dc.description.abstracteng | Isolating cells from a living organism and growing them in a Petri dish allowed scientists to
study the physiology and biochemistry of healthy and diseased cells. Today we have cell
cultures from almost any tissue type, including the brain. One brain region has been
fundamental for understanding neuronal and synaptic dynamics, both in vivo and in culture:
the hippocampus. Since the H.M. case, the hippocampus drew attention to itself with the
promise of understanding molecular and electrical mechanisms behind learning and memory.
This made the primary culture from hippocampus tissue one of the most commonly used
models in the neuroscience field. Despite being a very common preparation, it is still
imperfectly known.
For example, during preparation, multiple animals are sacrificed, and tissues are pooled,
regardless of their sex. That creates a female-male mixed culture in which the female to male
neuron ratio is unknown. It is still unclear whether the neurons of different genders behave
differently in these cultures. To address this question, I performed a systematic investigation
on cultured female and male neurons. I found differences in their electrical activity as well as
in their synaptic translation rate. First, I compared the firing rates with a calcium indicator and
found higher spontaneous electrical activity and larger response capacity to electrical
stimulation in male neurons than in female neurons. The following step was investigating the
dynamics of synaptic compartments with a synaptotagmin 1 (Syt1) uptake assay. It also proved
that male neurons have a larger active synaptic vesicle pool size and dynamics than female
neurons. An immunostaining survey with a focus on synaptic proteins did not show major
differences between the two sexes. Their transcriptomes also shown substantial differences.
Finally, I also examined the local translation, and found higher translation rate at the male
synapse, which could, at least in part, explain the functional differences. These results present
an extensive comparison for functional behavior and synaptic structure between female and
male neurons and encourage a first discussion on primary hippocampal culture preparation in
respect to female-male neuron ratio.
Another overlooked aspect of the primary hippocampal culture is the circadian effects on
cellular biology. It is now well established that circadian rhythm is kept in every mammalian
cell via the molecular clock, which consists of several transcription factors. However, without
a central pacemaker, which in vivo is located in the suprachiasmatic nucleus (SCN) of the
hypothalamus, it would be difficult to maintain a 24-hour rhythmicity in cell cultures. Therefore,
we expect that primary hippocampal neurons in culture will maintain a form of rhythmus in
culture, but this has never been studied. I performed a series of experiments indicating the
existence of a weak circadian rhythm in the firing patterns, the synaptic activity and mRNA
localization at the synapse, even after 20 day-long deprivation of external stimuli. I found a
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rhythmically expressed transcript, RNA-binding motif 3 (RBM3), whose knock-down results in
significant changes in the firing pattern and in the reduction of the active synaptic vesicle pool
dynamics, the post-synapse size, and the post-synaptic translation rate. This implies that
RBM3 is involved in sustaining the rhythmic abundance of synaptic proteins, and therefore in
sustaining rhythmic synaptic function.
Overall, these findings change the impression of the primary hippocampal culture. It is
essential to be aware of the female-male ratio and the timing of experiments. | de |