C. Lopez-Garcia

A new stabilizing agent for the tetramethyl benzidine (TMB) reaction product in the histochemical detection of horseradish peroxidase (HRP)

In this paper, an alternative procedure for the histochemical detection of HRP using amonium heptamolybdate (AHM) as a stabilizing agent and tetramethyl benzidine (TMB) as a chromogen is reported. This procedure avoids the two main problems that occur in previous methods using sodium nitroferricyanide (SNF) as the stabilizer, namely, the appearance of needle-shaped crystals at non-specific anatomical sites, and intensive tissue shrinkage. A comparative study of both, the TMB-AHM and TMB-SNF methods, was performed in the analysis of cerebral cortex afferent connections of the lizard Podarcis hispanica. This study demonstrates that the two methods are of similar sensitivity. The TMB-AHM reaction can be carried out at physiological pH (from 6 to 8), thus, avoiding tissue contraction. The reaction product is of an intense blue-green colour and, as with the TMB-SNF method, shows granulation. The appearance of non-specific precipitates is completely avoided when the incubation medium is maintained at a pH in excess of 5.

Delayed postnatal neurogenesis in the cerebral cortex of lizards.

Labelled cells were consistently observed in the medial cortex of the lizard brain after i.p. injections of tritiated thymidine (5 microCi/g b. wt.), 1, 7, 18 or 28 days of survival and posterior autoradiographic evaluation. In 3 groups of specimens (postnatal, young and adult) of the species Podarcis hispanica, after one day of survival, labelled cells were located in the ependymal cell layer underlying the medial cortex. After intermediate survival times (7, 18 days), labelled cells were found in 3 zones: the ependymal layer, the inner plexiform layer and the granular layer. After one month of survival, most labelled cells were observed in the granular layer. In the granular layer, these cells were distributed at random. These results show that postnatal neurogenesis in the medial cortex of the lizard occurs following a spatio-temporal pattern reminiscent of that found in the fascia dentata of the mammalian hippocampus.

Zinc-positive boutons in the cerebral cortex of lizards show glutamate immunoreactivity

SummaryZinc-positive boutons, originating in the medial cortex of lizards, exhibit glutamate immunoreactivity. This finding supports the presumed homology between lizard zinc-positive boutons and the hippocampal mossy fibres of mammals, which are also glutamate-immunoreactive and zinc-positive.Zinc-positive boutons of lizards contain a chelatable pool of zinc located in the synaptic vesicles, as occurs in the hippocampal mossy fibres of mammals. These synaptic systems also contain glutamate, which indicates a possible simultaneous action of zinc and glutamate during synaptic transmission.

Postnatal neurogenesis in the olfactory bulbs of a lizard. A tritiated thymidine autoradiographic study

Autoradiographically labelled cells were observed in the olfactory bulbs of perinatal, young and adult specimens of the lizard Podarcis hispanica following intraperitoneal injection of tritiated thymidine (5 muCi/g b.wt). After survival times of 7, 18 and 28 days labelled cells were found in the granular layer of both main and accessory bulbs. A few labelled cells were observed in the ependyma, mitral and glomerular layer. In the main olfactory bulb, one week of survival time resulted in labelling of cells in the innermost part of the granular layer. Longer survival times (up to 4 weeks), resulted in labelling of cells mainly in the outermost part of the granular layer. This spatio-temporal gradient was not observed in the accessory bulb. Nevertheless, longer survival times resulted in greater number of labelled cells located in the dorsal and ventral parts of the granular layer of the accessory bulb.

Fos-like expression and nuclear size in osmotically stimulated supraoptic nucleus neurons.

This study has analysed by immunocytochemistry the pattern of expression of Fos-related proteins, as well as variations in nuclear size, after the osmotically induced activation of supraoptic nucleus neurons of the rat. In control rats most supraoptic nucleus neurons were Fos-like negative. After acute and chronic dehydration by salt-loading, the number of Fos-like positive neurons increased dramatically. The level of Fos-like immunoreactivity was higher in chronically stimulated rats, and also the neurons of the ventral region of the supraoptic nucleus were more intensely stained than those of the dorsal region. The karyometric analysis was made on electron micrographs. The mean nuclear profile area showed a significant increase in dehydrated rats with respect to the controls (73 +/- 16 microns 2 in those dehydrated for six days vs 54 +/- 13 in controls, mean +/- S.D.). However, no significant differences in this parameter were found when one-day and six-day dehydrated groups were compared. The invagination factor of the nuclear membrane, a nuclear shape indicator, decreased significantly in dehydrated rats, indicating a tendency towards spherical nuclei. It is noteworthy that the nuclear profile perimeter was constant, about 32 microns, in control and osmotically simulated rats. The higher nuclear accumulation of Fos-related antigens after six days of dehydration suggests that in chronically stimulated supraoptic nucleus neurons there is a sustained induction of cell-specific genes. Moreover, the transcription rate of the target genes containing the consensus DNA sequence TGAC/GTCA or c-AMP responsive elements recognition sites may depend upon the nuclear concentration of Fos-related antigens in supraoptic nucleus neurons. Our results also suggest that the initial Fos-related antigen expression and nuclear size increase are triggered concomitantly in supraoptic nucleus neurons after a short period of osmotic stimulation. On the other hand, we propose that nuclear envelope invaginations represent a reservoir of nuclear membrane which allows dynamic changes in nuclear size and shape depending on the metabolic status of the supraoptic nucleus neurons.

