Calcium-permeable AMPA receptors mediate long-term potentiation in interneurons in the amygdala

Fear conditioning is a paradigm that has been used as a model for emotional learning in animals'. The cellular correlate of fear conditioning is thought to be associative N-methyl-D-aspartate (NMDA) receptor-dependent synaptic plasticity within the amygdala(1-3). Here we show that glutamatergic synaptic transmission to inhibitory interneurons in the basolateral amygdala is mediated solely by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. In contrast to AMPA receptors at inputs to pyramidal neurons, these receptors have an inwardly rectifying current-voltage relationship, indicative of a high permeability to calcium(4 5), Tetanic stimulation of inputs to interneurons caused an immediate and sustained increase in the efficacy of these synapses. This potentiation required a rise in postsynaptic calcium, but was independent of NMDA receptor activation. The potentiation of excitatory inputs to interneurons was reflected as an increase in the amplitude of the GABAA-mediated inhibitory synaptic current in pyramidal neurons. These results demonstrate that excitatory synapses onto interneurons within a fear conditioning circuit show NMDA-receptor independent long-term potentiation. This plasticity might underlie the increased synchronization of activity between neurons in the basolateral amygdala after fear conditioning(6).

Morphological and electrophysiological properties of principal neurons in the rat lateral amygdala in vitro

In this study, we characterize the electrophysiological and morphological properties of spiny principal neurons in the rat lateral amygdala using whole cell recordings in acute brain slices. These neurons exhibited a range of firing properties in response to prolonged current injection. Responses varied from cells that showed full spike frequency adaptation, spiking three to five times, to those that showed no adaptation. The differences in firing patterns were largely explained by the amplitude of the afterhyperpolarization (AHP) that followed spike trains. Cells that showed full spike frequency adaptation had large amplitude slow AHPs, whereas cells that discharged tonically had slow AHPs of much smaller amplitude. During spike trains, all cells showed a similar broadening of their action potentials. Biocytin-filled neurons showed a range of pyramidal-like morphologies, differed in dendritic complexity, had spiny dendrites, and differed in the degree to which they clearly exhibited apical versus basal dendrites. Quantitative analysis revealed no association between cell morphology and firing properties. We conclude that the discharge properties of neurons in the lateral nucleus, in response to somatic current injections, are determined by the differential distribution of ionic conductances rather than through mechanisms that rely on cell morphology.

Excitatory synaptic transmission in the lateral and central amygdala

The amygdala plays a major role in the acquisition and expression of fear conditioning. NMDA receptor-dependent synaptic plasticity within the basolateral amygdala has been proposed to underlie the acquisition and possible storage of fear memories. Here the properties of fast glutamatergic transmission in the lateral and central nuclei of the amygdala are presented. In the lateral amygdala, two types of neurons, interneurons and projection neurons, could be distinguished by their different firing properties. Glutamatergic inputs to interneurons activated AMPA receptors with inwardly rectifying current-voltage relations (I-Vs), whereas inputs to projection neurons activated receptors that had linear I-Vs, indicating that receptors on interneurons lack GluR2 subunits. Inputs to projection neurons formed dual component synapses with both AMPA and NMDA components, whereas at inputs to interneurons, the contribution of NMDA receptors was very small. Neurons in the central amygdala received dual component glutamatergic inputs that activated AMPA receptors with linear I-Vs. NMDA receptor-mediated EPSCs had slow decay time constants in the central nucleus. Application of NR2B selective blockers ifenprodil or CP-101,606 blocked NMDA EPSCs by 70% in the central nucleus, but only by 30% in the lateral nucleus. These data show that the distribution of glutamatergic receptors on amygdalar neurons is not uniform. In the lateral amygdala, interneurons and pyramidal neurons express AMPA receptors with different subunit compositions. Synapses in the central nucleus activate NMDA receptors that contain NR1 and NR2B subunits, whereas synapses in the lateral nucleus contain receptors with both NR2A and NR2B subunits.

Neuronal calcium-binding proteins and schizophrenia

Calcium-binding proteins (CBPs) such as calbindin, parvalbumin and calretinin are used as immunohistochemical markers for discrete neuronal subpopulations. They are particularly useful in identifying the various subpopulations of GABAergic interneurons that control output from prefrontal and cingulate cortices as well as from the hippocampus. The strategic role these interneurons play in regulating output from these three crucial brain regions has made them a focus for neuropathological investigation in schizophrenia. The number of pathological reports detailing subtle changes in these CBP-containing interneurons in patients with schizophrenia is rapidly growing. These proteins however are more than convenient neuronal markers. They confer survival advantages to neurons and can increase the neuron's ability to sustain firing. These properties may be important in the subtle pathophysiology of nondegenerative phenomena such as schizophrenia. The aim of this review is to introduce the reader to the functional properties of CBPs and to examine the emerging literature reporting alterations in these proteins in schizophrenia as well as draw some conclusions about the significance of these findings. (C) 2002 Elsevier Science B.V. All rights reserved.

