Elsevier

Neuroscience

Volume 169, Issue 1, 11 August 2010, Pages 132-142
Neuroscience

Cognitive, Behavioral, and Systems Neuroscience
Research Paper
In vivo voltammetric monitoring of catecholamine release in subterritories of the nucleus accumbens shell

https://doi.org/10.1016/j.neuroscience.2010.04.076Get rights and content
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Abstract

Fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes has been used to demonstrate that sub-second changes in catecholamine concentration occur within the nucleus accumbens (NAc) shell during motivated behaviors, and these fluctuations have been attributed to rapid dopamine signaling. However, FSCV cannot distinguish between dopamine and norepinephrine, and caudal regions of the NAc shell receive noradrenergic projections. Therefore, in the present study, we examined the degree to which norepinephrine contributes to catecholamine release within rostral and caudal portion of NAc shell. Analysis of tissue content revealed that dopamine was the major catecholamine detectable in the rostral NAc shell, whereas both dopamine and norepinephrine were found in the caudal subregion. To examine releasable catecholamines, electrical stimulation was used to evoke release in anesthetized rats with either stimulation of the medial forebrain bundle, a pathway containing both dopaminergic and noradrenergic projections to the NAc, or the ventral tegmental area/substantia nigra, the origin of dopaminergic projections. The catecholamines were distinguished by their responses to different pharmacological agents. The dopamine autoreceptor blocker, raclopride, as well as the monoamine and dopamine transporter blockers, cocaine and GBR 12909, increased evoked catecholamine overflow in both the rostral and caudal NAc shell. The norepinephrine autoreceptor blocker, yohimbine, and the norepinephrine transporter blocker, desipramine, increased catecholamine overflow in the caudal NAc shell without significant alteration of evoked responses in the rostral NAc shell. Thus, the neurochemical and pharmacological results show that norepinephrine signaling is restricted to caudal portions of the NAc shell. Following raclopride and cocaine or raclopride and GBR 12909, robust catecholamine transients were observed within the rostral shell but these were far less apparent in the caudal NAc shell, and they did not occur following yohimbine and desipramine. Taken together, the data demonstrate that catecholamine signals in the rostral NAc shell detected by FSCV are due to change in dopamine transmission.

Key words

norepinephrine
dopamine
rostral and caudal nucleus accumbens shell
carbon-fiber microelectrode
medial forebrain bundle
ventral tegmental area/substantia nigra

Abbreviations

CPu
caudate-putamen
DAT
dopamine transporter
DNB
dorsal noradrenergic bundle
FSCV
fast-scan cyclic voltammetry
HPLC
high performance liquid chromatography
MFB
medial forebrain bundle
NAc
nucleus accumbens
NET
norepinephrine transporter
VNB
ventral noradrenergic bundle
VTA/SN
ventral tegmental area/substantia nigra
Dopamine transmission within the nucleus accumbens (NAc) shell, a small region of the basal forebrain, is involved in information processing that mediates reward. Because of the role of dopamine in addiction and motivated behavior (Wise, 2002, Di Chiara, 1995, Aragona et al., 2008, Owesson-White et al., 2008), there have been extensive studies of dopamine transmission within this region. In our laboratories, we have used fast-scan cyclic voltammetry (FSCV) with a carbon-fiber microelectrode to correlate real-time dopamine transmission in the rostral NAc shell during associative learning and intake of drugs of abuse (Owesson-White et al., 2008, Phillips et al., 2003, Aragona et al., 2009). Dopamine was identified by the similarity of the cyclic voltammogram recorded in vivo with in vitro calibrations for dopamine. The cyclic voltammogram for dopamine is distinct from all neurochemicals that have been tested except norepinephrine (Baur et al., 1988, Heien et al., 2003). However, the cyclic voltammograms for dopamine and norepinephrine are virtually identical (Heien et al., 2004, Park et al., 2009), and thus independent confirmation of the specific catecholamine detected is required.
The dense dopaminergic innervation of the NAc shell is primarily from the cell bodies that originated in the ventral tegmental area (VTA). Anatomical studies have revealed that the NAc shell also receives a noradrenergic input to caudal portions of this structure (Berridge et al., 1997, Schroeter et al., 2000, Delfs et al., 1998). These noradrenergic inputs originate primarily from the A2 cell group in the nucleus tractus solitarius (NTS) (Delfs et al., 1998) and A6 cell group in the locus coeruleus (Berridge et al., 1997). The neurons from the NTS project through the ventral noradrenergic bundle (VNB) and the medial forebrain bundle (MFB) to the NAc shell. The neurons from the locus coeruleus project through the dorsal noradrenergic bundle (DNB) and the MFB to the NAc shell. Importantly, both noradrenergic projections to the NAc primarily terminate within the caudal NAc shell. Immunohisotchemical studies suggest that the rostral NAc shell receives little noradrenergic innervation (Berridge et al., 1997, Schroeter et al., 2000, Delfs et al., 1998), however, interpretation of cyclic voltammetric data with respect to the specific catecholamine detected requires that the dopamine/norepinephrine distribution is understood in more detail.
In the current study, we characterized catecholamine (dopamine and norepinephrine) content and overflow in both the medial rostral and caudal regions of the NAc shell. Because there is no delineating feature that defines the division of the medial rostral shell from the medial caudal shell, a variety of stereotaxic coordinates have been used by various groups (Heidbreder and Feldon, 1998, Hajnal and Norgren, 2004). We defined the medial rostral shell as the region of the NAc that extends from 1.45 to 1.90 mm anterior of bregma. The medial caudal NAc shell is defined here as the region from 0.70 to 1.20 mm anterior of bregma. Catecholamine release in anesthetized rats was evoked by electrical stimulation of the MFB, a fiber bundle that contains the majority of both the noradrenergic and dopaminergic fibers that project to the NAc shell. The other region stimulated was the VTA/substantia nigra (VTA/SN), a region that contains dopaminergic neurons and through which passes a portion of the VNB (Park et al., 2009, Ungerstedt, 1971). The combined anatomical, neurochemical, and pharmacological characterization show that the major catecholamine released in the rostral NAC shell is dopamine whereas a mixture of dopamine and norepinephrine is released in the caudal NAc shell.

