4 research outputs found
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
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
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
Endocytosis is altered by chronic alcohol exposure in cultured neurons
Endocytosis is required for many cellular pivotal processes
including membrane recycling, nutrient uptake and signal
transduction. This process is particularly relevant in
polarized cells such as neurons. Exposure to ethanol
causes a variety of anomalies in the developing brain
and 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 (BSA) and transferrin (Tf) 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.
The present study shows that the
exposure of neurons in primary
culture to chronic ethanol (0 to 14
DIV, 30 mM) affects both clathrindependent
and clathrinindependent
endocytosis by
probably acting not only on the
several proteins which are directly
involved in the different steps of
these processes, but also on the
proteins that participate in the
organization and dynamics of the
actin cytoskeleton as well as on
cholesterol cellular levels.
Therefore, a toxic effect of alcohol
on endocytosis could affect some of
the important neuronal activities
which depend on the endocytic
process, including synaptic vesicle
recycling, trafficking of
postsynaptic receptors, polarized
axon elongation, growth cone
navigation and neuronal
migration