Context-dependent limb movement encoding in neuronal populations of motor cortex.

Neuronal networks of the mammalian motor cortex (M1) are important for dexterous control of limb joints. Yet it remains unclear how encoding of joint movement in M1 depends on varying environmental contexts. Using calcium imaging we measured neuronal activity in layer 2/3 of the M1 forelimb region while mice grasped regularly or irregularly spaced ladder rungs during locomotion. We found that population coding of forelimb joint movements is sparse and varies according to the flexibility demanded from individual joints in the regular and irregular context, even for equivalent grasping actions across conditions. This context-dependence of M1 encoding emerged during task learning, fostering higher precision of grasping actions, but broke apart upon silencing of projections from secondary motor cortex (M2). These findings suggest that M1 exploits information from M2 to adapt encoding of joint movements to the flexibility demands of distinct familiar contexts, thereby increasing the accuracy of motor output.

High-density multi-fiber photometry for studying large-scale brain circuit dynamics.

Animal behavior originates from neuronal activity distributed across brain-wide networks. However, techniques available to assess large-scale neural dynamics in behaving animals remain limited. Here we present compact, chronically implantable, high-density arrays of optical fibers that enable multi-fiber photometry and optogenetic perturbations across many regions in the mammalian brain. In mice engaged in a texture discrimination task, we achieved simultaneous photometric calcium recordings from networks of 12-48 brain regions, including striatal, thalamic, hippocampal and cortical areas. Furthermore, we optically perturbed subsets of regions in VGAT-ChR2 mice by targeting specific fiber channels with a spatial light modulator. Perturbation of ventral thalamic nuclei caused distributed network modulation and behavioral deficits. Finally, we demonstrate multi-fiber photometry in freely moving animals, including simultaneous recordings from two mice during social interaction. High-density multi-fiber arrays are versatile tools for the investigation of large-scale brain dynamics during behavior.

Sensory representation of an auditory cued tactile stimulus in the posterior parietal cortex of the mouse.

Sensory association cortices receive diverse inputs with their role in representing and integrating multi-sensory content remaining unclear. Here we examined the neuronal correlates of an auditory-tactile stimulus sequence in the posterior parietal cortex (PPC) using 2-photon calcium imaging in awake mice. We find that neuronal subpopulations in layer 2/3 of PPC reliably represent texture-touch events, in addition to auditory cues that presage the incoming tactile stimulus. Notably, altering the flow of sensory events through omission of the cued texture touch elicited large responses in a subset of neurons hardly responsive to or even inhibited by the tactile stimuli. Hence, PPC neurons were able to discriminate not only tactile stimulus features (i.e., texture graininess) but also between the presence and omission of the texture stimulus. Whereas some of the neurons responsive to texture omission were driven by looming-like auditory sounds others became recruited only with tactile sensory experience. These findings indicate that layer 2/3 neuronal populations in PPC potentially encode correlates of expectancy in addition to auditory and tactile stimuli.

Pathway-specific reorganization of projection neurons in somatosensory cortex during learning.

In the mammalian brain, sensory cortices exhibit plasticity during task learning, but how this alters information transferred between connected cortical areas remains unknown. We found that divergent subpopulations of cortico-cortical neurons in mouse whisker primary somatosensory cortex (S1) undergo functional changes reflecting learned behavior. We chronically imaged activity of S1 neurons projecting to secondary somatosensory (S2) or primary motor (M1) cortex in mice learning a texture discrimination task. Mice adopted an active whisking strategy that enhanced texture-related whisker kinematics, correlating with task performance. M1-projecting neurons reliably encoded basic kinematics features, and an additional subset of touch-related neurons was recruited that persisted past training. The number of S2-projecting touch neurons remained constant, but improved their discrimination of trial types through reorganization while developing activity patterns capable of discriminating the animal's decision. We propose that learning-related changes in S1 enhance sensory representations in a pathway-specific manner, providing downstream areas with task-relevant information for behavior.

Chronic imaging of cortical sensory map dynamics using a genetically encoded calcium indicator.

In vivo optical imaging can reveal the dynamics of large-scale cortical activity, but methods for chronic recording are limited. Here we present a technique for long-term investigation of cortical map dynamics using wide-field ratiometric fluorescence imaging of the genetically encoded calcium indicator (GECI) Yellow Cameleon 3.60. We find that wide-field GECI signals report sensory-evoked activity in anaesthetized mouse somatosensory cortex with high sensitivity and spatiotemporal precision, and furthermore, can be measured repeatedly in separate imaging sessions over multiple weeks. This method opens new possibilities for the longitudinal study of stability and plasticity of cortical sensory representations.

Omigapil ameliorates the pathology of muscle dystrophy caused by laminin-alpha2 deficiency.