Experimental methods

Animals

Adult male Sprague–Dawley rats (320–400 g) were purchased from Charles Rivers (Wilmington, MA, USA) and housed in temperature and humidity controlled rooms with ad libitum food and water with a 12/12 h light/dark cycle. All procedures for handling and caring for the laboratory animals were in accordance with the NIH Guide for Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of North Carolina at Chapel Hill.

Surgery

Rats were anesthetized with urethane (1.5 g/kg) and immobilized in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA). Temperature was maintained at 37 °C with a heating pad (Harvard Apparatus, Holliston, MA, USA). Small holes on the skull were drilled for reference (Ag/AgCl) and stimulating electrodes as well as the carbon-fiber microelectrodes. Anteroposterior (AP), mediolateral (ML) and dorsoventral (DV) positions were referenced from bregma. The dura mater was punctured, carefully removed, and a single carbon-fiber microelectrode was lowered into the rostral (AP +1.7 mm, ML +0.8 mm, DV 6.0–8.0 mm) or caudal (AP +1.0 mm, ML +0.9 mm, DV 6.0–8.0 mm) NAc shell, coordinates taken from an atlas of (Paxinos and Watson, 2007). An Ag/AgCl reference electrode was placed in the contralateral cortex, and it was secured with dental cement. Electrical stimulation was accomplished with a bipolar, stainless-steel electrode (0.2 mm in diameter, Plastics One, Roanoke, VA, USA) insulated to the tip. The tips of the bipolar electrode were separated by ∼1.0 mm. It was placed into the MFB (AP −4.0 mm, ML +1.4 mm, DV 8.0–9.0 mm) or the VTA/SN (AP −5.2 mm, ML +1.0 mm, DV 8.0–9.0 mm).

Electrical stimulation

Computer-generated stimulus trains, (NL 800A, Neurolog, Medical Systems Corp., Great Neck, NY, USA), were optically isolated from the electrochemical system. The trains consisted of biphasic pulses (300 μA, 2 ms each phase) with stimulation frequencies between 10 and 60 Hz applied through the bipolar electrode. Unless noted otherwise, the number of stimulus pulses was 24. The time interval between electrical stimulations was 4 or 5 min to ensure reproducible catecholamine concentrations were released with each stimulation (Park et al., 2009).

Voltammetric procedures

Glass-encased, cylindrical carbon-fiber microelectrodes and Ag/AgCl reference electrodes were prepared as described previously (Cahill et al., 1996). T-650 carbon fibers (5.1 μm in nominal diameter, Thornel, Amoco Corporation, Greenville, SC, USA) with an exposed length of 75–100 μm were used. Fast-scan cyclic voltammetry was computer-controlled and has been described in detail previously (Heien et al., 2003). A triangular scan (−0.4 to +1.3 V, 400 V/s) was repeated every 100 ms. Data was digitized and stored on a computer using software written in LABVIEW (National Instruments). Background-subtracted cyclic voltammograms were obtained by digitally subtracting voltammograms collected during stimulation from those collected during baseline recording. Voltammetric responses were viewed as color plots with the abscissa as voltage, the ordinate as acquisition time, and the current encoded in color (Michael et al., 1998). Temporal responses were determined by monitoring the current at the peak oxidation potential for dopamine in successive voltammograms or by principal component regression (Heien et al., 2005) as noted in the text. The current was converted to concentration based on calibration curves obtained after the in vivo experiment with known concentrations of dopamine in vitro.

Histology

At the end of experiments, electrode placements were verified by stereotaxically lowering a tungsten electrode to the original position of the carbon-fiber microelectrode or directly with the carbon-fiber microelectrodes as described earlier (Garris and Wightman, 1994b, Park et al., 2009). When the carbon-fiber microelectrode (27 of 57 electrodes used in this study) was used to lesion the brain, this precluded post calibration of the electrode sensitivity and instead, we used a post calibration factor of 6.9±0.3 pA/(μM·μm2).
A lesion was made at the recording site by applying constant current (20 μA for 10 s) to the tungsten or carbon-fiber electrodes. Brains were removed from the skull and stored in 10% formaldehyde for at least 3 days, and coronally sectioned into 40–50 μm thick slices with a cryostat. The sections mounted on slides were stained with 0.2% thionin, and coverslipped before viewing under a light microscope.

