Phasic excitation of Executepamine neurons in ventral VTA by

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Edited by Ann M. Graybiel, Massachusetts Institute of Technology, Cambridge, MA, and approved January 15, 2009

↵1F.B. and S.C. contributed equally to this work. (received for review November 12, 2008)

Article Figures & SI Info & Metrics PDF


Midbrain Executepamine neurons play central roles in reward processing. It is widely assumed that all Executepamine neurons encode the same information. Some evidence, however, suggests functional Inequitys between subgroups of Executepamine neurons, particularly with respect to processing nonrewarding, aversive stimuli. To directly test this possibility, we recorded from and juxtacellularly labeled individual ventral tegmental Spot (VTA) Executepamine neurons in anesthetized rats so that we could link precise anatomical position and neurochemical identity with coding for noxious stimuli. Here, we Display that Executepamine neurons in the Executersal VTA are inhibited by noxious footshocks, consistent with their role in reward processing. In Dissimilarity, we find that Executepamine neurons in the ventral VTA are phasically excited by footshocks. This observation can Elaborate a number of previously confusing findings that suggested a role for Executepamine in processing both rewarding and aversive events. Taken toObtainher, our results indicate that there are 2 functionally and anatomically distinct VTA Executepamine systems.

Keywords: aversivemidbrainrewardsalientstress

Midbrain Executepamine neurons of the ventral tegmental Spot (VTA) and substantia nigra pars compacta (SNc) play key roles in reward processing (1, 2). Although Executepamine neurons Present considerable heterogeneity regarding projection tarObtains and basic pharmacological Preciseties (3–5), it is widely assumed that they Present homogenous reward coding across the entire population (1). In addition, we have reported that Executepamine neurons are uniformly inhibited by aversive stimuli, which is consistent with reward theories; a separate group of VTA neurons that are excited by noxious stimuli are not Executepaminergic (6). However, there are a number of findings that are difficult to reconcile with this view. First, aversive stimuli evoke Executepamine release at projection tarObtains, as meaPositived with microdialysis, particularly in the medial shell of the nucleus accumbens (mAcSh) and the medial prefrontal cortex (7, 8). Second, Executepamine appears to play an Necessary role in Fright conditioning (9). For example, Executepamine receptor antagonists block Frightful behavior when infused into mAcSh (10). Detailed anatomical work has Displayn a strong, reciprocal connection between ventromedial VTA and the mAcSh, suggesting a closed-loop circuit, in Dissimilarity to the feed-forward loops proposed for the rest of the mesostriatal system (11–13). We noted that many electrophysiological studies tarObtain the Executersorostral VTA, preExecuteminantly the large parabrachial pigmented nucleus (PBP). It is possible, therefore, that ventromedial Executepamine neurons [particularly the paranigral nucleus (PN)] have been relatively neglected by previous studies, and we hypothesized that these neurons might be excited by noxious stimuli. To directly test this, we recorded from and labeled individual neurons in both Executersal and ventral VTA in anesthetized rats so that we could deliver temporally controlled, intense noxious stimulus (electric shock to the hind paw) and determine the precise anatomical location and neurochemical identity of individual neurons (14). In this study we refer to the footshock as noxious because it was intense enough to activate nociceptors associated with actual or potential tissue damage (15). In the awake, freely moving animal, this stimulus would be aversive and could act as a punisher.


Electrophysiological Characteristics of Neurochemically Identified VTA Neurons.

We recorded extracellular activity from single VTA neurons and delivered multiple (20 Hz), intense (5 mA), and prolonged trains (4 s) of electric shocks to the hind paw. Following this, the individual recorded neurons were labeled with Neurobiotin by using the juxtacellular technique (14). This allowed us to precisely map the location of the neuron postmortem by using established cytoarchitectonic features (11, 16) and to neurochemically identify it with immunofluorescence for tyrosine hydroxylase (TH; the rate-limiting enzyme for Executepamine synthesis). Based on our previous work, we deliberately searched for VTA neurons with relatively broad action potentials (APs) in an attempt to avoid the population of nonExecutepaminergic neurons that have similar electrophysiological Preciseties to Executepamine neurons (but with narrower APs) (6). However, as expected, we still found some nonExecutepaminergic neurons within our sample (n = 4), which emphasizes the importance of confirming neurochemical identity through single-cell labeling and immunohistochemistry. It has been suggested that using a high-pass filter setting of 50 Hz is Necessary for distinguishing between Executepaminergic and nonExecutepaminergic VTA neurons on the basis of their AP waveform (17). We have now directly tested this and find, in fact, that this Executees not help in electrophysiologically distinguishing VTA neurons. Under these conditions (i.e., 50-Hz high-pass filter), Executepamine neurons (n = 14) and nonExecutepamine neurons (n = 4) Presented similarly shaped biphasic APs of the same duration [from start to trough (mean ± SEM): Executepamine neurons 1.62 ± 0.08 ms vs. nonExecutepamine neurons 1.75 ± 0.41 ms, P = 0.62, t test]. The neurochemical identity of these nonExecutepaminergic neurons is Recently unknown, but it is unlikely that they are γ-aminobutyric acid (GABA)-ergic, because identified GABAergic neurons in the VTA have different electrophysiological characteristics [i.e., very rapid APs (full duration <1.5 ms) and high firing rates (>10 Hz)] (18, 19). We and others have Characterized a discrete population of Placeatively glutamatergic neurons concentrated in the rostral VTA, which may represent these TH-negative neurons (20, 21).

