Sodium channels in dendrites of rat cortical pyramidal neurons.

The voltage-dependent properties that have been directly demonstrated in Purkinje cell and hippocampal pyramidal cell dendrites play an important role in the integrative capacities of these neurons. By contrast, the properties of neocortical pyramidal cell dendritic membranes have been more difficult to assess. Active dendritic conductances near sites of synaptic input would have an important effect on the input-output characteristics of these neurons. In the experiments reported here, we obtained direct evidence for the existence of voltage-dependent Na+ channels on the dendrites of neocortical neurons by using cell-attached patch and whole cell recordings from acutely isolated rat neocortical pyramidal cells. The qualitative and quantitative properties of dendritic and somatic currents were indistinguishable. Insofar as Na+ currents are concerned, the soma and primary apical dendrite can be considered as one relatively uniform compartment. Similar dendritic Na+ currents on dendrites in mature neurons would play an important role in determining the integrative properties of these cortical units.     

Multibranch activity in basal and tuft dendrites during firing of layer 5 cortical neurons in vivo

Layer 5 pyramidal neurons process information from multiple cortical layers to provide a major output of cortex. Because of technical limitations it has remained unclear how these cells integrate widespread synaptic inputs located in distantly separated basal and tuft dendrites. Here, we obtained in vivo two-photon calcium imaging recordings from the entire dendritic field of layer 5 motor cortex neurons. We demonstrate that during subthreshold activity, basal and tuft dendrites exhibit spatially localized, small-amplitude calcium transients reflecting afferent synaptic inputs. During action potential firing, calcium signals in basal dendrites are linearly related to spike activity, whereas calcium signals in the tuft occur unreliably. However, in both dendritic compartments, spike-associated calcium signals were uniformly distributed throughout all branches. Thus, our data support a model of widespread, multibranch integration with a direct impact by basal dendrites and only a partial contribution on output signaling by the tuft.     

Human metallothionein genes are clustered on chromosome 16.

The metallothioneins are a family of heavy-metal binding proteins of low molecular weight. They function in the regulation of trace metal metabolism and in the protection against toxic heavy metal ions. In man, the metallothioneins are encoded by at least 10-12 genes separated into two groups, MT-I and MT-II. To understand the genomic organization of these genes and their involvement in hereditary disorders of trace metal metabolism, we have determined their chromosomal location. Using human-mouse cell hybrids and hybridization probes derived from cloned and functional human MT1 and MT2 genes, we show that the functional human genes are clustered on human chromosome 16. Analysis of RNA from somatic cell hybrids indicated that hybrids that contained human chromosome 16 expressed both human MT1 and MT2 mRNA, and this expression is regulated by both heavy metal ions and glucocorticoid hormones.     

Isoforms of the Na,K-ATPase are present in both axons and dendrites of hippocampal neurons in culture.

The distributions of isoforms of the Na,K-ATPase alpha subunit were determined in mature cultured hippocampal neurons and in a polarized epithelial cell line. We find that hippocampal neurons express the alpha 1 and alpha 3 isoforms in the membranes of both axons and dendrites. In contrast the alpha 1 and alpha 3 proteins are exclusively basolateral when expressed endogenously or by stable transfection in renal epithelial cells. These data suggest that epithelial cells and hippocampal neurons localize these proteins by different mechanisms. These observations contrast with those made for the vesicular stomatitis virus and the influenza glycoproteins, which are polarized in both epithelial and neuronal cells.     

Alterations of cortical pyramidal neurons in mice lacking high-affinity nicotinic receptors

