Third Trimester Equivalent Alcohol Exposure Reduces Modulation of Glutamatergic Synaptic Transmission by 5-HT1A Receptors in the Rat Hippocampal CA3 Region

Abstract

Fetal alcohol exposure has been associated with many neuropsychiatric disorders that have been linked to altered serotonin (5-hydroxytryptamine; 5-HT) signaling, including depression and anxiety. During the first 2 weeks of postnatal life in rodents (equivalent to the third trimester of human pregnancy) 5-HT neurons undergo significant functional maturation and their axons reach target regions in the forebrain (e.g., cortex and hippocampus). The objective of this study was to identify the effects of third trimester ethanol (EtOH) exposure on hippocampal 5-HT signaling. Using EtOH vapor inhalation chambers, we exposed rat pups to EtOH for 4 h/day from postnatal day (P) 2 to P12. The average serum EtOH concentration in the pups was 0.13 ± 0.04 g/dl (legal intoxication limit in humans = 0.08 g/dl). We used brain slices to assess the modulatory actions of 5-HT on field excitatory postsynaptic potentials in the hippocampal CA3 region at P13-P15. Application of the GABAA/glycine receptor antagonist, picrotoxin, caused broadening of field excitatory postsynaptic potentials (fEPSPs), an effect that was reversed by application of 5-HT in slices from air exposed rats. However, this effect of 5-HT was absent in EtOH exposed animals. In slices from naïve animals, application of a 5-HT1A receptor antagonist blocked the effect of 5-HT on the fEPSPs recorded in presence of picrotoxin, suggesting that third trimester ethanol exposure acts by inhibiting the function of these receptors. Studies indicate that 5-HT1A receptors play a critical role in the development of hippocampal circuits. Therefore, inhibition of these receptors by third trimester ethanol exposure could contribute to the pathophysiology of fetal alcohol spectrum disorders.

Keywords: fetal alcohol spectrum disorders; fetal alcohol syndrome; hippocampus; rat model; serotonin; synaptic transmission.

Figure 1

Characterization of the third trimester-equivalent ethanol exposure paradigm: pup weight gain and serum ethanol levels. (A) Average pup weight for air and ethanol (EtOH) exposed animals. (B) Averaged pup serum EtOH concentration measured at the end of the 4 h-long exposure (n = 22 animals from 10 litters). Triangles represent values obtained in individual pups.

Figure 2

Application of the GABA_A/glycine receptor antagonist, picrotoxin, induces broadening of field excitatory postsynaptic potentials in the CA3 hippocampal region from un-exposed naïve animals. (A) Sample traces illustrating the effect of picrotoxin (PTX; 50 μM) and the lack of effect of the NMDA receptor antagonist, APV (100 μM). The field excitatory post-synaptic potentials were blocked by the non-NMDA receptor antagonist, NBQX (10 μM). The shaded hatched area indicates where we measured area under the curve (A.U.C.). (B) Summary of results obtained with eight slices from three pups from 2 litters (see text for results of statistical analyses).

Figure 3

Third trimester-equivalent ethanol (EtOH) exposure blocks the 5-HT-induced inhibition of field excitatory post-synaptic potentials recorded under conditions of GABA_A/glycine receptor inhibition. Representative traces from air (A) and EtOH (B) exposed animals at baseline, in the presence of 50 μM picrotoxin (PTX), and in the presence of 50 μM PTX and 100 μM 5-HT. The shaded hatched area indicates where we measured area under the curve (A.U.C.). (C) Time course of the normalized A.U.C.s in all three conditions. See text for results of statistical analyses (n = 9–10 animals from 5 litters).

Figure 4

Pharmacological blockade of 5-HT_1A receptors blocks the 5-HT-induced inhibition of field excitatory post-synaptic potentials recorded under conditions of GABA_A/glycine receptor inhibition in slices from naïve rats. (A) Representative traces from a naïve animal obtained at baseline, in the presence of 50 μM picrotoxin (PTX), and in the presence of 50 μM PTX + 100 μM 5-HT. (B) Same as in panel A but in the constant presence of the 5-HT_1A antagonist, WAY-100635 (10 μM). The shaded hatched area indicates where we measured area under the curve (A.U.C.). (C) Time course of the effect of 5-HT on the PTX-induced increase of the AUC in absence and presence of WAY-100635. See text for results of statistical analyses (n = 6 animals from 2 litters).