Chronic ethanol self-administration was associated with lower hippocampal SERT density in cynomolgus macaques, an effect that was most pronounced in the molecular layer of the dentate gyrus. This is the first study to investigate the effects of chronic ethanol self-administration on hippocampal SERT density in a layer by region specific manner in a macaque model that closely resembles human physiology, neurobiology, and voluntary alcohol drinking patterns. In addition, we have shown that self-administration and human interaction had no significant effect on hippocampal SERT density suggesting that housing controls are sufficient for future studies.
The lower hippocampal SERT density observed in ethanol drinkers may be the result of a compensatory down-regulation of the transporter secondary to decreased serotonin concentrations associated with chronic ethanol consumption (Borg et al., 1985
; Fils-Aime et al., 1996
). Indeed, the hippocampus appears to be particularly vulnerable to decreased serotonergic neurotransmission following chronic ethanol consumption (Wu et al., 1986
). Alternatively, lower hippocampal SERT density may indicate a loss of serotonergic innervation. The human literature both supports and challenges this suggestion. Halliday et al. (1993
) reported degeneration of midbrain serotonergic neurons identified by tryptophan hydroxylase (TPH) immunoreactivity in the brains of alcoholics with and without Wernicke–Korsakoff’s syndrome. This study was contradicted by a subsequent study in which midbrain serotonergic neuron degeneration was not observed in alcoholics without Wernicke–Korsakoff’s (Baker et al., 1996
). Recent studies suggest a more complex picture. In a comprehensive analysis, Underwood et al. (2007
) investigated a heterogenous population of alcoholics and reported no differences in dorsal raphe serotonin neuron number or size but greater TPH immunoreactivity and density of TPH+ neuron processes in the dorsal raphe of alcoholics than controls. Similar findings were reported by Bonkale et al. (2006
) who observed greater TPH immunoreactivity in a single subnucleus of the dorsal raphe of depressed alcoholic suicides than in healthy controls. Unfortunately, it is difficult to make comparisons among these studies due to small sample sizes and sample heterogeneity, but overall they suggest that chronic alcohol consumption is associated with serotonergic neuroplasticity. Importantly, however, recent work has shown that the SERT is a more accurate marker of serotonin positive neurons than serotonergic biosynthetic enzymes (Nielsen et al., 2006
), the use of which can frequently produce false negatives. In addition, differences (or lack thereof) in raphe neuron number are not necessarily indicative of the density of serotonergic projections to regions outside the midbrain, making it possible that previously reported results are region specific. Further investigation will be required to determine the impact of serotonergic neurodegeneration and the extent that brain regions receiving these midbrain serotonergic projections are affected.
No correlational relationship was observed between hippocampal SERT density and individual measures of ethanol intake or blood ethanol concentration. It is possible that the neurobiological adaptations observed are reached at a lower threshold level of alcohol intake. The animals used in the present study had an average daily ethanol intake ranging from 1.16 to 4.20
g/kg (Grant et al., 2008
) suggesting that changes in hippocampal SERT occur at alcohol intakes lower than 1.16
g/kg/day (about an average of four drinks per day). Several studies have reported correlations between amount of ethanol intake and neurobiological endpoints (Adell and Myers, 1995
; Heinz et al., 1998
; Acosta et al., 2010
; Cuzon Carlson et al., 2011
). However, in extrapolating to the human literature, accurate measures of intake are difficult to ascertain whereas duration of drinking is more quantifiable. Chronicity of drinking may in fact be a more accurate predictor of the neurobiological adaptations induced by of chronic ethanol consumption, as has been reported in previous studies examining effects on the serotonergic system (Preuss et al., 2000
; Berggren et al., 2002
; Johnson et al., 2008
). While the monkeys in the present study varied in overall levels of consumption, the duration of drinking was identical, which may explain the lack of correlation between intake and blood ethanol concentration measures and hippocampal SERT density.
With the identification of a SERT polymorphism affecting transcriptional control of the protein product (5-HTTLPR), a significant amount of research has investigated whether basal SERT levels play a role in the risk for alcohol dependence. The 5-HTTLPR is a 44 base pair insertion/deletion with the short allele conferring reduced SERT mRNA, density, and activity compared to the long variant (Lesch et al., 1996
). The alcohol literature is replete with contradictory findings, but a recent meta-analysis revealed a modest association between dependence and the presence of at least one short allele of the 5-HTTLPR polymorphism. Importantly, however, the study also uncovered the potential for publication bias toward positive results (McHugh et al., 2010
). We are unable to conclusively determine whether lower hippocampal SERT density is a risk factor for heavy or compulsive ethanol self-administration, but the data we report here suggest that basal SERT density may not be a risk factor for the amount an individual drinks. All of the subjects in this study were randomly assigned to drinking and control groups so there is no reason to expect that drinkers, as a group, would have lower SERT densities before they were exposed to ethanol. In addition, the absence of any correlation between individual consumption and hippocampal SERT density, while not conclusive, does not support the hypothesis that basal SERT density is a risk factor for or predictor of the amount consumed.
