Quantitative fine-structural analysis of olfactory cortical synapses

To determine the extent to which hippocampal synapses are typical of those found in other cortical regions, we have carried out a quantitative analysis of olfactory cortical excitatory synapses, reconstructed from serial electron micrograph sections of mouse brain, and have compared these new observations with previously obtained data from hippocampus. Both superficial and deep layer I olfactory cortical synapses were studied. Although individual synapses in each of the areas—CA1 hippocampus, olfactory cortical layer Ia, olfactory cortical area Ib—might plausibly have been found in any of the other areas, the average characteristics of the three synapse populations are distinct. Olfactory cortical synapses in both layers are, on average, about 2.5 times larger than their hippocampal counterparts. The layer Ia olfactory cortical synapses have fewer synaptic vesicles than do the layer Ib synapses, but the absolute number of vesicles docked to the active zone in the layer Ia olfactory cortical synapses is about equal to the docked vesicle number in the smaller hippocampal synapses. As would be predicted from studies on hippocampus that relate paired-pulse facilitation to the number of docked vesicles, the synapses in layer 1a exhibit facilitation, whereas the ones in layer 1b do not. Although hippocampal synapses provide as a good model system for central synapses in general, we conclude that significant differences in the average structure of synapses from one cortical region to another exist, and this means that generalizations based on a single synapse type must be made with caution.

Modulation of nicotinic acetylcholine receptors by strychnine

Strychnine, a potent and selective antagonist at glycine receptors, was found to inhibit muscle (α1β1γδ, α1β1γ, and α1β1δ) and neuronal (α2β2 and α2β4) nicotinic acetylcholine receptors (AcChoRs) expressed in Xenopus oocytes. Strychnine alone (up to 500 μM) did not elicit membrane currents in oocytes expressing AcChoRs, but, when applied before, concomitantly, or during superfusion of acetylcholine (AcCho), it rapidly and reversibly inhibited the current elicited by AcCho (AcCho-current). Although in the three cases the AcCho-current was reduced to the same level, its recovery was slower when the oocytes were preincubated with strychnine. The amount of AcCho-current inhibition depended on the receptor subtype, and the order of blocking potency by strychnine was α1β1γδ > α2β4 > α2β2. With the three forms of drug application, the Hill coefficient was close to one, suggesting a single site for the receptor interaction with strychnine, and this interaction appears to be noncompetitive. The inhibitory effects on muscle AcChoRs were voltage-independent, and the apparent dissociation constant for AcCho was not appreciably changed by strychnine. In contrast, the inhibitory effects on neuronal AcChoRs were voltage-dependent, with an electrical distance of ≈0.35. We conclude that strychnine regulates reversibly and noncompetitively the embryonic type of muscle AcChoR and some forms of neuronal AcChoRs. In the former case, strychnine presumably inhibits allosterically the receptor by binding at an external domain whereas, in the latter case, it blocks the open receptor-channel complex.

Direct adenovirus-mediated gene transfer of interleukin 1 and tumor necrosis factor α soluble receptors to rabbit knees with experimental arthritis has local and distal anti-arthritic effects

Adenoviral vectors were used to deliver genes encoding a soluble interleukin 1 (IL-1)-type I receptor-IgG fusion protein and/or a soluble type I tumor necrosis factor α (TNFα) receptor-IgG fusion protein directly to the knees of rabbits with antigen-induced arthritis. When tested individually, knees receiving the soluble IL-1 receptor had significantly reduced cartilage matrix degradation and white blood cell infiltration into the joint space. Delivery of the soluble TNFα receptor was less effective, having only a moderate effect on white blood cell infiltration and no effect on cartilage breakdown. When both soluble receptors were used together, there was a greater inhibition of white blood cell infiltration and cartilage breakdown with a considerable reduction of synovitis. Interestingly, anti-arthritic effects were also seen in contralateral control knees receiving only a marker gene, suggesting that sustained local inhibition of disease activity in one joint may confer an anti-arthritic effect on other joints. These results suggest that local intra-articular gene transfer could be used to treat systemic polyarticular arthritides.

