Juxtacellular Monitoring and Localization of Single Neurons within Sub-cortical Brain Structures of Alert,Head-restrained Rats

There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures in behaving animals remains a technical challenge, especially if the structures must be targeted stereotaxically. This protocol describes convenient surgical and electrophysiological techniques that maintain the animal’s head in the stereotaxic plane and unambiguously isolate the spiking activity of single neurons. The protocol combines head restraint of alert rodents, juxtacellular monitoring with micropipette electrodes, and iontophoretic dye injection to identify the neuron location in post-hoc histology. While each of these techniques is in itself well-established, the protocol focuses on the specifics of their combined use in a single experiment. These neurophysiological and neuroanatomical techniques are combined with behavioral monitoring. In the present example, the combined techniques are used to determine how self-generated vibrissa movements are encoded in the activity of neurons within the somatosensory thalamus. More generally, it is straightforward to adapt this protocol to monitor neuronal activity in conjunction with a variety of behavioral tasks in rats, mice, and other animals. Critically, the combination of these methods allows the experimenter to directly relate anatomically-identified neurophysiological signals to behavior.     

Calmodulin regulation of Nav1.4 current: role of binding to the carboxyl terminus

Calmodulin (CaM) regulates steady-state inactivation of sodium currents (Na(V)1.4) in skeletal muscle. Defects in Na current inactivation are associated with pathological muscle conditions such as myotonia and paralysis. The mechanisms of CaM modulation of expression and function of the Na channel are incompletely understood. A physical association between CaM and the intact C terminus of Na(V)1.4 has not previously been demonstrated. FRET reveals channel conformation-independent association of CaM with the C terminus of Na(V)1.4 (CT-Na(V)1.4) in mammalian cells. Mutation of the Na(V)1.4 CaM-binding IQ motif (Na(V)1.4(IQ/AA)) reduces cell surface expression of Na(V)1.4 channels and eliminates CaM modulation of gating. Truncations of the CT that include the IQ region abolish Na current. Na(V)1.4 channels with one CaM fused to the CT by variable length glycine linkers exhibit CaM modulation of gating only with linker lengths that allowed CaM to reach IQ region. Thus one CaM is sufficient to modulate Na current, and CaM acts as an ancillary subunit of Na(V)1.4 channels that binds to the CT in a conformation-independent fashion, modulating the voltage dependence of inactivation and facilitating trafficking to the surface membrane.     

Compound I formation is a partially rate-limiting process in chloroperoxidase-catalyzed bromination reactions

The kinetics of chloroperoxidase-catalyzed bromination and chlorination reactions were studied at various halide and hydrogen peroxide concentrations. At very high concentrations, both chloride (KI = 370 mM) and bromide (KI = 150 mM) are competitive substrate inhibitors versus hydrogen peroxide. Results at subinhibitory halide concentrations for bromination reactions (kcat = 4 ms-1, kcat/KPeroxide = 1.6 microM-1 x s-1 and kcat/KBr = 4.0 microM-1 x s-1) and chlorination reactions (kcat = 1.5 ms-1, kcat/Kperoxide = 2.3 microM-1 x s-1, and kcat/KBr = 0.32 microM-1 x s-1) indicate that halide oxidation is rate-limiting in chlorination reactions. However, in bromination reactions, both compound I formation and bromide oxidation are partially rate-limiting. This is the first documented case where compound I formation participates in determining the overall rate of a peroxidase reaction.     

Susceptibility to neurodegeneration in a glaucoma is modified by Bax gene dosage

In glaucoma, harmful intraocular pressure often contributes to retinal ganglion cell death. It is not clear, however, if intraocular pressure directly insults the retinal ganglion cell axon, the soma, or both. The pathways that mediate pressure-induced retinal ganglion cell death are poorly defined, and no molecules are known to be required. DBA/2J mice deficient in the proapoptotic molecule BCL2-associated X protein (BAX) were used to investigate the roles of BAX-mediated cell death pathways in glaucoma. Both Bax+/- and Bax-/- mice were protected from retinal ganglion cell death. In contrast, axonal degeneration was not prevented in either Bax+/- or Bax-/- mice. While BAX deficiency did not prevent axonal degeneration, it did slow axonal loss. Additionally, we compared the effects of BAX deficiency on the glaucoma to its effects on retinal ganglion cell death due to two insults that are proposed to participate in glaucoma. As in the glaucoma, BAX deficiency protected retinal ganglion cells after axon injury by optic nerve crush. However, it did not protect retinal ganglion cells from N-methyl-D-aspartate (NMDA)-induced excitotoxicity. BAX is required for retinal ganglion cell death in an inherited glaucoma; however, it is not required for retinal ganglion cell axon degeneration. This indicates that distinct somal and axonal degeneration pathways are active in this glaucoma. Finally, our data support a role for optic nerve injury but not for NMDA receptor-mediated excitotoxicity in this glaucoma. These findings indicate a need to understand axon-specific degeneration pathways in glaucoma, and they suggest that distinct somal and axonal degeneration pathways may need to be targeted to save vision.     

