A dendrodendritic reciprocal synapse provides a recurrent excitatory connection in the olfactory bulb

Neuronal synchronization in the olfactory bulb has been proposed to arise from a diffuse action of glutamate released from mitral cells (MC, olfactory bulb relay neurons). According to this hypothesis, glutamate spills over from dendrodendritic synapses formed between MC and granule cells (GC, olfactory bulb interneurons) to activate neighboring MC. The excitation of MC is balanced by a strong inhibition from GC. Here we show that MC excitation is caused by glutamate released from bulbar interneurons located in the GC layer. These reciprocal synapses depend on an unusual, 2-amino-5-phosphonovaleric acid-resistant, N-methyl-d-aspartate receptor. This type of feedback excitation onto relay neurons may strengthen the original sensory input signal and further extend the function of the dendritic microcircuit within the main olfactory bulb.

Regional and cellular fractionation of working memory

This chapter recounts efforts to dissect the cellular and circuit basis of a memory system in the primate cortex with the goal of extending the insights gained from the study of normal brain organization in animal models to an understanding of human cognition and related memory disorders. Primates and humans have developed an extraordinary capacity to process information “on line,” a capacity that is widely considered to underlay comprehension, thinking, and so-called executive functions. Understanding the interactions between the major cellular constituents of cortical circuits—pyramidal and nonpyramidal cells—is considered a necessary step in unraveling the cellular mechanisms subserving working memory mechanisms and, ultimately, cognitive processes. Evidence from a variety of sources is accumulating to indicate that dopamine has a major role in regulating the excitability of the cortical circuitry upon which the working memory function of prefrontal cortex depends. Here, I describe several direct and indirect intercellular mechanisms for modulating working memory function in prefrontal cortex based on the localization of dopamine receptors on the distal dendrites and spines of pyramidal cells and on interneurons in the prefrontal cortex. Interactions between monoamines and a compromised cortical circuitry may hold the key to understanding the variety of memory disorders associated with aging and disease.

Involvement of the amygdala in memory storage: Interaction with other brain systems

There is extensive evidence that the amygdala is involved in affectively influenced memory. The central hypothesis guiding the research reviewed in this paper is that emotional arousal activates the amygdala and that such activation results in the modulation of memory storage occurring in other brain regions. Several lines of evidence support this view. First, the effects of stress-related hormones (epinephrine and glucocorticoids) are mediated by influences involving the amygdala. In rats, lesions of the amygdala and the stria terminalis block the effects of posttraining administration of epinephrine and glucocorticoids on memory. Furthermore, memory is enhanced by posttraining intra-amygdala infusions of drugs that activate β-adrenergic and glucocorticoid receptors. Additionally, infusion of β-adrenergic blockers into the amygdala blocks the memory-modulating effects of epinephrine and glucocorticoids, as well as those of drugs affecting opiate and GABAergic systems. Second, an intact amygdala is not required for expression of retention. Inactivation of the amygdala prior to retention testing (by posttraining lesions or drug infusions) does not block retention performance. Third, findings of studies using human subjects are consistent with those of animal experiments. β-Blockers and amygdala lesions attenuate the effects of emotional arousal on memory. Additionally, 3-week recall of emotional material is highly correlated with positron-emission tomography activation (cerebral glucose metabolism) of the right amygdala during encoding. These findings provide strong evidence supporting the hypothesis that the amygdala is involved in modulating long-term memory storage.

Differential synaptic localization of two major gamma-aminobutyric acid type A receptor alpha subunits on hippocampal pyramidal cells.

Hippocampal pyramidal cells, receiving domain specific GABAergic inputs, express up to 10 different subunits of the gamma-aminobutyric acid type A (GABAA) receptor, but only 3 different subunits are needed to form a functional pentameric channel. We have tested the hypothesis that some subunits are selectively located at subsets of GABAergic synapses. The alpha 1 subunit has been found in most GABAergic synapses on all postsynaptic domains of pyramidal cells. In contrast, the alpha 2 subunit was located only in a subset of synapses on the somata and dendrites, but in most synapses on axon initial segments innervated by axo-axonic cells. The results demonstrate that molecular specialization in the composition of postsynaptic GABAA receptor subunits parallels GABAergic cell specialization in targeting synapses to a specific domain of postsynaptic cortical neurons.