High performance liquid chromatography (HPLC)

HPLC analysis of tissue content of catecholamines followed previously published procedures (Park et al., 2009). Briefly, the brain of an anesthetized rat was rapidly removed and placed on ice. Coronal brain slices (500 μm thick) were prepared in ice cold artificial cerebral spinal fluid (aCSF) using a Lancer Vibratome (World Precision Instruments, Sarasota, FL, USA.). The slice containing the rostral NAc shell was adjacent to the slice containing the caudal NAc shell. The dorsal shell region in these slices was dissected on a dry stainless steel surface while taking care to avoid inclusion of the adjacent NAc core and ventral pallidum. The resultant tissue was wet weighed in a pre-tared volume of 200 μL 0.1 N HClO4 containing 1 μM hydroquinone (HQ) and homogenized with a sonic dismembrator (Fisher Sci., model 60, Pittsburgh, PA, USA). The sample was then centrifuged at 6000 g for 10 min and the supernatant was removed and filtered using a 0.2-μm syringe microfilter (Millex-LG, Millipore, Bedford, MA, USA). Injections (10 μL) were made onto a reverse phase column (C-18, 5 μm, 4.6×250 mm, Waters symmetry 300, Waters Corporation, Milford, MA, USA). The mobile phase (prepared in HPLC grade water) contained 0.1 M citric acid, 1 mM hexyl sodium sulfate, 0.1 mm EDTA, and 10% methanol (pH 3.5) at a flow rate of 1 mL/min. Catecholamines were detected with a thin-layer radial electrochemical cell (BASi, West Lafayette, IN, USA) at 700 mV versus a Ag/AgCl reference electrode. Catecholamine standards were prepared from 10 mM stock solutions in 0.1 N HClO4. A Labview stripchart recorder program (Jorgenson Laboratory, UNC-CH) was used for data collection through home built electronic equipment. The ratio of the peak area of the analyte to that of the internal standard (HQ) was used to calculate the amount of catecholamine in the original tissue sample.

Drugs and reagents

All chemicals and drugs were reagent-quality and were used without additional purification. Drugs were obtained from Sigma-Aldrich (St. Louis, MO, USA). In vitro dopamine post calibration of carbon-fiber microelectrodes was done in a Tris buffer solution at pH 7.4 containing 15 mM Tris, 140 mM NaCl, 3.25 mM KCl, 1.2 mM CaCl2, 1.25 mM NaH2PO4, 1.2 mM MgCl2, and 2.0 mM Na2SO4 in double distilled water (Mega Pure System, Corning Glasswork, Corning, NY, USA). Desipramine-HCl, cocaine-HCl, raclopride-HCl, and yohimbine-HCl were dissolved in saline. GBR 12909-HCl was dissolved in a small volume of distilled water and diluted with saline. Injected volumes were 0.6 ml/kg and were given i.p.

Data analysis

Clampfit 8.1 as part of pCLAMP 8.1 software package (Axon Instruments, Foster City, CA, USA) was used to analyze time values such as tmax. and t1/2 and signal amplitude values according to procedures described in the literature (Park et al., 2007). tmax. is the time to reach the maximum concentration and t1/2, was taken as the time to descend from its maximum value to half of that value. [CA]max is the maximal evoked catecholamine concentration. Catecholamine transients were identified by principal component regression algorithm written into the Tar Heel CV software as descried earlier (Heien et al., 2004, Keithley et al., 2009). Catecholamine transients were defined as anything greater than five times the root-mean square noise level and were analyzed for frequency, amplitude, and duration using Mini Analysis Software (Synaptosoft, Decatur, GA, USA). Data are represented as mean±SEM and “n” values indicating the number of rats. Statistical significance of changes in [CA]max and t1/2-values before and after drugs was determined using a 2×2×2 repeated measures ANOVA followed by a t-test post hoc analysis with a Bonferroni correction for multiple comparison for brain region (rostral and caudal shell), drugs (yohimbine and yohimbine + desipramine, raclopride and raclopride + cocaine or raclopride + cocaine and raclopride+GBR 12909) and stimulation region (MFB and VTA/SN) as the three variables. Statistical significance of [CA]max, t1/2, and tmax.-values was determined using a 2×2 repeated ANOVA followed by a t-test post hoc analysis with a Bonferroni correction for multiple comparison for brain region (rostral and caudal shell) and stimulation region (MFB and VTA/SN) as the two variables. Statistical analysis employed GraphPad Software version 4.0 (San Diego, CA, USA) or SPSS 14.0 software (SPPS, Chicago, IL, USA). Differences were considered significant when the P-value was <0.05.

Results

Catecholamine content of the rostral and caudal NAc shell

The rostral and caudal NAc shell were dissected from fresh brains and their catecholamine content determined by HPLC (Table 1). The dopamine tissue content in the rostral and caudal NAc regions was not significantly different (t=0.99, df =25, P=0.33). Dopamine was a major catecholamine in the rostral NAc shell. Only a very low amount of norepinephrine was found (norepinephrine was present in 1 of 15 samples from five rats), but both dopamine and norepinephrine were present in significant amounts in the caudal NAc shell. The ratio of norepinephrine to dopamine content for the caudal NAc shell was ∼1 to 3. Serotonin was not detected in either subregion.

Table 1. Monoamine content in the NAc shell

NAc shellRostral (μg/g, n=15 samples)Caudal (μg/g, n=12 samples)
Norepinephrine0.04±0.041.14±0.55⁎
Dopamine3.99±0.733.09±0.57
Values represent the mean±SEM.
⁎
Significantly different from norepinephrine in the caudal NAc shell (P<0.05, unpaired t-test).