The neuronal nicotinic acetylcholine receptors (nAChRs) are allosteric membrane proteins involved in multiple cognitive processes, including attention, learning, and memory. The most abundant form of heterooligomeric nAChRs in the brain contains the β2- and α4- subunits and binds nicotinic agonists with high affinity. In the present study, we investigated in the mouse the consequences of the deletion of one of the nAChR components: the β2-subunit (β2^−/−) on the microanatomy of cortical pyramidal cells. Using an intracellular injection method, complete basal dendritic arbors of 650 layer III pyramidal neurons were sampled from seven cortical fields, including primary sensory, motor, and associational areas, in both β2^−/− and WT animals. We observed that the pyramidal cell phenotype shows significant quantitative differences among different cortical areas in mutant and WT mice. In WT mice, the density of dendritic spines was rather similar in all cortical fields, except in the prelimbic/infralimbic cortex, where it was significantly higher. In the absence of the β2-subunit, the most significant reduction in the density of spines took place in this high-order associational field. Our data suggest that the β2-subunit is involved in the dendritic morphogenesis of pyramidal neurons and, in particular, in the circuits that contribute to the high-order functional connectivity of the cerebral cortex.     

Ethanol-nicotine interactions at alpha-bungarotoxin-insensitive nicotinic acetylcholine receptors in rat cortical neurons

Numerous studies have indicated a correlation between ethanol intake and cigarette smoking in heavy drinkers. We have studied the underlying pharmacological basis of this relationship using cultured rat cortical neurons. These neurons express nicotinic receptors having characteristics similar to those described for the alpha4beta2 subunit combination. In the presence of alpha-bungarotoxin both acetylcholine (ACh) and nicotine evoked currents with respective EC50 values of 4.3 and 3.4 microM. The maximal nicotine-activated response, however, was only 56% that of the maximal ACh current. It was previously shown that 10 to 100 mM of ethanol potentiated ACh-mediated currents in these neurons. We demonstrate that 100 mM ethanol similarly potentiates currents evoked by 300 nM (40%) and 1 microM nicotine 61%). This suggests that an ethanol-induced potentiation of nicotinic currents may enhance the acute positive reinforcement associated with nicotine and could increase tobacco use during heavy ethanol intake. However, further experimentation indicated that the continuous perfusion of 30, 100, or 300 nM nicotine desensitizes ACh-evoked currents by 38, 54, and 62%, respectively, with little direct receptor-channel activation. The residual ACh currents of nicotine-desensitized receptor channels were potentiated by 100 mM ethanol to nearly the extent as were the undesensitized control responses. We propose that the opposing effect of ethanol on nicotine-induced desensitization could also explain the increased tobacco use observed with excessive drinking. Thus, ethanol has a dual effect regarding nicotine. It enhances acute nicotine-mediated receptor activation, although opposing the net effect of nicotine-induced receptor channel desensitization.     

Resistance gene analogs are conserved and clustered in soybean.

Sequences of cloned resistance genes from a wide range of plant taxa reveal significant similarities in sequence homology and structural motifs. This is observed among genes conferring resistance to viral, bacterial, and fungal pathogens. In this study, oligonucleotide primers designed for conserved sequences from coding regions of disease resistance genes N (tobacco), RPS2 (Arabidopsis) and L6 (flax) were used to amplify related sequences from soybean [Glycine max (L.) Merr.]. Sequencing of amplification products indicated that at least nine classes of resistance gene analogs (RGAs) were detected. Genetic mapping of members of these classes located them to eight different linkage groups. Several RGA loci mapped near known resistance genes. A bacterial artificial chromosome library of soybean DNA was screened using primers and probes specific for eight RGA classes and clones were identified containing sequences unique to seven classes. Individual bacterial artificial chromosomes contained 2-10 members of single RGA classes. Clustering and sequence similarity of members of RGA classes suggests a common process in their evolution. Our data indicate that it may be possible to use sequence homologies from conserved motifs of cloned resistance genes to identify candidate resistance loci from widely diverse plant taxa.     