High functional diversity in deep‐sea fish communities and increasing intraspecific trait variation with increasing latitude

Variation in both inter‐ and intraspecific traits affects community dynamics, yet we know little regarding the relative importance of external environmental filters versus internal biotic interactions that shape the functional space of communities along broad‐scale environmental gradients, such as latitude, elevation, or depth. We examined changes in several key aspects of functional alpha diversity for marine fishes along depth and latitude gradients by quantifying intra‐ and interspecific richness, dispersion, and regularity in functional trait space. We derived eight functional traits related to food acquisition and locomotion and calculated seven complementary indices of functional diversity for 144 species of marine ray‐finned fishes along large‐scale depth (50–1200 m) and latitudinal gradients (29°–51° S) in New Zealand waters. Traits were derived from morphological measurements taken directly from footage obtained using Baited Remote Underwater Stereo‐Video systems and museum specimens. We partitioned functional variation into intra‐ and interspecific components for the first time using a PERMANOVA approach. We also implemented two tree‐based diversity metrics in a functional distance‐based context for the first time: namely, the variance in pairwise functional distance and the variance in nearest neighbor distance. Functional alpha diversity increased with increasing depth and decreased with increasing latitude. More specifically, the dispersion and mean nearest neighbor distances among species in trait space and intraspecific trait variability all increased with depth, whereas functional hypervolume (richness) was stable across depth. In contrast, functional hypervolume, dispersion, and regularity indices all decreased with increasing latitude; however, intraspecific trait variation increased with latitude, suggesting that intraspecific trait variability becomes increasingly important at higher latitudes. These results suggest that competition within and among species are key processes shaping functional multidimensional space for fishes in the deep sea. Increasing morphological dissimilarity with increasing depth may facilitate niche partitioning to promote coexistence, whereas abiotic filtering may be the dominant process structuring communities with increasing latitude.     

Peroxisome proliferator-activated receptor-gamma activators inhibit IFN-gamma-induced expression of the T cell-active CXC chemokines IP-10, Mig, and I-TAC in human endothelial cells

Peroxisome proliferator-activated receptor-gamma (PPARgamma), a member of the nuclear hormone receptor superfamily originally shown to play an important role in adipocyte differentiation and glucose homeostasis, is now known to regulate inflammatory responses. Given the importance of endothelial cell (EC)-derived chemokines in regulating leukocyte function and trafficking, we studied the effects of PPARgamma ligands on the expression of chemokines induced in ECs by the Th1 cytokine IFN-gamma. Treatment of ECs with PPARgamma activators significantly inhibited IFN-gamma-induced mRNA and protein expression of the CXC chemokines IFN-inducible protein of 10 kDa (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha-chemoattractant (I-TAC), whereas expression of the CC chemokine monocyte chemoattractant protein-1 was not altered. PPARgamma activators decreased IFN-inducible protein of 10 kDa promoter activity and inhibited protein binding to the two NF-kappaB sites but not to the IFN-stimulated response element ISRE site. Furthermore, PPARgamma ligands inhibited the release of chemotactic activity for CXC chemokine receptor 3 (CXCR3)-transfected lymphocytes from IFN-gamma-stimulated ECs. These data suggest that anti-diabetic PPARgamma activators might attenuate the recruitment of activated T cells at sites of Th1-mediated inflammation.     

