A. Molowny

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.

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.

Postnatal neurogenesis in the nucleus sphericus of the lizard, Podarcis hispanica

Autoradiography was used to demonstrate the genesis and migration of cells in the nucleus sphericus of perinatal, juvenile and adult lizards, Podarcis hispanica (Sauria, Lacertidae). Following intraperitoneal injections of [3H]thymidine (5 microCi/g b. w.) and survival times of 1, 7, 18 and 28 days, labelled cells were found in the ependyma, marginal layer and mural layer of the nucleus sphericus in specimens of all ages. After short survival times, most labelled cells were located in the ependymal layer. Longer survival times resulted in labelling of cells mainly in the mural layer. At intermediate survival times, a substantial number of labelled cells was also found in the marginal layer. The time course of labelling suggests that neuronal proliferation takes place in the ependyma surrounding the ventricle. Subsequently, the newly formed neurons migrate centripetally and are recruited into the mural layer.

Late generated neurons in the medial cortex of adult lizards send axons that reach the Timm-reactive zones

Double labelling autoradiography-HRP experiments were performed to examine whether late generated neurons in the medial cortex of lizards develop and send axons to their targets. One to two months after receiving a series of tritiated thymidine ([3H]T) injections to label recently generated neurons, lizards (Podarcis hispanica) were subjected to a HRP labelling experiment. HRP was stereotaxically injected into the projection areas of the medial cerebral cortex, i.e. the cortical Timm-reactive areas. Following a short survival time, lizards were sacrificed and their brains processed first for HRP histochemical detection and then for autoradiography. Many cell somata in the cell layer of the medial cortex were retrogradely labelled. A few of the HRP labelled somata also displayed autoradiographic silver granules labelling their nuclei. This indicates that their time of origin had coincided with the tritiated thymidine pulse. These doubly labelled somata are evidence that newly formed neurons grow axons that reach the areas injected with HRP.

The lizard cerebral cortex as a model to study neuronal regeneration

The medial cerebral cortex of lizards, an area homologous to the hippocampal fascia dentata, shows delayed postnatal neurogenesis, i.e., cells in the medial cortex ependyma proliferate and give rise to immature neurons, which migrate to the cell layer. There, recruited neurons differentiate and give rise to zinc containing axons directed to the rest of cortical areas, thus resulting in a continuous growth of the medial cortex and its zinc-enriched axonal projection. This happens along the lizard life span, even in adult lizards, thus allowing one of their most important characteristics: neuronal regeneration. Experiments in our laboratory have shown that chemical lesion of the medial cortex (affecting up to 95% of its neurons) results in a cascade of events: first, massive neuronal death and axonal-dendritic retraction and, secondly, triggered ependymal-neuroblast proliferation and subsequent neo-histogenesis and regeneration of an almost new medial cortex, indistinguishable from a normal undamaged one. This is the only case to our knowledge of the regeneration of an amniote central nervous centre by new neuron production and neo-histogenesis. Thus the lizard cerebral cortex is a good model to study neuronal regeneration and the complex factors that regulate its neurogenetic, migratory and neo-synaptogenetic events.

Postnatal neurogenesis in the medial cortex of the tropical lizard Tropidurus hispidus.

Young, adult and presumed old specimens of the tropical lizard Tropidurus hispidus, living in an almost steady warm habitat, have been the subjects of a 5-bromodeoxiuridine immunocytochemical study to label proliferating brain cells. All animals showed abundant 5-bromodeoxiuridine-labeled nuclei in the ependyma of their telencephalic lateral ventricles, with these being especially abundant in the medial cortex ependyma. Surprisingly, adult animals displayed higher numbers of labeled nuclei when compared with those of young specimens. In a second experiment, in order to check the evolution of ependymal-labeled nuclei, adult specimens were allowed 4 h or 2, 4, 7, 15 or 30 days of survival after the 5-bromodeoxiuridine pulse. Most labeled nuclei appeared isolated at short survival times (4 h and 2 days after the 5-bromodeoxiuridine pulse) but from day 4 and beyond, labeled nuclei appeared in couples or groups usually located in the ependyma. Labeled nuclei with vertical fusiform appearance in the inner plexiform layer or even recruited in the medial cortex cell layer were assumed to be migratory. These presumed migratory nuclei were unexpectedly few (less than 30%) when compared with other lizards, and they appeared much later; at 15 and 30 days after the pulse. This situation resembles that of mammals where only a small proportion of postnatally generated neurons can develop and survive.

Imaging synaptic zinc release in living nervous tissue.