Neurofibrillary tangle-bearing neurons are functionally integrated in cortical circuits in vivo

Alzheimer''s disease (AD) is pathologically characterized by the deposition of extracellular amyloid-β plaques and intracellular aggregation of tau protein in neurofibrillary tangles (NFTs) (1, 2). Progression of NFT pathology is closely correlated with both increased neurodegeneration and cognitive decline in AD (3) and other tauopathies, such as frontotemporal dementia (4, 5). The assumption that mislocalization of tau into the somatodendritic compartment (6) and accumulation of fibrillar aggregates in NFTs mediates neurodegeneration underlies most current therapeutic strategies aimed at preventing NFT formation or disrupting existing NFTs (7, 8). Although several disease-associated mutations cause both aggregation of tau and neurodegeneration, whether NFTs per se contribute to neuronal and network dysfunction in vivo is unknown (9). Here we used awake in vivo two-photon calcium imaging to monitor neuronal function in adult rTg4510 mice that overexpress a human mutant form of tau (P301L) and develop cortical NFTs by the age of 7–8 mo (10). Unexpectedly, NFT-bearing neurons in the visual cortex appeared to be completely functionally intact, to be capable of integrating dendritic inputs and effectively encoding orientation and direction selectivity, and to have a stable baseline resting calcium level. These results suggest a reevaluation of the common assumption that insoluble tau aggregates are sufficient to disrupt neuronal function.Neurofibrillary tangles (NFTs) containing aggregated tau protein (1) have long been considered key players in the progressive neural dysfunction and neurodegeneration observed in Alzheimer’s disease (AD) (2, 3) and other tauopathies (4, 5). It is commonly assumed that NFT-bearing neurons exhibit deficits in synaptic integration and eventually lead to neurodegeneration (11, 12). However, the actual functional properties of NFT-bearing neurons in intact neural circuits have not been explored previously (13). We addressed this question directly using awake in vivo two-photon calcium imaging in a mouse model of NFT formation (rTg4510) by applying recently developed imaging approaches allowing for single-neuron–level and population-level assessment of neural activity in awake mice (14). Because two-photon calcium imaging allows for measurement of response properties in many neurons simultaneously, we were able to directly isolate the impact of NFT deposition in a neuronal microcircuit by evaluating population-level network dynamics and, more specifically, by differentiating the function of individual NFT-bearing and neighboring non–NFT-bearing neurons.To assess the functional properties of neurons in the visual cortex, we used a genetically encoded ratiometric calcium indicator, yellow cameleon 3.6 (YC3.6), packaged in an adeno-associated viral vector (15, 16). To assess functional responses, we exploited the well-characterized functional architecture of visual cortex whereby neurons in mouse visual cortex modulate their activity during presentation of drifting gratings moving at specific orientations and directions (orientation and direction selectivity) (17, 18). We further used YC3.6 as a FRET-based ratiometric indicator to make quantitative measurements of resting calcium (15). Resting calcium is tightly regulated in the brain, and slight deviations can trigger chronic and severe degenerative pathways (15). Thus, measurement of resting calcium is an important and complementary functional assay for evaluating neuronal health. Importantly, performing experiments in awake, head-fixed animals eliminates the impact of anesthesia on response properties, resting calcium, and tau aggregation (Fig. 1 A and B and Movie S1) (19).Open in a separate windowFig. 1.YC3.6 calcium imaging in awake cortex of control mice reveals robust visual response tuning. (A) Awake and head-fixed mice expressing AAV-YC3.6 in visual cortex were presented with drifting gratings to record visually evoked calcium responses through a chronic cranial window (Movie S1). (B) In vivo two-photon image of YC3.6-expressing neurons in layer 2/3 (∼180 µm below the brain surface) with four example neurons (white circles). (Scale bar: 50 μm.) (C) Drifting gratings (eight stimulus types, 45° apart) were presented for 7–10 s, followed by 7–10 s without stimulation (gray screen). This sequence was repeated 10 times. For cell 1 (orientation- and direction-selective), three representative single-trial time courses (YFP:CFP ratio) and the average trace across 10 trials reveal its predominant response to a grating direction of 225°. SEM is shown in gray; arrowhead indicates preferred direction. (D) Polar plot of cell 1 demonstrating selectivity of this neuron for the 225° stimuli. (E) Average traces of three other neurons (white circles in A) illustrate the diversity in orientation and direction tuning. Cell 2 is orientation-selective but not direction-selective, cell 3 responds to all directions (broadly tuned), and cell 4 does not respond to any direction (not responding). (F) OSI and DSI for cells 1–4.     

