Disruptions in serotonergic regulation of cortical glutamate release in primate insular cortex in response to chronic ethanol and nursery rearing

Abstract

Early-life stress has been shown to increase susceptibility to anxiety and substance abuse. Disrupted activity within the anterior insular cortex (AIC) has been shown to play a role in both of these disorders. Altered serotonergic processing is implicated in controlling the activity levels of the associated cognitive networks. We therefore investigated changes in both serotonin receptor expression and glutamatergic synaptic activity in the AIC of alcohol-drinking rhesus monkeys. We studied tissues from male rhesus monkeys raised under two conditions: Male rhesus monkeys (1) "mother reared" (MR) by adult females (n=9) or (2) "Nursery reared" (NR), that is, separated from their mothers and reared as a separate group under surrogate/peer-reared conditions (n=9). The NR condition represents a long-standing and well-validated nonhuman primate model of early life stress. All monkeys were trained to self-administer ethanol (4% w/v) or an isocaloric maltose-dextrin control solution. Subsets from each rearing condition were then given daily access to ethanol, water, or maltose-dextrin for 12 months. Tissues were collected at necropsy and were further analyzed. Using real time RT-PCR we found that ethanol-naive, NR monkeys had lower AIC levels of 5-HT(1A) and 5-HT(2A) receptor mRNA compared with ethanol-naive, MR animals. Although NR monkeys consumed more ethanol over the 12-month period compared with MR animals, both MR and NR animals expressed greater 5-HT(1A) and 5-HT(2A) receptor mRNA levels following chronic alcohol self-administration. The interaction between nursery-rearing conditions and alcohol consumption resulted in a significant enhancement of both 5-HT(1A) and 5-HT(2A) receptor mRNA levels such that lower expression levels observed in nursery-rearing conditions were not found in the alcohol self-administration group. Using voltage clamp recordings in the whole cell configuration we recorded excitatory postsynaptic currents in both ethanol-naive and chronic self-administration groups of NR and MR monkeys. Both groups that self-administered ethanol showed greater glutamatergic activity within the AIC. This AIC hyperactivity in MR alcohol-consuming monkeys was accompanied by an increased sensitivity to regulation by presynaptic 5-HT(1A) receptors that was not apparent in the ethanol-naive, MR group. Our data indicate that chronic alcohol consumption leads to greater AIC activity and may indicate a compensatory upregulation of presynaptic 5-HT(1A) receptors. Our results also indicate that AIC activity may be less effectively regulated by 5-HT in ethanol-naive NR animals than in NR monkeys in response to chronic ethanol self-administration. These data suggest possible mechanisms for increased alcohol seeking and possible addiction potential among young adults who had previously experienced early-life stress that include disruptions in both AIC activity and serotonin system dynamics.

Figure 1

A, Daily ethanol intake (g/kg) during 12 months of unlimited access to ethanol (4% w/v) in MR and NR monkeys. Two data sets are plotted for MR monkeys: one set with all four MR alcohol exposed monkeys used in the study and one set with three MR alcohol exposed monkeys that were used for gene expression studies. Data are smoothed using weighted average of nine nearest data points. In comparing consumption of NR monkeys with each set of MR, NR monkeys consumed significantly greater ethanol than either set of MR monkeys throughout the study period (two-way repeated measures ANOVA, p<0.01). B, Blood ethanol concentrations during 22-hour free access and corresponding ethanol consumption at the time of sampling (sample taken 6 hours post session start time) revealing a significant correlation (r²=0.67, p<0.0001).

Figure 2

Relative mRNA expression levels of GAPDH and various 5-HT receptor subtypes for ethanol naïve and ethanol exposed MR or NR monkeys. A, Comparison of ethanol’s effects on MR and NR groups reveals a significant increase in GADPH, 5-HT_1A and 5-HT_2A mRNA levels (n=3, *p<0.05 unpaired t test). Chronic ethanol significantly upregulated GAPDH, 5-HT_1A and 5-HT_2A mRNA levels in the NR monkeys (n=4, *p<0.05 unpaired t test), with a significant interaction between chronic ethanol exposure and rearing conditions for 5-HT_1A and 5-HT_2A mRNA levels (#p<0.05, 2-way ANOVA). B, When comparing rearing conditions, nursery-rearing alone produced a significant decrease in expression of both 5-HT_1A and 5-HT_2A when compared to MR, ethanol-naive monkeys. No differences were seen between MR and NR animals chronically-exposed to ethanol.

Figure 3

Synaptic properties of AIC neurons upon white matter or intracortical electrical stimulation in MR control animals. A, EPSCs consisted of APV-sensitive NMDA component and NBQX-sensitive AMPA/kainate component. B, Electrical stimulation evoked a rapid synaptic response followed by a delayed synaptic barrage. Bi, Single sweep showing white matter evoked short-latency synaptic response followed by delayed synaptic barrage. Arrows show individual synaptic responses that comprise the barrage response. Asterisk shows persistent depolarization resulting from synaptic stimulation. Depolarization persisted for ~1500 msec. Bii, Averaged sweeps showing white matter stimulation-evoked and intracortical stimulation-evoked responses from the same neuron. Asterisk shows short-latency synaptic response that was elicited by white matter stimulation but not intracortical stimulation. Arrow shows difference in latency to onset of synaptic barrage arising from white matter stimulation and intracortical stimulation. C, Short-term plasticity of white-matter stimulation evoked synaptic responses showing frequency-dependent depression for an individual neuron (i) and for the population (n=6 cells; ii).

Figure 4

AMPA/kainate mediated spontaneous EPSCs recorded from AIC neurons in MR and NR control and chronic ethanol-consuming monkeys. A, MR animals that chronically self-administered ethanol showed significantly greater frequency (lower IEI) of sEPSCs as compared to MR ethanol-naïve controls (*p<0.05). NR ethanol-naïve monkeys also showed significantly lower sEPSC IEIs (higher frequency) as compared to MR ethanol-naïve controls (*p<0.05). B, Cumulative IEI and amplitude histograms for the population of cells in each group of animals. C, Example traces for all four groups are also shown, along with a trace demonstrating that these sEPSC events could be eliminated with the AMPA/kainate receptor blocker DNQX in a MR control animal.

Figure 5

The 5-HT_1A receptor agonist 8-OH DPAT reduced the amplitude of white matter-evoked EPSCs in layer 4/5 AIC neurons. A–B, Time course of the effect of 8-OH DPAT (2 μM) on EPSC amplitude recorded from neurons from control MR monkeys (A) or ethanol-exposed MR monkeys (B). C, 8-OH DPAT reduced the amplitude of EPSCs from ethanol-exposed MR monkeys significantly more than EPSCs from MR control monkeys. The magnitude of the decreased EPSC amplitude is paralleled by the magnitude of the increase in the paired pulse depression ratio (PPDR) (*p<0.05, Wilcoxon rank test).

Figure 6

Proposed model of synaptic changes in animals exposed to alcohol and/or nursery rearing based on experimental findings. A–B, Chronic exposure to alcohol may increase synaptic strength and expression of 5-HT_1A and 5-HT_2A receptors when compared to MR controls. Nursery rearing may also increase synaptic strength but decrease expression of 5-HT_1A and 5-HT_2A receptors. D, Chronic alcohol exposure may increases 5-HT_1A and 5-HT_2A receptor expression in nursery reared monkeys, returning serotoninergic tone closer to baseline levels.