Long-lasting reduction of inhibitory function and gamma-aminobutyric acid type A receptor subunit mRNA expression in a model of temporal lobe epilepsy.

This study evaluated hippocampal inhibitory function and the level of expression of gamma-aminobutyric acid type A (GABAA) receptor mRNA in an in vivo model of epilepsy. Chronic recurrent limbic seizures were induced in rats using injections of pilocarpine. Electrophysiological studies performed on hippocampal slices prepared from control and epileptic animals 1 to 2 months after pilocarpine injections demonstrated a significant hyperexcitability in the epileptic animals. Reduced levels of mRNA expression for the alpha 2 and alpha 5 subunits of the GABAA receptors were evident in the CA1, CA2, and CA3 regions of the hippocampus of epileptic animals. No decrease in mRNA encoding alpha 1, beta 2, or gamma 2 GABAA receptor subunits was observed. In addition, no change in the mRNA levels of alpha CaM kinase II was seen. Selective decreases in mRNA expression did not correlate with neuronal cell loss. The results indicate that selective, long-lasting reduction of GABAA subunit mRNA expression and increased excitability, possibly reflecting loss of GABAergic inhibition, occur in an in vivo model of partial complex epilepsy.

Presence of mRNA for glutamic acid decarboxylase in both excitatory and inhibitory neurons.

Neurons in very low density hippocampal cultures that are physiologically identified as either GABAergic inhibitory or glutamatergic excitatory all contain mRNA for the gamma-aminobutyric acid (GABA) synthetic enzyme, glutamic acid decarboxylase (GAD), as detected by single cell mRNA amplification and PCR. However, consistent with the physiology, immunocytochemistry revealed that only a subset of the neurons stain for either GAD protein or GABA. A similar fraction hybridize with RNA probes for GAD65 and GAD67. Hippocampal CA1 pyramidal neurons in slice preparations, which are traditionally thought to be excitatory, also contain mRNA for GAD65 and GAD67. Hippocampal neurons in culture did not contain mRNA for two other neurotransmitter synthesizing enzymes, tyrosine hydroxylase, and choline acetyl transferase. These data suggest that in some neurons, presumably the excitatory neurons, GAD mRNA is selectively regulated at the level of translation. We propose that neurotransmitter phenotype may be posttranscriptionally regulated and neurons may exhibit transient phenotypic plasticity in response to environmental influences.

Mixed synapses discovered and mapped throughout mammalian spinal cord.

Previously, synaptic activity in the spinal cord of adult mammals was attributed exclusively to chemical neurotransmission. In this study, evidence was obtained for the existence, relative abundance, and widespread distribution of "mixed" (chemical and electrical) synapses on neurons throughout the spinal cords of adult mammals. Using combined confocal microscopy and "grid-mapped freeze fracture," 36 mixed synapses containing 88 "micro" gap junctions (median = 45 connexons) were found and mapped to 33 interneurons and motor neurons in Rexed laminae III-IX in cervical, thoracic, and lumbosacral spinal cords of adult male and female rats. Gap junctions were adjacent to presumptive active zones, where even small gap junctions would be expected to increase synaptic efficacy. Two morphological types of mixed synapse were discerned. One type contained distinctive active zones consisting of "nested" concentric toroidal deformations of pre- and postsynaptic membranes, which, because of their unusual topology, were designated as "synaptic sombreros." A second type had gap junctions adjacent to active zones consisting of broad, flat, shallow indentations of the plasma membrane. Morphometric analysis indicates that mixed synapses correspond to 3-5% of all synapses on the somata and proximal dendrites, but, because of their subcellular location and morphology, they could represent 30-100% of excitatory synapses. The relative abundance of mixed synapses on several classes of neurons in spinal cords of adult rats suggests that mixed synapses provide important but previously unrecognized pathways for bidirectional communication between neurons in the mammalian central nervous system.