Identification of sites for exponential translation in living dendrites

Neuronal processes contain mRNAs and membrane structures, and some forms of synaptic plasticity seem to require protein synthesis in dendrites of hippocampal neurons. To quantitate dendritic protein synthesis, we used multiphoton microscopy of green fluorescent protein synthesized in living isolated dendrites. Transfection of dendrites with mRNA encoding green fluorescent protein resulted in fluorescence that exponentially increased on stimulation with a glutamate receptor agonist; a reaction attenuated by the translation inhibitors anisomycin and emetine. Comparable experiments on whole neurons revealed that (RS)-3,5-dihydroxy-phenylglycine 0.5 H(2)O (DHPG)-stimulated fluorescence was linear in cell bodies relative to the exponential increase seen in dendrites. Detailed spatial analysis of the subdendritic distribution of fluorescence revealed "hotspots," sites of dendritic translation that were spatially stable. However, detailed temporal analysis of these hotspots revealed heterogeneous rates of translation. A double-label protocol counterstaining for ribosomes indicated that sites of "fastest" translation correlated with increased ribosome density, consistent with ribosome subunit assembly for initiation, the first step of translation. We propose that dendrites have specific sites specialized for fast translation.     

Color-tuned neurons are spatially clustered according to color preference within alert macaque posterior inferior temporal cortex

Large islands of extrastriate cortex that are enriched for color-tuned neurons have recently been described in alert macaque using a combination of functional magnetic resonance imaging (fMRI) and single-unit recording. These millimeter-sized islands, dubbed “globs,” are scattered throughout the posterior inferior temporal cortex (PIT), a swath of brain anterior to area V3, including areas V4, PITd, and posterior TEO. We investigated the micro-organization of neurons within the globs. We used fMRI to identify the globs and then used MRI-guided microelectrodes to test the color properties of single glob cells. We used color stimuli that sample the CIELUV perceptual color space at regular intervals to test the color tuning of single units, and make two observations. First, color-tuned neurons of various color preferences were found within single globs. Second, adjacent glob cells tended to have the same color tuning, demonstrating that glob cells are clustered by color preference and suggesting that they are arranged in color columns. Neurons separated by 50 μm, measured parallel to the cortical sheet, had more similar color tuning than neurons separated by 100 μm, suggesting that the scale of the color columns is <100 μm. These results show that color-tuned neurons in PIT are organized by color preference on a finer scale than the scale of single globs. Moreover, the color preferences of neurons recorded sequentially along a given electrode penetration shifted gradually in many penetrations, suggesting that the color columns are arranged according to a chromotopic map reflecting perceptual color space.     

Estrogen receptors in dendrites and axon terminals in the guinea pig hypothalamus.

The ultrastructural localization of steroid hormone receptors has been made possible by the development of immunocytochemical procedures using monoclonal antibodies. Estrogen receptor-immunoreactivity (ER-IR) in the brain is present most abundantly in neuronal nuclei when observed with light microscopy. However, we have also observed ER-IR in the perikarya and cytoplasmic processes of neurons. To determine the organelles with which the cytoplasmic ER-IR is associated, we developed a technique for ultrastructural visualization of ER-IR. Ovariectomized guinea pigs were perfused, brains vibratome-sectioned, and estrogen receptors immunostained by either an immunoperoxidase-diaminobenzidine technique or by an immunogold-streptavidin procedure, each followed by silver intensification. Electron microscopic analysis confirmed distribution of ER-IR throughout cell nuclei, but ER-IR was also observed in proximal and distal dendrites and rough endoplasmic reticulum. Most surprisingly, however, ER-IR was found in many axon terminals containing predominantly round, and in some cases, flattened clear synaptic vesicles. Parallel experiments examining the distribution of progestin receptors confirmed the localization at the same subcellular sites as for estrogen receptors. The results of this experiment corroborate our earlier findings of extranuclear steroid receptor-immunoreactivity in the brain, and they suggest potential nongenomic sites of action for estradiol and progesterone in dendrites and axon terminals.     

Ectopic dendrites occur only on cortical pyramidal cells containing elevated GM2 ganglioside in alpha-mannosidosis.