Laminin alpha2-deficient congenital muscular dystrophy, called MDC1A, is a rare, devastating genetic disease characterized by severe neonatal hypotonia ("floppy infant syndrome"), peripheral neuropathy, inability to stand or walk, respiratory distress, and premature death in early life. Transgenic overexpression of the apoptosis inhibitor protein BCL-2, or deletion of the proapoptotic Bax gene in a mouse model for MDC1A prolongs survival and mitigates pathology, indicating that apoptotic events are involved in the pathology. Here we demonstrate that the proapoptotic glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-Siah1-CBP/p300-p53 pathway is activated in a mouse model for MDC1A. Moreover, we show that omigapil, which inhibits GAPDH-Siah1-mediated apoptosis, ameliorates several pathological hallmarks in the MDC1A mouse model. Specifically, we demonstrate that treatment with omigapil inhibits apoptosis in muscle, reduces body weight loss and skeletal deformation, increases locomotive activity, and protects from early mortality. These data qualify omigapil, which is in late phase of clinical development for human use, as a drug candidate for the treatment of MDC1A.

Functional expression of mammalian receptors and membrane channels in different cells.

In native tissues, the majority of medically important membrane proteins is only present at low concentrations, making their overexpression in recombinant systems a prerequisite for structural studies. Here, we explore the commonly used eukaryotic expression systems-yeast, baculovirus/insect cells (Sf9) and Semliki Forest Virus (SFV)/mammalian cells-for the expression of seven different eukaryotic membrane proteins from a variety of protein families. The expression levels, quality, biological activity, localization and solubility of all expressed proteins are compared in order to identify the advantages of one system over the other. SFV-transfected mammalian cell lines provide the closest to native environment for the expression of mammalian membrane proteins, and they exhibited the best overall performance. But depending on the protein, baculovirus-infected Sf9 cells performed almost as well as mammalian cells. The lowest expression levels for the proteins tested here were obtained in yeast.

Molecular fingerprinting of carbohydrate structure phenotypes of three porifera proteoglycan-like glyconectins.

Glyconectins (GNs) represent a new class of proteoglycan-like cell adhesion and recognition molecules found in several Porifera species. Physico-chemical properties of GN carbohydrate moieties, such as size, composition, and resistance to most glycosaminoglycan-degrading enzymes, distinguish them from any other type of known glycoproteins. The molecular mechanism of GN-mediated self/non-self discrimination function is based on highly species-specific and Ca(2+)-dependent GN to GN associations that approach the selectivity of the evolutionarily advanced immunoglobulin superfamily. Carbohydrates of glyconectins 1, 2, and 3 are essential for species-specific auto-aggregation properties in three respective Porifera species. To obtain a structural insight into the molecular mechanisms, we performed carbohydrate structural analyses of glyconectins isolated from the three sponge model systems, Microciona prolifera (GN1), Halichondria panicea (GN2), and Cliona celata (GN3). The glycan content of all three GNs ranged between 40 and 60% of their total mass. Our approach using sequential and selective chemical degradation of GN glycans and subsequent mass spectrometric and NMR analyses revealed that each glyconectin presents novel and highly species-specific carbohydrate sequences. All three GNs include distinct acid-resistant and acid-labile carbohydrate domains, the latter composed of novel repetitive units. We have sequenced four short sulfated and one pyruvilated unit in GN1, eight larger and branched pyruvilated oligosaccharides in GN2, which represent a heterogeneous but related family of structures, and four sulfated units in GN3.

Molecular recognition between glyconectins as an adhesion self-assembly pathway to multicellularity.

The appearance of multicellular forms of life has been tightly coupled to the ability of an organism to retain its own anatomical integrity and to distinguish self from non-self. Large glycoconjugates, which make up the outermost cell surface layer of all Metazoans, are the primary candidates for the primordial adhesion and recognition functions in biological self-assembly systems. Atomic force microscopy experiments demonstrated that the binding strength between a single pair of Porifera cell surface glyconectin 1 glycoconjugates from Microciona prolifera can hold the weight of 1600 cells, proving their adhesion functions. Here, measurement of molecular self-recognition of glyconectins (GNs) purified from three Porifera species was used as an experimental model for primordial xenogeneic self/non-self discrimination. Physicochemical and biochemical characterization of the three glyconectins, their glycans, and peptides using gel electrophoresis, ultracentrifugation, NMR, mass spectrometry, glycosaminoglycan-degrading enzyme treatment, amino acid and carbohydrate analyses, and peptide mapping showed that GNs define a new family of proteoglycan-like molecules exhibiting species-specific structures with complex and repetitive acidic carbohydrate motives different from the classical proteoglycans and mucins. In functional self-assembly color-coded bead, cell, and blotting assays, glyconectins displayed species-specific recognition and adhesion. Affinity-purified monospecific polyclonal antibodies prepared against GN1, -2, and -3 glycans selectively inhibited cell adhesion of the respective sponge species. These results together with species-specific coaggregation of GN carbohydrate-coated beads with cells showed that GN glycans are functional in cell recognition and adhesion. The specificity of carbohydrate-mediated homophilic GN interactions in Porifera approaches the binding selectivity of the evolutionarily advanced immunoglobulin superfamily. Xenoselectivity of primordial glyconectin to glyconectin recognition may be a new paradigm in the self-assembly and non-self discrimination pathway of cellular adhesion leading to multicellularity.

