In vivo voltammetric monitoring of norepinephrine release in the rat ventral bed nucleus of the stria terminalis and anteroventral thalamic nucleus

Abstract

The role and contribution of the dense noradrenergic innervation in the ventral bed nucleus of the stria terminalis (vBNST) and anteroventral thalamic nucleus (AV) to biological function and animal behaviors is poorly understood due to the small size of these nuclei. The aim of this study was to compare norepinephrine release and uptake in the vBNST with that in the AV of anesthetized rats. Measurements were made in vivo with fast-scan cyclic voltammetry following electrical stimulation of noradrenergic projection pathways, either the dorsal noradrenergic bundle (DNB) or the ventral noradrenergic bundle (VNB). The substance detected was identified as norepinephrine based upon voltammetric, anatomical, neurochemical and pharmacological evidence. Fast-scan cyclic voltammetry enables the selective monitoring of local norepinephrine overflow in the vBNST evoked by the stimulation of either the DNB or the VNB while norepinephrine in the AV was only evoked by DNB stimulation. The alpha2-adrenoceptor antagonist yohimbine and the norepinephrine uptake inhibitor desipramine increased norepinephrine overflow and slowed its disappearance in both regions. However, control of extracellular norepinephrine by both autoreceptors and uptake was greater in the AV. The greater control exerted by autoreceptors and uptake in the AV resulted in reduced extracellular concentration compared with the v BNST when large numbers of stimulation pulses were employed. The differences in noradrenergic transmission observed in the terminal fields of the v BNST and the AV may differentially regulate activity in these two regions that both contain high densities of norepinephrine terminals.

Figure 1

Maps of maximal stimulated release of catecholamines measured in the AV and the vBNST as a function of depth of the stimulating electrode. A. The dotted lines show the approximate track of each of the two poles of the stimulating electrode. They are superimposed on a coronal section containing the DNB and into the VTA/SN and VNB. Abbreviations used: DNB, dorsal noradrenergic bundle; VNB, ventral noradrenergic bundle; VTA, ventral tegmental area; SN, substantia nigra. The coronal section is from the atlas of Paxinos and Watson (2007). B. Maximal stimulated release during electrical stimulations (60 Hz, 60 pulses) measured in the vBNST (—) and the AV (---) as the stimulating electrode was lowered in small increments through the regions shown in (A). The relative response is the response at a particular depth (I_d^x) divided by the maximum response (I_d^max). Note that the distance scale is different in panels A and B. C. Representative individual responses at the peak current for catecholamines in the BNST (left) and the AV (right) measured at the depth indicated in mm. The bars under the current traces denote the period of electrical stimulation. Insets: Background-subtracted cyclic voltammograms measured during the evoked responses. The concentration bars are based on postcalibration in norepinephrine solutions.

Figure 2

Background-subtracted cyclic voltammograms measured in the BNST, the AV and the CPu at the end of DNB or VTA/SN and VNB stimulations (60 Hz, 60 pulses). In the upper trace, cyclic voltammograms are also shown for authentic norepinephrine (⋯) and dopamine (---, both at 4 μM) in a Tris buffer solution at a pH 7.4 recorded after in vivo measurement.

Figure 3

Maps of stimulated norepinephrine release within the AV and BNST with stimulation of the VNB and DNB. A. The dotted line in the coronal section (AP 0.0 mm from bregma) illustrate the approximate path of the carbon-fiber microelectrodes in the BNST aimed at the dorsal (d, hatched area above the ACA) and ventral (v, filled region below the ACA) BNST. The right part of the figure shows maximal evoked responses detected in the cyclic voltammograms following electrical stimulation (60 Hz, 60 pulses) of the VNB (⋯) or the DNB (—). Data are normalized as in Figure 1. B. The dotted line in the coronal section (AP -2.2 mm from bregma) shows the approximate path of the carbon-fiber microelectrodes in the AV (filled region). The right part of the figure shows maximal evoked responses from the cyclic voltammograms recorded after electrical stimulation (60 Hz, 60 pulses) of the VNB (⋯) or the DNB (—). Data normalized as in Figure 1. The coronal sections were taken from the atlas of Paxinos and Watson (2007). Abbreviations used: ACA, anterior commissure; CPu, caudate-putamen; LV, lateral ventricle; VTN, ventral thalamic nucleus.

Figure 4

Effect of yohimbine (Yo, 5 mg/kg), desipramine (DMI, 15 mg/kg), raclopride (Ra, 2 mg/kg) and GBR 12909 (GBR, 15 mg/kg) on catecholamine overflow in the AV and the CPu. A. Recordings of extracellular catecholamine in the AV (left) evoked by DNB stimulation with 60 pulses and in the CPu (right) evoked by VTA/SN stimulation with 24 pulses in the absence and presence of Yo (---) and Yo + DMI (⋯) at 60 Hz. B. Recordings of extracellular catecholamine in the AV (left) evoked by the DNB stimulation with 60 pulses and in the CPu (right) evoked by VTA/SN stimulation with 24 pulses in the absence and presence of Ra (---) and Ra + GBR (⋯) at 60 Hz. The bars under the current traces indicate the period of electrical stimulation.

Figure 5

Effect of clonidine (Cl, 0.2 mg/kg), yohimbine (Yo, 5 mg/kg), desipramine (DMI, 15 mg/kg), raclopride (Ra, 2 mg/kg) and GBR 12909 (GBR, 15 mg/kg) on catecholamine overflow in the vBNST. Representative recordings of extracellular catecholamine in the vBNST evoked by DNB stimulation (left) or VNB stimulation (right) in the absence and presence of (A) Cl (---) and Cl + Yo (⋯), (B) Yo (---) and Yo + DMI (⋯) and (C) Ra (---) and Ra + GBR 12909 (⋯) at 60 Hz with 60 pulses. The bars under the current traces indicate the period of electrical stimulation.

Figure 6

Effect of lidocaine on catecholamine response in the vBNST after drug study (Yo + DMI). Catecholamine response in the vBNST before (left), 5 min after (middle) and 30 min after (right) lidocaine (350 nmol/min) infused into the sites of the electrical stimulation (DNB, VNB, LC and NTS – one site per animal). An infusion cannula was combined with the bipolar stimulating electrodes. Abbreviations used: DNB, dorsal noradrenergic bundle; VNB, ventral noradrenergic bundle; LC, locus coeruleus; NTS, nucleus tractus solitarius. The bars under the current traces indicate the period of electrical stimulation.

Figure 7

Effect of frequency and pulse number (stimulus duration) on catecholamine overflow. A. Individual catecholamine overflows in the AV (left) evoked by DNB stimulation and in the vBNST evoked by DNB and VNB stimulation (right) for pulse frequency (10, 20, 40 and 60 Hz) with 60 pulses. B. Effect of pulse number (10, 20, 40, 80 and 120 for vBNST) on catecholamine in the AV (left) and in the vBNST (right) is shown. Initiation and termination of stimuli were indicated by the boxes. C. Maximal catecholamine responses in the AV and the vBNST as a function of stimulation frequency. D. Maximal catecholamine responses in the AV and vBNST as a function of pulse number. Relative responses in C and D are the monitored response (I^x) divided by the maximum response (I^max).