An isoform of the rod photoreceptor cyclic nucleotide-gated channel β subunit expressed in olfactory neurons

Sensory transduction in olfactory neurons involves the activation of a cyclic nucleotide-gated (CNG) channel by cAMP. Previous studies identified a CNG channel α subunit (CNG2) and a β subunit (CNG5), which when heterologously expressed form a channel with properties similar but not identical to those of native olfactory neurons. We have cloned a new type of CNG channel β subunit (CNG4.3) from rat olfactory epithelium. CNG4.3 derives from the same gene as the rod photoreceptor β subunit (CNG4.1) but lacks the long, glutamic acid-rich domain found in the N terminus of CNG4.1. Northern blot and in situ hybridization revealed that CNG4.3 is expressed specifically in olfactory neurons. Expression of CNG4.3 in human embryonic kidney 293 cells did not lead to detectable currents. Coexpression of CNG4.3 with CNG2 induced a current with significantly increased sensitivity for cAMP whereas cGMP affinity was not altered. Additionally, CNG4.3 weakened the outward rectification of the current in the presence of extracellular Ca2+, decreased the relative permeability for Ca2+, and enhanced the sensitivity for l-cis diltiazem. Upon coexpression of CNG2, CNG4.3, and CNG5, a conductance with a cAMP sensitivity greater than that of either the CNG2/CNG4.3 or the CNG2/CNG5 channel and near that of native olfactory channel was observed. Our data suggest that CNG4.3 forms a subunit of the native olfactory CNG channel. The expression of various CNG4 isoforms in retina and olfactory epithelium indicates that the CNG4 subunit may be necessary for normal function of both photoreceptor and olfactory CNG channels.

Region-dependent dynamics of cAMP response element-binding protein phosphorylation in the basal ganglia

The cAMP response element-binding protein (CREB) is an activity-dependent transcription factor that is involved in neural plasticity. The kinetics of CREB phosphorylation have been suggested to be important for gene activation, with sustained phosphorylation being associated with downstream gene expression. If so, the duration of CREB phosphorylation might serve as an indicator for time-sensitive plastic changes in neurons. To screen for regions potentially involved in dopamine-mediated plasticity in the basal ganglia, we used organotypic slice cultures to study the patterns of dopamine- and calcium-mediated CREB phosphorylation in the major subdivisions of the striatum. Different durations of CREB phosphorylation were evoked in the dorsal and ventral striatum by activation of dopamine D1-class receptors. The same D1 stimulus elicited (i) transient phosphorylation (≤15 min) in the matrix of the dorsal striatum; (ii) sustained phosphorylation (≤2 hr) in limbic-related structures including striosomes, the nucleus accumbens, the fundus striati, and the bed nucleus of the stria terminalis; and (iii) prolonged phosphorylation (up to 4 hr or more) in cellular islands in the olfactory tubercle. Elevation of Ca2+ influx by stimulation of L-type Ca2+ channels, NMDA, or KCl induced strong CREB phosphorylation in the dorsal striatum but not in the olfactory tubercle. These findings differentiate the response of CREB to dopamine and calcium signals in different striatal regions and suggest that dopamine-mediated CREB phosphorylation is persistent in limbic-related regions of the neonatal basal ganglia. The downstream effects activated by persistent CREB phosphorylation may include time-sensitive neuroplasticity modulated by dopamine.

Tyrosine phosphorylation of paxillin and focal adhesion kinase by activation of muscarinic m3 receptors is dependent on integrin engagement by the extracellular matrix

The G protein-coupled m1 and m3 muscarinic acetylcholine receptors increase tyrosine phosphorylation of several proteins, including the focal adhesion-associated proteins paxillin and focal adhesion kinase (FAK), but the mechanism is not understood. Activation of integrins during adhesion of cells to extracellular matrix, or stimulation of quiescent cell monolayers with G protein-coupled receptor ligands including bradykinin, bombesin, endothelin, vasopressin, and lysophosphatidic acid, also induces tyrosine phosphorylation of paxillin and FAK and formation of focal adhesions. These effects are generally independent of protein kinase C but are inhibited by agents that prevent cytoskeletal assembly or block activation of the small molecular weight G protein Rho. This report demonstrates that tyrosine phosphorylation of paxillin and FAK elicited by stimulation of muscarinic m3 receptors with the acetylcholine analog carbachol is inhibited by soluble peptides containing the arginine–glycine–aspartate motif (the recognition site for integrins found in adhesion proteins such as fibronectin) but is unaffected by peptides containing the inactive sequence arginine–glycine–glutamate. Tyrosine phosphorylation elicited by carbachol, but not by cell adhesion to fibronectin, is reduced by the protein kinase C inhibitor GF 109203X. The response to carbachol is dependent on the presence of fibronectin. Moreover, immunofluorescence studies show that carbachol treatment induces formation of stress fibers and focal adhesions. These results suggest that muscarinic receptor stimulation activates integrins via a protein kinase C-dependent mechanism. The activated integrins transmit a signal into the cell’s interior leading to tyrosine phosphorylation of paxillin and FAK. This represents a novel mechanism for regulation of tyrosine phosphorylation by muscarinic receptors.