GATA proteins identify a novel ventral interneuron subclass in the developing chick spinal cord

Members of the GATA transcription factor gene family have been implicated in a variety of developmental processes, including that of the vertebrate central nervous system. However, the role of GATA proteins in spinal cord development remains unresolved. In this study, we investigated the expression and function of two GATA proteins, GATA2 and GATA3, in the developing chick spinal cord. We show that both proteins are expressed by a distinct subpopulation of ventral interneurons that share the same dorsoventral position as CHX10-positive V2 interneurons. However, no coexpression is observed between the two GATA proteins and CHX10. By in vivo notochord grafting and cyclopamine treatment, we demonstrate that the spatially restricted pattern of GATA3 expression is regulated, at least in part, by the signaling molecule Sonic hedgehog. In addition, we further show that Sonic hedgehog induces GATA3 expression in a dose-dependent manner. Using in ovo electroporations, we also demonstrate that GATA2 is upstream of GATA3 in the same epigenetic cascade and that GATA3 is capable of inducing GATA2 expression in vivo. Furthermore, the ectopically expressed GATA proteins can repress differentiation of other ventral cell fates, but not the development of progenitor populations identified by PAX protein expression. Taken together, our findings strongly suggest an important role for GATA2 and GATA3 proteins in the establishment of a distinct ventral interneuron subpopulation in the developing chick spinal cord. (C) 2002 Elsevier Science (USA).

Patterning of the vertebrate ventral spinal cord

We review investigations that have lead to a model of how the ventral spinal cord of higher vertebrate embryos is patterned during development. Central to this model is the secreted morphogen protein, Sonic hedgehog. There is now considerable evidence that this molecule acts in a concentration-dependent manner to direct the development of the spinal cord. Recent studies have suggested that two classes of homeodomain proteins are induced by threshold concentrations of Sonic hedgehog. Reciprocal inhibition between the two classes acts to convert the continuous gradient of Sonic hedgehog into defined domains of transcription factor expression. However, a number of aspects of ventral spinal cord patterning remain to be elucidated. Some issues currently under investigation involve temporal aspects of Shh-signalling, the role of other signals in ventral patterning and the characterisation of ventral interneurons. In this review, we discuss the current state of knowledge of these issues and present some preliminary studies aimed at furthering understanding of these processes in spinal cord patterning.

In ovo electroporation of Crim1 in the developing chick spinal cord

The novel mammalian gene Crim1 encodes a transmembrane bound protein with similarity to the secreted bone morphogenetic protein (BMP) antagonists, vertebrate Chordin, and its Drosophila homologue short gastrulation. Crim1 is expressed in the neural tube in mouse in a restricted pattern, but its function in central nervous system development is largely unknown. We isolated the chicken Crim1 orthologue and analyzed its expression in the developing neural tube. Chicken CRIM1 shares strong homology to human/mouse CRIM1 and C. elegans CRIM1-like proteins. Crim1 is expressed in a similar but not identical pattern to that in the developing spinal cord of mouse, including the notochord, floor plate, motor neurons, and the roof plate. Unlike follistatin, a secreted inhibitor of BMPs, in ovo electroporation of CRIM1, as a full-length transmembrane bound or secreted ectodomain was not sufficient to disrupt early patterning of the neural tube. However, ectodomain CRIM1 overexpression leads to an approximate 50% decrease in populations of specific ventral neuronal populations, including ISL-1(+) motor neurons, CHX-10(+) V1, and EN-1(+) V2 interneurons.

Invertebrate D2 type dopamine receptor exhibits age-based plasticity of expression in the mushroom bodies of the honeybee brain

We have isolated a cDNA clone from the honeybee brain encoding a dopamine receptor, AmDop2, which is positively coupled to adenylyl cyclase. The transmembrane domains of this receptor are 88% identical to the orthologous Drosophila D2 dopamine receptor, DmDop2, though phylogenetic analysis and sequence homology both indicate that invertebrate and vertebrate D2 receptors are quite distinct. In situ hybridization to mRNA in whole-mount preparations of honeybee brains reveals gene expression in the mushroom bodies, a primary site of associative learning. Furthermore, two anatomically distinct cell types in the mushroom bodies exhibit differential regulation of AmDop2 expression. In all nonreproductive females (worker caste) and reproductive males (drones) the receptor gene is strongly and constitutively expressed in all mushroom body interneurons with small cell bodies. In contrast, the large cell-bodied interneurons exhibit dramatic plasticity of AmDop2 gene expression. In newly emerged worker bees (cell-cleaning specialists) and newly emerged drones, no AmDop2 transcript is observed in the large interneurons whereas this transcript is abundant in these cells in the oldest worker bees (resource foragers) and older drones. Differentiation of the mushroom body interneurons into two distinct classes (i.e., plastic or nonplastic with respect to AmDop2 gene expression) indicates that this receptor contributes to the differential regulation of distinct neural circuits. Moreover, the plasticity of expression observed in the large cells implicates this receptor in the behavioral maturation of the bee.

