In vivo voltammetry monitoring of electrically evoked extracellular norepinephrine in subregions of the bed nucleus of the stria terminalis

Abstract

Norepinephrine (NE) is an easily oxidized neurotransmitter that is found throughout the brain. Considerable evidence suggests that it plays an important role in neurocircuitry related to fear and anxiety responses. In certain subregions of the bed nucleus of the stria terminalis (BNST), NE is found in large amounts. In this work we probed differences in electrically evoked release of NE and its regulation by the norepinephrine transporter (NET) and the α(2)-adrenergic autoreceptor (α(2)-AR) in two regions of the BNST of anesthetized rats. NE was monitored in the dorsomedial BNST (dmBNST) and ventral BNST (vBNST) by fast-scan cyclic voltammetry at carbon fiber microelectrodes. Pharmacological agents were introduced either by systemic application (intraperitoneal injection) or by local application (iontophoresis). The iontophoresis barrels were attached to a carbon fiber microelectrode to allow simultaneous detection of evoked NE release and quantitation of iontophoretic delivery. Desipramine (DMI), an inhibitor of NET, increased evoked release and slowed clearance of released NE in both regions independent of the mode of delivery. However, the effects of DMI were more robust in the vBNST than in the dmBNST. Similarly, the α(2)-AR autoreceptor inhibitor idazoxan (IDA) enhanced NE release in both regions but to a greater extent in the vBNST by both modes of delivery. Since both local application by iontophoresis and systemic application of IDA had similar effects on NE release, our results indicate that terminal autoreceptors play a predominant role in the inhibition of subsequent release.

Fig. 1.

Anatomical mapping of catecholamine release sites in the dorsomedial (dm) and ventral (v) bed nucleus of the stria terminalis (BNST). A, left: anatomical positioning of electrode in BNST (dashed line represents target electrode tract). Right: lesion made with carbon fiber microelectrodes provides histological evidence that the electrode was positioned in the correct dorsal-ventral axis to target the dmBNST and vBNST. AP, anterior-posterior; ML, medial-lateral; CPu, caudate putamen; VP, ventral pallidum; PA, preoptic areas. B: the BNST is divided into dorsal and ventral regions by the anterior commissure (AC). Mapping of release on the dorsal-ventral axis shows that the most robust signal is observed ventral to the commissure (vBNST), although norepinephrine (NE) release is also observed in the dmBNST. Inset: representative cyclic voltammogram that confirms the increase in current observed is catecholaminergic in nature. C: representative time course of electrically evoked NE release in the dmBNST and vBNST. Maximal evoked NE concentration ([NE]_max) is indicated by vertical dashed lines for each region. Horizontal red bar under current traces in B and C indicates the time and duration of electrical stimulation.

Fig. 2.

Effects on electrically evoked NE release in the dmBNST and vBNST after intraperitoneal (ip) injection of idazoxan (IDA, 5 mg/kg), desipramine (DMI, 15 mg/kg), raclopride (RA, 2 mg/kg), and GBR 12909 (GBR, 15 mg/kg). A: effect on [NE]_max. B: effect on clearance half-life (t_1/2). *Significantly different from predrug (P < 0.05); #significantly different from IDA (P < 0.05); $significantly different from vBNST (P < 0.05); &values adopted from Park et al. (2009).

Fig. 3.

The electrophoretic mobilities of the electroactive compounds uric acid, acetaminophen (AP), and dopamine (DA) (■) are positively correlated to previously reported iontophoretic rates (Herr et al. 2008). The linear regression from this correlation was used to determine iontophoretic rates for DMI or IDA (□) relative to AP based on electrophoretic mobilities (μ_ep) calculated by capillary electrophoresis. EOF, electroosmotic flow.

Fig. 4.

Stimulated NE release before and after a localized ejection of AP and IDA. Shown is the current as a function of time at the oxidation potential for NE. A: representative baseline current trace for the stimulated release of NE. Boxes indicate the beginning and end of stimulation. B: representation of iontophoretic ejection of AP and IDA. The measured signal is due solely to AP and is used to estimate the concentration of IDA. Here, 5 μM AP is the average concentration across the electrode and is equivalent to 12 μM IDA. C: current trace for stimulated release 120 s after ejection seen in B. At the time of stimulation (open box), the concentration of AP has decreased to 2% of its original value, corresponding to a decrease in IDA concentration to 240 nM. The extracellular concentration of NE seen in C is significantly increased from that observed predrug.

Fig. 5.

Effects on electrically evoked NE in the dmBNST and vBNST after iontophoretic delivery of RA, IDA, and DMI. A: effect on [NE]_max. B: effect on t_1/2. *Significantly different from predrug (P < 0.05); $significantly different from vBNST (P < 0.05).

Fig. 6.

Time course of drug effect onset due to systemic (ip, A) and iontophoretic (B) delivery of NE drugs DMI and IDA. Drugs were delivered either by ip injection or iontophoretically at t = 0. Since washout of drugs is not possible for systemic delivery, IDA and DMI were evaluated in separate animals. In contrast, iontophoretic drug effects are short-lived; thus administration of DMI followed administration of IDA after evoked NE release returned to its predrug value.