Transmission Electron Microscopy for Analysis of Mitochondria in Mouse Skeletal Muscle.

Skeletal muscle is the most abundant tissue in the human body and regulates a variety of functions including locomotion and whole-body metabolism. Skeletal muscle has a plethora of mitochondria, the organelles that are essential for aerobic generation of ATP which provides the chemical energy to fuel vital functions such as contraction. The number of mitochondria in skeletal muscle and their function decline with normal aging and in various neuromuscular diseases and in catabolic conditions such as cancer, starvation, denervation, and immobilization. Moreover, compromised mitochondrial function is also associated with metabolic disorders including type 2 diabetes mellitus. It is now clear that maintaining mitochondrial content and function in skeletal muscle is vital for sustained health throughout the lifespan. While a number of staining methods are available to study mitochondria, transmission electron microscopy (TEM) is still the most important method to study mitochondrial structure and health in skeletal muscle. It provides critical information about mitochondrial content, cristae density, organization, formation of autophagosomes, and any other abnormalities commonly observed in various disease conditions. In this article, we describe a detailed protocol for sample preparation and analysis of mouse skeletal muscle mitochondria by TEM.

Synaptic organization of thalamocortical axon collaterals in the perigeniculate nucleus and dorsal lateral geniculate nucleus.

We examined the synaptic targets of large non-gamma-aminobutyric acid (GABA)-ergic profiles that contain round vesicles and dark mitochondria (RLD profiles) in the perigeniculate nucleus (PGN) and the dorsal lateral geniculate nucleus (dLGN). RLD profiles can provisionally be identified as the collaterals of thalamocortical axons, because their ultrastrucure is distinct from all other previously described dLGN inputs. We also found that RLD profiles are larger than cholinergic terminals and contain the type 2 vesicular glutamate transporter. RLD profiles are distributed throughout the PGN and are concentrated within the interlaminar zones (IZs) of the dLGN, regions distinguished by dense binding of Wisteria floribunda agglutinin (WFA). To determine the synaptic targets of thalamocortical axon collaterals, we examined RLD profiles in the PGN and dLGN in tissue stained for GABA. For the PGN, we found that all RLD profiles make synaptic contacts with GABAergic PGN somata, dendrites, and spines. In the dLGN, RLD profiles primarily synapse with GABAergic dendrites that contain vesicles (F2 profiles) and non-GABAergic dendrites in glomerular arrangements that include triads. Occasional synapses on GABAergic somata and proximal dendrites were also observed in the dLGN. These results suggest that correlated dLGN activity may be enhanced via direct synaptic contacts between thalamocortical cells, whereas noncorrelated activity (such as that occurring during binocular rivalry) could be suppressed via thalamocortical collateral input to PGN cells and dLGN interneurons.

Relative distribution of synapses in the pulvinar nucleus of the cat: implications regarding the "driver/modulator" theory of thalamic function.

To provide a quantitative comparison of the synaptic organization of "first-order" and "higher-order" thalamic nuclei, we followed bias-corrected sampling methods identical to a previous study of the cat dorsal lateral geniculate nucleus (dLGN; Van Horn et al. [2000] J. Comp. Neurol. 416:509-520) to examine the distribution of terminal types within the cat pulvinar nucleus. We observed the following distribution of synaptic contacts: large terminals that contain loosely packed round vesicles (RL profiles), 3.5%; presynaptic profiles that contain densely packed pleomorphic vesicles (F1 profiles), 7.3%; profiles that could be both presynaptic and postsynaptic that contain loosely packed pleomorphic vesicles (F2 profiles), 5.0%; and small terminals that contain densely packed round vesicles (RS profiles), 84.2%. Postembedding immunocytochemistry for gamma-aminobutyric acid (GABA) was used to distinguish the postsynaptic targets as thalamocortical cells or interneurons. The distribution of synaptic contacts on thalamocortical cells was as follows: RL profiles, 2.1%; F1 profiles, 6.9%; F2 profiles, 5.4%; and RS profiles, 85.6%. The distribution of synaptic contacts on interneurons was as follows: RL profiles, 11.8%; F1 profiles, 9.7%; F2 profiles, 2.8%; and RS profiles, 75.6%. These distributions are similar to that found within the dLGN in that the RS inputs (the presumed "modulators") far outnumber the RL inputs (the presumed "drivers"). However, in comparison to the dLGN, the pulvinar nucleus receives significantly fewer numbers of RL, F1, and F2 contacts and significantly higher numbers of RS contacts. Thus, the RS/RL synapse ratio in the pulvinar nucleus is 24:1, in contrast to the 5:1 RS/RL synapse ratio in the dLGN (Van Horn et al., 2000). In first-order nuclei, the lower RS/RL synapse ratio may result in the transfer of visual information that is largely unmodified. In contrast, in higher-order nuclei, the higher RS/RL synapse ratio may allow for a finer modulation of driving inputs.