Electrically stimulated catecholamine release in the rostral NAc shell

Catecholamine release was measured by FSCV in the rostral NAc shell during electrical stimulation (60 Hz, 24 pulses, 300 μA) of either the VTA/SN or the MFB. When the stimulating electrode was above or below these stimulation sites, catecholamine release in the NAc shell was not observed. Representative examples of the time courses are shown in Fig. 1A. With either location of the stimulating electrode, catecholamine release began immediately upon stimulus initiation, reached a similar maximum, and the concentration returned to its initial value after the stimulation. Cyclic voltammograms recorded at the maximum were identical to those for catecholamines recorded after in vivo use (Fig. 1A, right panel), with the maximum oxidation current observed at ∼+0.65 V and the peak for the reduction of the electrochemically formed quinone at ∼−0.2 V on the reverse scan. Shown in the right panel of Fig. 1B is a photograph of a brain slice used for histological verification of the location of the carbon-fiber microelectrode. The black arrow points to the location of the electrode tip when the lesion was made. The lesion sites for all from carbon-fiber electrodes in the rostral NAc shell are shown in are shown in Suppl. Fig. 1, left panel.
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Fig. 1. Electrically evoked catecholamine responses measured in the rostral NAc shell. (A) Representative maximal catecholamine responses in the rostral NAc shell were evoked by MFB (solid line) or VTA/SN (dotted line) stimulation (60 Hz, 24 pulses). The bars under the current traces denote the period of electrical stimulation. Insets: background-subtracted cyclic voltammograms measured during the evoked responses. (B) Solid line in the schematic diagram illustrate the approximate path of the carbon-fiber microelectrodes through the rostral NAc shell (left). The location of the carbon-fiber microelectrode tip in the rostral NAc shell (right) was visualized by the electrolytic lesion (black arrow). The boundaries of the NAc shell and core (right) are indicated by the dotted white line. The coronal section is adapted from the atlas of Paxinos and Watson (2007). (C) Maximal stimulated release during electrical stimulation measured in the rostral NAc shell as the carbon-fiber microelectrode was lowered in small increments through the regions shown in (B). The relative response is the concentration at a particular depth (Cdx) divided by the maximum concentration (Cdmax). The catecholamine response in the rostral NAc shell was evoked by MFB or VTA/SN stimulation (60 Hz, 24 pulses). Abbreviations used: CPu, caudate-putamen.

To characterize the distribution of catecholamine release sites in the rostral NAc shell, release evoked by 60 Hz stimulation was measured at different depths with fast-scan cyclic voltammetry with stimulating electrodes placed either in the MFB or VTA/SN. Fig. 1B (left panel) shows the coronal plane in which measurements were made (AP +1.8 mm) and the approximate electrode track is shown by the solid line. The carbon-fiber microelectrode was lowered within the rostral NAc shell in 200–300 μm increments beginning at 3.3 mm from the skull, with release measured at each location. A plot of [CA]max normalized to the maximum value observed along the track is shown in Fig. 1C. Robust release was not observed until a depth of 5.3 mm below the skull. Release reached a maximum at a depth of 6.5 mm and decreased to negligible values at 8.0 mm. Responses from misplaced electrodes (determined via histology to be located in the caudate-putamen (CPu) near the ventricle or in the NAc core) were discarded.

Electrically stimulated catecholamine release in the caudal NAc shell

Identical experiments were conducted in the caudal NAc shell. Electrical stimulation of the MFB (example in Fig. 2A) resulted in catecholamine release as identified by the cyclic voltammograms (Fig. 2A, right panel). The catecholamine concentration remained elevated for a longer time than in the rostral NAc shell. When release in the caudal NAc shell was evoked with VTA/SN stimulation, its amplitude was about one third of that evoked by MFB stimulation. A histological record of one carbon-fiber microelectrode placement in the caudal NAc shell (AP+1.0 mm) is shown in Fig. 2B (right panel, black arrow indicates the lesion site).
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Fig. 2. Electrically evoked catecholamine responses measured in the caudal NAc shell. (A) Representative maximal catecholamine responses at the peak current for catecholamines in the caudal NAc shell. The bars under the current traces denote the period of electrical stimulation (60 Hz, 24 pulses). Insets: background-subtracted cyclic voltammograms measured during the evoked responses. (B) Solid line in the schematic diagram illustrate the approximate path of the carbon-fiber microelectrodes in the caudal NAc shell (left). The boundaries of the NAc shell and core (right) are indicated by the dotted white line. The placement of the carbon-fiber microelectrode tip in the caudal NAc shell (right) was visualized by the electrolytic lesion (black arrow). The coronal section is adapted from the atlas of Paxinos and Watson (2007). (C) Maximal stimulated release during electrical stimulation measured in the caudal NAc shell as the carbon-fiber microelectrode was lowered in small increments through the regions shown in B. The relative response is the response at particular depth (Cdx) divided by the maximum concentration (Cdmax). The catecholamine response in the caudal NAc shell was evoked by MFB or VTA/SN stimulation (60 Hz, 24 pulses). Abbreviations used: CPu, caudate-putamen.

The vertical distribution of release sites in this region was also characterized with both MFB and VTA/SN stimulation. The approximate vertical track is shown in Fig. 2B (left panel). The measured [CA]max, normalized to the maximum value observed along the track, is shown in Fig. 2C. With MFB stimulation, release began to become apparent at ∼5.5 mm below the skull. Maximal release was observed between 6.2 and 6.8 mm and then it decreased. With VTA/SN stimulation, release was much lower and reached a maximum at about 6.4 mm below the skull.

Comparison of catecholamine overflow in the rostral and caudal NAc shell

Catecholamine release and its time course in the rostral and caudal NAc shell was quantitated and compared in the sites where maximal release was observed (Table 2). Evoked [CA]max in the rostral NAc shell was statistically identical with stimulation of the MFB or VTA/SN. The time to reach the maximum concentration (tmax.) was ∼0.6 s in the rostral NAc shell with stimulation in either location, 0.2 s longer than the duration of the 60 Hz stimulation (0.4 s). The response time of the electrode is ∼0.2 s, accounting for the delay (Owesson-White et al., 2008). Following the stimulation, the time to return to half of the maximal value (t1/2) in the rostral NAc shell was approximately the same with stimulation in each region. Since the descending part of the curves is due to neuronal uptake (Giros et al., 1996), the similar clearance time suggests that the same transporter is responsible for the removal of catecholamine in this region.

