The present data indicate that a period of natural cell death for midbrain dopamine neurons in non-human primates peaks at mid-gestation (E80). In the rat, natural death in midbrain DA neurons has been identified during the late prenatal and early postnatal period, with the rate maximal at P2 (Oo Burke, 1997
). While the mid-gestational peak of this event in the monkey contrasts with its postnatal occurrence in the rat, it is important to note that there is a poor relationship between the timing of birth and the maturational state of the brain in different species. However, there is a strong conservation in the sequence of neural events across mammalian species. Using the data provided by Clancy et al. (2001)
, it is striking that the developmental time of P2 in the rat is equivalent to E78 in the rhesus monkey, a species with the same gestational period as the vervet monkey. Thus it would appear that natural cell death of midbrain dopamine neurons occurs at the same developmental stage in rodents and primates, although the timing with respect to gestation and birth is very different.
In the rat a second, smaller peak of NCD in midbrain DA neurons occurs at about P14 (Oo Burke, 1997
), an age in the rat that is estimated to be equivalent to E127 in the monkey (Clancy et al., 2001
). We observed no evidence of cell death in our samples between E120 and E130. We also examined a series of neonatal monkey brains that covered the same early neonatal period (P11-P25) in which NCD was seen in rat DA neurons and did not observe any TH-ir neurons with signs of apoptosis. Thus, it appears that the second wave of apoptosis in rat DA neurons does not have a counterpart in the monkey.
In order to understand the significance of the NCD for midbrain DA neurons, it is relevant to ask what developmental events coincide with these periods. Oo and Burke (1997)
speculated that the later wave of NCD in the rat may be related to the period of target contact and competition for synapse formation, as the rate of synaptogenesis is greatest rate between P13-17 (Hattori McGeer, 1973
). In the rhesus monkey, synaptogenesis in the striatum begins at E65, has the greatest rate of increase at about the time of birth, and synaptic density achieves a plateau at about P30 (Brand Rakic, 1984
). However, it is also important to refer to estimates of the time at which terminals of developing DA neurons reach the striatum, as the data for striatal synapse formation is not specific to dopaminergic contacts (Goldman-Rakic, 1981
). In the vervet monkey we have observed that TH-ir fibers first impinge upon the posterior striatum between E55-60, and continue to increase in density until after birth (Bundock, 1999
, Sladek et al., 1995
). As shown in , the greatest rate of increase in striatal DA concentration occurs between E70-90, the time when our data indicates that apoptosis is peaking in substantia nigra DA neurons. During this time, striatal DA concentration rises from 3% to 35% of adult levels. The actual trigger of apoptosis cannot be discerned from this study. However as striatal DA levels are a good marker of dopaminergic terminal density (Onn et al., 1986
, Wilson et al., 1996
), our data suggest that apoptosis in this population coincides with the time at which the dopaminergic innervation of the striatum is increasing at its greatest rate. The establishment of the next 35% of adult striatal DA levels (35-70%), which occurs more slowly, is not associated with any detectable natural cell death in this population. This phase of maturation may reflect an increase in DA production in each neuron rather than an increase in the number of terminals (Morrow et al., 2005
In addition to determining when natural cell death occurs in primate midbrain DA neurons, another goal of the present study was to estimate the extent of apoptosis that occurs during this time. The peak rate of apoptosis was 5% of the TH+ neurons, at about E80. It should be noted that this percentage is based on the number of cells TH+ at the time of examination, rather than as a percentage of the eventual number of cells expressing detectable TH immunoreactivity. If the latter denominator is used the rate of apoptosis at E80 is 2%. This estimate is higher than in the rat, where the highest incidence of apoptosis was observed to be 10 TH+ cells per substantia nigra and 60 apoptotic profiles per substantia nigra (Oo Burke, 1997
). This apparent difference could be due either to a real difference in the proportion of developing DA neurons that undergo apoptosis or to a difference in the duration of the apoptotic process in rodents and primates. The peak rate of apoptosis (5%) in our study is somewhat higher than that found in the one study using human fetal brain (periaqueductal gray, 2.85%), although the samples in this latter study (Chan Yew, 1998
) were spaced several weeks apart, so a definite peak could not be identified. Over the 25 day span in which we detected apoptosis in TH+ cells, the mean rate was 2.6%/day. However, it is not possible to translate reliably this number into the proportion of the population that is sacrificed by NCD. This is because of the time a cell can retain the characteristics of apoptosis is not known. Generally the apoptotic process is thought to be a rather rapid event, taking a only few hours to complete (Bursch et al., 1990
, Oppenheim, 1991
). However, NCD of central neurons in vivo may be more protracted. In fact, it has been shown in the rat that the elimination of the apoptotic granule hippocampus cells induced by adrenalectomy took 72 hours (Hu et al., 1997
). The end stages of cell disassembly would not meet our criteria for inclusion, but based on Hu et al. (1997)
it is possible that our study included cells in which apoptosis started 24-48 hours earlier. Using this figure, we estimate that at least 50% of the final population was removed during this window of NCD.
While it appears that the timing of apoptotic cell death in primate nigrostriatal DA neurons is associated with the initial innervation of the striatum and formation of synapses, the actual biochemical triggers for the process are not known. However, recent investigations of NCD in DA neurons of mice strongly implicate a role for GDNF as a critical target-derived factor in regulating the magnitude of NCD. Thus, intrastriatal injection of anti-GDNF neutralizing antibodies were found to augment the extent of NCD, while over-expression of GDNF led to an increased number of DA neurons after the first phase of NCD (Kholodilov et al., 2004
, Oo et al., 2003
The extent of NCD in nigrostriatal DA neurons conceivably may have an impact on later susceptibility to disorders such as Parkinson’s disease. As the degree of NCD is a determinant of an individual’s endowment of nigrostriatal DA neurons and as these neurons appear to be progressively lost during aging (Fearnley Lees, 1991
, Forno, 1996
), it is possible that an unusually large degree of NCD may increase the risk of acquiring Parkinson’s disease later in life. It is also possible that while undergoing NCD, neurons may be more susceptible to insults than at other times, and our preliminary data appear to support this (Elsworth et al., 2005
). Previous investigations have revealed that the period of most rapid brain growth (“the brain growth spurt”) is a time when rats respond to NMDA antagonists and GABA-A agonists with apoptotic neurodegeneration in many brain regions (Ikonomidou et al., 2001
). In the rat the brain growth spurt peaks at approximately 7 days after birth, for rhesus monkeys the peak growth takes place after approximately 115 days of gestation, and in man this occurs during the first month of life (Dobbing Sands, 1979
). It is quite possible that the brain growth spurt and the time of NCD are related; both have been linked with synaptogenesis. However, the time of peak brain growth does not correspond exactly with the occurrence of NCD and synaptogenesis for DA neurons in rats or monkeys.
The present data show that in fetal monkey brains, a distinct peak of apoptotic NCD occurs in midbrain DA neurons at about E80, midway through gestation. We estimate that at least 50% of the population is lost in this 25-day period. Other data reported here suggests that the onset of apoptosis in DA neurons is linked with the time of greatest rate of increase in striatal DA innervation. This is consistent with the theory that apoptosis in this population is triggered by inadequate supply of striatal growth factors.