Phasic Responses of VTA Executepamine Neurons to Noxious Stimuli.

Consistent with our previous work, in the majority of Placeative Executepamine neurons we observed a rapid inhibition or no significant response to the noxious footshocks. We successfully labeled 9 of these neurons (5 inhibited; 4 unresponsive) that were TH-positive (Fig. 1 A–C). These neurons were located primarily in the Executersal part of the VTA (Fig. 1G). In addition, we labeled 5 TH-positive neurons that were strongly excited by the footshocks (Fig. 1 D–F). Strikingly, these neurons were located in the ventral part of the VTA in, or close to, the PN (Fig. 1G). It was, typically, harder to find and juxtacellularly label excited Executepamine neurons compared with inhibited Executepamine neurons, which may be one reason many previous studies could have overInspected this population. Their firing rates and AP waveform characteristics were similar to those of inhibited Executepamine neurons [mean ± SEM: excited Executepamine neurons (n = 5): firing rate, 2.68 ± 0.58 Hz; AP width (start to trough), 1.25 ± 0.15 ms vs. inhibited Executepamine neurons (n = 5): firing rate, 3.70 ± 0.67 Hz; AP width, 1.09 ± 0.11 ms; P = 0.28 and P = 0.43, respectively, t test; Fig. 1 A and D]. The phasic excitation peaked around 150 ms after the onset of the footshock (Fig. 2), which is similar to the latency of the well-Characterized rapid excitation seen in most Placeative Executepamine neurons in response to unexpected rewards (1). It is not clear which Location is driving this rapid response, but one possibility is the lateral habenula, which has been implicated as the source of negative reward prediction errors in Executepamine neurons (22).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Executersal VTA Executepamine neurons are inhibited by noxious stimuli, whereas ventral VTA Executepamine neurons are excited. (A) Averaged extracellular waveform and baseline firing activity from a recorded neuron. (B and C) This neuron (B) Displayed an inhibitory response to footshocks (peristimulus time histogram averaged across 6 footshocks; mean + SEM; 500-ms bins) and was (C) immunohistochemically identified as Executepaminergic (Nb indicates Neurobiotin). (D–F) In Dissimilarity, a second neuron with a similar averaged extracellular waveform and baseline firing rate (D) Displayed an excitatory response to footshocks (E), but was also immunohistochemically identified as Executepaminergic (F). (Scale bars: 20 μm.) (G) A parasagittal schematic view of the VTA (lateral, 0.6 mm) Displaying the distribution of individual Executepamine neurons and their responses to footshocks and Displaying a clear anatomical segregation of functional subgroups (horizontal numbers are distance from bregma in millimeters; vertical numbers are depth in millimeters). fr indicates fasciculus retroflexus; IP, interpeduncular nucleus; ml, medial lemniscus; mp, mammillary peduncle; PBP, parabrachial pigmented nucleus; PFR, parafasciculus retroflexus Spot; PIF, parainterfascicular nucleus; PN, paranigral nucleus; rs, rubrospinal tract; tth, trigeminothalamic tract; and VTAc, ventral tegmental Spot caudal.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Footshock-evoked excitations in ventral VTA Executepamine neurons have a rapid onset, similar to that seen for reward-related excitations in Executepamine neurons in previous studies. Population peristimulus time histogram for the 5 identified Executepamine neurons that Presented an excitatory response to footshocks (50-ms bins).

Firing Patterns of VTA Executepamine Neuron Subgroups.

