Sensory and behavioral modulation of thalamic head-direction cells.

Head-direction (HD) neurons are thought to exclusively encode directional heading. In awake mice, we found that sensory stimuli evoked robust short-latency responses in thalamic HD cells, but not in non-HD neurons. The activity of HD cells, but not that of non-HD neurons, was tightly correlated to brain-state fluctuations and dynamically modulated during social interactions. These data point to a new role for the thalamic compass in relaying sensory and behavioral-state information.

Opto-juxtacellular interrogation of neural circuits in freely moving mice.

Neural circuits are assembled from an enormous variety of neuronal cell types. Although significant advances have been made in classifying neurons on the basis of morphological, molecular and electrophysiological properties, understanding how this diversity contributes to brain function during behavior has remained a major experimental challenge. Here, we present an extension to our previous protocol, in which we describe the technical procedures for performing juxtacellular opto-tagging of single neurons in freely moving mice by using Channelrhodopsin-2-expressing viral vectors. This method allows one to selectively target molecularly defined cell classes for in vivo single-cell recordings. The targeted cells can be labeled via juxtacellular procedures and further characterized via post-hoc morphological and molecular analysis. In its current form, the protocol allows multiple recording and labeling attempts to be performed within individual animals, by means of a mechanical pipette micropositioning system. We provide proof-of-principle validation of this technique by recording from Calbindin-positive pyramidal neurons in the mouse hippocampus during spatial exploration; however, this approach can easily be extended to other behaviors and cortical or subcortical areas. The procedures described here, from the viral injection to the histological processing of brain sections, can be completed in ~4-5 weeks.This protocol is an extension to: Nat. Protoc. 9, 2369-2381 (2014): https://doi.org/10.1038/nprot.2014.161.

Modular microcircuit organization of the presubicular head-direction map.

Our internal sense of direction is thought to rely on the activity of head-direction (HD) neurons. We find that the mouse dorsal presubiculum (PreS), a key structure in the cortical representation of HD, displays a modular "patch-matrix" organization, which is conserved across species (including human). Calbindin-positive layer 2 neurons within the "matrix" form modular recurrent microcircuits, while inputs from the anterodorsal and laterodorsal thalamic nuclei are non-overlapping and target the "patch" and "matrix" compartments, respectively. The apical dendrites of identified HD cells are largely restricted within the "matrix," pointing to a non-random sampling of patterned inputs and to a precise structure-function architecture. Optogenetic perturbation of modular recurrent microcircuits results in a drastic tonic suppression of firing only in a subpopulation of HD neurons. Altogether, our data reveal a modular microcircuit organization of the PreS HD map and point to the existence of cell-type-specific microcircuits that support the cortical HD representation.

Juxtacellular opto-tagging of hippocampal CA1 neurons in freely moving mice.

Neural circuits are made of a vast diversity of neuronal cell types. While immense progress has been made in classifying neurons based on morphological, molecular, and functional properties, understanding how this heterogeneity contributes to brain function during natural behavior has remained largely unresolved. In the present study, we combined the juxtacellular recording and labeling technique with optogenetics in freely moving mice. This allowed us to selectively target molecularly defined cell classes for in vivo single-cell recordings and morphological analysis. We validated this strategy in the CA1 region of the mouse hippocampus by restricting Channelrhodopsin expression to Calbindin-positive neurons. Directly versus indirectly light-activated neurons could be readily distinguished based on the latencies of light-evoked spikes, with juxtacellular labeling and post hoc histological analysis providing 'ground-truth' validation. Using these opto-juxtacellular procedures in freely moving mice, we found that Calbindin-positive CA1 pyramidal cells were weakly spatially modulated and conveyed less spatial information than Calbindin-negative neurons - pointing to pyramidal cell identity as a key determinant for neuronal recruitment into the hippocampal spatial map. Thus, our method complements current in vivo techniques by enabling optogenetic-assisted structure-function analysis of single neurons recorded during natural, unrestrained behavior.

Morphological and biochemical analyses of otoliths of the ice-fish Chionodraco hamatus confirm a common origin with red-blooded species.

The morphology and composition of the three otoliths of the Antarctic ice-fish Chionodraco hamatus were studied by scanning electron microscopy and X-ray diffraction. The composition of the sagitta, lapillus and asteriscus protein matrices was also analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, western blots and confocal laser scanning microscopy to reveal the presence of and to localize the calcium-binding proteins calmodulin, calbindin and S-100. Morphological results indicated that the otoliths in this ice-fish were similar to those of Trematomus bernacchii, a red-blooded Antarctic species [B. Avallone et al. (2003) J. Submicrosc. Cytol. Pathol. 35, 69-76], but rather different from those of other teleosts. These two Antarctic species possessed a completely vateritic asteriscus, whereas their sagitta and lapillus were made mostly of aragonite. Parallel analysis of protein patterns in C. hamatus and T. bernacchii revealed that the sagitta significantly differed from the lapillus and asteriscus in both species. The sagitta did not contain the S-100 protein and showed calmodulin and calbindin located in discontinuous or incremental zones, respectively. These results demonstrate that the otoliths of C. hamatus and T. bernacchii share more resemblances than differences and support the idea of a common origin of these species.

Gentamicin ototoxicity in the saccule of the lizard Podarcis Sicula induces hair cell recovery and regeneration.