In a variety of neuronal storage diseases, cortical pyramidal cells elaborate ectopic dendrites at the axon hillock. A feature common to all the diseases characterized by ectopic dendrites is an elevated level of GM2 ganglioside in cerebral cortex. In cats with one such disease, alpha-mannosidosis, the number of pyramidal cells bearing ectopic dendrites is small; the present study shows that GM2 ganglioside is stored only in those pyramidal neurons exhibiting ectopic dendrites. Using a Golgi-electron microscopy method with periodic acid-Schiff (PAS) staining, we first established that pyramidal cells bearing ectopic dendrites contained PAS+ membranous inclusions, consistent with storage of glycolipids. In contrast, those with smooth axon hillocks accumulated PAS- floccular inclusions, consistent with storage of oligosaccharides. Next, application of a monoclonal antibody against GM2 ganglioside revealed that subsets of both pyramidal and intrinsic neurons contained GM2-like immunoreactivity. Every GM2+ cell contained PAS+ membranous inclusions, indicating that pyramidal cells bearing ectopic dendrites stored GM2 ganglioside. In cats with alpha-mannosidosis induced by swainsonine, some pyramidal neurons showed GM2-like immunoreactivity after 4 weeks of treatment, whereas ectopic dendrites only became evident after 7 weeks of treatment. Thus, GM2 ganglioside accumulated in pyramidal neurons before ectopic dendrites emerged from the axon hillock. We propose that the reinitiation of dendrite growth on mature pyramidal cells is brought about by accumulated GM2 ganglioside.     

Glucocorticoid receptors in LHRH neurons.

Luteinizing hormone-releasing hormone (LHRH)-producing neurons constitute the final pathway for regulation of reproductive endocrine function by the central nervous system. Chronically elevated levels of glucocorticoids exert an inhibitory effect on reproductive function. Although this is thought to be mediated in part via modulation of classical transmitters and peptides which regulate LHRH synthesis and release, it is also possible that glucocorticoids may regulate LHRH neurons directly. We localized glucocorticoid receptors (GR) in LHRH neurons in the rat central nervous system using immunocytochemistry. In males and randomly cycling females 10-24% of LHRH neurons in the medial septum-diagonal bands of Broca, and preoptic regions colocalized nuclear GR. Ovariectomy increased the percentage of GR/LHRH neurons at the level of the organum vasculosum of the lamina terminalis to 34%, half of which showed both nuclear and cytoplasmic GR. Treatment with estradiol reversed this effect. We suggest that the actions of glucocorticoids on reproductive endocrine function are mediated partly through direct modulation of LHRH gene expression and/or release by activated GR. Moreover, GR in LHRH neurons may provide a mechanism by which the gonadal steroid progesterone can affect LHRH neurons directly, despite a lack of progesterone receptors in these neurons.     

Nonequivalence of alpha-bungarotoxin receptors and acetylcholine receptors in chick sympathetic neurons.

alpha-Bungarotoxin binds selectively to chick sympathetic neurons that are responsive iontophoretically applied acetylcholine. alpha-Bungarotoxin (125 nM) does not affect the response of cultured neurons to acetylcholine, nor does it affect a cholinergic synaptic potential recorded from sympathetic ganglia. d-Tubocurarine (100 muM) inhibits alpha-bungarotoxin binding and blocks acetylcholine receptor function in both preparations, but alpha-bungarotoxin does not protect acetylcholine receptors against d-tubocurarine blockade of acetylcholine responses. The receptor for alpha-bungarotoxin can be extracted from neuronal membranes with nonionic detergents and, when assayed by velocity sedimentation in sucrose gradients, sediments at a rate faster than that of skeletal muscle acetylcholine receptors. Treatment of alpha-bungarotoxin-receptor complexes with glutaraldehyde (0.1%, wt/vol) increases their stability from a half-time for dissociation of 3.5 hr to greater than 6 days at 23 degrees. This permits a quantitative assay of alpha-bungarotoxin-receptor complexes after relatively long periods of velocity sedimentation. It is concluded that alpha-bungarotoxin does not bind to the acetylcholine-binding site of neuronal acetylcholine receptors. These results compel a reevaluation of studies that assume that alpha-bungarotoxin is a specific ligand for neuronal acetylcholine receptors.     