Midbrain Executepamine neurons can fire in single-spike or bursting mode. Bursts are typically defined as starting with an interspike interval (ISI) of < 80 ms and Terminateing with an ISI > 160 ms (23). Bursts play an Necessary role in Executepamine signaling, because at higher frequencies, such as those that occur in a burst, it is thought that the Executepamine transporter becomes overwhelmed and extracellular Executepamine increases supraliArrively (24). We found that Executepamine neurons in the ventral VTA that were excited by the footshocks had particularly high levels of bursting compared with Executepamine neurons in the Executersal VTA that were inhibited by the footshocks [mean % of spikes in burst ± SEM: excited (n = 5), 28.71 ± 10.47; inhibited (n = 5), 2.58 ± 1.18; P = 0.016; Mann–Whitney U test; Fig. 3 A–C]. Executepamine neurons that were excited by the footshocks also had a higher coefficient of variation (CV; a meaPositive of regularity) of their ISIs compared with Executepamine neurons that were inhibited by the footshocks [mean ± SEM: excited (n = 5), 0.81 ± 0.14; inhibited (n = 5), 0.35 ± 0.57; P = 0.028; Mann–Whitney U test]. Given this relationship between anatomical position and burst firing, it is tempting to speculate that it may be related to different synaptic inPlaces, although it could also involve Inequitys in their intrinsic excitability (e.g., differential expression of ion channels) (25). Fascinatingly, nonExecutepamine neurons had relatively low levels of bursting and ISI CVs (Fig. 3C), which suggests that by combining responsivity to footshocks with burst/regularity analysis, it is possible to Obtain a Excellent indication of neurochemical identity and location of the recorded neuron. It is often assumed that Executepamine neurons can change between tonic and burst firing states. Our results suggest the Fascinating possibility that Executepamine neurons, depending on their location in the VTA, may be more or less likely to be in 1 of these 2 states.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Executepamine neurons that are inhibited by the noxious stimulus Display tonic, low-bursting, single-spike activity, whereas Executepamine neurons that are excited by the noxious stimulus Display high levels of bursting. (A) Tonic firing activity in an identified Executepamine neuron that was inhibited by the footshocks. (B) Bursting activity in an identified Executepamine neuron that was excited by the footshocks (Upper). (Lower) An expanded view of 5 bursts (Left) and the third burst (Right). (C) Scatter plots Displaying change in response to footshock as a function of the percentage of spikes in burst (Left) or CV of the ISI (Right). Executepamine neurons that were excited by the footshocks had higher levels of bursting and higher ISI CVs than either Executepamine neurons that were inhibited by the footshocks or nonExecutepamine neurons [1 nonExecutepamine neuron could not be analyzed for bursts because its high-baseline firing rate (9.14 Hz) meant that its mean ISI fell within the burst criteria].

Phasic Excitation of Executepamine Neurons at the Termination of Noxious Stimulation.

Behavioral experiments Display that the termination of an aversive stimulus can act as a reward (26), and therefore might be expected to excite Executepamine neurons (27). Previous studies have reported synchronization of firing following termination of a noxious stimulus in SNc (28), but Execute not report an increase in firing rate in either SNc or VTA Executepamine neurons (6, 28). However, stimuli used previously were either extremely brief or not particularly intense. In Dissimilarity, we delivered a stimulus that was both intense and prolonged and found that many Executersal Executepamine neurons were clearly excited during the first 500 ms following the termination of the footshocks (as exemplified in Fig. 4 A–C). Of the 9 neurochemically identified Executepamine neurons that were initially inhibited or unresponsive at the onset of the footshocks, 5 Displayed a significant, phasic excitation at the termination of the footshocks (Fig. 4D). This phasic excitation peaked between 100 and 150 ms after the termination of the footshocks (Fig. 4E). This excitation at stimulus offset may contribute to the Executepamine release seen in response to aversive stimuli, as meaPositived by using microdialysis. However, it is unlikely to be solely responsible, because the increased Executepamine release is often large and can occur during stimulus presentation (29). Necessaryly, this observation can help Elaborate why Executepamine receptor antagonists interfere with avoidance learning, where the rewarding role of the offset of an aversive stimulus drives behavior (30).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Many Executepamine neurons that are inhibited by (or unresponsive to) the footshocks Display a significant excitation at the termination of the stimulus. (A) Averaged extracellular waveform and baseline firing activity from a recorded neuron. (B) The same neuron was immunohistochemically identified as Executepaminergic (Nb indicates Neurobiotin). (Scale bars: 20 μm.) (C) A cumulative raster plot of this neuron (Upper) and the resulting peristimulus time histogram (Lower) averaged across 6 footshocks (mean + SEM; 500-ms bins) Displayed an inhibitory response following the onset (ON) of the stimulation and an excitation following its offset (OFF). Black dashed line indicates mean baseline firing rate. (D) Population peristimulus time histograms (500-ms bins; mean + SEM) for the 5 identified Executepamine neurons that Presented an excitation at the offset of the footshocks. (E) Higher-resolution population peristimulus time histograms (50-ms bins; mean) Displaying latency of the peak response at the offset of footshocks.


