We first examined cultured hippocampal neurons. We took micrographs of the neurons (from three different cultures) that had clearly visible mitochondria in their synaptic regions. Axons with presynaptic terminals in the synaptic regions were identified on the basis of the presence of synaptic vesicle clusters and a dark presynaptic active zone. Dendrites with postsynaptic structures were identified based on the presence of a postsynaptic density and enrichment of microtubules (for examples see Fig. 1A, 1B).
From these electron micrographs, we first measured the “darkness” or intensity of individual mitochondria. Comparing the mitochondria in the presynaptic terminals with those in the postsynaptic dendrites, the intensity measurements showed neuron-to-neuron or synapse-to-synapse variations. In some synapses, the mitochondria were noticeably darker in the presynaptic terminals than in the postsynaptic dendrites (Fig. 1A, 1B), whereas in other neurons, presynaptic mitochondria appeared similar to postsynaptic mitochondria (data not shown). However, the values averaged over ~600 randomly selected mitochondria exhibited a clear trend: the intensity of the presynaptic mitochondria was statistically significantly greater than the intensity of the postsynaptic mitochondria (Fig. 1C, presynaptic 168.3 ± 2.3 vs postsynaptic 161.0 ± 1.8, p < 0.0001, Mann-Whitney U test).
The size of the mitochondria was also different between the two synaptic compartments. The average size, as determined by area, of the presynaptic mitochondria was roughly half the size of the postsynaptic mitochondria (Fig. 1D, presynaptic 0.077 µm2 ± 0.010 vs postsynaptic 0.146 µm2 ± 0.012, p < 0.0001, Mann-Whitney U test). As an additional assessment of the physical size of the mitochondria, we measured the smallest Feret diameter of the mitochondria. Feret diameter was calculated as the distance between two superimposed parallel lines tangential to the surface of the mitochondria. We found a much higher proportion of presynaptic mitochondria that had a small minimum Feret diameter compared to postsynaptic mitochondria (Fig. 1E). Consistently, the difference between the average minimum Feret diameter of the presynaptic and postsynaptic mitochondria was statistically highly significant (Fig. 1F, presynaptic 0.188 µm ± 0.009 vs postsynaptic 0.239 µm ± 0.009, p < 0.0001, Mann-Whitney U test).
We next examined the neurons in the hippocampus. We took micrographs from the stratum lucidum of the CA3 region of the hippocampus (from three rats). We chose mossy terminal synapses because they typically have abundant presynaptic mitochondria. The postsynaptic processes were the proximal portions of the apical dendrites of the pyramidal cell neurons, including the basal connection of the dendrites to the soma.
In these hippocampal synapses, examples of dark presynaptic mitochondria were readily visible (Fig. 1G, 1H; and supplemental Fig1 for additional examples), although some variations existed. Despite the variations, the intensity values averaged from the measurement of ~2,500 mitochondria (from three rats) showed that the difference between presynaptic and postsynaptic compartments was statistically significant: the presynaptic mitochondria were significantly darker than the postsynaptic mitochondria (Fig. 1I, presynaptic 169.4 ± 0.7 vs postsynaptic 165.0 ± 0.9, p < 0.0001, Mann-Whitney U test). Notably, assessments of the mitochondrial size showed that average area (Fig. 1J, presynaptic 0.038 µm2 ± 0.001 vs postsynaptic 0.087 µm2 ± 0.005, p < 0.0001, Mann-Whitney U test) and minimum Feret diameter (Fig. 1K, 1L, presynaptic 0.167 µm ± 0.002 vs postsynaptic 0.210 µm ± 0.004, p <0.0001, Mann-Whitney U test) were smaller in presynaptic mitochondria, indicating that presynaptic mitochondria are thinner than postsynaptic mitochondria.
Early ultrastructural studies have noted darker mitochondria in some axonal terminals of a few neuronal types, including neurons in lateral geniculate nucleus and the spinal cord. While the hippocampal neurons and the mitochondria within have been examined under electron microscopy, the physical properties of the mitochondria, such as intensity, were not specifically investigated. In a study of electron microscopic characterization of cultured hippocampal neurons, some mitochondria were visibly darker and thinner in axons than in dendrites (see Figure 5 in). But this difference was incidental to the main focus of the study; it was neither quantitatively analyzed nor explicitly discussed.
The present study provides a thorough comparison of the differences in intensity and size of mitochondria between the presynaptic and postsynaptic compartments of hippocampal synapses. Our analyses of two different systems – in vitro and in vivo – have demonstrated a remarkably similar trend: overall the presynaptic mitochondria are narrower in diameter and overall darker in electron density than the postsynaptic mitochondria. Do darker mitochondria indicate their higher activity? A definitive answer to this question would require tools that can simultaneously assess both the physical properties such as electron density or intensity and biochemical activities, such as ATP production of mitochondria, at the resolution of a single mitochondrion in real time in live neurons. Before such accurate and powerful tools are devised, though, we can infer from studies on the correlation between the ultrastructural features and function of mitochondria. Among these correlative studies is a recent report on cultured hippocampal neurons in which populations of darker mitochondria were directly proportional to an overall higher activity of mitochondria. If the “darkness” signifies activity, the existence of darker mitochondria in the presynaptic terminals of the hippocampal neurons (this study) and of other neurons most likely reflects high demands in energy supply and Ca2+ buffering, both of which are attributed to the functions of mitochondria, and are results of presynaptic transmission.
It is worth noting that not every presynaptic terminal of the hippocampal neurons has darker mitochondria. We have observed variations at the level of synapses, neurons, or even animals. For example, of the three rats examined, the hippocampi from two of them had visibly darker presynaptic mitochondria, but the third rat did not exhibit a statistically significant difference in the intensity between the presynaptic and postsynaptic mitochondria (supplemental Fig. 2). This variability may simply reflect the fact that mitochondria are ‘multitasking’ as well as the dynamic nature of energy demands and consumption in different compartments of a neuron under diverse conditions at any given time.
The difference in the size of mitochondria, on the other hand, is almost invariable between the two synaptic compartments. This is particularly true in terms of the diameter of the mitochondria: the presynaptic mitochondria, by and large, are thinner than the postsynaptic mitochondria (Fig. 1J, 1K). This finding is consistent with a previous 3D reconstruction study reporting that axonal mitochondria are small and thin. A critical next step will be to determine whether thinner presynaptic mitochondria are merely an adaptation to the generally narrower axonal space, or whether their thinness might, or might also, indicate a population of mitochondria that are functionally more active.