Parvalbumin-, calbindin-, and calretinin-immunoreactive neurons in the prefrontal cortex of the owl monkey (Aotus trivirgatus): A standardized quantitative comparison with sensory and motor areas

Recent studies have revealed regional variation in the density and distribution of inhibitory neurons in different cortical areas, which are thought to reflect area-specific specializations in cortical circuitry. However, there are as yet few standardized quantitative data regarding how the inhibitory circuitry in prefrontal cortex (PFC), which is thought to be involved in executive functions such as cognition, emotion and decision making, compares to that in other cortical areas. Here we used immunohistochemical techniques to determine the density and distribution of parvalbumin (PV)-, calbindin (CB)-, and calretinin (CR)-immunoreactive (ir) neurons and axon terminals in the dorsolateral and orbital PFC of the owl monkey (Aotus trivirgatus), and compared them directly with data obtained using the same techniques in 11 different visual, somatosensory and motor areas. We found marked differences in the density of PV-ir, CB-ir, and CR-ir interneurons in several cortical areas. One hundred and twenty eight of all 234 possible between-area pairwise comparisons were significantly different. The density of specific subpopulations of these cells also varied among cortical areas, as did the density of axon terminals. Comparison of PFC with other cortical areas revealed that 40 of all 66 possible statistical comparisons of the density of PV-ir, CB-ir, and CR-ir cells were significantly different. We also found evidence for heterogeneity in the pattern of labeling of PV-ir, CB-ir, and CR-ir cells and axon terminals between the dorsolateral and orbital subdivisions of PFC. These data are likely to reflect basic differences in interneuron circuitry, which are likely to influence inhibitory function in the cortex. Copyright (C) 2003 S. Karger AG, Basel.

Gap-junction communication between subtypes of direction-selective ganglion cells in the developing retina

The On-Off direction-selective ganglion cells (DSGCs) in the rabbit retina comprise four distinct subtypes that respond preferentially to image motion in four orthogonal directions; each subtype forms a regular territorial array, which is overlapped by the other three arrays. In this study, ganglion cells in the developing retina were injected with Neurobiotin, a gap-junction-permeable tracer, and the DSGCs were identified by their characteristic type 1 bistratified (BiS1) morphology. The complex patterns of tracer coupling shown by the BiSl ganglion cells changed systematically during the course of postnatal development. BiSl cells appear to be coupled together around the time of birth, but, over the next 10 days, BiSl cells decouple from each other, leading to the mature pattern in which only one subtype is coupled. At about postnatal day 5, before the ganglion cells become visually responsive, each of the BiSl cells commonly showed tracer coupling both to a regular array of neighboring BiSl cells, presumably destined to be DSGCs of the same subtype, and to a regular array of overlapping BiSl cells, presumably destined to be DSGCs of a different subtype. The gap-junction intercellular communication between subtypes of DSGCs with different preferred directions may play an important role in the differentiation of their synaptic connectivity, with respect to either the inputs that DSGCs receive from retinal interneurons or the outputs that DSGCs make to geniculate neurons. (C) 2004 Wiley-Liss, Inc.

Comparative analysis of CNS populations in knockout mice with altered growth hormone responsiveness

Recently we have shown that growth hormone (GH) inhibits neuronal differentiation and that this process is blocked by suppressor of cytokine signalling-2 (SOCS2). Here we examine several cortical and subcortical neuronal populations in GH hyper-responsive SOCS2 null (-/-) mice and GH non-responsive GH receptor null (GHR-/-) mice. While SOCS2-/- mice showed a 30% decrease in density of NeuN positive neurons in cortex compared to wildtype, GHR-/- mice showed a 25% increase even though brain size was decreased. Interneuron sub-populations were variably affected, with a slight decrease in cortical parvalbumin expressing interneurons in SOCS2-/- mice and an increase in cortical calbindin and calretinin and striatal cholinergic neuron density in GHR-/- mice. Analysis of glial cell numbers in cresyl violet or glial fibrillary acidic protein (GFAP) stained sections of cortex showed that the neuron: glia ratio was increased in GHR-/- mice and decreased in SOCS2-/- mice. The astrocytes in GHR-/- mice appeared smaller, while they were larger in SOCS2-/- mice. Neuronal soma size also varied in the different genotypes, with smaller striatal cholinergic neurons in GHR-/- mice. While the size of layer 5 pyramidal neurons was not significantly different from wildtype, SOCS2-/- neurons were larger than GHR-/- neurons. In addition, primary dendritic length was similar in all genotypes but dendritic branching of pyramidal neurons in the cortex appeared sparser in GHR-/- and SOCS2-/- mice. These results suggest that GH, possibly regulated by SOCS2, has multiple effects on central nervous system (CNS) development and maturation, regulating the number and size of multiple neuronal and glial cell types.

