In summary, axon terminals forming axosomatic synapses are larger than axodendritic ones, and both sets of terminals increase in size with age. However, in profiles of axodendritic terminals, the mean number of synaptic vesicles does not change with age, and neither does the number of vesicles per unit area of the terminal profiles. For axosomatic terminals the situation is somewhat different, since with age the number of synaptic vesicles in the profiles of these terminals increases by about 20%. Furthermore, these increases are correlated with the cognitive impairment indices of the same monkeys. However, the numbers of vesicles per unit area of the terminal profiles does not change significantly with age, due to the increase in size of the terminals. Also, the synaptic junctions are similar in length at axodendritic and axosomatic synapses and there is no change in the lengths of the junctions with age in either set of terminals. The other component of axon terminals is the mitochondria, which provide the energy for sustained neurotransmitter secretion. The mean size of mitochondrial profiles does not change with age in axodendritic terminals, but in axosomatic terminals, there is a significant increase in mitochondria volume of about 14% with age.
The difference in axon terminal and mitochondria size between axodendritic and axosomatic synapses in young monkeys is consistent with similar observations reported for symmetric synapses in the adult rat hippocampus (Miles et al., 1996
) and in the rat substantia nigra, pars reticulata (von Krosigk et al., 1992
). This suggests that these differences are widespread throughout the brain and occur in different species, differences that may be linked to a higher metabolism of axosomatic compared to axodendritic terminals. The finding that the size of axon terminals and mitochrondria remains larger in old monkeys in axosomatic compared to axodendritic symmetric synapses suggests that axosomatic synapses are also metabolically more active in aged animals.
For axodendritic inhibitory terminals, the only morphological change with age is a 21% increase in mean size. In our earlier study (Peters et al., 2008
) it was found that there is a 22% loss of axodendritic symmetric synapses in area 46 with age. Therefore, the increase in axodendritic axon terminal size may be occurring in response to the net loss of inhibitory input to dendrites in area 46. It is possible that the loss involves only one subclass of axon terminals, since axodendritic terminals originate from a variety of inhibitory neurons such as double bouquet cells, bitufted cells, a variety of smooth multipolar cells, and neurogliaform cells (e.g. Somogyi et al., 1998
), but this has not been established. The only study comparable to the present one appears to be that of Bertoni-Freddari et al. (2007)
who compared the mitochondria in the neuropil of frontal and temporal cortices of adult (mean age 10.3 years) and old (mean age 21 years) long-tailed macaques. However, they examined all axon terminals in the neuropil of these cortices and found that there were no significant differences with age in the volume density (number of mitochondria per μm3
of tissue), numeric density, average volume of individual mitochondria, or average long diameter of mitochondria in axon terminals in the two cortical areas. They conclude that there is a preservation of mitochondria in axon terminals with increasing age. This conclusion is consistent with our results, since no differences in the sizes of mitochondria or ratio of mitochondria area per axon terminal area were found for axodendritic synapses.
Compared to axodendritic terminals, axosomatic terminals forming symmetric synapses are more affected by age. Axosomatic terminals not only increase in size, but they also show an increase in the mean number of synaptic vesicles and in the sizes of individual mitochondria. Presumably this reflects the interaction between these two structural elements, since mitochondria are involved in providing energy for the cycling of synaptic vesicles and for neurotransmitter synthesis and uptake in synaptic vesicles. The age-related increase in size of axosomatic terminals could occur in response to the loss of other axosomatic terminals, but as far as can be ascertained there is no information about whether axosomatic terminals in prefrontal cortex are lost with age, or even about the number of axosomatic terminals present on layer 2/3 pyramidal cells. The only study of axosomatic terminals and the effects of age appears to be that of Tigges et al. (1992)
, who examined Betz cells in motor cortex of the rhesus monkey. They concluded that the total number of axosomatic terminals does not change with age and found no change in their sizes or in their mitochondrial content. Our results appear to conflict with this previous report, although it is possible that aging affects axosomatic inhibitory synapses in the motor cortex and in area 46 differently.
The main source of axosomatic synapses on pyramidal cells is the fast-spiking parvalbumin-positive basket cell of the cortex (e.g. Somogyi et al., 1998
). It is possible that the changes affecting axosomatic synapses with age are part of a homeostatic mechanism aimed at maintaining the high-energy needs involved in neurotransmitter release by fast-spiking neurons. Interestingly, it has been recently shown that, in area 46 of old monkeys, the frequency of spontaneous but not miniature IPSCs recorded in pyramidal neurons is increased (Luebke et al., 2004
). Our finding that aging results in an increased size of axosomatic terminals, mitochondria area and synaptic vesicle numbers suggests that basket cells providing axosomatic inhibition become more active with age, an effect that could explain the increased frequency of spontaneous IPSCs (Luebke et al., 2004
). On the other hand, the lack of effect of aging on the frequency or amplitude of miniature IPSCs (Luebke et al., 2004
) suggests that the increased number of synaptic vesicles per terminal reported here may not be paralleled by an increased loading of inhibitory neurotransmitter per synaptic vesicle and frequency and amplitude of quantal neurotransmitter release.
Axosomatic inhibition by parvalbumin-expressing GABAergic axon terminals on pyramidal neurons is critically involved in the generation and temporal organization of gamma and theta cortical oscillatory rhythms, which are indispensable for cortical processing (Freund, 2003
; Freund and Katona, 2007
). Axosomatic synapses are thought to be especially important in controlling the output of pyramidal neurons, as well as synchronizing the activity of groups of pyramidal cells (e.g. Freund, 2003
). In mice, it was recently found that parvalbumin-positive neurons modulate gamma-frequency and signal transmission in neocortex by reducing noise and amplifying circuit signals (Sohal et al., 2009
). Therefore, the increased size and, presumably, increased metabolic activity of axosomatic axon terminals in aging could have a critical impact on the actions of fast-spiking neurons and their modulation of gamma oscillatory rhythms. Furthermore, the finding that the numbers of synaptic vesicles in axon terminals forming axosomatic, but not axodendritic, synapses are correlated with impaired cognitive performance suggests that altered axosomatic inhibition with age has a detrimental effect on the function of neocortical circuits. Interestingly, studies in aged humans show that reduced cognitive function, as assessed in the auditory oddball paradigm, is associated with increased gamma synchrony in frontal regions (Paul et al., 2005
). In addition, recent evidence indicates that compared to children or young adults, aged adults have increased gamma synchrony during a visual simple choice-reaction task (Werkle-Bergner et al., 2009
). Based on our and these previous findings, it is tempting to speculate that enhanced axosomatic GABAergic inhibition with age would increase gamma synchrony and decrease cognitive function.
In conclusion, the age-related increases in axon terminal size, mitochondria size and synaptic vesicle numbers in axosomatic terminals suggest an enhanced axosomatic GABAergic input to pyramidal neurons in the aged cerebral cortex and this may represent one neuronal mechanism contributing to decreased cognitive performance and, possibly, increased gamma synchrony detected in older individuals. In this context, it would be of interest to determine if similar morphological alterations of axosomatic terminals can be detected in cortical areas involved in sensory processing and if these correlate with specific cognitive alterations.