Table 2. Parameters measured during the electrically evoked (300 μA, 60 Hz, 24 pulses) catecholamine responses in the rostral and caudal NAc shell

(n≥8) stimulationCA in rostral shellCA in caudal shell
MFBVTA/SNMFBVTA/SN
[CA]max μM2.00±0.26⁎1.78±0.28⁎0.65±0.060.18±0.03
tmax. (s)0.65±0.02⁎0.60±0.02⁎0.83±0.070.97±0.09
t1/2 (s)1.12±0.05⁎1.01±0.04⁎1.84±0.241.52±0.17
[CA]max is the maximal evoked catecholamine concentration; tmax is the time to reach to the peak response from the start of the stimulation; t1/2 is the time required for catecholamine overflow to decay to 50% of the maximum. Values represent the mean±SEM.
⁎
(P<0.05, t-test with Bonferroni correction) indicates significantly different from caudal NAc shell.
[CA]max in the caudal NAc shell was significantly lower than that in the rostral NAc shell (F1,38=1459, P<0.0001). In the caudal NAc shell, [CA]max was approximately three times greater when evoked in the MFB than when evoked in the VTA/SN (t=6.7, df =15, P<0.0001). The values of tmax. in the caudal NAc shell were statistically longer than those measured in the rostral NAc shell (F1,41=11.35, P<0.01). Similarly, the t1/2-values in the caudal NAc shell were statistically longer than the values measured in the rostral NAc shell (F1,41=12.27, P<0.005). The delayed tmax., coupled with the higher values of t1/2, indicates uptake is weaker in the caudal NAc shell allowing greater diffusional transport of catecholamines from their release site (Venton et al., 2003). Again, the similarity of the t1/2-values in the caudal NAc shell suggests the transporter responsible for clearance is independent of the stimulation site.

Influence of dopamine and norepinephrine drugs on extracellular catecholamine evoked by VTA/SN and MFB stimulation in rostral and caudal NAc shell

The tissue content measurements (Table 1) show that norepinephrine tissue content is very low within the rostral NAc shell, and thus the catecholamine responses in this region would be expected to be due to dopamine. However, those in the caudal NAc shell could be due to either dopamine, norepinephrine, or both. To provide evidence for the specific catecholamine(s) measured in each region, pharmacological agents were employed. For dopamine, the D2 receptor antagonist, raclopride (2 mg/kg), and the monoamine transporter blocker, cocaine (15 mg/kg), were used to enhance release and reduce uptake respectively. For norepinephrine, the norepinephrine α2-adrenoceptor antagonist, yohimbine (6 mg/kg), and the norepinephrine transporter blocker, desipramine (desmethylimipramine, 15 mg/kg), were used to enhance release and reduce uptake respectively. The doses selected were based on our prior work in the CPu, a dopamine rich region, and the ventral bed nucleus of the stria terminalis, a norepinephrine rich region (Park et al., 2009).
The responses in the rostral NAc shell evoked by MFB or VTA/SN stimulation (60 Hz, 24 pulses) were unchanged 30 min following administration of yohimbine and desipramine (representative example in Fig. 3A). Neither [CA]max nor t1/2, a measure of the clearance rate, were affected by the norepinephrine drugs in the rostral NAc shell (Table 3). In contrast, raclopride and cocaine increased the responses in the rostral NAc shell (representative example in Fig. 3B). Raclopride significantly increased both [CA]max and t1/2 (Table 3) and these increases were enhanced by cocaine. The increases were found irrespective of the stimulus location (MFB or VTA/SN). Thus, although evoked catecholamine in the rostral NAc shell is insensitive to drugs that alter norepinephrine neurotransmission, it is sensitive to drugs that act on dopaminergic terminals.
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Fig. 3. Effect of yohimbine (Yo, 5 mg/kg), desipramine (DMI, 15 mg/kg), raclopride (Ra, 2 mg/kg) and cocaine (Co, 15 mg/kg) on catecholamine overflow in the rostral NAc shell. Representative recordings of extracellular catecholamine in the rostral NAc shell evoked by MFB (left) or VTA/SN stimulation (right) in the presence of (A) Yo (---) and Yo+DMI (…), (B) Ra (---) and Ra+Co (…) at 60 Hz with 24 pulses. The bars under the current traces indicate the period of electrical stimulation.

Table 3. Effects of yohimbine (Yo, 6 mg/kg), Yo+desipramine (DMI, 15 mg/kg), raclopride (Ra, 2 mg/kg), Ra+cocaine (Co,15 mg/kg) and Ra+GBR 12909 (GBR, 15 mg/kg) on evoked catecholamine release