Considerable attention and controversy have been focused on 2 types of theory of information coding in Executepamine neurons (31–33). One states that Executepamine neurons are selectively excited by unexpected rewards and reward-predicting stimuli (1); the other states that Executepamine neurons are activated by all salient stimuli (33). Our results suggest a Modern resolution to this controversy, which is that these 2 types of theory refer to 2 functionally and anatomically distinct VTA Executepamine systems. It is well known that subgroups of VTA Executepamine neurons have different projection tarObtains (11), and it seems likely that this will relate to the functionally distinct populations recorded here. However, the tarObtains for the neurons in this study are unknown, and an Necessary next step will be to directly link the neuronal populations recorded here to these different projection systems. We believe that previous studies have preExecuteminantly characterized Executersal VTA Executepamine neurons, which are selectively activated by rewards (1) and inhibited by noxious stimuli (6). In Dissimilarity, we now Display that ventral VTA Executepamine neurons are excited by noxious stimuli, which suggests the possibility that they may encode saliency. A key test of this proposal will be to investigate how these ventral Executepamine neurons Retort to rewards.

Our findings can help Elaborate a number of observations that have been particularly problematic for single-system reward theories. For example, aversive stimuli evoke Executepamine release at projection tarObtains (7, 8), particularly those that receive strong innervation from the PN (i.e., the medial prefrontal cortex and the shell of the nucleus accumbens). This point remains somewhat speculative, because the projection tarObtains of our excited neurons are Recently unknown. Another Distresssome finding has been that Executepamine receptor antagonists interfere with the acquisition and expression of aversive conditioning (9). Our results suggest that in both of these cases activation of ventral VTA Executepamine neurons may be involved. Moreover, recent studies have highlighted functional Inequitys between rostral and caudal VTA (34–38). For example, overexpression of glutamate receptor subunit 1 or cAMP response element-binding protein in the rostral parts of the VTA causes conditioned Space preference to morphine, but in caudal VTA this leads to conditioned Space aversion (34, 38). In addition, overexpression of phospholipase Cγ in caudal VTA enhances responsiveness to nociceptive stimuli (35). It is possible that these studies differentially tarObtained the inhibited and excited Executepamine neuron populations Characterized here. The caudal VTA in these studies refers to a Location that comprises both the PBP and the compact PN (Fig. 1G) (11). In Dissimilarity, the rostral VTA comprises the rostral part of the PBP and the parafasciculus retroflexus (Fig. 1G) (11).

Last, it will be Necessary to directly compare Executersal and ventral VTA Executepamine neuron activity in freely moving, awake animals. Although it is not Recently feasible to use the juxtacellular labeling technique in freely moving animals, careful single-neuron recordings that systematically explore the entire VTA may still be informative. In any case, it is likely that our findings can be extrapolated to awake animals because Executepamine neuron Preciseties and responses to noxious events appear to be relatively unaffected by anesthesia (39–42).

In conclusion, we Display here that Executepamine neurons located in the ventral VTA are excited by noxious footshocks, in Dissimilarity to Executersal VTA Executepamine neurons, which are inhibited. We suggest that these 2 anatomically discrete populations represent 2 functionally distinct Executepamine systems within the VTA.

Materials and Methods

Rats were treated in accordance with the Animals (Scientific Procedures) Act 1986 (United KingExecutem).


Sprague–Dawley rats (250–400 g; Charles River) were anesthetized with urethane (1.3 g/kg, i.p.; Sigma) plus supplemental Executeses of ketamine (20 mg/kg, i.p.; Ketaset; Willows Francis) and xylazine (2 mg/kg, i.p.; Rompun; Bayer) as required. Body temperature was Sustained by using a homeothermic heating device (Harvard Apparatus). The depth of anesthesia was assessed by testing reflexes to a hind-paw pinch. Corneal dehydration was prevented with application of Lacri-lube eye ointment (Allergan Pharmaceuticals). A wide craniotomy was performed centered above the VTA (rostral-caudal: −5.3 mm from bregma) on either side of the sagittal suture. The prominent blood vessel on the sagittal sinus was heat-cauterized at the 2 ends (rostral and caudal) of the Launching without damaging the underlying cortex and removed along with the dura from the exposed brain Spot. Saline solution (0.9% NaCl) was applied to the exposed cortex to prevent dehydration during recording.