Characterization of neogenin-expressing neural progenitor populations and migrating neuroblasts in the embryonic mouse forebrain

Many studies have demonstrated a role for netrin-1-deleted in colorectal cancer (DCC) interactions in both axon guidance and neuronal migration. Neogenin, a member of the DCC receptor family, has recently been shown to be a chemorepulsive axon guidance receptor for the repulsive guidance molecule (RGM) family of guidance cues [Rajagopalan S, Deitinghoff L, Davis D, Conrad S, Skutella T, Chedotal A, Mueller B, Strittmatter S (2004) Neogenin mediates the action of repulsive guidance molecule. Nat Cell Biol 6:755-762]. Here we show that neogenin is present on neural progenitors, including neurogenic radial glia, in the embryonic mouse forebrain suggesting that neogenin expression is a hallmark of neural progenitor populations. Neogenin-positive progenitors were isolated from embryonic day 14.5 forebrain using flow cytometry and cultured as neurospheres. Neogenin-positive progenitors gave rise to neurospheres displaying a high proliferative and neurogenic potential. In contrast, neogenin-negative forebrain cells did not produce long-term neurosphere cultures and did not possess a significant neurogenic potential. These observations argue strongly for a role for neogenin in neural progenitor biology. In addition, we also observed neogenin on parvalbumin- and calbindin-positive interneuron neuroblasts that were migrating through the medial and lateral ganglionic eminences, suggesting a role for neogenin in tangential migration. Therefore, neogenin may be a multi-functional receptor regulating both progenitor activity and neuroblast migration in the embryonic forebrain. (c) 2006 IBRO. Published by Elsevier Ltd. All rights reserved.

Robo1 regulates the development of major axon tracts and interneuron migration in the forebrain

The Slit genes encode secreted ligands that regulate axon branching, commissural axon pathfinding and neuronal migration. The principal identified receptor for Slit is Robo ( Roundabout in Drosophila). To investigate Slit signalling in forebrain development, we generated Robo1 knockout mice by targeted deletion of exon 5 of the Robo1 gene. Homozygote knockout mice died at birth, but prenatally displayed major defects in axon pathfinding and cortical interneuron migration. Axon pathfinding defects included dysgenesis of the corpus callosum and hippocampal commissure, and abnormalities in corticothalamic and thalamocortical targeting. Slit2 and Slit1/2 double mutants display malformations in callosal development, and in corticothalamic and thalamocortical targeting, as well as optic tract defects. In these animals, corticothalamic axons form large fasciculated bundles that aberrantly cross the midline at the level of the hippocampal and anterior commissures, and more caudally at the medial preoptic area. Such phenotypes of corticothalamic targeting were not observed in Robo1 knockout mice but, instead, both corticothalamic and thalamocortical axons aberrantly arrived at their respective targets at least 1 day earlier than controls. By contrast, in Slit mutants, fewer thalamic axons actually arrive in the cortex during development. Finally, significantly more interneurons ( up to twice as many at E12.5 and E15.5) migrated into the cortex of Robo1 knockout mice, particularly in both rostral and parietal regions, but not caudal cortex. These results indicate that Robo1 mutants have distinct phenotypes, some of which are different from those described in Slit mutants, suggesting that additional ligands, receptors or receptor partners are likely to be involved in Slit/Robo signalling.

GABAergic excitation in the basolateral amygdala

GABA-containing interneurons are a diverse population of cells whose primary mode of action in the mature nervous system is inhibition of postsynaptic target neurons. Using paired recordings from parvalbumin-positive interneurons in the basolateral amygdala, we show that, in a subpopulation of interneurons, single action potentials in one interneuron evoke in the postsynaptic interneuron a monosynaptic inhibitory synaptic current, followed by a disynaptic excitatory glutamatergic synaptic current. Interneuron-evoked glutamatergic events were blocked by antagonists of either AMPA/kainate or GABA(A) receptors, and could be seen concurrently in both presynaptic and postsynaptic interneurons. These results show that single action potentials in a GABAergic interneuron can drive glutamatergic principal neurons to threshold, resulting in both feedforward and feedback excitation. In interneuron pairs that both receive glutamatergic inputs after an interneuron spike, electrical coupling and bidirectional GABAergic connections occur with a higher probability relative to other interneuron pairs. We propose that this form of GABAergic excitation provides a means for the reliable and specific recruitment of homogeneous interneuron networks in the basal amygdala.