Signal enhancement in distal cortical dendrites by means of interactions between active dendritic spines.

Pyramidal neurons in the cerebral cortex characteristically give rise to an apical dendrite, whose distal dendritic branches in layer I are covered with spines. These spines are known to be sites of synaptic connections, but the physiological properties of the spines and the functional significance of their responses are still largely unknown. The main function attributed thus far to these synaptic responses, situated at a great distance from the neuronal cell body, is slow background modulation of impulse output in the axon. In pursuing computer simulation analysis of electrical properties of dendrites, we have obtained results suggesting interactions between distal dendritic spines. If the heads of dendritic spines have excitable membrane properties, the spread of current from one or several spines could bring adjacent spines to their thresholds for impulse generation. This could give rise to a sequence of spine head action potentials, representing a saltatory propagation, from one or more excitable spine heads to nearby excitable spine heads, in the distal dendritic branches. Both the amplification due to several spine action potentials and the possibility of propagation into more proximal branches would increase the efficacy of distal synaptic inputs. Because of nonlinear dependence upon several modifiable parameters (such as spine stem resistance and membrane excitability) and upon the spatio-temporal pattern of synaptic input, such contingent synaptic enhancement would be particularly relevant to cortical functions underlying information processing and to plasticity underlying learning and memory.     

Multicopy plasmids are clustered and localized in Escherichia coli

We localized the multicopy plasmid RK2 in Escherichia coli and found that the number of fluorescent foci observed in each cell was substantially less than the copy number of the plasmid, suggesting that many copies of RK2 are grouped into a few multiplasmid clusters. In minimal glucose media, the majority of cells had one or two foci, with a single focus localized near midcell, and two foci near the 1/4 and 3/4 cell positions. The number of foci per cell increased with cell length and with growth rate, and decreased upon entering stationary phase, suggesting a coordination of RK2 replication or segregation with the bacterial cell cycle. Time-lapse microscopy demonstrated that partitioning of RK2 foci is achieved by the splitting of a single focus into two or three smaller foci, which are capable of separating with rapid kinetics. A derivative of the high-copy-number plasmid pUC19 containing the lacO array was also localized by tagging with GFP-LacI. Whereas many of the cells contained numerous, randomly diffusing foci, most cells exhibited one or two plasmid clusters located at midcell or the cell quarter positions. Our results suggest a model in which multicopy plasmids are not always randomly diffusing throughout the cell as previously thought, but can be replicated and partitioned in clusters targeted to specific locations.     

Striatal GABAergic and cortical glutamatergic neurons mediate contrasting effects of cannabinoids on cortical network synchrony

Activation of type 1 cannabinoid receptors (CB1R) decreases GABA and glutamate release in cortical and subcortical regions, with complex outcomes on cortical network activity. To date there have been few attempts to disentangle the region- and cell-specific mechanisms underlying the effects of cannabinoids on cortical network activity in vivo. Here we addressed this issue by combining in vivo electrophysiological recordings with local and systemic pharmacological manipulations in conditional mutant mice lacking CB1R expression in different neuronal populations. First we report that cannabinoids induce hypersynchronous thalamocortical oscillations while decreasing the amplitude of faster cortical oscillations. Then we demonstrate that CB1R at striatonigral synapses (basal ganglia direct pathway) mediate the thalamocortical hypersynchrony, whereas activation of CB1R expressed in cortical glutamatergic neurons decreases cortical synchrony. Finally we show that activation of CB1 expressed in cortical glutamatergic neurons limits the cannabinoid-induced thalamocortical hypersynchrony. By reporting that CB1R activations in cortical and subcortical regions have contrasting effects on cortical synchrony, our study bridges the gap between cellular and in vivo network effects of cannabinoids. Incidentally, the thalamocortical hypersynchrony we report suggests a potential mechanism to explain the sensory “high” experienced during recreational consumption of marijuana.