Dynamic mapping at the laminar level of odor-elicited responses in rat olfactory bulb by functional MRI

We have applied functional MRI (fMRI) based on blood oxygenation level-dependent (BOLD) image-contrast to map odor-elicited olfactory responses at the laminar level in the rat olfactory bulb (OB) elicited by iso-amyl acetate (10−2 dilution of saturated vapor) with spatial and temporal resolutions of 220×220×1,000 μm and 36 s. The laminar structure of the OB was clearly depicted by high-resolution in vivo anatomical MRI with spatial resolution of 110×110×1,000 μm. In repeated BOLD fMRI measurements, highly significant (P < 0.001) foci were located in the outer layers of both OBs. The occurrence of focal OB activity within a domain at the level of individual glomeruli or groups of glomeruli was corroborated on an intra- and inter-animal basis under anesthetized conditions with this noninvasive method. The dynamic studies demonstrated that the odor-elicited BOLD activations were highly reproducible on a time scale of minutes, whereas over tens of minutes the activations sometimes varied slowly. We found large BOLD signal (ΔS/S = 10–30%) arising from the olfactory nerve layer, which is devoid of synapses and composed of unmyelinated fibers and glial cells. Our results support previous studies with other methods showing that odors elicit activity within glomerular layer domains in the mammalian OB, and extend the analysis to shorter time periods at the level of individual glomeruli or groups of glomeruli. With further improvement, BOLD fMRI should be ideal for systematic analysis of the functional significance of individual glomeruli in olfactory information encoding and of spatiotemporal processing within the olfactory system.

Dopamine-dependent responses to morphine depend on glucocorticoid receptors

Previous work has shown that glucocorticoid hormones facilitate the behavioral and dopaminergic effects of morphine. In this study we examined the possible role in these effects of the two central corticosteroid receptor types: mineralocorticoid receptor (MR), and glucocorticoid receptor (GR). To accomplish this, specific antagonists of these receptors were infused intracerebroventricularly and 2 hr later we measured: (i) locomotor activity induced by a systemic injection of morphine (2 mg/kg); (ii) locomotor activity induced by an infusion of morphine (1 μg per side) into the ventral tegmental area, which is a dopamine-dependent behavioral response to morphine; (iii) morphine-induced dopamine release in the nucleus accumbens, a dopaminergic projection site mediating the locomotor and reinforcing effects of drugs of abuse. Blockade of MRs by spironolactone had no significant effects on locomotion induced by systemic morphine. In contrast, blockade of GRs by either RU38486 or RU39305, which is devoid of antiprogesterone effects, reduced the locomotor response to morphine, and this effect was dose dependent. GR antagonists also reduced the locomotor response to intraventral tegmental area morphine as well as the basal and morphine-induced increase in accumbens dopamine, as measured by microdialysis in freely moving rats. In contrast, spironolactone did not modify dopamine release. In conclusion, glucocorticoids, via GRs, facilitate the dopamine-dependent behavioral effects of morphine, probably by facilitating dopamine release. The possibility of decreasing the behavioral and dopaminergic effects of opioids by an acute administration of GR antagonists may open new therapeutic strategies for treatment of drug addiction.