DOTA alpha-melanocyte-stimulating hormone analogues for imaging metastatic melanoma lesions.

Scintigraphic imaging of metastatic melanoma lesions requires highly tumor-specific radiopharmaceuticals. Because both melanotic and amelanotic melanomas overexpress melanocortin-1 receptors (MC1R), radiolabeled analogues of alpha-melanocyte-stimulating hormone (alpha-MSH) are potential candidates for melanoma diagnosis. Here, we report the in vivo performance of a newly designed octapeptide analogue, [betaAla(3), Nle(4), Asp(5), D-Phe(7), Lys(10)]-alpha-MSH(3-10) (MSH(OCT)), which was conjugated through its N-terminal amino group to the metal chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) to enable incorporation of radiometals (e.g., indium-111) into the peptide. DOTA-MSH(OCT) displayed high in vitro MC1R affinity (IC(50) 9.21 nM). In vivo [(111)In]DOTA-MSH(OCT) exhibited a favorable biodistribution profile after injection in B16-F1 tumorbearing mice. The radiopeptide was rapidly cleared from blood through the kidneys and, most importantly, accumulated preferentially in the melanoma lesions. Lung and liver melanoma metastases could be clearly imaged on tissue section autoradiographs 4 h after injection of [(111)In]DOTA-MSH(OCT). A comparative study of [(111)In]DOTA-MSH(OCT) with [(111)In]DOTA-[Nle(4), D-Phe(7)]-alpha-MSH ([(111)In]-DOTA-NDP-MSH) demonstrated the superiority of the DOTA-MSH(OCT) peptide, particularly for the amount of radioactivity taken up by nonmalignant organs, including bone, the most radiosensitive tissue. These results demonstrate that [(111)In]DOTA-MSH(OCT) is a promising melanoma imaging agent.

A novel DOTA-alpha-melanocyte-stimulating hormone analog for metastatic melanoma diagnosis.

UNLABELLED: Scintigraphic imaging of metastatic melanoma lesions requires highly tumor-specific radiolabeled compounds. Because both melanotic and amelanotic melanomas overexpress receptors for alpha-melanocyte-stimulating hormone (alpha-MSH; receptor name: melanocortin type 1 receptor, or MC1R), radiolabeled alpha-MSH analogs are potential candidates for melanoma diagnosis. The aim of this study was to develop a melanoma-selective radiolabeled alpha-MSH analog suitable for melanoma diagnosis. METHODS: The very potent alpha-MSH analog [Nle(4), D-Phe(7)]-alpha-MSH (NDP-MSH) and a newly designed alpha-MSH octapeptide analog, [betaAla(3), Nle(4), Asp(5), D-Phe(7), Lys(10)]-alpha-MSH(3-10) (MSH(oct)), were conjugated to the metal chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) to enable radiometal incorporation. The resulting DOTA conjugates were evaluated in vitro for their MC1R-binding affinity and melanogenic activity in isolated mouse B16F1 cells and in vivo for their biodistribution in mouse models of primary and metastatic melanoma after labeling with (111)In. RESULTS: DOTA-MSH(oct) was shown to bind with high affinity (inhibitory concentration of 50% [IC(50)] = 9.21 nmol/L) to the MC1R, although with lower potency than does DOTA-NDP-MSH (IC(50) = 0.25 nmol/L). In B16F1 melanoma-bearing mice, both (111)In-DOTA-NDP-MSH and (111)In-DOTA-MSH(oct) exhibited high MC1R-mediated uptake by melanoma, which differed by a factor of only 1.5 at 4 h after injection. The main route of excretion for both radioconjugates was the kidneys, whereby (111)In-DOTA-MSH(oct) led to somewhat higher kidney values than did (111)In-DOTA-NDP-MSH. In contrast, the latter was much more poorly cleared from other nonmalignant tissues, including bone, the most radiosensitive organ. Therefore, (111)In-DOTA-MSH(oct) displayed higher uptake ratios of tumor to nontarget tissue (e.g., tumor-to-bone ratio 4 h after injection was 4.9 for (111)In-DOTA-NDP-MSH and 53.9 for (111)In-DOTA-MSH(oct)). Lung and liver melanoma metastases could easily be visualized on tissue section autoradiographs after injection of (111)In-DOTA-MSH(oct). Radio-reversed-phase high-performance liquid chromatography analysis of urine samples revealed that most (111)In-DOTA-MSH(oct) is excreted intact 4 h after injection, indicating good in vivo stability. CONCLUSION: (111)In-DOTA-MSH(oct) exhibits more favorable overall performance than does (111)In-DOTA-NDP-MSH in murine models of primary and metastatic melanoma, making it a promising melanoma imaging agent.