Circuitry for color coding in the primate retina.

Human color vision starts with the signals from three cone photoreceptor types, maximally sensitive to long (L-cone), middle (M-cone), and short (S-cone) wavelengths. Within the retina these signals combine in an antagonistic way to form red-green and blue-yellow spectral opponent pathways. In the classical model this antagonism is thought to arise from the convergence of cone type-specific excitatory and inhibitory inputs to retinal ganglion cells. The circuitry for spectral opponency is now being investigated using an in vitro preparation of the macaque monkey retina. Intracellular recording and staining has shown that blue-ON/yellow-OFF opponent responses arise from a distinctive bistratified ganglion cell type. Surprisingly, this cone opponency appears to arise by dual excitatory cone bipolar cell inputs: an ON bipolar cell that contacts only S-cones and an OFF bipolar cell that contacts L- and M-cones. Red-green spectral opponency has long been linked to the midget ganglion cells, but an underlying mechanism remains unclear. For example, receptive field mapping argues for segregation of L-and M-cone signals to the midget cell center and surround, but horizontal cell interneurons, believed to generate the inhibitory surround, lack opponency and cannot contribute selective L- or M-cone input to the midget cell surround. The solution to this color puzzle no doubt lies in the great diversity of cell types in the primate retina that still await discovery and analysis.

7-Chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine S,S-dioxide (IDRA 21), a congener of aniracetam, potently abates pharmacologically induced cognitive impairments in patas monkeys.

We report here on the ability of IDRA 21 and aniracetam, two negative allosteric modulators of glutamate-induced DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor desensitization, to attenuate alprazolam-induced learning deficit in patas monkeys working in a complex behavioral task. In one component of a multiple schedule (repeated acquisition or "learning"), patas monkeys acquired a different four-response chain each session by responding sequentially on three keys in the presence of four discriminative stimuli (geometric forms or numerals). In the other component (performance) the four-response chain was the same each session. The response chain in each component was maintained by food presentation under a fixed-ratio schedule. When alprazolam (0.1 or 0.32 mg/kg p.o.) was administered alone, this full allosteric modulator of gamma-aminobutyric acid type A (GABAA) receptors produced large decreases in the response rate and accuracy in the learning component of the task. IDRA 21 (3 or 5.6 mg/kg p.o.) and aniracetam (30 mg/kg p.o.) administered 60 min before alprazolam, having no effect when given alone, antagonized the large disruptive effects of alprazolam on learning. From dose-response studies, it can be estimated that IDRA 21 is approximately 10-fold more potent than aniracetam in antagonizing alprazolam-induced learning deficit. We conclude that IDRA 21, a chemically unrelated pharmacological congener of aniracetam, improves learning deficit induced in patas monkeys by the increase of GABAergic tone elicited by alprazolam. Very likely IDRA 21 exerts its behavioral effects by antagonizing AMPA receptor desensitization.

Inhibitory influence of frontal cortex on locus coeruleus neurons.

The functional influence of the frontal cortex (FC) on the noradrenergic nucleus locus coeruleus (LC) was studied in the rat under ketamine anesthesia. The FC was inactivated by local infusion of lidocaine or ice-cold Ringer's solution while recording neuronal activity simultaneously in FC and LC. Lidocaine produced a transient increase in activity in FC, accompanied by a decrease in LC unit and multiunit activity. This was followed by a total inactivation of FC and a sustained increase in firing rate of LC neurons. Subsequent experiments revealed antidromic responses in the FC when stimulation was applied to the LC region. The antidromic responses in FC were found in a population of neurons (about 8%) restricted to the dorsomedial area, FR2. The results indicate that there is a strong inhibitory influence of FC on the tonic activity of LC neurons. The antidromic responses in FC to stimulation of the LC region suggest that this influence is locally mediated, perhaps through interneurons within the nucleus or neighboring the LC.

Nitric oxide inhibits hypothalamic luteinizing hormone-releasing hormone release by releasing gamma-aminobutyric acid.

