Brain Development: Normal Processes and the Effects of Alcohol ...

1986; Bonthiu s and West, 1988) . Exposur e to moder -
ate amounts of ethanol (bloo d ethanol concentration s
of-150 mg/dl) causes reductions (15%-20%) in cerebellar
siz e similar to tha t eviden t fo r cerebral corte x
(Miller, 1996b) . Followin g exposur e to high amount s
(sufficient t o produc e bloo d ethano l concentration s
of >250 mg/dl), the size of the cerebellum i s more adversely
affected .
Cerebellar neuron s ar e generate d i n tw o site s —
the VZ and a derived proliferative zone, th e externa l
granular layer (EGL). Attention ha s been directed toward
the EGL because i t is most prominent in the rat
postnatally an d i t give s birth t o a populou s neuro n
type i n th e centra l nervou s system (CNS) , th e cere -
bellar granul e neuron . Postnata l exposur e t o hig h
amounts o f ethano l prolong s th e existenc e o f th e
EGL an d delay s its depletion (Bauer-Moffet t an d Altman,
1977 ; Korngut h e t al. , 1979) . Moreover , cel l
proliferation i n the EG L i s reduced. These data suggest
that the EG L i s different fro m othe r derive d proliferative
zone s i n tha t it s proliferative activity is no t
stimulated by ethanol. Th e EG L differ s fro m th e S Z
and IHZ insofar as it is the sol e source of granule neurons,
whereas the S Z and IH Z produce neuron s that
can be generated i n the VZ. Nonetheless, thi s conclu -
sion remain s unconfirme d unti l dose-respons e dat a
analysis of the EG L i s performed.
CELL CYCLE KINETICS
In vivo and In Situ Studies
As cells proliferate the y pass through a defined set of
steps, cumulativel y known a s the cell cycle. The cel l
cycle has four phases: Gl, S, G2, and M. During th e
Gl phas e th e cell s eithe r prepar e fo r anothe r pass
through the cell cycle or exit from the cell cycle. Durings,
cells replicate their nuclear DNA . G 2 i s a short
period whe n preparatio n fo r mitosi s take s place ,
which occur s durin g M . Dat a o n th e cel l cycl e ar e
best appreciated for VZ cells. The somat a of VZ cells
move rhythmically as the cell s pass through th e fou r
phases (Sauer , 1936 ; Watterso n e t al. , 1956 ; Saue r
and Chittenden , 1959 ; Atla s an d Bond , 1965) .
Through thi s process , calle d interkinetic nuclear migration,
mitotic cell s tha t ar e a t th e ventricula r surface
reconstitute and move their nuclei to positions in
the external third of the VZ where they replicate their
DNA during S.
ETHANOL AND PROLIFERATION OF NEURONA L PRECURSORS 18 7
Ethanol doe s no t affec t th e patter n o f interkinetic
nuclear migration in the VZ per se (Kennedy and Elliott,
1985; Miller, 1989). On the other hand, ethanol
does affec t th e tim e a VZ cel l take s to pass throug h
the cell cycle. For example, in the neocortical V Z of a
21-day-old rat fetus (Miller and Nowakowski, 1991) or
a slic e o f corte x explante d fro m a 17-day-ol d fetu s
(Siegenthaler an d Miller , 2005a) , the tota l length of
the cel l cycl e (T c) i s increased 26%-29% . Thi s in -
crease ha s a direct negativ e effec t o n th e numbe r o f
cells produce d i n th e VZ. An increase i n th e Tc is
also eviden t i n th e EG L o f the cerebellu m (Borge s
and Lewis , 1983) . Ethanol-induce d increase s in Tc
primarily result from a lengthening o f Gl. Thes e effects
o f ethanol ar e no t th e sam e fo r all proliferative
populations. Fo r example, th e Tc of the neocortica l
SZ i s not affecte d b y moderate exposure s to ethano l
(Miller and Nowkowski, 1991).
In addition to cell cycle kinetics, the production of
new cells is defined by three othe r features of proliferating
populations: th e growt h fraction (GF), th e pro -
portion of cells that exit the cell cycle, and the leaving
fraction (LF) . The G F i s the proportio n o f total cel l
population tha t i s actively cycling. Overall , th e G F
for th e cerebra l wal l extends ove r th e perio d o f neo -
cortical neuronongenesi s (Mille r an d Kuhn , 1995 ;
Takahashi e t al, 1995 , 1996) . Althoug h ther e i s no
ethanol-induced differenc e i n th e G F a t th e begin -
ning of this period, as neuronal generation proceeds a
significant differenc e takes place . Ethano l cause s a n
increase in the GF. The effec t of ethanol on the overall
GF i s largely due t o ethanol-induced increase s in
the SZ . Both the specifi c G F fo r the S Z (Miller and
Nowkowski, 1991 ) and th e numbe r o f cells in the S Z
(Miller, 1989 ) ar e increase d b y ethanol. Th e G F for
the VZ is either unaffecte d by ethanol in vivo (Miller
and Nowkowski , 1991) or subtly, but significantly , reduced
i n th e V Z o f an organotypi c slice preparation
(Siegenthaler an d Miller , 2005a) . The effec t o f th e
increases i n GF i n the S Z is accentuated wit h time as
the S Z become s th e majo r neocortica l proliferativ e
zone.
After cells pass through M, the daughter cells either
re-enter the cycle or exit the cell cycle. The numbe r of
cells tha t exi t th e cel l cycl e ca n b e determine d b y
tracing the proportio n o f cells that have incorporate d
a DN A precursor during S relative to the tota l popu -
lation o f cycling cells, for example, cells that express
Ki-67 (Chen n an d Walsh , 2002 , 2003 ; Siegenthale r
and Miller , 2005a ; 2005b) . Accordingly , a n i n sit u