High temporal resolution of amino acid levels in rat nucleus accumbens during operant ethanol self-administration: involvement of elevated glycine in anticipation.

One of the most valid preclinical models of alcoholism is that of self-administration, particularly operant procedures (the use of consequences to modify the occurrence and form of behavior), which allow quantification of the reinforcing effects of drugs (Poling and Bryceland 1979). These procedures allow the study of different contingencies for alcohol reward with good temporal resolution of self-administration behavior in a highly controlled environment and low day-to-day variability within both subject and condition (Sanchis-Segura and Spanagel 2006). Many studies suggest that the mesolimbic dopaminergic (DA) system plays a very important role in the ethanol self-administration process. Operant self-administration of ethanol increases extracellular levels of DA in the nucleus accumbens (NAC) (Weiss et al. 1993; Doyon et al. 2003). Conversely, drugs such as naltrexone, which attenuate the rewarding properties of ethanol, attenuate ethanol-induced stimulation of DA activity in NAC (Gonzales and Weiss 1998). However, the precise role of DA in the rewarding properties of ethanol is far from clear. A recent study pointed out that a transient increase in DA in NAC may be related to the stimulus properties of ethanol presentation rather than its pharmacological or reinforcing actions (Doyon et al. 2004). Alternative neurochemical mechanisms for regulation of operant ethanol self-administration have not been thoroughly explored. The few studies measuring amino acid changes following forced ethanol administration report increased basal levels of extracellular glutamate as measured by microdialysis (Dahchour and De Witte 2003). Taurine levels also are increased in a dose-dependent manner by ethanol (Dahchour et al. 2000; Smith et al. 2004) and this escalation is enhanced after repeated ethanol treatment (Dahchour and De Witte 2000). This finding suggests that taurine may be a possible neurobiological molecule involved in ethanol reinforcement. This hypothesis is supported by findings that acamprosate (a calcium homolog of taurine) and homotaurine (a taurine precursor) both modulate the mesolimbic DA system and decrease ethanol reinforcement processes (Olive et al. 2002; Cowen et al. 2005). However, no study has reported changes in taurine or other amino acids during operant ethanol self-administration. Changes in neurotransmitter release are thought to occur over a short time period during most behavioral events. Thus, in vivo detection of neurotransmitters under these conditions should have a high temporal resolution. To date, fast-scan cyclic voltammetry (FSCV) and microdialysis sampling with HPLC have been used most often. FSCV provides good chemical selectivity as well as sub-second temporal resolution (Robinson et al. 2003). However, it is not able to determine basal concentrations or to detect DA changes for periods longer than 90 s (Heien et al. 2005). In contrast to FSCV, microdialysis sampling with HPLC can estimate basal concentrations and detect both DA and amino acid changes over longer times, but it requires longer sampling times, usually 5–20 min per sample. Each sample represents an average concentration during this period and so precludes detection of more rapid changes of extracellular chemicals associated with neurotransmission. A newer method, capillary electrophoresis (CE), possesses a high mass sensitivity (Zhou et al. 1995; Robert et al. 1998; Shou et al. 2006) and so requires only a very low volume of sample (≈20 nL), decreasing sampling time to 1 s. Coupled with an on-line separation time of about 14 s, this brings the sampling frequency within the range to allow monitoring of rapid changes that may correlate with behavioral episodes. Ethanol in water is not extremely palatable, and many protocols in rodents require some form of deprivation and/or sucrose fading to establish high levels of intake. However, these are quite unlike the way that humans start to use ethanol. To better emulate this, we developed an ethanol-containing gelatin (10% ethanol, 10% Polycose®, 0.25% gelatin, wt/vol) that induces reliable and robust self-administration without food or water restriction (Rowland et al. 2005; Peris et al. 2007). Voluntary consumption of this ‘jello shot’ results in pharmacologically relevant concentrations of ethanol in brain (Peris et al. 2007) comparable to those previously reported after similar amounts of voluntary ethanol drinking (Nurmi et al. 1999). In the present study, we used CE coupled with laser-induced fluorescence detection (CE-LIF), to provide for the first time, almost real time measurement of amino acid changes in microdialysate during operant responding for ethanol gel. This approach allows exploration of neural mechanisms underlying ethanol self-administration as well as those involved in positive reinforcement and anticipation of reward.