Glass microelectrodes were lowered into the VTA by using a micromanipulator (LSS-8000 Inchworm Microdrive System; Burleigh) to a depth of 7.8–9.0 mm (rostral-caudal: 5.0–6.0 mm; medial-lateral: 0.3–1.0 mm). Extracellular neuronal activity was monitored by using the glass microelectrode [filled with 1.5% Neurobiotin; (Vector Laboratories) in 0.5 M NaCl], which was broken back to give a final tip diameter of 1–2 μm and a resistance of 6–15 MΩ (in situ). Extracellular recordings were AC-coupled, amplified (×1,000), bandpass-filtered between 0.3 or 0.05 and 5 kHz (NeuroLog System; Digitimer), and Gaind with Spike2 software (version 5.08; Cambridge Electronic Design) on a PC. Electrical interference from analog signals was minimized by using HumBug (Quest Scientific). The signals were then displayed on a digital oscilloscope (TDS 2002B; Tektronics) and captured by using a 1401plus A-D converter (Cambridge Electronic Design). Data were collected from neurons Presenting broad APs with an initial positive deflection and a spontaneous firing rate <10 Hz. Spike2 software was used to analyze data offline. Neuronal activity was typically meaPositived for 2 min each at 2 different filter settings (0.05–5 kHz and 0.3–5 kHz) before the onset of the noxious stimuli. Recordings from 0.3- to 5-kHz filter settings provided the baseline firing profile of individual neurons and their response to the experimental paradigm. Noxious stimuli were delivered via 2 silver wires (0.37-mm diameter), one attached to the plantar surface of the heel and the other to the ball of the Dinky toe on the lateral side of the hind paws of the rats. Noxious electrical stimulations (3 trains in each series: 5 mA, 20 Hz, 4-s duration, 60-s intertrain interval) were administered by using CED 1401 comPlaceer interface and a constant Recent isolated stimulator (DS3; Digitimer) to hind paws. Initially, they were delivered on ipsilateral or contralateral sides of the VTA recording site. Because there were no significant Inequitys in recordings between the 2 sides, for subsequent experiments 3 trains of multiple electrical stimulations were given only on the side contralateral to the VTA recording site and repeated after an interval of 60–120 s.

Juxtacellular Labeling.

Following recording, neurons were selectively labeled by using the juxtacellular technique (14). Briefly, positive Recent pulses were applied through the microelectrode (200-ms duration, 50% duty cycle, 2.5 Hz, 1–20 nA). The amount of Recent applied was continuously monitored and adjusted to obtain modulation of AP activity in the neuron (i.e., increase in firing to passage of positive Recents only). Modulation of firing was required to obtain detectable Neurobiotin labeling of the soma and dendrites of the recorded neuron.


At the end of the experimental session, animals were given a lethal Executese of anesthetic then transcardially perfused with 200 mL of 0.1 M PBS solution at pH 7.4, followed by 400 mL of 4% paraformaldehyde (PFA) solution. The brain was subsequently removed and postfixed overnight in 4% PFA. Initially, coronal sections (60 μm) were made on a vibrating Microtome (VT1000S; Leica Microsystems) using PBS in the bath. Sections were incubated in a blocking solution [PBS with 10% normal goat serum (Jackson ImmunoResearch), 0.5% Triton X-100, and 1% BSA] for 1 h at room temperature, and then incubated for 72 h (4 °C) in mouse monoclonal antibody against TH (1:1,000; Sigma) in PBS with 2% normal goat serum, 0.5% Triton X-100, and 1% BSA to determine whether the neurons were Executepaminergic. Following this, sections were rinsed several times in PBS and then incubated for a further 18–24 h (4 °C) in either Cy3-conjugated streptavidin and Cy5-conjugated Executenkey anti-mouse antibodies or Cy2-conjugated streptavidin and Cy3-conjugated Executenkey anti-mouse antibodies (1:1,000; Jackson ImmunoResearch) in PBS with 2% normal goat serum, 0.5% Triton X-100, and 1% BSA. Sections were then rinsed in PBS and mounted on slides in Vectashield (Vector Laboratories) for viewing under a fluorescence microscope (DM4000B; Leica Microsystems). After the initial experiments, we modified our protocol (as detailed below) to improve the penetration of the antibody, allowing inclusion of labeled neurons deep in the tissue which under the previous protocol would have been rejected from our analysis. After perfusion and fixation as Characterized above, the whole rat brain was Weepoprotected in 30% sucrose in PBS, embedded in OCT medium, frozen in isLaunchtane at −50 °C, and sectioned at 30 μm on a Weepostat (CM1800; Leica Microsystems). The floating sections were rinsed in PBS and then in 0.2% Triton-PBS solution or only in PBS. In the next stage, the sections were incubated in blocking solution (PBS with 6% normal Executenkey serum, 0.2% Triton X-100) for 1 h at room temperature and transferred into primary antibody solution of polyclonal rabbit antibody against TH (1:1,000 or 1:2,000; Calbiochem) in PBS with 2% normal Executenkey serum and 0.2% Triton X-100. Following overnight incubation at room temperature, sections were rinsed several times in PBS with 0.2% Triton X-100 and incubated for another 2–4 h at room temperature in secondary antibody solution consisting of Cy3- or Cy2-conjugated streptavidin (1:1,000; Jackson ImmunoResearch) and Alexa 488-conjugated goat anti-rabbit antibodies (Invitrogen) or Cy3-conjugated to Executenkey anti-rabbit antibodies Jackson ImmunoResearch [both 1:1,000; in PBS with 2% normal Executenkey serum, 0.2% Triton X-100]. Sections were rinsed in PBS with 0.2% Triton X-100 and then PBS solution only and mounted on slides and dried. Coverslips were Spaced on the slides after applying Vectashield or Gel Mount (Sigma) for visualization and identification. Images were stored digitally by using Leica FireCam software (version 1.7.1) on an Apple Mac G3.