Drug (n≥4)Region stimulation(%) of control
Rostral NAc shellCaudal NAc shell
MFBVTA/SNMFBVTA/SN
Yo[CA]max94.5±4.8100±5.3121±3.3⁎110±4.4
t1/295.6±6.5101±5.6109±4.0108±7.2
Yo+DMI[CA]max93.3±5.487.8±8.7159±9.7⁎#&135±4.7⁎#&
t1/292.4±5.9101±9.2147±17⁎#&138±9.1⁎#&
Ra[CA]max167±17⁎185±24⁎140.6±8.8⁎159±13⁎
t1/2120±4.2⁎155±7.6⁎156.4±13.6⁎159±27⁎
Ra+Co[CA]max261±28⁎#457±130⁎#227±15⁎#406±58⁎#
t1/2215±12⁎#306±22⁎#569±101⁎#497±64⁎#&
Ra+GBR[CA]max309±41⁎#––202±17⁎#—
t1/2378±72⁎#—338±122⁎#—
Data are mean±SEM and were obtained during 60 Hz stimulations (24 pulses). The maximal catecholamine concentration, [CA]max, and the time required for catecholamine overflow decay to 50% of the maximum, t1/2, are shown.
⁎
Indicates significantly different from control values (P<0.05, t-test with Bonferroni correction).
#
Significantly different versus a single drug (yohimbine or raclopride) (P<0.05, t-test with Bonferroni correction).
&
Significantly different from rostral NAc shell values (P<0.05, t-test with Bonferroni correction).
In the caudal NAc shell, catecholamine signaling was altered by both dopaminergic and noradrenergic drugs in contrast to the rostral NAc shell. The response in the caudal NAc shell region evoked by the MFB stimulation showed a modest increase 30 min after injection of yohimbine (representative result in Fig. 4A, left, data from multiple animals summarized in Table 3). Desipramine administered after yohimbine, further increased the evoked response (representative example in Fig. 4A, left). Raclopride and cocaine also increased the responses in the caudal NAc shell evoked by either MFB or VTA/SN stimulation (representative example Fig. 4B, data from multiple animals summarized in Table 3). Following these dopaminergic drugs, both [CA]max and t1/2 were increased in the caudal NAc shell when evoked by either stimulation site (Table 3). With stimulation of the VTA/SN, the evoked catecholamine response in the caudal NAc shell was not changed following yohimbine alone, but did increase with the combination of desipramine and yohimbine (representative example in Fig. 4A, right, Table 3). This combination of noradrenergic drugs led to significant increases in [CA]max and t1/2 in the caudal NAc shell with both stimulation sites (Table 3) in contrast to the rostral NAc shell (F1,44=55.42, P<0.0001 for [CA]max, and F1,43=14.43, P<0.005 for t1/2). The combination of raclopride and cocaine increased t1/2 of the response evoked by either the MFB or by the VTA/SN stimulation more in the caudal NAc shell than in the rostral NAc shell (Fig. 4B, Table 3) (F1,42=10.40, P<0.01). Thus in the caudal NAc shell, catecholamine transmission is sensitive to both noradrenergic and dopaminergic drugs.
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Fig. 4. Effect of yohimbine (Yo, 5 mg/kg), desipramine (DMI, 15 mg/kg), raclopride (Ra, 2 mg/kg) and cocaine (Co, 15 mg/kg) on catecholamine overflow in the caudal NAc shell. Representative recordings of extracellular catecholamine in the rostral shell evoked by MFB stimulation (left) or VTA/SN (right) in the presence of (A) Yo (---) and Yo+DMI (…), (B) Ra (---) and Ra+Co (…) at 60 Hz with 24 pulses. The bars under the current traces indicate the period of electrical stimulation.

In this study, cocaine was used as a catecholamine uptake inhibitor because the effects of cocaine seeking and self-administration on dopamine neurotransmission were previously studied in the NAc (Phillips et al., 2003, Owesson-White et al., 2009). However, cocaine is not a selective dopamine transporter (DAT) inhibitor. Therefore, we also studied the effect of GBR 12909, a selective DAT inhibitor, on catecholamine release evoked by MFB stimulation in the rostral and caudal NAc shell. GBR 12909, administered after raclopride, further increased the response in the rostral and caudal NAc shell region evoked by the MFB stimulation (representative result in Suppl. Fig. 2, data from multiple animals summarized in Table 3). There were no significant differences in the increases of [CA]max and t1/2 by raclopride followed by GBR 12909 between rostral and caudal NAc shell. The effects of GBR 12909 on evoked dopamine occur more slowly than with cocaine (Espana et al., 2008, Budygin et al., 2000). Therefore, comparison of the GBR 12909 and cocaine effects after administration of raclopride can differ because of their different time courses of action.

Catecholamine transient release in the rostral and caudal NAc shell after administration of raclopride and cocaine

Fig. 5 shows catecholamine fluctuations 15 min before and after raclopride and cocaine in both the rostral and caudal NAc shell. The fluctuations before drug in the color plots represent the electrical noise of the system. Following administration of both raclopride (2 mg/kg) and cocaine (15 mg/kg), fluctuations in the voltammetric responses were observed (Fig. 5B) while there was no detectable signal before the administration of the drugs (Fig. 5A). The color plots, and cyclic voltammograms extracted from them (Fig. 5B) clearly reveal that these are transient increases in catecholamines that occur without electrical stimulation. This was confirmed by principal component regression of the voltammograms (Heien et al., 2005). These catecholamine fluctuations are similar to the dopamine transients that we previously reported in the CPu of anesthetized rats following similar drug treatments (Venton and Wightman, 2007). As in the CPu, dopamine autoreceptor blockade alone was insufficient to evoke transients in anesthetized rats. However, 5 min following a subsequent injection of cocaine, transients were induced that reached a maximum amplitude within 15 min and continued for 20–30 min. Transients were observed following the dopaminergic drugs in the rostral NAc shell in 9 out of 12 rats while only 4 of 16 rats showed weak transients in the caudal NAc shell. Drug-evoked transients were not observed following administration of the noradrenergic drugs, yohimbine (6 mg/kg) and desipramine (15 mg/kg), in either the rostral or caudal NAc shell.
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Fig. 5. Drug induced catecholamine concentration transients in the rostral and caudal NAc shell after raclopride (2 mg/kg i.p.) and cocaine (15 mg/kg i.p.). Two-dimensional color plot representation of the background-subtracted cyclic voltammograms collected over 30 s (A) before and (B) following administration of drugs in the rostral and caudal NAc shell. Catecholamine concentration changes are apparent in the color plots (B) at the potential for its oxidation (∼0.65 V) and its reduction (−0.2 V). Principal component regression was used to extract the time course of the catecholamine concentration transients (lower traces in A, B). Times are indicated by the vertical bars. Inset: Cyclic voltammograms recorded at the time indicated by the arrows were identical to those for a catecholamine.

