Ethanol impairs extracellular zinc intake in cultured astrocytes

Zinc (Zn) deficiency is present in many physiological and health problems. Among circumstances involved in Zn deficiency, ethanol consumption appears as a prominent cause. In the CNS substantial amounts of Zn appear accumulated in synaptic vesicles of a particular class of neurons: the Zn enriched neurons very abundant in the telencephalon and cerebral cortex. This is the so called synaptic Zn which is simultaneously released with the neurotransmitter thus exerting a neuromodulator role during synaptic transmission. Neighbour astrocytic processes have to capture the excess of both extracellular Zn and neurotransmitter in order to maintain efficient synaptic transmission between neurons. In this work we analyze the effect of exposure to 30 mM ethanol for 7 days in the ability of cultured rat astrocytes to capture and manage extracellular Zn. Intracellular Zn levels were visualized by using the TSQ Zn fluorochrome, either in normal culture conditions or after supplementary addition of 50 μM ZnSO4 to the culture. Fluorescence was recorded with an Olympus microscope BX50WI, equipped with a Hamamatsu ORCA digital camera controlled with the Aquacosmos software. Basal Zn levels in cultured astrocytes was greatly and significantly lower in ethanol treated cells (about 30% of control cultures). These differences were consistently maintained after addition of extracellular Zn to cell monolayers, resulting in a lower ability to uptake or retain Zn. The Zn was uptaked by the endocytic pathway, as demonstrated by the marker FM1-43 and was mainly confined to bright organelles that were more abundant in control cells. In conclusion, ethanol impairs astrocyte Zn management resulting in a lower capacity for extracellular Zn intake in resting conditions and after extracellular addition. It has been proposed that an efficient method to palliate Zn deficiency it could be a dietary supplement. Nevertheless, this study suggests that a dietary Zn supplementation may not be enough for recovery of cellular normal function in alcoholic cultured astrocytes

Searching free zinc at the ultrastructural level in cultured astrocytes

Zinc is an element that is necessary for many physiological functions in the body but may play an important role in diseases affecting most systems in the body if its balance is altered by environmental, toxicological or idiosyncrasy of subjects. We have centred our investigation in central nervous system, using cultured astrocytes since they are involved in clearance of zinc exocytated to the extracellular medium during synaptic transmission. In previous works we have used a zinc fluorochrome, i.e., the TSQ (6-Methoxy-(8-p-toluenesulfonamido)quinoline) to in vivo zinc uptake in cultured astrocytes and its accumulation in organelles named zincosomes. However, the precise location of these zinc-enriched structures (zincosomes) at the ultrastructural level is a very hard task. In a previous attempt at the electron microscopy level, only topographical approximation by combining light and electron microscopy allowed us to identify selected zincosomes previously marked with TSQ. Now, our objective is to adapt zinc autometallography (Timm’s method) to TSQ labelled cultured astrocytes. For the electron microscopic detection of zincosomes, the first important step is to achieve a good zinc precipitation during or previous to glutaraldehyde fixation. Surprisingly, neither ditizone nor selenite were successful as zinc precipitating agents; only sodium sulphide gave us good results. We also found that while glutaraldehyde is the best option for animal experimentation, paraformaldehyde prefixation gave us best results. Paraformaldehyde prefixation allowed both ultrastructure preservation as well as zinc-precipitated-detection with Timm autometallography in semithin sections. These semithin sections were included again and zincosomes become clearly visible in ultrathin sections

Neurons of the Dentate Molecular Layer in the Rabbit Hippocampus

The molecular layer of the dentate gyrus appears as the main entrance gate for information into the hippocampus, i.e., where the perforant path axons from the entorhinal cortex synapse onto the spines and dendrites of granule cells. A few dispersed neuronal somata appear intermingled in between and probably control the flow of information in this area. In rabbits, the number of neurons in the molecular layer increases in the first week of postnatal life and then stabilizes to appear permanent and heterogeneous over the individuals’ life span, including old animals. By means of Golgi impregnations, NADPH histochemistry, immunocytochemical stainings and intracellular labelings (lucifer yellow and biocytin injections), eight neuronal morphological types have been detected in the molecular layer of developing adult and old rabbits. Six of them appear as interneurons displaying smooth dendrites and GABA immunoreactivity: those here called as globoid, vertical, small horizontal, large horizontal, inverted pyramidal and polymorphic. Additionally there are two GABA negative types: the sarmentous and ectopic granular neurons. The distribution of the somata and dendritic trees of these neurons shows preferences for a definite sublayer of the molecular layer: small horizontal, sarmentous and inverted pyramidal neurons are preferably found in the outer third of the molecular layer; vertical, globoid and polymorph neurons locate the intermediate third, while large horizontal and ectopic granular neurons occupy the inner third or the juxtagranular molecular layer. Our results reveal substantial differences in the morphology and electrophysiological behaviour between each neuronal archetype in the dentate molecular layer, allowing us to propose a new classification for this neural population

Extracellular zinc intake in cultured astrocytes is altered by ethanol exposure

Ethanol reduces the amount of intracellular zinc detectable with TSQ. Ethanol impairs astrocyte Zn management. It results in a lower capacity for exogenous Zn intake and delivering to zincosomes. Thus, Zn supplementation (dietary?) may not be enough for recovery of cellular normal function; as it happens in alcohol treated astrocytes. Zincosomes are a kind of low density primary endosomes