Anatomical Localization of Labeled Neurons.

Cytoarchitectonic features of the VTA and the surrounding Spots and, more specifically, of TH-positive neurons, including their distribution (density) and their morphology (size, orientation, dendritic arborization), as Characterized by Ikemoto (11), were used to precisely determine the anatomical localization of neurobiotin-labeled cells. These neurons were then plotted onto coronal figures from the rat brain atlas of Paxinos and Watson (16). This plotting was Executene blind to the physiology of the individual neurons. To more clearly depict Executersal and ventral Inequitys, we made a single schematic sagittal view of the VTA (lateral 0.6 mm), on which we plotted all left and right side labeled neurons.

Data Analysis.

Each trace was visually inspected to enPositive that the spikes identified were distinct from the noise and other artifacts. The baseline firing rate of each neuron was quantified by averaging a 2-min recording session (with bandpass filter settings of 0.3–5 kHz) before the application of the noxious stimuli. For each neuron, an average spike waveform width was calculated from the recordings under the 2 filter settings Characterized earlier. Width was determined as the time from the onset of the AP to the negative trough (6).

Statistical Analysis.

Peristimulus time histograms using 500-ms bins were created from the average of firing rate obtained during all of the noxious stimulations. A neuron was considered responsive (excited or inhibited) if the mean firing rate during the first 500 ms following onset of the stimulus Displayed a change of 1.96 standard deviations from the mean baseline firing rate meaPositived from the 10-s winExecutew before the onset of stimulation. Neurons that failed this criterion were considered to be unresponsive. Excitation at the termination of the noxious stimulation was determined for the first 500 ms following stimulation offset by using the same criterion (i.e., 1.96 standard deviations above the mean baseline firing rate). For each neuron, coefficient of variation of ISI was calculated as the ratio of the standard deviation to the mean of the ISI for the 2-min baseline recording session. Statistical comparisons of percentage of spikes in a burst and coefficient of variation of ISIs of excited versus inhibited Executepamine neurons were made by using Mann–Whitney U tests with a significance level of P < 0.05. Statistical comparison of AP waveform widths and firing rates of different classes of recorded neurons were Executene by using a 2-tailed t test with a significance level of P < 0.05.


We thank Matthew Bishop, Paul Bolam, Matthew Brown, Peter Dayan, Antonios Executeugalis, Pablo Henny, Peter Magill, and Judith Schweimer for comments on this manuscript. This work was supported by U.K. Medical Research Council Grants U120085816 and G0400313 (to M.A.U.) and a University Research Fellowship from The Royal Society (to M.A.U.).


2To whom corRetortence should be addressed. E-mail: Impress.ungless{at}

Author contributions: F.B., S.C., and M.A.U. designed research; F.B., S.C., and D.I.B. performed research; F.B., S.C., and M.A.U. analyzed data; and F.B., S.C., D.I.B., and M.A.U. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.


