The soma and the nucleus do not play an active role in the transmission of the neural signal. Instead, these two structures serve to maintain the cell and keep the neuron functional. Think of the cell body as a small factory that fuels the neuron. The soma produces the proteins that the other parts of the neuron, including the dendrites, axons, and synapses, need to function properly.
The support structures of the cell include mitochondria, which provide energy for the cell, and the Golgi apparatus, which packages products created by the cell and dispatches them to various locations inside and outside the cell. The axon hillock is located at the end of the soma and controls the firing of the neuron. If the total strength of the signal exceeds the threshold limit of the axon hillock, the structure will fire a signal known as an action potential down the axon.
The axon hillock acts as something of a manager, summing the total inhibitory and excitatory signals. If the sum of these signals exceeds a certain threshold, the action potential will be triggered and an electrical signal will then be transmitted down the axon away from the cell body. This action potential is caused by changes in ion channels which are affected by changes in polarization.
When a signal is received by the cell, it causes sodium ions to enter the cell and reduce the polarization. If the axon hillock is depolarized to a certain threshold, an action potential will fire and transmit the electrical signal down the axon to the synapses. It is important to note that the action potential is an all-or-nothing process and that signals are not partially transmitted. The neurons either fire or they do not. The axon is the elongated fiber that extends from the cell body to the terminal endings and transmits the neural signal.
The larger the diameter of the axon, the faster it transmits information. Some axons are covered with a fatty substance called myelin that acts as an insulator. These myelinated axons transmit information much faster than other neurons. The myelin surrounding the neurons protects the axon and aids in the speed of transmission. The myelin sheath is broken up by points known as the nodes of Ranvier or myelin sheath gaps.
Electrical impulses are able to jump from one node to the next, which plays a role in speeding up the transmission of the signal. Axons connect with other cells in the body including other neurons, muscle cells, and organs. These connections occur at junctions known as synapses. The synapses allow electrical and chemical messages to be transmitted from the neuron to the other cells in the body.
The terminal buttons are located at the end of the neuron and are responsible for sending the signal on to other neurons. At the end of the terminal button is a gap known as a synapse. Neurotransmitters are used to carry the signal across the synapse to other neurons.
When an electrical signal reaches the terminal buttons, neurotransmitters are then released into the synaptic gap. Neurons serve as basic building blocks of the nervous system and are responsible for communicating messages throughout the body.
Knowing more about the different parts of the neuron can help you to better understand how these important structures function as well as how different problems, such as diseases that impact axon myelination, might impact how messages are communicated throughout the body. The axonal EGFP signal was used to verify that each mitochondrion was inside the axon only after it was scored. This calculation resulted in an estimated sample size of newly-formed boutons.
The number of animals required to achieve this was estimated from pilot studies to be 10—15 animals. In this study, 21 animals were used, 15 were imaged and 12 produced high-quality data that was included see exclusion criteria below. The final dataset was obtained from three different batches of littermates.
A total of 51 ROIs and axons were tracked. The total number of mitochondria counted across all time-points was 11, along with 4, unique boutons. Mitochondria were not linked between timepoints because they lacked individuality due to their ability to move, fuse and split Lewis et al.
Some data were excluded from the final dataset. Any ROI that was too dim for accurate axon tracing subjectively based on analyst experience within the first four time points was not tracked. Any axon where the signal-to-noise in a session was low enough that the scorer could not be confident in bouton scoring was removed.
Data from one animal that had only two axons tracked was also removed. The bouton dynamic fraction was calculated as the proportion of unique boutons on an axon that were either lost or gained. Mitochondria were classified as being present at a bouton if the distance between their centroids defined by points placed by the analyst was less than or equal to 1.
A dichotomous variable mitochondria present or not was chosen for analyses rather than a continuous variable distance from nearest mitochondrion because the axonal segment was a small sample of the axonal arbor and the true distance to the nearest mitochondria from each bouton could not be accurately measured, especially for boutons at the edge of the field of view.
The distance of 1. Stronger effects on synaptic ultrastructure were seen with closer distances of mitochondria. The Randomization of bouton or mitochondrion position was carried out in a similar fashion to Smit-Rigter et al.