A SCA7 CAG/CTG repeat expansion is stable in Drosophila melanogaster despite modulation of genomic context and gene dosage

CAG and CTG repeat expansions are the cause of at least a dozen inherited neurological disorders. In these so-called "dynamic mutation" diseases, the expanded repeats display dramatic genetic instability, changing in size when transmitted through the germline and within somatic tissues. As the molecular basis of the repeat instability process remains poorly understood, modeling of repeat instability in model organisms has provided some insights into potentially involved factors, implicating especially replication and repair pathways. Studies in mice have also shown that the genomic context of the repeat sequence is required for CAG/CTG repeat instability in the case of spinocerebellar ataxia type 7 (SCA7), one of the most unstable of all CAG/CTG repeat disease loci. While most studies of repeat instability have taken a candidate gene approach, unbiased screens for factors involved in trinucleotide repeat instability have been lacking. We therefore attempted to use Drosophila melanogaster to model expanded CAG repeat instability by creating transgenic flies carrying trinucleotide repeat expansions, deriving flies with SCA7 CAG90 repeats in cDNA and genomic context. We found that SCA7 CAG90 repeats are stable in Drosophila, regardless of context. To screen for genes whose reduced function might destabilize expanded CAG repeat tracts in Drosophila, we crossed the SCA7 CAG90 repeat flies with various deficiency stocks, including lines lacking genes encoding the orthologues of flap endonuclease-1, PCNA, and MutS. In all cases, perfect repeat stability was preserved, suggesting that Drosophila may not be a suitable system for determining the molecular basis of SCA7 CAG repeat instability.     

Prostaglandin E receptor type 4-associated protein interacts directly with NF-kappaB1 and attenuates macrophage activation

Macrophage activation participates pivotally in the pathophysiology of chronic inflammatory diseases, including atherosclerosis. Through the receptor EP4, prostaglandin E(2) (PGE(2)) exerts an anti-inflammatory action in macrophages, suppressing stimulus-induced expression of certain proinflammatory genes, including chemokines. We recently identified a novel EP4 receptor-associated protein (EPRAP), whose function in PGE(2)-mediated anti-inflammation remains undefined. Here we demonstrate that PGE(2) pretreatment selectively inhibits lipopolysaccharide (LPS)-induced nuclear factor kappaB1 (NF-kappaB1) p105 phosphorylation and degradation in mouse bone marrow-derived macrophages through EP4-dependent mechanisms. Similarly, directed EPRAP expression in RAW264.7 cells suppresses LPS-induced p105 phosphorylation and degradation, and subsequent activation of mitogen-activated protein kinase kinase 1/2. Forced expression of EPRAP also inhibits NF-kappaB activation induced by various proinflammatory stimuli in a concentration-dependent manner. In co-transfected cells, EPRAP, which contains multiple ankyrin repeat motifs, directly interacts with NF-kappaB1 p105/p50 and forms a complex with EP4. In EP4-overexpressing cells, PGE(2) enhances the protective action of EPRAP against stimulus-induced p105 phosphorylation, whereas EPRAP silencing in RAW264.7 cells impairs the inhibitory effect of PGE(2)-EP4 signaling on LPS-induced p105 phosphorylation. Additionally, EPRAP knockdown as well as deficiency of NF-kappaB1 in macrophages attenuates the inhibitory effect of PGE(2) on LPS-induced MIP-1beta production. Thus, PGE(2)-EP4 signaling augments NF-kappaB1 p105 protein stability through EPRAP after proinflammatory stimulation, limiting macrophage activation.     

Analysis of the mitogenic effect of fetuin preparations on arterial smooth muscle cells: the role of contaminant platelet-derived growth factor

Fetuin, a major protein of fetal calf serum, partially purified by the method of Pedersen, stimulated growth of aortic smooth muscle cells. More highly purified fetuin preparations stimulated growth less than Pedersen fetuin, as previously described for other cell types, suggesting that this activity is due to a contaminant. Recently bovine alpha 2-macroglobulin or "Embryonin" has been proposed as the mitogenic component of crude fetuin preparations. We found that active fetuin preparations did contain alpha 2-macroglobulin that stimulated smooth muscle cell growth. However, alpha 2-macroglobulin purified directly from platelet-poor bovine plasma or fetuin purified from Pedersen fetuin by gel filtration lacked appreciable mitogenic effect on smooth muscle cells. Since alpha 2-macroglobulin can bind platelet-derived growth factor (PDGF), and since highly acidic fetuin might bind the very basic PDGF molecule non-specifically, we measured the PDGF content of various fetuin preparations and found a good correlation between the PDGF content and mitogenic activity. Gel filtration experiments demonstrated that in Pedersen fetuin PDGF occurred both free, and in association with alpha 2-macroglobulin. We conclude that the principal mitogenic component for smooth muscle cells in crude fetuin preparations is PDGF, since purified bovine alpha 2-macroglobulin or fetuin do not appreciably affect growth of these cells. These results help to resolve a long-standing controversy regarding the nutrition of cultured cells. In addition, we suggest that before alpha 2-macroglobulin or "Embryonin" is accepted as a bona fide growth factor for a given cell type, the role of contamination with PDGF should be assessed.