The maximal concentration of transients following cocaine and raclopride within the caudal NAc shell, 0.05±0.01 μM (n=4 rats), was much lower than in the rostral NAc shell, 0.17±0.02 μM (n=9 rats, t=4.7, df = 11, P=0.0006). The average transient frequency was ∼0.35 Hz (10.5±0.5 transients per 30 s, n=8) for rostral NAc shell. In addition, the maximal concentration of transient (0.14±0.03 μM, n=5) and average transient frequency (0.38 Hz, 11.5±0.4 transients per 30 s, n=5) following GBR 12909 and raclopride in the rostral NAc shell were not significantly different from the values following cocaine and raclopride (Suppl. Fig. 2). It was difficult to compile a frequency average for transients in the caudal NAc shell due to the absence of catecholamine transients in most rats. The drug-induced transients in the rostral NAc shell were observed at similar depths (6.0–7.5 mm from the skull surface) as stimulated release (compare Suppl. Fig. 3 with Figs. 1C and 2C).

Discussion

Here, we examined the evoked release of catecholamines in two NAc sub regions with FSCV. The sensor employed, the carbon-fiber microelectrode, has micron dimensions allowing investigation of these two sub regions that are only separated by ∼800 μm. Catecholamine release was evoked by either stimulation of the MFB, a major ascending pathway that contains both dopaminergic and noradrenergic neurons (Ungerstedt, 1971), or the VTA/SN region. Stimulation in the latter region activates dopamine cell bodies (Garris and Wightman, 1994a) as well as the VNB that passes through these regions (Ungerstedt, 1971). The major finding of this work is that catecholamine release in the rostral NAc shell is predominantly from dopaminergic neurons whereas in the caudal NAc shell release of both catecholamines can be evoked. This finding is consistent with the neurochemical content of the subregions of the NAc shell. Our findings also show that measurable fluctuations in extracellular dopamine arise following combined administration of a dopamine autoreceptor antagonist and a catecholamine uptake inhibitor, cocaine, or a dopamine uptake inhibitor, GBR 12909. In contrast, simultaneous inhibition of noradrenergic autoreceptors and uptake does not evoke measurable transients in anesthetized rats. Our data are consistent with previous immunohistochemistry studies have shown that noradrenergic neurons innervate the caudal NAc shell to a greater degree than the rostral region (Delfs et al., 1998), whereas dopaminergic neurons originating in the VTA project to both sub regions (Ikemoto, 2007, Swanson, 1982). The current study not only provides more definitive evidence that catecholamine transmission within the rostral NAc shell measured by FSCV is due primarily to dopamine transmission, but also indicates that norepinephrine signaling is restricted to the caudal NAc shell. This is of interest because it has been suggested that the noradrenergic projection in the caudal NAc shell may modulate processes associated with stress, behavior state, or reinforcement via its actions within this region (Hajnal and Norgren, 2004).

Extracellular measurements of catecholamines in the NAc shell

Our chromatographic analysis of tissue content (Table 1) confirmed that dopamine is the predominant catecholamine in the rostral NAc shell whereas ∼30% of the catecholamine in the caudal NAc shell was norepinephrine. Extracellular norepinephrine and dopamine measurements by microdialysis within the NAc also showed that dopamine is the predominant catecholamine in the NAc core whereas both norepinephrine and dopamine are present in dialysate from the NAc shell (McKittrick and Abercrombie, 2007, Hajnal and Norgren, 2004, Carboni et al., 2006). In this study, our voltammetric measurements confirmed that stimulation of ascending pathways evokes catecholamine release in both the rostral and caudal NAc shell. Stimulation at 60 Hz at both of the sites investigated evokes a rapid increase in catecholamines throughout the NAc shell. [CA]max in the caudal NAc shell was significantly lower than that in the rostral NAc shell although tissue content is similar. This discrepancy between [CA]max and dopamine content may reflect our failure to dissect exactly the same nuclei each time. Furthermore, norepinephrine release has been shown to be a smaller fraction of total stores than is dopamine release (Garris and Wightman, 1995). Thus, tissue content is also not always proportional to catecholamine transmission in the terminal region. Consistent with our evoked release, extracellular dopamine has been shown to have a rostral to caudal gradient (Heidbreder and Feldon, 1998). The time to reach the maximum concentration (tmax.) in the rostral NAc shell is 0.2 s longer than the stimulus duration, a consequence of the finite response time of the electrode (Owesson-White et al., 2008). The clearance rates (measured as t1/2, Table 2) were slower in the caudal NAc shell than in the rostral region. Consistent with this, the dopamine transporter (DAT) is less densely distributed in the caudal NAc shell than in the rostral region (Pilotte et al., 1996). The norepinephrine transporter (NET) exhibits a similar distribution to that of the noradrenergic terminals in both the NAc shell of rats (Schroeter et al., 2000) and humans (Tong et al., 2007). Thus, in the rostral NAc shell, uptake is primarily controlled by the DAT whereas in the caudal region both NET and DAT contribute to the observed clearance.
To establish the nature of the catecholamine detected we used pharmacological agents. In prior work we and others have shown that release from dopaminergic terminals is enhanced by antagonists of the dopamine autoreceptor and prolonged by inhibition of the DAT, but is insensitive to drugs that act on noradrenergic neurons (Park et al., 2009, Carboni et al., 2006, Gobert et al., 1998, Palij and Stamford, 1992).
Conversely, release from noradrenergic terminals is insensitive to dopamine specific drugs but is enhanced by antagonists of the norepinephrine autoreceptor and inhibition of NET (Park et al., 2009, Gobert et al., 1998). Here, such pharmacological studies confirmed that dopamine was the predominant species released in the rostral NAc shell, an observation consistent with the microdialysis, anatomical, and tissue content studies. In contrast, release in the caudal NAc shell was affected by both a norepinephrine autoreceptor antagonist and an inhibitor of the NET as well as the dopaminergic drugs. Therefore, we conclude that stimulated release in the caudal NAc shell is a mixture of dopamine and norepinephrine.

