It is now well-known that offspring of parents with alcohol dependence (AD) have an increased risk for developing alcohol and drug dependence by young adulthood (Hill et al., 2008
; Kendler et al., 2008
). With a number of morphological differences now being identified in high-risk offspring (see Tessner and Hill, 2010
, for review), it is clear that some of these variations may provide clues to the neurobiological underpinnings for addiction. It is also clear that alcohol has neuropathological effects on neuronal integrity in adults (Sullivan and Pfefferbaum, 2005
) and adolescents (DeBellis et al., 2005
). Reduced hippocampal volume (DeBellis et al., 2000
) and smaller prefrontal cortex volume (DeBellis et al., 2005
) along with neurocognitive changes (Brown et al., 2000
) have been reported in adolescent-onset alcohol use disorders. What is not always clear is whether the differences observed are a consequence of exposure or, through the effects of familial risk factors, are antecedent to the development of alcohol dependence. Alternatively, the effects of risk and exposure may be in opposite directions so that removal of cases with substance use disorders may be necessary to observe familial risk group differences as was the case in the present study. To illustrate, adolescents with significant alcohol exposure show reduced hippocampal volume (DeBellis et al., 2000
) that is not seen in high-risk male offspring with minimal alcohol exposure (Hill et al., 2001
). However, other regional alterations in morphology have been observed in high-risk offspring with only minimal alcohol or drug exposure including reduced volume of the right amygdala (Hill et al., 2001
), and the right orbitofrontal cortex (OFC) (Hill et al., 2009
Both total and gray matter volumes of the cerebellum are greater in high-risk offspring from multiplex for alcohol dependence families in comparison to controls. The results are unlikely to be due to the personal diagnosis of offspring as no single diagnosis contributed more than 25% of cases with the exception of substance use disorders. Analyses controlling for the effects of exposure to alcohol and drugs in these adolescent and young adult participants suggest that the greater volume seen in individuals with high familial loading for alcohol dependence is present before drug and alcohol use are initiated. How these morphological changes may be related to greater susceptibility to alcohol dependence and other substance use disorders is currently unknown. However, it is clear that adults with long histories of alcohol dependence display disruption of frontocerebellar circuitry (Sullivan et al., 2003
). Other regions such as the orbitofrontal cortex have been studied far more extensively as candidate regions for addiction susceptibility because of their involvement in disinhibitory processes (Dom et al., 2005
). The present results suggest that the cerebellum may play an important role in addiction susceptibility as well.
In addition to structural alterations, functional changes in the cerebellum have also been observed in association with addiction. In a PET study using a visual–spatial recognition task with delayed response and monetary reward, Martin-Soelch et al. (2001)
found greater activation of the left cerebellum in an opiate addicted group in comparison to controls. Also, greater activation in the right superior cerebellum and left frontal cortex has been reported for alcohol dependent subjects in comparison to controls when performing a Sternberg working memory task (Desmond et al., 2003
). Additionally, cocaine users in comparison to controls have more difficulty inhibiting their responses in a GO/NO GO inhibition task with varying working memory load which is accompanied by greater activation in the left cerebellum (Hester and Garavan, 2004
). Taken together, these studies suggest that individuals susceptible to substance use disorders may have altered cognitive functioning involving the cerebellum.
In addition to the structural and functional cerebellar alterations seen across different addictions, there is reason to believe that cerebellar development may be altered in those with highest risk for developing an addiction through familial/genetic loading. Cortical development has been studied extensively allowing for description of the overall process. Briefly, during adolescence and young adulthood changes in brain structure and refinement of brain organization lead to significant changes in cognitive, social, and emotional behavior (Casey et al., 2005
; Yurgelun-Todd, 2007
). White matter volume appears to increase well into adulthood while gray matter volume tends to increase in childhood and adolescence followed by a decrease (Jernigan et al., 1991
; Pfefferbaum et al., 1994
; Giedd et al., 1996a
; Sowell et al., 2004
), with females reaching their peak 1–2 years earlier than males (Lenroot and Giedd, 2006
). Because cortical development appears to follow a pattern that subserves the needs of the organism, primary motor, sensory and visual areas mature earlier than those supporting more complex cognitive functions such as the association areas (Gogtay et al., 2004
). Although these developmental changes in cortical organization and functioning have been studied extensively, developmental changes in cerebellar morphology and functioning have received less attention.
Previous reports indicate that the cerebellum undergoes substantial development during childhood and adolescence, first increasing to a peak volume and then subsequently declining (Castellanos et al., 2002
; Keller et al., 2003
; Raz et al., 2003
; Mackie et al., 2007
; Tiemeier et al., 2010
), peaking approximately 2 years later than cerebral volume (Giedd et al., 2009
). In addition to an overall larger volume seen in males than females, peak volumes differ by gender with girls peaking at approximately 12 years and boys at about 15 years of age (Tiemeier et al., 2010
). White matter volume of the cerebellum increases through adolescence with a peak of approximately 17 years in females and 22 years in males. In the present study, we find that total cerebellar volume for males appears to peak later than it does for females in agreement with the Tiemeier et al. findings. Moreover, we find that when the contaminating influence of alcohol and drug exposure that is brought about by a substance use disorder diagnosis is removed, the developmental curves for high- and low-risk offspring more clearly show a difference in developmental trajectories by risk group. This difference appears to show that low-risk males reach peak volumes earlier than high-risk males. A similar trend can be observed for the females, with high-risk females showing a somewhat different developmental trajectory than low-risk females.