Nitric oxide synthase (NOS)-containing neurons, termed NOergic neurons, occur in various regions of the hypothalamus, including the median eminence-arcuate region, which plays an important role in controlling the release of luteinzing hormone-releasing hormone (LHRH). We examined the effect of NO on release of gamma-aminobutyric acid (GABA) from medial basal hypothalamic (MBH) explants incubated in vitro. Sodium nitroprusside (NP) (300 microM), a spontaneous releaser of NO, doubled the release of GABA. This release was significantly reduced by incubation of the tissue with hemoglobin, a scavenger of NO, whereas hemoglobin alone had no effect on the basal release of GABA. Elevation of the potassium concentration (40 mM) in the medium increased GABA release 15-fold; this release was further augmented by NP. Hemoglobin blocked the increase in GABA release induced by NP but had no effect on potassium-induced release, suggesting that the latter is not related to NO. As in the case of hemoglobin, NG-monomethyl-L-arginine (NMMA), a competitive inhibitor of NOS, had no effect on basal release of GABA, which indicates again that NO is not significant to basal GABA release. However, NMMA markedly inhibited the release of GABA induced by high potassium, which indicates that NO plays a role in potassium-induced release of GABA. In conditions in which the release of GABA was substantially augmented, there was a reduction in GABA tissue stores as well, suggesting that synthesis of GABA in these conditions did not keep up with release of the amine. Although NO released GABA, there was no effect of the released GABA on NO production, for incubation of MBH explants with GABA had no effect on NO release as measured by [14C]citrulline production. To determine whether GABA had any effect on the release of LHRH from these MBH explants, GABA was incubated with the tissue and the effect on LHRH release was determined. GABA (10(-5) or 10(-6) M) induced a 70% decrease in the release of LHRH, indicating that in the male rat GABA inhibits the release of this hypothalamic peptide. This inhibition in LHRH release induced by GABA was blocked by NMMA (300 microM), which indicates that GABA converts the stimulatory effect of NO on LHRH release into an inhibitory one, presumably via GABA receptors, which activate chloride channels that hyperpolarize the cell. Previous results have indicated that norepinephrine stimulates release of NO from the NOergic neurons, which then stimulates the release of LHRH. The current results indicate that the NO released also induces release of GABA, which then inhibits further LHRH release. Thus, in vivo the norepinephrinergic-driven pulses of LHRH release may be terminated by GABA released from GABAergic neurons via NO.

Characterization of subtype-specific antibodies to the human D5 dopamine receptor: studies in primate brain and transfected mammalian cells.

To achieve a better understanding of how D5 dopamine receptors mediate the actions of dopamine in brain, we have developed antibodies specific for the D5 receptor. D5 antibodies reacted with recombinant baculovirus-infected Sf9 cells expressing the D5 receptor but not with the D1 receptor or a variety of other catecholaminergic and muscarinic receptors. Epitope-tagged D5 receptors expressed in mammalian cells were reactive with both D5 antibodies and an epitope-specific probe. A mixture of N-linked glycosylated polypeptides and higher molecular-mass species was detected on immunoblots of membrane fractions of D5-transfected cells and also of primate brain. D5 receptor antibodies intensely labeled pyramidal neurons in the prefrontal cortex, whereas spiny medium-sized neurons and aspiny large interneurons of the caudate nucleus were relatively lightly labeled. Antibodies to the D5 dopamine receptor should prove important in experimentally determining specific roles for the D5 and D1 receptors in cortical processes and diseases.