Ethanol impairs extracellular zinc intake in cultured astrocytes

Zinc is an ion that participates in increasing described cellular and tissular functions. On the other hand, zinc deficiency is also present in many physiological and health problems affecting most organs along the body, including teratological problems. Among circumstances involved in zinc deficiency, ethanol consumption is probably one of the most frequent. In the central nervous system zinc is also present and it is especially important in neurons that include zinc in the transmission synaptic vesicles. This zinc is delivered as the neurotransmitter exerting a neuromodulator role in the synaptic transmission. Neighbour astrocytes have to maintain the extracellular zinc homeostasis in order to maintain neurons continuously able to perform synaptic transmission. It has been proposed that an efficient method to palliate zinc deficiency it could be a dietary supplement. In this work we analyze the ability of cultured astrocytes in the management of extracellular zinc. Cultured rat astrocytes were used in this work, incubated in presence or not of 30 mM Ethanol for 7 days. Intracellular zinc levels were visualized by using the TSQ zinc fluorochrome, after addition or not of 50 µM ZnSO4. Fluorescence was recorded with an Olympus microscope BX50WI, equipped with a Hamamatsu ORCA digital camera controlled with the Aquacosmos software. Basal zinc levels in cultured astrocytes was greatly and significantly lower in ethanol treated cells (about 30% of control cultures). These differences were consistently maintained after addition of extracellular zinc to cell monolayers, resulting in a lower ability to uptake or retain zinc. The zinc was uptaked by the endocitic pathway, as demonstrated with the endosome marker FM 1-43 and was mainly confined to bright organelles that were more abundant in control cells. In conclusion, ethanol impairs astrocyte zinc management resulting in a lower capacity for extracellular zinc intake in resting conditions and after extracellular addtion. Consequently the efficiency of a dietary zinc supplementation may not be enough for recovery of cellular normal function

Stainings of ectopic granular and small horizontal neurons.

<p><i>A,</i> NADPH diaphorase histochemical staining of an ectopic granular neuron. <i>B,</i> ABC-diaminobenzidine-nickel staining of biocytin injected into a granular ectopic neuron with an axon (arrow head) crossing the granular layer. <i>C,</i> NADPH diaphorase histochemical staining of a small horizontal neuron. <i>D,</i> idem, parvalbumin immunostaining. <i>E,</i> idem, calretinin immunostaining. <i>F,</i> Golgi impregnated small horizontal neuron (arrow head) and two granule cells (arrows). Scale bars, <i>A–F</i>: 25 µm.</p

Morphometry and distribution of the neuronal somata in the molecular layer of the dentate gyrus.

<p><i>A,</i> Profiles of neuronal somata from different neurons taken as prototypes; the length of the two principal axes and the perimeter length were the main parameters analyzed, (v- vertical neuron, g- globular neuron, e- ectopic granular neuron, sh- small horizontal neuron, lh- large horizontal neuron, s- sarmentous neuron, P- polymorphic neuron). <i>B,</i> Frequency of location of neuronal types in the inner, middle and outer molecular layer strata (from a pool of 120 neurons from a 30-day-old rabbit; 15 neurons/type). <i>C,</i> Plots of the minor axis value against the major axis value; observe how specific neurons segregate in distinct populations. <i>D,</i> Idem, plots of the surface value of the profile (soma area) (ordinate) against the axes ratio (minor/major) and some types also segregate. <i>E,</i> Plots of the minor axis values (abscise) in front of the major axis values (ordinate) of the neurons which are more frequent in the outer molecular: sarmentous, inverted pyramidals and small horizontals. <i>F,</i> Idem, for the more frequent neurons in the middle molecular: verticals, globoid and polymorphic. <i>G,</i> Idem, for those more frequently located in the inner molecular: ectopic granular and large horizontal. Scale bars, <i>A–G</i>: 25 µm.</p

Stainings of inverted pyramidal and large horizontal neurons.

<p><i>A,</i> NADPH diaphorase histochemical staining of an inverted pyramidal neuron. <i>B,</i> ABC-DAB-nickel staining of biocytin injected into an inverted pyramidal neuron. <i>C,</i> NADPH diaphorase histochemical staining of a large horizontal neuron in the inferior blade of the dentate gyrus. <i>D,</i> Golgi impregnation of a large horizontal neuron. <i>E,</i> parvalbumin immunostaining of a large horizontal neuron in the superior blade of the dentate gyrus; note the counterstained granular layer. Scale bars, <i>A–E</i>, 25 µm.</p

Camera lucida drawings and electrophysiological recordings of granular ectopic and small horizontal neurons.

<p><i>A, B,</i> drawings of ectopic granular neurons in A, the axon crossing the granular layer, bifurcates and arborization in the hilus (<b>hi</b>). <i>C,</i> electrophysiological record from an ectopic granular neuron. <i>D, E,</i> drawings of the small horizontal neurons. Observed in <i>D</i>, the profuse axonal arborization close to the hippocampal fissure (<b>hp</b>). <i>F,</i> electrophysiological recording of a small horizontal neuron. Scale bars, <i>A–F:</i> 25 µm.</p

Camera lucida drawings and electrophysiological recordings of sarmetous and polymorphic neurons.

<p><i>A, B,</i> Drawings of sarmentous neurons. <i>C,</i> electrophysiological recording of a sarmentous neuron. <i>D, E,</i> drawings of polymorphic neurons; note the dense axonal arborization in <i>D</i> distributed by the outer and middle molecular layers. <i>F,</i> elecrophysiological record of a polymorphic neuron. Scale bars, <i>A–E:</i> 25 µm.</p