Activity differentially regulates the surface expression of synaptic AMPA and NMDA glutamate receptors

Distinct subtypes of glutamate receptors often are colocalized at individual excitatory synapses in the mammalian brain yet appear to subserve distinct functions. To address whether neuronal activity may differentially regulate the surface expression at synapses of two specific subtypes of ionotropic glutamate receptors we epitope-tagged an AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor subunit (GluR1) and an NMDA (N-methyl-d-aspartate) receptor subunit (NR1) on their extracellular termini and expressed these proteins in cultured hippocampal neurons using recombinant adenoviruses. Both receptor subtypes were appropriately targeted to the synaptic plasma membrane as defined by colocalization with the synaptic vesicle protein synaptophysin. Increasing activity in the network of cultured cells by prolonged blockade of inhibitory synapses with the γ-aminobutyric acid type A receptor antagonist picrotoxin caused an activity-dependent and NMDA receptor-dependent decrease in surface expression of GluR1, but not NR1, at synapses. Consistent with this observation identical treatment of noninfected cultures decreased the contribution of endogenous AMPA receptors to synaptic currents relative to endogenous NMDA receptors. These results indicate that neuronal activity can differentially regulate the surface expression of AMPA and NMDA receptors at individual synapses.

Adenosine acts by A1 receptors to stimulate release of prolactin from anterior-pituitaries in vitro

Adenosine has been identified in the anterior pituitary gland and is secreted from cultured folliculostellate (FS) cells. To determine whether adenosine controls the secretion of anterior pituitary hormones in vitro, adenosine was incubated with anterior pituitaries. It stimulated prolactin (PRL) release at the lowest concentration used (10−10 M); the stimulation peaked at 10−8 M with a threefold increase in release and declined to minimal stimulation at 10−4 and 10−3 M. Follicle-stimulating hormone release was maximally inhibited at 10−8 M, whereas luteinizing hormone release was not significantly inhibited. Two selective A1 adenosine receptor antagonists (10−7 or 10−5 M) had no effect on basal PRL release, but either antagonist completely blocked the response to the most effective concentration of adenosine (10−8 M). In contrast, a highly specific A2 receptor antagonist (10−7 or 10−5 M) had no effect on basal PRL release or the stimulation of PRL release induced by adenosine (10−8 M). We conclude that adenosine acts to stimulate PRL release in vitro by activating A1 receptors. Since the A1 receptors decrease intracellular-free calcium, this would decrease the activation of nitric oxide synthase in the FS cells, resulting in decreased release of nitric oxide (NO). NO inhibits PRL release by activating guanylate cyclase that synthesizes cGMP from GTP; cGMP concentrations increase in the lactotrophs leading to inhibition of PRL release. In the case of adenosine, NO release from the FS cells decreases, resulting in decreased concentrations of NO in the lactotrophs, consequent decreased cGMP formation, and resultant increased PRL release.

Ionophores and receptors using cation-π interactions: Collarenes

Cation-π interactions are important forces in molecular recognition by biological receptors, enzyme catalysis, and crystal engineering. We have harnessed these interactions in designing molecular systems with circular arrangement of benzene units that are capable of acting as ionophores and models for biological receptors. [n]Collarenes are promising candidates with high selectivity for a specific cation, depending on n, because of their structural rigidity and well-defined cavity size. The interaction energies of [n]collarenes with cations have been evaluated by using ab initio calculations. The selectivity of these [n]collarenes in aqueous solution was revealed by using statistical perturbation theory in conjunction with Monte Carlo and molecular dynamics simulations. It has been observed that in [n]collarenes the ratio of the interaction energies of a cation with it and the cation with the basic building unit (benzene) can be correlated to its ion selectivity. We find that collarenes are excellent and efficient ionophores that bind cations through cation-π interactions. [6]Collarene is found to be a selective host for Li+ and Mg2+, [8]collarene for K+ and Sr2+, and [10]collarene for Cs+ and Ba2+. This finding indicates that [10]collarene and [8]collarene could be used for effective separation of highly radioactive isotopes, 137Cs and 90Sr, which are major constituents of nuclear wastes. More interestingly, collarenes of larger cavity size can be useful in capturing organic cations. [12]Collarene exhibits a pronounced affinity for tetramethylammonium cation and acetylcholine, which implies that it could serve as a model for acetylcholinestrase. Thus, collarenes can prove to be novel and effective ionophores/model-receptors capable of heralding a new direction in molecular recognition and host-guest chemistry.

Odor space and olfactory processing: Collective algorithms and neural implementation

Several basic olfactory tasks must be solved by highly olfactory animals, including background suppression, multiple object separation, mixture separation, and source identification. The large number N of classes of olfactory receptor cells—hundreds or thousands—permits the use of computational strategies and algorithms that would not be effective in a stimulus space of low dimension. A model of the patterns of olfactory receptor responses, based on the broad distribution of olfactory thresholds, is constructed. Representing one odor from the viewpoint of another then allows a common description of the most important basic problems and shows how to solve them when N is large. One possible biological implementation of these algorithms uses action potential timing and adaptation as the “hardware” features that are responsible for effective neural computation.