Role of the cotransporter KCC2 in cortical excitatory synapse development and febrile seizure susceptibility

Le co-transporteur KCC2 spécifique au potassium et chlore a pour rôle principal de réduire la concentration intracellulaire de chlore, entraînant l’hyperpolarisation des courants GABAergic l’autorisant ainsi à devenir inhibiteur dans le cerveau mature. De plus, il est aussi impliqué dans le développement des synapses excitatrices, nommées aussi les épines dendritiques. Le but de notre projet est d’étudier l’effet des modifications concernant l'expression et la fonction de KCC2 dans le cortex du cerveau en développement dans un contexte de convulsions précoces. Les convulsions fébriles affectent environ 5% des enfants, et ce dès la première année de vie. Les enfants atteints de convulsions fébriles prolongées et atypiques sont plus susceptibles à développer l’épilepsie. De plus, la présence d’une malformation cérébrale prédispose au développement de convulsions fébriles atypiques, et d’épilepsie du lobe temporal. Ceci suggère que ces pathologies néonatales peuvent altérer le développement des circuits neuronaux irréversiblement. Cependant, les mécanismes qui sous-tendent ces effets ne sont pas encore compris. Nous avons pour but de comprendre l'impact des altérations de KCC2 sur la survenue des convulsions et dans la formation des épines dendritiques. Nous avons étudié KCC2 dans un modèle animal de convulsions précédemment validé, qui combine une lésion corticale à P1 (premier jour de vie postnatale), suivie d'une convulsion induite par hyperthermie à P10 (nommés rats LHS). À la suite de ces insultes, 86% des rats mâles LHS développent l’épilepsie à l’âge adulte, au même titre que des troubles d’apprentissage. À P20, ces animaux presentent une augmentation de l'expression de KCC2 associée à une hyperpolarisation du potentiel de réversion de GABA. De plus, nous avons observé des réductions dans la taille des épines dendritiques et l'amplitude des courants post-synaptiques excitateurs miniatures, ainsi qu’un déficit de mémoire spatial, et ce avant le développement des convulsions spontanées. Dans le but de rétablir les déficits observés chez les rats LHS, nous avons alors réalisé un knock-down de KCC2 par shARN spécifique par électroporation in utero. Nos résultats ont montré une diminution de la susceptibilité aux convulsions due à la lésion corticale, ainsi qu'une restauration de la taille des épines. Ainsi, l’augmentation de KCC2 à la suite d'une convulsion précoce, augmente la susceptibilité aux convulsions modifiant la morphologie des épines dendritiques, probable facteur contribuant à l’atrophie de l’hippocampe et l’occurrence des déficits cognitifs. Le deuxième objectif a été d'inspecter l’effet de la surexpression précoce de KCC2 dans le développement des épines dendritiques de l’hippocampe. Nous avons ainsi surexprimé KCC2 aussi bien in vitro dans des cultures organotypiques d’hippocampe, qu' in vivo par électroporation in utero. À l'inverse des résultats publiés dans le cortex, nous avons observé une diminution de la densité d’épines dendritiques et une augmentation de la taille des épines. Afin de confirmer la spécificité du rôle de KCC2 face à la région néocorticale étudiée, nous avons surexprimé KCC2 dans le cortex par électroporation in utero. Cette manipulation a eu pour conséquences d’augmenter la densité et la longueur des épines synaptiques de l’arbre dendritique des cellules glutamatergiques. En conséquent, ces résultats ont démontré pour la première fois, que les modifications de l’expression de KCC2 sont spécifiques à la région affectée. Ceci souligne les obstacles auxquels nous faisons face dans le développement de thérapie adéquat pour l’épilepsie ayant pour but de moduler l’expression de KCC2 de façon spécifique.

Novel antennal lobe substructures revealed in the small hive beetle Aethina tumida

The small hive beetle, Aethina tumida, is an emerging pest of social bee colonies. A. tumida shows a specialized life style for which olfaction seems to play a crucial role. To better understand the olfactory system of the beetle, we used immunohistochemistry and 3-D reconstruction to analyze brain structures, especially the paired antennal lobes (AL), which represent the first integration centers for odor information in the insect brain. The basic neuroarchitecture of the A. tumida brain compares well to the typical beetle and insect brain. In comparison to other insects, the AL are relatively large in relationship to other brain areas, suggesting that olfaction is of major importance for the beetle. The AL of both sexes contain about 70 olfactory glomeruli with no obvious size differences of the glomeruli between sexes. Similar to all other insects including beetles, immunostaining with an antiserum against serotonin revealed a large cell that projects from one AL to the contralateral AL to densely innervate all glomeruli. Immunostaining with an antiserum against tachykinin-related peptides (TKRP) revealed hitherto unknown structures in the AL. Small TKRP-immunoreactive spherical substructures are in both sexes evenly distributed within all glomeruli. The source for these immunoreactive islets is very likely a group of about 80 local AL interneurons. We offer two hypotheses on the function of such structures.

