activity-dependent increases in the intracellular concentration of Cl- ([Cl-]i) were investigated by describing active and passive pathways for Cl- influx and efflux. 2. During 99-s applications of GABA, driving force declined by 51% due to increases in [Cl-]i; thus passive Cl- influx through GABA-activated pathways can significantly affect [Cl-]i. 3. Decreasing the extracellular K+ concentration ([K+]o) from 5.8 to 1 mM caused a rapid hyperpolarizing shift in the mean IPSC reversal potential (EIPSC) from -67.6 to -81.9 mV, even when membrane potential (Vm) was maintained constant and depolarized with respect to EIPSC. 4. Decreasing [K+]o from 5.8 to 1 mM caused a rapid hyperpolarizing shift in the mean GABA reversal potential (EGABA) from -64.7 to -81.1 mV, even when Vm was maintained constant and depolarized with respect to EGABA. Reducing the extracellular Cl- concentration from 153 to 89 mM, while maintaining [K+]o constant at 1 mM, shifted the mean EGABA from -81.1 to -66.2 mV, an amount close to that predicted by the Nernst equation for Cl-. We conclude that reducing [K+]o caused a hyperpolarizing shift in EGABA and EIPSC by decreasing [Cl-]i. 5. The shift of EIPSC and EGABA upon alteration of [K+]o did not result from contamination of the responses by additional K+-mediated components because it was unaffected by block of K+ channels with intracellular Cs+. 6. Reducing the extracellular Na+ concentration from 141 to 70 mM had no effect on EGABA. 7. Furosemide, bath-applied at 5 X 10(-4) M while holding Vm depolarized with respect to EIPSC, caused a rapid, reversible decrease in IPSC driving force averaging 69%, consistent with the presence of a furosemide-sensitive outward Cl- -transport system. 8. Reducing [K+]o from 5.8 to 1 mM in the presence of 5 X 10(-4) M furosemide produced a smaller shift of EIPSC from -61.0 to -71.2 mV, however, after washout of furosemide from [K+]o = 1 mM saline, EIPSC shifted further to -89.8 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

The extent of the intra- and infrapyramidal mossy fiber projection (IIP-MF) in the hippocampus of mice and rats is strain-specific, and correlates negatively with the strain-specific capacity of avoidance learning. If variations of the IIP-MF influence the capacity for 2-way avoidance learning, then developmental modification of the IIP-MF projection in an individual member of a strain should remain correlated with its adult behavior. Pups of strains with high avoidance capacity and small IIP-MF projections (RHA rats, DBA/2 and BALB/c mice) were injected with varying doses of thyroxine during the postnatal period. This transient hyperthyroidism resulted in a strong, yet largely unpredictable, variability of the IIP-MF projection in the adult animals. Furthermore, postnatal saline injections also increased the variability of the IIP-MF projection; however, this was to a lesser degree than when using thyroxine. The animals were tested for 2-way avoidance learning at the age of 90-120 d. Many showed strain-atypical avoidance scores. These deviations from the inherited level of 2-way avoidance learning were strongly correlated with the magnitude of the IIP-MF projection, regardless of whether the structural changes resulted from thyroxine or saline injections. A multivariate analysis showed that the observed correlations could neither be explained by thyroxine-induced changes in brain weight nor by individual differences of other terminal fields in the hippocampal region CA3. These results suggest that the extent of the IIP-MF projection is influenced by several genetic and epigenetic factors. Irrespective of the underlying causes, the magnitude of the IIP-MF (or of an unknown but well-correlated variable) appears to bias the adult capacity for 2-way avoidance learning predictably.