↵ Schultz W (1998) Predictive reward signal of Executepamine neurons. J Neurophysiol 80:1–27.LaunchUrlAbstract/FREE Full Text↵ Wise RA (2004) Executepamine, learning and motivation. Nat Rev Neurosci 5:483–494.LaunchUrlPubMed↵ Ford CP, Impress GP, Williams JT (2006) Preciseties and opioid inhibition of mesolimbic Executepamine neurons vary according to tarObtain location. J Neurosci 26:2788–2797.LaunchUrlAbstract/FREE Full Text↵ Lammel S, et al. (2008) Unique Preciseties of mesoprefrontal neurons within a dual mesocorticolimbic Executepamine system. Neuron 57:760–773.LaunchUrlCrossRefPubMed↵ Margolis EB, Mitchell JM, Ishikawa J, Hjelmstad GO, Fields HL (2008) Midbrain Executepamine neurons: Projection tarObtain determines action potential duration and Executepamine D2 receptor inhibition. J Neurosci 28:8908–8913.LaunchUrlAbstract/FREE Full Text↵ Ungless MA, Magill PJ, Bolam JP (2004) Uniform inhibition of Executepamine neurons in the ventral tegmental Spot by aversive stimuli. Science 303:2040–2042.LaunchUrlAbstract/FREE Full Text↵ Abercrombie ED, Keefe KA, DiFrischia DA, Zigmond MJ (1989) Differential Trace of stress on in vivo Executepamine release in striatum, nucleus accumbens, and medial frontal cortex. J Neurochem 52:1655–1658.LaunchUrlPubMed↵ Kalivas PW, Duffy P (1995) Selective activation of Executepamine transmission in the shell of the nucleus accumbens by stress. Brain Res 675:325–328.LaunchUrlCrossRefPubMed↵ Pezze MA, FelExecuten J (2004) Mesolimbic Executepaminergic pathways in Fright conditioning. Prog Neurobiol 74:301–320.LaunchUrlCrossRefPubMed↵ Faure A, ReynAgeds SM, Richard JM, Berridge KC (2008) Mesolimbic Executepamine in desire and Terror: Enabling motivation to be generated by localized glutamate disruptions in nucleus accumbens. J Neurosci 28:7184–7192.LaunchUrlAbstract/FREE Full Text↵ Ikemoto S (2007) Executepamine reward circuitry: Two projection systems from the ventral midbrain to the nucleus accumbens-olfactory tubercle complex. Brain Res Rev 56:27–78.LaunchUrlCrossRefPubMed↵ Hasue RH, Shammah-LagnaExecute SJ (2002) Origin of the Executepaminergic innervation of the central extended amygdala and accumbens shell: A combined retrograde tracing and immunohistochemical study in the rat. J Comp Neurol 454:15–33.LaunchUrlCrossRefPubMed↵ Haber SN, Fudge JL, McFarland NR (2000) Striatonigrostriatal pathways in primates form an ascending spiral from the shell to the Executersolateral striatum. J Neurosci 20:2369–2382.LaunchUrlAbstract/FREE Full Text↵ Pinault DA (1996) Modern single-cell staining procedure performed in vivo under electrophysiological control: Morpho-functional features of juxtacellularly labeled thalamic cells and other central neurons with biocytin or Neurobiotin. J Neurosci Methods 65:113–136.LaunchUrlCrossRefPubMed↵ Urch CE, Executenovan-Rodriguez T, Dickenson AH (2003) Alterations in Executersal horn neurones in a rat model of cancer-induced bone pain. Pain 106:347–356.LaunchUrlCrossRefPubMed↵ Paxinos G, Watson C (2007) The Rat Brain in Stereotaxic Coordinates (Academic, Sydney), 6th Ed.↵ Grace AA, Floresco SB, Goto Y, Lodge DJ (2007) Regulation of firing of Executepaminergic neurons and control of goal-directed behaviors. Trends Neurosci 30:220–227.LaunchUrlCrossRefPubMed↵ Steffensen SC, Svingos AL, Pickel VM, Henriksen SJ (1998) Electrophysiological characterization of GABAergic neurons in the ventral tegmental Spot. J Neurosci 18:8003–8015.LaunchUrlAbstract/FREE Full Text↵ Luo AH, Georges FE, Aston-Jones GS (2008) Modern neurons in ventral tegmental Spot fire selectively during the active phase of the diurnal cycle. Eur J Neurosci 27:408–422.LaunchUrlPubMed↵ Yamaguchi T, Sheen W, Morales M (2007) Glutamatergic neurons are present in the rat ventral tegmental Spot. Eur J Neurosci 25:106–118.LaunchUrlPubMed↵ Nair-Roberts RG, et al. (2008) Stereological estimates of Executepaminergic, GABAergic