There is little information available on the susceptibility of reptilian saccule hair cells to ototoxin-induced sensory damage. In this study, we report morphological evidence of hair cell recovery and regeneration after damage induced by gentamicin in the saccule of a lizard. We perform morphological analysis using scanning electron microscopy and confocal laser scanning microscopy with actin and calbindin as markers for hair cells and tubulin as a marker for supporting cells. The data were consistent: gentamicin induced damage in the hair cells, and the damage increased with increasing duration of treatment. Initially, the saccule appeared unhealthy. Subsequently, the sensory hair cells became compromised, with fused stereovilli, followed by widespread loss of hair cell bundles from the hair cells. Finally, numerous hair cells were lost. Morphologically, the saccule appeared normal 28days after gentamicin treatment. Using a mitogenic marker, we tested whether or not there is hair cell regeneration following administration of gentamicin. We found evidence of bromodeoxyuridine incorporation first in supporting cell nuclei and subsequently in hair cell nuclei. This indicates that a process of sensory epithelium repair and hair cell regeneration occurred, in both extrastriolar and striolar regions, and that the recovery was due to both the proliferation of supporting cells and, as seems likely, self-repair of hair cell bundles.

The membranous labyrinth during larval development in lamprey (Lampetra planeri, Bloch, 1784).

SEM and CLSM studies were performed on the membranous labyrinth of Lampetra planeri, a threatened species of brook lamprey, spanning from the 1st to the 4th year of ammocoetes larval stages and on the adults. In all the examined stages, the entire membranous labyrinth does not show any morphologic differences, but only a progressive increase in size. SEM and CLSM observations show that the ciliated chamber is lined with numerous unsensorial multiciliated cells. In the early stages, the ciliary bundles were approximately 15 microm long, while in the late stages they reached 30 microm. In the crista sensory area, we observed two populations of hair cells. "Type II" cells are peculiar for this species and show both long stereocilia decreasing in length and a long kinocilium (10-12 microm). Two other types of ciliary bundles have been found on the sensory hair cells of the Macula communis: the first one has both kinocilium and stereocilia about 4-5 microm long; the second shows a long kinocilium (7-10 microm in length) and short stereocilia bundles with a gradual increase in length. In the early stages of development, the three macular areas show few and sparsely distributed hair cells. In the late developmental stages, hair cells become more numerous and densely populated.

Localization of calbindin D-28K in the otoconia of lizard Podarcis sicula.

The membranous labyrinth of lizard Podarcis sicula contains calcite and aragonite crystals. Saccule, utricle and lagena contain calcite crystals while aragonite crystals are present only in the saccule where they are very abundant. We have recently demonstrated the presence of calbindin D-28K in the organic matrix of lizard P. sicula otoconia. In order to define its localization, since calbindin modulates cellular Ca2+ level, otoconia from utricle and lagena were collected separately from those from saccule and then otoconial proteins were extracted. Immunoblot assay on proteins extracted from the otoconia and confocal laser scanning microscope analyses of otoconia using monoclonal anti-calbindin D-28K antibodies indicated that calbindin D-28K is a protein typical of aragonite crystals.

Evidence for hair cell regeneration in the crista ampullaris of the lizard Podarcis sicula.

We studied hair cell regeneration in the crista ampullaris of the lizard Podarcis sicula both in untreated animals and at early and late time intervals following a single high dose of gentamicin. The study was carried out using the S-phase marker 5-bromo-2'-deoxyuridine. Our ultrastructural and immunofluorescence studies showed that both apoptosis and hair cell regeneration happen in the lizard crista ampullaris in untreated animals, and that regenerative processes are greatly accelerated after treatment with the aminoglycoside antibiotic gentamicin. Our observations indicate that hair cell regeneration is strongly implicated in the repair of damaged sensory epithelium, and that new hair cells appear likely to arise from supporting cells.

Calbindin D28K is a component of the organic matrix of lizard Podarcis sicula otoconia.

The factors controlling otoconia growth are not well known but it seems that the type of proteins contained in the otoconia regulates the initiation and/or the subsequent rates of crystal growth determining the morphology and the size of the final crystal. In order to clarify the mechanism of otoconia formation and their turnover, major proteins contained in the otoconia from the maculae of the saccule, utricle and lagena of inner ear of lizard Podarcis sicula were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Coomassie staining of SDS-PAGE resulted in a major broad band of 15 kDa and four other bands of 21, 28, 45 and 97 kDa. The proteins of 15, 21, 28 and 45 kDa were separated by high-pressure liquid chromatography on a C-4-reverse-phase column and the incubation of blots with monoclonal anti-Calbindin D28K antibodies indicated that the band of 28 kDa was Calbindin D28K, a calcium-binding protein.

Enfermedad biliar y pancreática producida por ascaris. Hospital Universitario de los Andes / Biliar and pancreatic disease produced by ascaris. Hospital Universitario of Los Andes

Se informa sobre un caso de enfermedad biliar y pancreática producida por ascaris, presentado en el Hospital Universitario de Los Andes, Mérida, en Febrero de 1990. Se utilizó como único método diagnóstico y de seguimiento la ecosonografía, que reportó: forma lineal, intravesicular con movimientos propios que no deja sombra acústica posterior y, edema de páncreas. El control: cuerpo extraño (ascaris) en vías de desintegración y pancreas normal. Se administró tratamiento médico sintomático y antihelmíntico, se reserva el tratamiento quirúrgico solo en caso de litiasis vesicular secundaria a huevos de ascaris o sus restos y el cual se realizará en forma electiva