The present results support our previous work with a sample of 34 male participants in finding larger cerebellar volume (total and gray matter) in high-risk offspring from multiplex for alcohol dependence families (Hill et al., 2007
). Importantly, variation in SNPs within the BDNF and GABRA2 genes show evidence for gene/gene interaction in altering cerebellar volume. In order to better understand the relationship between cerebellar volume, genotypic variance and familial risk, preliminary analyses were performed relating BDNF and GABRA2 genotypes and familial risk. An association for BDNF was not found. For GABRA2, the rs279871 SNP showed a significant association between risk status and presence of the “2” allele (χ2
= 7.34, d.f.=1, p
=0.007). Because of modest sample size, we view this result as preliminary. However, it does suggest that risk group differences in GABRA2 in association with BDNF variation may promote cerebellar growth resulting in larger cerebellar volume in high-risk offspring.
It is possible that risk group differences at the time these participants were scanned might represent developmental differences in the high- and low-risk groups. Age-regression analyses showed significant age-related changes over the age range studied with total cerebellar volume and gray matter volume following a quadratic pattern and white matter a more linear trend. As noted in previous reports (Giedd et al., 2009
), cerebellar volumes of males and females differed significantly. Comparison of high- and low-risk subjects without substance use disorder shows that high-risk subjects, especially males, may be delayed in reaching full cerebellar volume before a subsequent reduction in volume with age is seen. These results support our previous results in showing what appeared to be a delay in gray matter reduction with age in the high-risk male sample studied.
Of particular interest were our findings concerning the association between variation in GABRA2 and BDNF genes and cerebellar volume. These data provide for the first time an indication that specific genes that have been associated with alcohol dependence risk (Edenberg et al., 2004
; Janak et al., 2006
) may be influential in determining cerebellar growth and differentiation. In addition, there is abundant evidence that these two genes may be operating in early development and beyond to influence cerebellar development. Although GABA is the principal inhibitory neurotransmitter in the nervous system, GABA appears to have excitatory effects, inducing immature cerebellar granule cells to proliferate. This is accomplished when GABA depolarizes these cells allowing intracellular calcium to be increased and phosphorylation of MAPK and CAMKII to occur which then leads to trophic effects in these neurons (Fiszman, 2005
). BDNF mRNA expression increases by approximately one-third from infancy to adulthood, rising during adolescence and peaking during young adulthood (Webster et al., 2002
). Accordingly, it appeared plausible that BDNF might influence growth and development of cells within the cerebellum, a region that does not reach peak volume until adolescence or young adulthood. BDNF has also been reported to influence the volume of the hippocampus (Szeszko et al., 2005
; Bueller et al., 2006
) and posterior superior cerebellar vermis (Agartz et al., 2006
) through its trophic effects on neurons.
In addition to the specific effects that each of these genes have on neuronal development, a number of studies point to the interaction of these two genes in neuronal development of the cerebellum. Evidence for these epistatic effects includes in vitro studies showing that BDNF accelerates cerebellar granule cell gene expression and modulates cell differentiation (Lin et al., 1998
; Bulleit and Hsieh, 2000
). Also, there is evidence that BDNF may be involved in the maturation of the GABAergic system, particularly in the hippocampus, by promoting differentiation and formation of synapses and cell survival (Berninger et al., 1995
). A direct effect of BDNF on GABAA receptors has now been demonstrated. In a novel approach, GABAA receptors were microtransplanted from human epileptic brain tissue to Xenopus
oocytes (Palma et al., 2005
). With this technique, the oocytes acquire human GABAA receptors which can then be tested for response to BDNF. In this experiment, exposure to BDNF increased the currents generated by the “epileptic” GABAA receptors. With demonstrated effects for each of these genes alone and together in cerebellar development, we view the present results showing an interaction of these genes on cerebellar gray matter volume as quite plausible. What is unclear is how the presence of the GABRA2 minor allele (1 or 2 copies) protects cerebellar cells from tissue reduction effects often associated with the BDNF Met allele.
4.1. Limitations of the study
One limitation of the present report is that age-related cerebellar relationships were determined using cross-sectional data. However, data were collected for 131 high- and low-risk individuals across a broad age range (8–28) allowing for description of developmental patterns for total, gray and white matter in childhood, adolescence and young adulthood. Another limitation of the study was its focus on total cerebellar volumes for the statistical analyses presented. Functional variation among the cerebellar lobes is well documented (Andreasen and Pierson, 2008
). Use of a single SNP to characterize genetic variation of the BDNF and GABRA2 genes might also be viewed as a limitation. However, among several GABRA2 SNPs studied, the rs279871 SNP was highly related to alcohol dependence (Edenberg et al., 2004
). Similarly, allelic variation in the BDNF SNP rs6265 and regional variation in brain volume is well documented (Szeszko et al., 2005
; Agartz et al., 2006
; Bueller et al., 2006
Another possible limitation of our analysis was that some offspring were exposed to alcohol, cigarettes or other drugs used by the mothers while they were in utero. Although the ideal situation would be to have offspring without such exposure when evaluating the effects of familial risk, this may not be possible due to the base rate of such exposures in the general population. However, the rates of alcohol use for mothers during their pregnancies observed in this sample are similar to those seen in the general population. Using data from a survey of eight obstetric clinics in Southeastern Michigan, Flynn et al. (2003)
found that 81.9% of the mothers reported drinking less than 1 drink per week. In our sample, 76.3% reported no drinking during pregnancy. Smoking during pregnancy was reported by 22.2% of our sample in comparison to national survey data for the US and England in which 12–20% of mothers continue to smoke during pregnancy (ONS, 2006
; Gilman et al., 2008
). These considerations suggest that the risk group differences observed for cerebellar volume are minimally influenced by the effects of prenatal use of substances by the mothers of these offspring.
In summary, these results suggest that familial risk group differences are associated with differences in cerebellar morphology that may be due to altered developmental trajectories that appear to be influenced by genetic variation in BDNF and its influence on GABRA2 signaling in early development and/or altered gene expression through adolescence and young adulthood.