Role of the cotransporter KCC2 in cortical excitatory synapse development and febrile seizure susceptibility

Le co-transporteur KCC2 spécifique au potassium et chlore a pour rôle principal de réduire la concentration intracellulaire de chlore, entraînant l’hyperpolarisation des courants GABAergic l’autorisant ainsi à devenir inhibiteur dans le cerveau mature. De plus, il est aussi impliqué dans le développement des synapses excitatrices, nommées aussi les épines dendritiques. Le but de notre projet est d’étudier l’effet des modifications concernant l'expression et la fonction de KCC2 dans le cortex du cerveau en développement dans un contexte de convulsions précoces. Les convulsions fébriles affectent environ 5% des enfants, et ce dès la première année de vie. Les enfants atteints de convulsions fébriles prolongées et atypiques sont plus susceptibles à développer l’épilepsie. De plus, la présence d’une malformation cérébrale prédispose au développement de convulsions fébriles atypiques, et d’épilepsie du lobe temporal. Ceci suggère que ces pathologies néonatales peuvent altérer le développement des circuits neuronaux irréversiblement. Cependant, les mécanismes qui sous-tendent ces effets ne sont pas encore compris. Nous avons pour but de comprendre l'impact des altérations de KCC2 sur la survenue des convulsions et dans la formation des épines dendritiques. Nous avons étudié KCC2 dans un modèle animal de convulsions précédemment validé, qui combine une lésion corticale à P1 (premier jour de vie postnatale), suivie d'une convulsion induite par hyperthermie à P10 (nommés rats LHS). À la suite de ces insultes, 86% des rats mâles LHS développent l’épilepsie à l’âge adulte, au même titre que des troubles d’apprentissage. À P20, ces animaux presentent une augmentation de l'expression de KCC2 associée à une hyperpolarisation du potentiel de réversion de GABA. De plus, nous avons observé des réductions dans la taille des épines dendritiques et l'amplitude des courants post-synaptiques excitateurs miniatures, ainsi qu’un déficit de mémoire spatial, et ce avant le développement des convulsions spontanées. Dans le but de rétablir les déficits observés chez les rats LHS, nous avons alors réalisé un knock-down de KCC2 par shARN spécifique par électroporation in utero. Nos résultats ont montré une diminution de la susceptibilité aux convulsions due à la lésion corticale, ainsi qu'une restauration de la taille des épines. Ainsi, l’augmentation de KCC2 à la suite d'une convulsion précoce, augmente la susceptibilité aux convulsions modifiant la morphologie des épines dendritiques, probable facteur contribuant à l’atrophie de l’hippocampe et l’occurrence des déficits cognitifs. Le deuxième objectif a été d'inspecter l’effet de la surexpression précoce de KCC2 dans le développement des épines dendritiques de l’hippocampe. Nous avons ainsi surexprimé KCC2 aussi bien in vitro dans des cultures organotypiques d’hippocampe, qu' in vivo par électroporation in utero. À l'inverse des résultats publiés dans le cortex, nous avons observé une diminution de la densité d’épines dendritiques et une augmentation de la taille des épines. Afin de confirmer la spécificité du rôle de KCC2 face à la région néocorticale étudiée, nous avons surexprimé KCC2 dans le cortex par électroporation in utero. Cette manipulation a eu pour conséquences d’augmenter la densité et la longueur des épines synaptiques de l’arbre dendritique des cellules glutamatergiques. En conséquent, ces résultats ont démontré pour la première fois, que les modifications de l’expression de KCC2 sont spécifiques à la région affectée. Ceci souligne les obstacles auxquels nous faisons face dans le développement de thérapie adéquat pour l’épilepsie ayant pour but de moduler l’expression de KCC2 de façon spécifique.