1. Single-electrode voltage-clamp recordings were made from CA3 pyramidal cells in organotypic hippocampal slice cultures for measurement of membrane currents underlying both the gamma-aminobutyric acid (GABA)-mediated, Cl- -dependent inhibitory postsynaptic potential (IPSC), evoked in response to stimulation of the mossy fiber pathway, and responses to iontophoretically applied GABA. Pre- and postsynaptic mechanisms mediating activity-dependent reductions in the conductance underlying the IPSC (gIPSC) were investigated. 2. During 99-s applications of GABA, the mean evoked conductance (gGABA) decreased 43% with an initial time constant of 51 s. Desensitization was never complete. 3. Ca2+-influx, activated with depolarizing voltage commands of 100-ms to 15-s duration in the presence of intracellular Cs+, had no effect on GABA responses. 4. Iontophoretic application of the GABAA-receptor agonist muscimol caused a rapid decrease of 80-100% in the amplitude of IPSCs evoked at depolarized membrane potentials (Vm). Recovery was 80% complete in 30 s. The second of two paired applications of muscimol, delivered at the same iontophoretic intensity, was reduced in amplitude 35%. This was shown to result from a decrease in driving force rather than from desensitization. We conclude that muscimol decreases IPSCs by causing an increase in the intracellular Cl- concentration. 5. Iontophoretic application of the GABAB-receptor agonist (+/-)-baclofen caused a decrease of only 30% in the amplitude of IPSCs evoked at depolarized Vms. This effect outlasted the post-synaptic effects of baclofen; recovery was 80% complete between 60 and 90 s. 6. Bath application of (-)-baclofen was found to decrease gIPSC without affecting the IPSC reversal potential. This effect was rapid in onset, could be observed at concentrations as low as 1 X 10(-7) M, and recovered quickly. The EC50 was roughly 5 X 10(-7) M and appeared similar to that for the baclofen-activated increase in postsynaptic conductance. No effect on responses to iontophoretically applied GABA was observed, demonstrating that baclofen decreases gIPSC by reducing presynaptic release via GABAB receptors. 7. Iontophoretic application of GABA reduced IPSCs in a dose-dependent manner. At low iontophoretic intensities, IPSCs were reduced only 30% and recovered slowly, as with baclofen iontophoresis. At higher iontophoretic intensities, IPSCs were more completely blocked. Recovery was initially fast, but took 60-90 s to be complete.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Intracellular recording techniques were used to investigate the mechanisms underlying the activity-dependent lability of inhibitory synaptic potentials indirectly evoked in CA3 pyramidal neurons by stimulation of the mossy fiber afferent pathway in organotypic slice cultures of hippocampus. 2. Repetitive stimulation (3-10 Hz, 30-60 s) was found to reduce the amplitude of the inhibitory postsynaptic potential (IPSP) and occasionally lead to repetitive, epileptiform discharge. 3. Under single-electrode voltage-clamp, the current underlying the inhibitory postsynaptic potential (IPSC) was found to have the same reversal potential (EIPSC) as the response to iontophoretically applied gamma-aminobutyric acid (EGABA), and both were blocked by bicuculline. Reducing the extracellular Cl- concentration from 153 to 89 mM shifted EGABA in the depolarizing direction by 9 mV from -64.7 to -55.6 mV, an amount close to that predicted by the Nernst equation. We therefore presume that the IPSC is mediated by GABA and that the reversal potentials of both are equal to ECl-. 4. Under single-electrode voltage-clamp, repetitive stimulation (3-10 Hz, 30-60 s) was found to cause a mean decrease in the conductance underlying the IPSC (gIPSC) of 22%. This decrease was independent of the membrane potential at which stimuli were delivered. 5. Under single-electrode voltage-clamp, repetitive stimulation (3-10 Hz, 30-60 s) was found to cause a 2-8 mV depolarizing shift in EIPSC when the membrane potential was held constant 5-15 mV depolarized from EIPSC. The mean decrease in IPSC driving force was 49%. If membrane potential was held 10-20 mV hyperpolarized from EIPSC, there was no change in driving force. 6. Currents activated by iontophoretically applied GABA were decreased in amplitude following repetitive stimulation at depolarized, but not hyperpolarized, holding potentials. 7. The decrease in IPSC driving force following repetitive stimulation at depolarized holding potentials was less after decreasing the extracellular K+ concentration from 5.8 to 1 mM. 8. We conclude that the decrease in driving force following repetitive stimulation results from an increase in the intracellular Cl- concentration, and that the activity-dependent decrease in gIPSC results from a decrease in presynaptic release rather than from postsynaptic receptor desensitization.

Organotypic cultures of histaminergic tuberomammillary (TM) neurons were grown using explants obtained from newborn rats. The cultures were examined after immunohistochemical localization of the histamine synthetic enzyme, L-histidine decarboxylase (HDC). The morphological properties of the somata, dendrites and axons of HDC-immunoreactive TM neurons in organotypic culture were virtually indistinguishable from those seen in situ. Extensive plexuses of HDC-immunopositive axons, including growth cones, were seen within the hypothalamus, the plasma surrounding the explant and co-cultured hippocampus. Organotypic cultures of TM histamine neurons, and co-cultures with their targets, provide a useful model system for studying several aspects of central histaminergic neurobiology.

The petit-mal seizures of the "tottering" mutant mouse (tg) have been attributed to an exaggerated noradrenergic projection from locus coeruleus to the telencephalon (Noebels 1984). In order to investigate the possible epileptogenic mechanisms involved, we have compared hippocampal slices from epileptic (tg/tg) and phenotypically healthy (tg/+) mice. Resting potentials, action potentials and afterpotentials, membrane impedances and time constants were not significantly different in 11 neurons from each group. Bath application of noradrenaline, isoproterenol and histamine or a transient exposure to Mg++-free medium caused a long lasting increase in extracellularly recorded population spikes induced in CA1 by electrical stimulation of stratum radiatum. Isoproterenol blocked the calcium dependent afterhyperpolarization and accommodation of firing. Tetanization of afferent fibres evoked post-tetanic potentiation and long-term potentiation. All these results are qualitatively similar to those previously described in rats and guinea pigs and have revealed no significant difference between tg/tg and tg/+ mice.