Catecholamine projections to the rostral and caudal NAc shell

To evoke dopamine release in the rostral NAc shell, we chose the MFB and the VTA/SN region for placement of the stimulating electrode. Stimulation of both regions resulted in similar amounts of dopamine release in the rostral NAc shell, suggesting the same neurons were stimulated in each case. This is likely the case because VTA dopaminergic neurons project through the MFB en route to terminal regions (Ungerstedt, 1971, Ikemoto, 2007). To evoke catecholamine release in the caudal NAc shell, we stimulated the same two regions but the amounts released were quite different. As well as dopamine release, norepinephrine release might be expected with stimulation of the VTA/SN region because the VNB originating from A2 and A1 neurons passes through this region (Ungerstedt, 1971). We have shown previously that electrical stimulation of the VTA/SN activates at least a portion of the VNB because norepinephrine release was detected in the ventral bed nucleus of the stria terminalis (Park et al., 2009). Release in the caudal NAc shell was greater with MFB stimulation indicating that more catecholamine terminals were activated. These could include the fibers of the DNB that also pass through the MFB (Berridge et al., 1997).
The data obtained in the caudal NAc shell cannot be used to distinguish the amount of norepinephrine and dopamine that are released because the cyclic voltammograms for dopamine and norepinephrine are virtually identical (Heien et al., 2004, Park et al., 2009). Confounding the pharmacological data is the fact that NET can modulate a portion of the extracellular dopamine because it has a greater affinity for dopamine than the DAT (Moron et al., 2002). However, the results in the rostral NAc shell can be interpreted with much more confidence. The data clearly show that dopamine is by far the predominant catecholamine detected. Thus, this work validates our prior measurements of dopamine concentration fluctuations during intracranial self-stimulation (Owesson-White et al., 2008) and during cocaine administration (Aragona et al., 2009).

Dopamine transients occur following combined inhibition of dopamine uptake and autoreceptors

A key feature of dopamine neurotransmission are the naturally occurring dopamine concentration transients that are observed in awake animals (Wightman et al., 2007). They occur as a consequence of burst firing of dopaminergic neurons that can be evoked by activation of NMDA receptors in the VTA (Sombers et al., 2009). However, in deeply anesthetized animals these are rarely seen (Venton and Wightman, 2007), a finding that is consistent with the reduction in burst firing that occurs in anesthetized animals (Fa et al., 2003). Previously we showed that dopamine transients can occur within the CPu of an anesthetized rat following administration of a D2 receptor antagonist and a catecholamine transporter blocker (Venton and Wightman, 2007). The results shown here indicate that dopamine transients of similar origin can occur in the NAc shell. Electrophysiological studies of VTA dopaminergic studies have shown that the combined regimen of cocaine and a D2 antagonist lead to increased burst firing and slow rhythmic oscillations in the firing rate of dopamine neurons in anesthetized rats (Shi et al., 2004). Even though the anesthesia, dose of drugs used, and route of administration differed, the frequency of the cell firing oscillations (0.74 Hz) was remarkably similar to the frequency (0.35 Hz) of the dopamine concentration transients reported here. Inhibition of autoreceptors and uptake remove two of the key, negative-feedback components that regulate dopaminergic neurons. Removal of negative feedback frequently leads to oscillatory behavior. Note, however, that similar inhibition of noradrenergic negative feedback does not cause oscillatory behavior. Sequential administration of a noradrenergic autoreceptor antagonist and noradrenergic uptake inhibitor do not give rise to transients in the caudal NAc shell or the bed nucleus of the stria terminalis (Park et al., 2009) even though there is sufficient norepinephrine present in those regions to contribute to robust electrically evoked responses.

Conclusion

The present results show that norepinephrine is regulated in the caudal NAc shell by norepinephrine autoreceptors and uptake transporters. This result suggests that dopaminergic and noradrenergic transmission within the caudal NAc shell may play significantly different roles in regulating the behavioral and physiologic responses associated with drug abuse, physical stressors and other rewarding and aversive stimuli, compared to the rostral NAc shell.

Acknowledgments

We thank Khristy Fontillas for providing technical assistance and thank Richard B. Keithley and Dr. Nii Addy for supporting data analysis. This work was supported by NIH (NS 15841 to RMW and DA 17318 to RMC and RMW).

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