The length of the axon was then estimated using Euclidean distances and a line was created and split into segments of 74 nm the original pixel size of the 2P images. The real positions of the objects of interest mitochondria or boutons were plotted to the closest segment of the axon based on where they were in the original image using nearest neighbor distance calculations.
This was repeated 1, times for each axon and the range plotted. Confidence intervals for proportions were calculated using the formula for single samples Newcombe, Therefore, the formula is as follows:. For repeated measures statistical tests, group sizes were matched by only including axons present in all relevant time-points for the particular test. To avoid pseudo-replication, samples were not pooled together across time from repeated measures. An assumed number of two Gaussian components were defined by the analyst.
Axons that fell under the threshold of 0. Kaplan-Meier curves were created for survival analysis, based on time-to-event data. For bouton survival, this was the time from first observation until the bouton was no longer observed.
By placing a cranial window over the primary somatosensory cortex S1 , we imaged segments of these long-range axons within layer 1 using in vivo 2P microscopy. As this is the first characterization of boutons in this axonal pathway, we compared the density and turnover of boutons in individual axonal branches.
Figure 1. Tracking bouton plasticity and mitochondrial positioning in axons of motor cortex neurons. Only the ipsilateral half of the brain sections are shown. D top Imaging timeline for tracking bouton structural plasticity bouton loss and gain. Viral injection and cranial window implantation were performed 24 days prior to initial 2P imaging.
Arrowheads indicate imaging time-points. Some boutons are labeled with arrowheads to show examples of stable yellow , lost red or gained green boutons. Contour lines indicate the slope of the GMM distribution. As mitochondria can support presynaptic function, we assessed whether the numerical density of boutons and mitochondria are correlated in axons population mean overtime was 1.
To assess this, we compared the fraction of dynamic boutons proportion lost and gained divided by the total number of unique boutons across daily and weekly intervals to the mean mitochondrial density between the two time-points Figure 2C , Supplementary Table S1.
Figure 2. Mitochondrial density along an axonal segment is correlated to bouton density but not bouton dynamics. C Example correlations between the fraction of boutons on each axon that were dynamic lost or gained; bouton dynamic fraction and either: left the number of mitochondria relative to the number of boutons mito:bouton ratio , or right mitochondrial density. As the overall density of axonal mitochondria was related to bouton density, but not to bouton dynamics, we assessed if there was instead a more local relationship between individual boutons and mitochondria near them.
Based on previous studies and effective resolution limits of our imaging, we chose 1. This local organization did not occur by chance, as randomizing or mirroring positions of either mitochondria or boutons along the axon backbone resulted in greater distances between them Figures 3A—C.
To determine if the structure of boutons affected the ability of mitochondria to localize there, we divided the bouton population into EPBs and TBs.
It is possible that mitochondria reside near TBs, but do not traverse their neck region. Therefore, we estimated the location of the base of TBs by re-plotting them to where they joined the axon backbone Figure 3G. Figure 3. A The distribution of distances between each mitochondrion and its nearest bouton was plotted against the results from 1, rounds of randomized positioning of boutons for comparison to chance levels.
Kolmogorov—Smirnov K—S test between real data and the median of randomized positioning. As a further control, the real bouton positions were mirrored along the axon backbone to maintain the inter-bouton distances black line resulting in a similar distribution to the randomized positioning. C Illustration of the routine for randomizing positions.
The original image was manually traced and a 2D skeleton interpolated from the segmented line trace. TBs were approximately placed at the nearest point on the axon backbone their base for randomizing in 1D. The 2D skeleton was then straightened to 1D and either mitochondria were randomly positioned alongside real bouton positions or vice versa.
F A greater proportion of EPBs have mitochondria within a biologically relevant distance 1. When mitochondrial localization was considered from the TB base instead of the head the difference was lost Chi-squared test. G Estimated location of TB bases was achieved by finding the nearest neighbor point on the axon backbone that was closest to the TB head and re-plotting the TB to that position.
Given that mitochondria have been implicated in the control of presynaptic function, we hypothesized that mitochondrial presence may relate to bouton maturity. To test this hypothesis, we separated boutons by age new or pre-existing ; Figure 4A. It was also not due to a general trend towards increased synaptic localization of mitochondria over time, as this was stable across the imaging paradigm Supplementary Figure S6.