Zinc enriched neurons have a pool of synaptic vesicles which contain free or loosely-bound zinc ions. The movement of the vesicular zinc ions into the synaptic clefts has been previously studied by microdialysis, fluorescence postmortem staining for zinc and radioactive zinc isotope. In this study the zinc fluorescence probe N-6-metoxy-p-toluensulfonamide quinoline (TSQ) has been applied as a tracer of synaptic release of zinc ions. This fluorochrome permeates cell membranes and when exposed to living brain slices gives rise to a staining pattern similar to that seen with autometallography. In the living brain slices, fluorescence emission persists after exposure to calcium saturated ethylen diamino-tetra-acetic acid (Ca-EDTA) because this chelator does not penetrate cell membranes, while sodium dethyldithiocarbamate (DEDTC), that does penetrate membranes, partially suppressed the fluorescence emission. Stimulation of slices bathed in the non-permeant chelator Ca-EDTA with 50 mM potassium chloride leads to a rapid and complete disappearance of fluorescence. In the absence of Ca-EDTA, however, potassium stimulation induces a sudden transitory increase in fluorescence. This increase is caused by a translocation of the fluorochrome (TSQ) zinc molecules from the weakly acid interior of the synaptic vesicles to the neutral extracellular space, whereby the fluorescence emission of the molecules is enhanced sufficiently to be recorded by a high sensitivity TV camera.

Ontogeny of somatostatin immunoreactive neurons in the medial cerebral cortex and other cortical areas of the lizard Podarcis hispanica

The ontogeny of somatostatin immunoreactive interneurons in the cerebral cortex of the lizard Podarcis hispanica has been studied in histological series of embryos, perinatal specimens, and adults. Somatostatin immunoreactive interneurons appear in the early stages of lizard cerebral cortex ontogeny, their number increases during embryonary development, reaches a peak in early postnatal life, and decreases in adult lizards. The first somatostatin immunoreactive somata in the lizard forebrain appeared on E36, and they were located in non cortical areas. Then, on E39 and later, somatostatin immunoreactive neurons were seen in the lizard cortex in a rostral‐to‐caudal spatial gradient, which parallels that of the normal histogenesis of the lizard cerebral cortex. On E39, labelled somata were seen in the medial and dorsal cortex inner plexiform layers; immunoreactive puncta and dendritic processes were detectable in the inner plexiform layer of the medial cortex. On E40, labelled neurons were observed in the inner plexiform layer of the lateral cortex; labelled processes were found in the inner plexiform layers (dorsomedial, dorsal, and lateral cortices) and the outer plexiform layers (medial and dorsomedial cortices). At hatching (PO), some somatostatin immunoreactive neurons populated the external plexiform layer of the dorsomedial cortex. On P28, groups of labelled neurons appeared in the cell layer of dorsal and lateral cortices, reaching the adult‐mature pattern of somatostatin immunoreactivity in the lizard cerebral cortex, i.e., labelled somata and dendritic processes populating the inner plexiform layers in addition to an axonic labelled plexus in the outermost part of the outer plexiform layers. Immunoreactive somata and processes occupied all the cortical areas, but they were especially abundant in the dorsomedial cortex. Proliferating Cell Nuclear Antigen (PCNA) immunostaining in the same histological series revealed that the number of PCNA immunoreactive nuclei in the subjacent proliferative neuroepithelium followed an inverse‐complementary evolution to somatostastin, suggesting some temporal relationship between somatostatin immunoreactive cells and neurogenesis in the lizard cerebral cortex.

Endocytosis in Cultured Neurons Is Altered by Chronic Alcohol Exposure

Endocytosis is required for many cellular pivotal processes, including membrane recycling, nutrient uptake, and signal transduction. This complex process is particularly relevant in polarized cells, such as neurons. Previous studies have demonstrated that alcohol alters intracellular traffic, including endocytosis, in several cell types. However, information on the effect of chronic alcohol exposure on this process in neurons is scarce. As an approach, we investigated the effect of alcohol exposure on the internalization of two widely used endocytic markers, albumin and transferrin, in developing hippocampal neurons in primary culture. The effect of this treatment on the levels of several representative proteins involved in the endocytic process was also analyzed. Some of these proteins are also involved in the organization of the actin cytoskeleton. Pretreatment of cells with inhibitors chlorpromazine or nystatin indicates that albumin is internalized mainly by caveolin-dependent endocytosis. On the other hand, alcohol decreases the endocytosis of both markers, although no qualitative changes in the distribution of either of these molecules were observed. Finally, the effect of ethanol on the proteins analyzed was heterogeneous. Alcohol decreases the levels of clathrin, AP-2, SNX9, Rab5, Rab11, EEA1, Cdc42, or RhoA but increases the amount of Arf6. Moreover, alcohol does not affect the levels of caveolin1, dynamin1, Rab7, and LAMP2. This toxic effect of alcohol on endocytosis could affect some of the important neuronal activities, which depend on this process, including cell signaling. Our results in neurons also stress the notion that one of the main targets of ethanol is intracellular transport.