Interleukin (IL)-2 is a brain-derived cytokine that influences mesocorticolimbic dopamine release, and is associated with pathological outcomes that are mediated, at least in part, by aberrations in mesolimbic neurotransmission. The mechanisms by which IL-2 modulates mesolimbic transmission, however, are not known. The NMDA receptor/channel (NMDAR) plays an essential role in neuronal excitability of mesolimbic neurons; we thus examined in neonatal rats the effects of IL-2 on NMDA-activated current (I) in NMDA voltage-clamped neurons freshly isolated from the ventral tegmental area (VTA), the site of origin of the mesolimbic system. IL-2 (0.01-500ng/ml) alone had no effect on membrane conductance. When co-applied with NMDA, IL-2 (50-500ng/ml) significantly potentiated I. In contrast, doses as low as 0.01ng/ml markedly decreased the NMDA response. Dose–response analysis showed that NMDA IL-2 (.50 ng/ml) increased the maximal I, without changing the EC, indicating that IL-2 potentiates I by increasing the NMDA 50 NMDA efficacy of the NMDAR. Moreover, current-voltage analysis revealed that IL-2 potentiation of I was voltage-dependent, being NMDA greater at negative potentials. In contrast, IL-2 inhibition of I was voltage-independent, and IL-2 did not alter the reversal potential. NMDA Additionally, IL-2 (1ng/ml) shifted the NMDA concentration-response curve to the right, significantly increasing the EC for NMDA 50 without changing the maximal I, suggesting that IL-2 inhibits the NMDAR by a competitive mechanism. IL-2 thus acts as a potent NMDA modulator of the NMDAR. IL-2-induced alterations of responses to NMDAR activation may contribute to synaptic plasticity in the mesolimbic system and to pathological outcomes associated with this system.

The brain is particularly sensitive to alcohol during its growth spurt period. To better understand the mechanism(s) involved, we studied the effects of ethanol on neurons freshly dissociated from the ventral tegmental area (VTA) in neonatal rats. Ethanol enhanced (35%) or depressed (45%) glycine-induced responses in VTA neurons (Ye et al., 2001a, 2001b). In this report, we investigated the role of protein kinase C (PKC) and protein kinase A (PKA) in ethanol-induced inhibition of glycine-activated current, using whole-cell patch-clamp technique. Ethanol inhibited glycine-activated current when it was coapplied with the agonist. This inhibition was enhanced when neurons were pretreated with ethanol before the subsequent coapplication of ethanol and glycine. Ethanol's inhibition of glycine-activated currents increased with the length of ethanol pretreatment time (ranging from 1 to 30 s), and reached the maximum at 30 s. However, this enhanced inhibition was not seen in the absence of internal ATP. In addition, phorbol-12-myristate-13-acetate (PMA, 100 nM), a PKC activator, markedly inhibited glycineactivated current. Blockade of PKC by chelerythrine or by PKC inhibitor peptide significantly attenuated ethanol-induced inhibition. Although partial increase of PKC activity by 1 nM PMA enhanced ethanol inhibition, pretreatment of ethanol did not increase ethanol inhibition after the neurons were treated with 100 nM PMA. These data suggest that ethanol and PKC share the same pathway to suppress glycine receptors. H-89 (1

1 The inhibitory effects of n-alcohols (methanol to dodecanol) on glycine-activated currents were studied in neurons freshly dissociated from the ventral tegmental area of neonatal rats using wholecell patch-clamp recording technique. 2 Ethanol enhanced and depressed glycine-activated currents in 35% and 45%, respectively, of neurons of ventral tegmental area of neonatal rats. In this report, we extended our focus of ethanolinduced inhibition of glycine currents to other straight-chain alcohols. 3 Aliphatic n-alcohols, which have carbon numbers less than nine, suppressed glycine currents in 45% (71/158) of the neurons. All results from this study are obtained from the 45% of cells displaying inhibition; the other 55% of the neurons were not studied. 4 Alcohol potency increased as the number of carbon atoms increased from one to five, and was at a maximal plateau from five to nine; alcohols with 10 or more carbons did not inhibit glycineactivated currents. Thus, a cutoff' point in their potency for inhibition of glycine receptor function occurred at about decanol. 5 A coapplication of dodecanol with ethanol eliminated the inhibition resulting from ethanol. Thus, dodecanol may bind to the receptor silently and compete with ethanol. 6 These observations indicate that straight-chain n-alcohols exhibit a cutoff' point in their potency for inhibition of the glycine receptor function between nine and 10 carbon atoms. The inability of longer alcohols to change the activation properties of the receptors may contribute to the cutoff effect.

The potentiation of glycine-induced responses by ethanol (EtOH) was studied in neurons freshly dissociated from the ventral tegmental area (VTA) of 5- to 14-day-old postnatal rats using whole-cell and gramicidin-perforated patch-clamp techniques. Under current-clamp conditions, EtOH increased glycine-induced membrane depolarization and action potential firing. Under voltage-clamp conditions, EtOH (0.1-40mM) alone did not elicit a current. When coapplied with glycine, EtOH enhanced the glycine-induced current in 35% (180 of 474) of the neurons. The EtOH-induced enhancement of glycine current was independent of membrane potential (between 260 and 160 mV); the reversal potential was not changed. Concentration-response analysis showed that in the presence of EtOH (10 mM), the EC50 for glycine decreased from 25 6 4 to 14 6 3 mM; the Hill coefficient increased from 1.5 6 0.2 to 1.9 6 0.3. Kinetic analysis of glycine currents indicated that EtOH decreased the time constant of activation and increased the time constant of deactivation of glycine-gated chloride channels. EtOH may accelerate glycine association with its receptor at the agonist binding site and increase the apparent agonist affinity. Our observations suggest that, at pharmacologically relevant concentrations, EtOH alters the function of glycine receptors and thus the excitability of neonatal VTA neurons. This action of EtOH may contribute to the neurobehavioral disturbances associated with fetal alcohol syndrome.

The brain is particularly sensitive to alcohol during the period of its rapid growth. To better understand the mechanism(s) involved, we studied ethanol effects on glycine-activated responses of ventral tegmental area (VTA) neurons isolated from the newborn rat, using whole cell and gramicidin perforated patch-clamp techniques. Previously we reported that 0.1-40m M ethanol enhances glycineinduced responses of 35% of VTA neurons (Ye et al. 2001). We now direct our attention to the inhibitory effects of ethanol observed in 45% (312 of 694) of neonatal VTA neurons. Under current-clamp conditions, 1 mM ethanol had no effect on the membrane potential of these cells, but it decreased glycine-induced membrane depolarization and the frequency of spontaneous action potentials. Under voltageclamp conditions, 0.1-10 mM ethanol did not elicit a current but depressed the glycine-induced currents. The ethanol-induced inhibition of glycine current was independent of membrane potential (between 260 and 160 mV). Likewise, ethanol did not alter the reversal potential of the glycine-activated currents. Ethanol-mediated inhibition of glycine current depended on the glycine concentration. While ethanol strongly depressed currents activated by 30μM glycine, it had no appreciable effect on maximal currents activated by 1 mM glycine. In the presence of ethanol (1 mM), the EC50 for glycine increased from 32±5 to 60±3μM. Thus ethanol may decrease the agonist affinity of glycine receptors. A kinetic analysis indicated that ethanol shortens the time constant of glycine current deactivation but has no effect on activation. In conclusion, by altering VTA neuronal function, ethanol-induced changes in glycine receptors may contribute to neurobehavioral manifestations of the fetal alcohol syndrome.