Our findings contradict those of Shibasaki et al. (2010
) who reported greater hippocampal SERT immunoreactivity in mice exposed to ethanol chronically using a vapor chamber apparatus, but support Tagliaferro et al. (2002
) who observed lower hippocampal SERT immunoreactivity in rats exposed to ethanol in their drinking water for 6
weeks. These contradictory results are likely due to strain/species differences, exposure methods, and perhaps the stress induced by involuntary ethanol exposure. Various rodent species and strains exhibit marked differences in neurochemistry and neuroanatomy (Ingram and Corfman, 1980
; Nguyen et al., 2000
; Chen et al., 2006
; Kapasova and Szumlinski, 2008
). Furthermore, voluntary drug self-administration can produce drastically different effects from non-contingent drug exposure (Hemby et al., 1997
; Jacobs et al., 2003
). Both of these factors may contribute to the disparate findings reported in rodent models.
Two studies have assessed the effects of alcohol on hippocampal SERT density in humans. The first examined the hippocampal formation in a regionally specific manner and reported greater SERT density in individuals with a positive blood ethanol concentration at time of death as measured by [3
H]imipramine binding (Gross-Isseroff and Biegon, 1988
). These results are difficult to attribute to an effect of chronic alcohol consumption, however, because [3
H]imipramine binds to both the SERT and a second, low affinity binding site (D’Amato et al., 1987
) and subjects were assigned to the alcohol group simply on the basis of the presence of alcohol at time of death, which is not necessarily indicative of chronic consumption. In contrast, a more recent study reported lower SERT density in the brains of alcoholics than controls using [3
H]paroxetine (Chen et al., 1991
). These authors, however, failed to examine the hippocampus by its functionally distinct subregions as reported in the present study.
Other studies have reported lower SERT binding or availability in brain regions outside the hippocampus in chronic drinkers (Heinz et al., 1998
; Szabo et al., 2004
; Storvik et al., 2006a
). It is tempting to suggest that SERT density is lower globally in animals and humans exposed to chronic ethanol, but comparisons between the existing literature and our own data are difficult to make due to significant species and methodological differences, most notably the heterogeneity of the samples investigated. Further work is needed to determine whether the differences observed in the hippocampal formation are also observed elsewhere in the brain, how these differences may affect neurotransmission and the responsivity to pharmacotherapeutics aimed at reducing drinking.
If the lower hippocampal SERT density we observed in the present study following chronic ethanol consumption is related to serotonergic fiber degeneration, then the hippocampus may be operating under a serotonin deficit in chronic ethanol drinkers. The dentate gyrus, which we found was most vulnerable to this effect, is the first step in the unidirectional circuit that makes up the hippocampal formation (Insausti and Amaral, 2004
), receiving all incoming sensory information from the entorhinal cortex (Insausti and Amaral, 2004
). Because of the complexity of the serotonin system and the abundance of functionally distinct receptors in the serotonin family (Barnes and Sharp, 1999
) it is difficult to speculate on the functional consequences of the changes we observed. The 5-HT1A
receptor, however, is particularly abundant in the hippocampal formation in the same regions where the SERT is localized (Stuart et al., 1986
). The 5-HT1A
receptor is coupled to Gi/o and consequently inhibits adenylate cyclase ultimately resulting cellular inhibition and decreased neurotransmission (De Vivo and Maayani, 1986
; Pugliese et al., 1998
). Lower serotonin concentrations would likely reduce the activation of the 5-HT1A
receptor and ultimately result in a relief of the inhibitory modulation that the hippocampal circuit is typically under. An alteration at the beginning of this circuit is likely to have downstream effects and ultimately modify the circuit’s final output.
Drugs targeting the serotonin system, including selective serotonin reuptake inhibitors (SSRIs), which increase extracellular serotonin levels in the brain by blocking SERT activity, have been proposed for the treatment of alcohol dependence (Johnson, 2004
). Unfortunately, these drugs have had only limited success despite an abundance of promising preclinical data (Kranzler et al., 1995
; Kabel and Petty, 1996
). This preclinical data has relied, however, mainly on rodent models, which exhibit important serotonergic differences compared to primates (Azmitia and Gannon, 1986
; Duncan et al., 1998
). As such, the field can benefit from work using primate models, including the present study, to better understand the interaction between chronic alcohol consumption and serotonergic perturbations in the brain. The results reported here suggest that continued exploration of drugs targeting the serotonin system is warranted for the treatment of alcohol use disorders.