The product of a thyroid hormone-responsive gene interacts with thyroid hormone receptors

Thyroid hormone is a critical mediator of central nervous system (CNS) development, acting through nuclear receptors to modulate the expression of specific genes. Transcription of the rat hairless (hr) gene is highly up-regulated by thyroid hormone in the developing CNS; we show here that hr is directly induced by thyroid hormone. By identifying proteins that interact with the hr gene product (Hr), we find that Hr interacts directly and specifically with thyroid hormone receptor (TR)—the same protein that regulates its expression. Unlike previously described receptor-interacting factors, Hr associates with TR and not with retinoic acid receptors (RAR, RXR). Hr can act as a transcriptional repressor, suggesting that its interaction with TR is part of a novel autoregulatory mechanism.

Postsynaptic clustering of γ-aminobutyric acid type A receptors by the γ3 subunit in vivo

Synaptic localization of γ-aminobutyric acid type A (GABAA) receptors is a prerequisite for synaptic inhibitory function, but the mechanism by which different receptor subtypes are localized to postsynaptic sites is poorly understood. The γ2 subunit and the postsynaptic clustering protein gephyrin are required for synaptic localization and function of major GABAA receptor subtypes. We now show that transgenic overexpression of the γ3 subunit in γ2 subunit-deficient mice restores benzodiazepine binding sites, benzodiazepine-modulated whole cell currents, and postsynaptic miniature currents, suggesting the formation of functional, postsynaptic receptors. Moreover, the γ3 subunit can substitute for γ2 in the formation of GABAA receptors that are synaptically clustered and colocalized with gephyrin in vivo. These clusters were formed even in brain regions devoid of endogenous γ3 subunit, indicating that the factors present for clustering of γ2 subunit-containing receptors are sufficient to cluster γ3 subunit-containing receptors. The GABAA receptor and gephyrin-clustering properties of the ectopic γ3 subunit were also observed for the endogenous γ3 subunit, but only in the absence of the γ2 subunit, suggesting that the γ3 subunit is at a competitive disadvantage with the γ2 subunit for clustering of postsynaptic GABAA receptors in wild-type mice.

A point mutation in the γ2 subunit of γ-aminobutyric acid type A receptors results in altered benzodiazepine binding site specificity

Benzodiazepines allosterically modulate γ-aminobutyric acid (GABA) evoked chloride currents of γ-aminobutyric acid type A (GABAA) receptors. Coexpression of either rat γ2 or γ3, in combination with α1 and β2 subunits, results both in receptors displaying high [3H]Ro 15-1788 affinity. However, receptors containing a γ3 subunit display a 178-fold reduced affinity to zolpidem as compared with γ2-containing receptors. Eight chimeras between γ2 and γ3 were constructed followed by nine different point mutations in γ2, each to the homologous amino acid residue found in γ3. Chimeric or mutant γ subunits were coexpressed with α1 and β2 in human embryonic kidney 293 cells to localize amino acid residues responsible for the reduced zolpidem affinity. Substitution of a methionine-to-leucine at position 130 of γ2 (γ2M130L) resulted in a 51-fold reduction in zolpidem affinity whereas the affinity to [3H]Ro 15-1788 remained unchanged. The affinity for diazepam was only decreased by about 2-fold. The same mutation resulted in a 9-fold increase in Cl 218872 affinity. A second mutation (γ2M57I) was found to reduce zolpidem affinity by about 4-fold. Wild-type and γ2M130L-containing receptors were functionally expressed in Xenopus oocytes. Upon mutation allosteric coupling between agonist and modulatory sites is preserved. Dose–response curves for zolpidem and for diazepam showed that the zolpidem but not the diazepam apparent affinity is drastically reduced. The apparent GABA affinity is not significantly affected by the γ2M130L mutation. The identified amino acid residues may define part of the benzodiazepine binding pocket of GABAA receptors. As the modulatory site in the GABAA receptor is homologous to the GABA site, and to all agonist sites of related receptors, γ2M130 may either point to a homologous region important for agonist binding in all receptors or define a new region not underlying this principle.