Endothelial, but not the inducible, nitric oxide synthase is detectable in normal and portal hypertensive rats.

BACKGROUND: Chronic portal hypertension is accompanied by a nitric oxide (NO) dependent vasodilation. Three isoforms of NO producing synthases (NOS) are characterized: neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). Sources of increased NO levels in chronic hypertension is disputed. METHODS: To determine eNOS and iNOS expression in different organs of portal hypertensive and control rats, we divided Sprague-Dawley rats in 6 groups: (1). Partial portal vein ligated rats, (2). Bile duct ligated rats, (3). Carbon tetrachloride treated rats, (4). Sham operated rats, (5). Untreated control rats, and (6). LPS treated rats. Immunohistochemistry (IHC) and immunoblotting (IB) using antibodies against eNOS or iNOS were carried out on samples from thymus, aorta, heart, lung, oesophagus, liver, spleen, kidney, pancreas, small and large intestine. RESULTS: IHC revealed an even eNOS expression in all groups. Expression of iNOS was restricted to macrophages in organs of LPS treated and the thymus of rats. IB mirrored these results. CONCLUSION: In chronic portal hypertension, the main source for NO production depends on eNOS activity.

Inhibition of NO biosynthesis, but not elevated blood pressure, reduces angiogenesis in rat models of secondary hypertension.

Arterial hypertension (AH) is characterized by reduced nitric oxide (NO) biosynthesis, vasoconstriction, and reduced microvascular density. In this study we asked whether AH also reduces the number of microvessels by impairing angiogenesis. AH was induced in Dahl salt-sensitive rats (DSS) with a salt diet and in Wistar-Kyoto rats by inhibiting NO formation with Nomega-nitro-L-arginine (NNA). Three weeks after induction of AH, two wound chambers containing collagen I (Vitrogen) were sutured into the mesenteric cavity of each animal. After additional 14 days, wound chamber neovascularization and the extent of vascularized connective tissue ingrowth were quantified. In NNA-induced AH, the number of newly formed vessels and the ingrowth of vascularized connective tissue into the wound chamber decreased as compared to controls. However, the number of newly formed vessels and the ingrowth of vascularized connective tissue did not change with increasing blood pressure in salt-fed DSS rats as compared to those fed a normal diet. Inhibition of NO biosynthesis, but not necessarily elevating blood pressure, reduces angiogenesis. Microvascular rarefaction in AH may be partially due to reduced angiogenesis because of impaired NO biosynthesis.

Neuroendocrine tumor targeting: study of novel gallium-labeled somatostatin radiopeptides in a rat pancreatic tumor model.

Somatostatin analogs labeled with radionuclides are of considerable interest in the diagnosis and therapy of SSTR-expressing tumors, such as gastroenteropancreatic, small cell lung, breast and frequently nervous system tumors. In view of the favorable physical characteristics of the Ga isotopes (67)Ga and (68)Ga, enabling conventional tumor scintigraphy, PET and possibly internal radiotherapy, we focused on the development of a Ga-labeled somatostatin analog suitable for targeting SSTR-expressing tumors. For this purpose, 3 somatostatin analogs, OC, TOC and TATE were conjugated to the metal chelator DOTA and labeled with the radiometals (111)In, (90)Y and (67)Ga. They were then evaluated for their performance in the AR4-2J pancreatic tumor model by testing SSTR2-binding affinity, internalization/externalization in isolated cells and biodistribution in tumor-bearing nude mice. Surprisingly, we found that, compared to (111)In or (90)Y, labeling with (67)Ga considerably improved the biologic performance of the tested somatostatin analogs with respect to SSTR2 affinity and tissue distribution. (67)Ga-labeled DOTA-somatostatin analogs were rapidly excreted from nontarget tissues, leading to excellent tumor-to-nontarget tissue uptake ratios. Of interest for radiotherapeutic application, [(67)Ga]DOTATOC was strongly internalized by AR4-2J cells. Furthermore, our results suggest a link between the radioligand charge and its kidney retention. The excellent tumor selectivity of Ga-DOTA somatostatin analogs together with the different applications of Ga in nuclear oncology suggests that Ga-DOTA somatostatin analogs will become an important tool in the management of SSTR-positive tumors.