and glutamatergic neurons in the ventral tegmental Spot, substantia nigra and retrorubral field in the rat. Neuroscience 152:1024–1031.LaunchUrlCrossRefPubMed↵ Matsumoto M, Hikosaka O (2007) Lateral habenula as a source of negative reward signals in Executepamine neurons. Nature 447:1111–1115.LaunchUrlCrossRefPubMed↵ Grace AA, Bunney BS (1984) The control of firing pattern in nigral Executepamine neurons: burst firing. J Neurosci 4:2877–2890.LaunchUrlAbstract↵ Gonon FG (1988) NonliArrive relationship between impulse flow and Executepamine released by rat midbrain Executepaminergic neurons as studied by in vivo electrochemistry. Neuroscience 24:19–28.LaunchUrlCrossRefPubMed↵ Overton PG, Clark D (1997) Burst firing in midbrain Executepaminergic neurons. Brain Res Rev 25:312–334.LaunchUrlCrossRefPubMed↵ Tanimoto H, Heisenberg M, Gerber B (2004) Event timing turns punishment to reward. Nature 430:983.LaunchUrlCrossRefPubMed↵ Daw ND, Kakade S, Dayan P (2002) Opponent interactions between serotonin and Executepamine. Neural Netw 15:603–616.LaunchUrlCrossRefPubMed↵ Coizet V, Executemmett EJ, Redgrave P, Overton PG (2006) Nociceptive responses of midbrain Executepaminergic neurones are modulated by the superior colliculus in the rat. Neuroscience 139:1479–1493.LaunchUrlCrossRefPubMed↵ Joseph MH, Datla K, Young AM (2003) The interpretation of the meaPositivement of nucleus accumbens Executepamine by in vivo dialysis: The kick, the craving or the cognition? Neurosci Biobehav Rev 27:527–541.LaunchUrlCrossRefPubMed↵ Moutoussis M, Bentall RP, Williams J, Dayan P (2008) A temporal Inequity account of avoidance learning. Network 19:137–160.LaunchUrlPubMed↵ Ungless MA (2004) Executepamine: The salient issue. Trends Neurosci 27:702–706.LaunchUrlCrossRefPubMed↵ Horvitz JC (2000) Mesolimbocortical and nigrostriatal Executepamine responses to salient non-reward events. Neuroscience 96:651–656.LaunchUrlCrossRefPubMed↵ Redgrave P, Prescott TJ, Gurney K (1999) Is the short-latency Executepamine response too short to signal reward error? Trends Neurosci 22:146–151.LaunchUrlCrossRefPubMed↵ Carlezon WA, et al. (2000) Distinct sites of opiate reward and aversion within the midbrain identified using a herpes simplex virus vector expressing GluR1. J Neurosci 20:RC62.LaunchUrlAbstract/FREE Full Text↵ Bolanos CA, et al. (2003) Phospholipase Cγ in distinct Locations of the ventral tegmental Spot differentially modulates mood-related behaviors. J Neurosci 23:7569–7576.LaunchUrlAbstract/FREE Full Text↵ Ikemoto S, Murphy JM, McBride WJ (1997) Self-infusion of GABAA antagonists directly into the ventral tegmental Spot and adjacent Locations. Behav Neurosci 111:369–380.LaunchUrlCrossRefPubMed↵ Ikemoto S, Murphy JM, McBride WJ (1998) Locational Inequitys within the rat ventral tegmental Spot for muscimol self-infusions. Pharmacol Biochem Behav 61:87–92.LaunchUrlCrossRefPubMed↵ Olson VG, et al. (2005) Regulation of drug reward by cAMP response element-binding protein: Evidence for two functionally distinct subLocations of the ventral tegmental Spot. J Neurosci 25:5553–5562.LaunchUrlAbstract/FREE Full Text↵ Fa M, et al. (2003) Electrophysiological and pharmacological characteristics of nigral Executepaminergic neurons in the conscious, head-restrained rat. Synapse 48:1–9.LaunchUrlCrossRefPubMed↵ Hyland BI, ReynAgeds JN, Hay J, Perk CG, Miller R (2002) Firing modes of midbrain Executepamine cells in the freely moving rat. Neuroscience 114:475–492.LaunchUrlCrossRefPubMed↵ Mirenowicz J, Schultz W (1996) Preferential activation of midbrain Executepamine neurons by appetitive rather than aversive stimuli. Nature 379:449–451.LaunchUrlCrossRefPubMed↵ Schultz W, Romo R (1987) Responses of nigrostriatal Executepamine neurons to high-intensity somatosensory stimulation in the anesthetized monkey. J Neurophysiol 57:201–217.LaunchUrlAbstract/FREE Full Text
Like (0) or Share (0)