Early ERK1/2 activation promotes DRP1-dependent mitochondrial fission necessary for cell reprogramming

During the process of reprogramming to induced pluripotent stem (iPS) cells, somatic cells switch from oxidative to glycolytic metabolism, a transition associated with profound mitochondrial reorganization. Neither the importance of mitochondrial remodelling for cell reprogramming, nor the molecular mechanisms controlling this process are well understood. Here, we show that an early wave of mitochondrial fragmentation occurs upon expression of reprogramming factors. Reprogramming-induced mitochondrial fission is associated with a minor decrease in mitochondrial mass but not with mitophagy. The pro-fission factor Drp1 is phosphorylated early in reprogramming, and its knockdown and inhibition impairs both mitochondrial fragmentation and generation of iPS cell colonies. Drp1 phosphorylation depends on Erk activation in early reprogramming, which occurs, at least in part, due to downregulation of the MAP kinase phosphatase Dusp6. Taken together, our data indicate that mitochondrial fission controlled by an Erk-Drp1 axis constitutes an early and necessary step in the reprogramming process to pluripotency.

Neurons in the medial cortex give rise to Timm-positive boutons in the cerebral cortex of lizards

The origin of Timm-positive presynaptic boutons in the cerebral cortex of the lizard, Podarcis hispanica, was investigated by injections of horseradish peroxidase (HRP)-saponine in Timm-positive areas, i.e. the dorsal and dorsomedial cortices. A broad retrograde labelling of cell somata in the medial cortex was found. Injections of HRP-saponine in the medial cortex resulted in broad anterograde labelling of boutons located in the Timm-positive zones. A double-labelling of the HRP labelled boutons was obtained by using the Neo-Timm or the sulphide-osmium methods. The present results suggest that neurons of the medial cortex send axons that terminate in Timm-positive boutons in the cerebral cortex of lizards.

Volumetric and densitometric study in the cerebral cortex and septum of a lizard (Lacerta galloti) using the Timm method.

The Timm sulfide-silver method for the histochemical detection of heavy metals has been applied to the study of cortical and septal regions in the telencephalon of the lizard Lacerta galloti. Reacting areas are located in the anterodorsal septum, in the inner plexiform layer of the medial cortex, and in the plexiform layers of the dorsomedial and dorsal cortices. A very specific kind of axonal ending is responsible for this reactivity. Volumetric and densitometric measures have been performed in each reacting area. Usefulness of both measures as indicators of the number of labeled axonal endings projecting on a zone is discussed. Resemblance between these axonal endings and hippocampal mossy fiber boutons in mammals is suggested.

Reactive neurogenesis during regeneration of the lesioned medial cerebral cortex of lizards.

This study reports that lesion of the adult lizard medial cortex (lizard hippocampal fascia dentata) induces a short period of intensive neurogenesis which we have termed reactive neurogenesis; a cell proliferation event that occurs in the subjacent ependyma. Specific lesion of the medial cortex was achieved by intraperitoneal injection of the neurotoxin 3-acetylpyridine and proliferating cells were detected using tritiated thymidine or 5-bromodeoxiuridine pulse labelling. After lesion, granule neurons in the lizard medial cortex cell layer appeared pyknotic and died; they were then removed and progressively replaced by a set of new neurons. These neurons were mostly generated from the second to the seventh day post-lesion. A dramatic temporal increment of labelled ependymal cells was detected when either tritiated thymidine or 5-bromodeoxiuridine pulses were delivered in that period. The maximum of about five thousand labelled cells per hemisphere was reached by the fourth day after the lesion. Beyond the seventh day post-lesion, the numbers of labelled cells returned to a level of about four hundred per hemisphere, similar to that of the control specimens. Electron microscopy revealed that the recently generated cells were neuroblasts or immature neurons with a characteristic pattern of chromatin condensation and a high number of ribonucleic granules.

Timm-staining intensity is correlated with the density of Timm-positive presynaptic structures in the cerebral cortex of lizards

SummaryIn cortical areas of the lizard, Podarcis hispanica, Timm staining reveals a distinct pattern of lamination. At the electron-microscope level, virtually all of the reaction product is located in the synaptic vesicles of Timm-positive boutons. Using linear-regression analysis, the area density of Timm-positive bouton profiles as well as the numerical and volume density of stained vesicles were found to be closely correlated with the light-microscopic densitometric values obtained for each Timm-positive cortical zone. We discuss the possibility of estimating stereological electron-microscopic data parameters from densitometric measurements at the light-microscope level.