These data show that the longer a bouton survives, the more likely it is to have a mitochondrion nearby. Figure 4. Mitochondrial presence at individual boutons is positively related to bouton age and longevity. A Timeline indicating the classification of pre-existing boutons first identified on day 0 and newly-formed boutons first identified on days 1, 2 or 3.
Newly-formed boutons had more mitochondria present than with randomized positioning of mitochondria, as did pre-existing boutons Chi-squared test. C Boutons that persisted in every time point after day 2 after all newly-formed boutons were identified had their mitochondrial localization tracked.
D Survival of boutons was measured as the time until bouton loss. Pre-existing boutons were significantly more stable than new boutons Log-rank test. The new bouton population was pooled from day 1—3 light red lines.
G The proportion of new boutons with or without mitochondria that were lost after their first day. It has been shown that newly-formed cortical boutons tend to be lost more quickly than pre-existing boutons Qiao et al. To determine if mitochondria relate to the stability of individual boutons locally, we assessed the survival of boutons with and without mitochondria.
As localization of mitochondria at TBs and EPBs appeared to be different, we assessed the impact of having resident mitochondria on the survival of the two bouton types separately.
This relationship between mitochondrial proximity and enhanced bouton survival was consistent across time for pre-existing boutons Supplementary Figure S7. Again, we assessed whether the stabilization of boutons was dependent on bouton type. Overall, these results suggest that the immediate survival of new boutons is weakly related to local mitochondrial presence, but this relationship becomes stronger and more consistent as boutons age.
It has long been reported that many, but not all, presynaptic release sites have mitochondria in close proximity to them Gray, ; Shepherd and Harris, ; Chang et al. The fact that mitochondria can modulate synaptic function suggests that having a resident mitochondrion may also relate to the activity-dependent plasticity of the synapse. Here, we have shown that there is indeed an association between mitochondrial positioning at presynaptic terminals and their structural longevity.
As with other axons De Paola et al. Newly-formed boutons are more likely to possess mitochondria within their first 24 h our smallest imaging interval than by chance Figure 4B , suggesting a link between synaptic and mitochondrial function even in the early stages of the synaptic lifecycle. Long-lasting boutons are even more likely to have resident mitochondria Figures 4B,C and this decreases the chance of those boutons being removed by half Figure 4H.
This results in a persisting population of synaptic boutons that are more likely to contain mitochondria. As such, it seems likely that mitochondrial recruitment links to some synaptic function that promotes synaptic longevity Rangaraju et al. This aligns with the previous finding that, in local axons within the visual cortex, boutons without mitochondria are more likely to be lost over a 4-day period Smit-Rigter et al.
Presynaptic mitochondria can modulate short-term plasticity of neurotransmitter release via their sequestration and slow release of calcium Billups and Forsythe, ; Sun et al. However, it remains unknown whether mitochondria directly influence long-term plasticity of synaptic function, as recently shown within dendrites Smith et al.
Our data suggest that any link between mitochondria and plasticity is local to neighboring synapses. This is because, although the density of mitochondria along different axonal branches varied considerably, it did not correlate with rates of bouton plasticity at the branch level Figure 2.
In contrast, the close proximity of mitochondria within 1. Mitochondria can be highly dynamic, undergoing rapid rounds of fusion and fission alongside axonal trafficking Lewis et al. However, it has been reported that, despite the overall axonal positioning of mitochondria being unstable, there are more likely to be mitochondria stably retained near boutons than non-synaptic locations Smit-Rigter et al.
In this study, we found that the spatial arrangement of mitochondria and synaptic boutons depends on bouton type as mitochondria were generally located closer to EPBs than to TBs Figure 3.
This may be because physical access to the bouton head is restricted by the neck of TBs or could reflect functional differences between bouton types. Indeed, the difference in proximity was mirrored by the fact that local mitochondria were strongly linked to the survival of EPBs but not TBs. The neurons were labelled by retrograde transport of horseradish peroxidase and GABA or glutamate-containing boutons were revealed by performing postembedding immunogold reactions on electron microscope sections.
Five neurons were examined and all of them were postsynaptic to boutons which contained either GABA or glutamate. Analysis of series of alternate sections, which were reacted for either GABA or glutamate, showed that there was no overlap in the populations of immunoreactive boutons.
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