Behavioural studies

Male Lister Hooded rats (Charles River Laboratories, Kent, UK) weighing 396±5 g were singly housed under a 12-h reverse light/dark cycle (off 0700 hours). Following recovery from surgery and throughout the experiment, rats were fed 20 g chow per day with water available ad libitum. Experiments were performed 5–7 days per week during the dark phase. All procedures were conducted in accordance with the United Kingdom 1986 Animals (Scientific Procedures) Act, Project License 80/2234. The experiments were carried out with approval from the Home Office.

Drugs

Cocaine hydrochloride (Macfarlan-Smith, Edinburgh, UK) and α-flupenthixol (Sigma-Aldrich, Poole, UK) were prepared as previously described13. Quinolinic acid (Sigma-Aldrich) was dissolved in sterile phosphate-buffered saline (PBS) and infused at 0.09 M, pH=7.4. Ibotenic acid (Abcam Biochemicals, Cambridge, UK) was dissolved in PBS at a concentration of 10 μg μl−1, pH=7.4 . The mixture of the GABA-B and GABA-A receptor agonists, baclofen, muscimol (B/M) (Sigma-Aldrich) was dissolved in PBS at the final concentration of 0.6 and 0.06 mM50. The selective D1 and D2 receptor antagonists SCH23390 and raclopride (Sigma-Aldrich) were dissolved in PBS at the concentration of 0.5, 1 or 1.5 μg μl−1 and 1, 2 and 3 μg μl−1, respectively. Drug doses are reported in the salt form.

Apparatus

Experiments were conducted using 12 standard operant conditioning chambers (Med Associates, St Albans, VT, USA) each fitted with a syringe pump and equipped with 2 cue lights above 2 levers and a houselight on the opposite wall, and were housed in sound- and light-attenuating cubicles as described previously41. Personal computers with Whisker software (Cardinal and Aitken, http://www.whiskercontrol.com) controlled infusions and light presentations and recorded lever presses.

Functional disconnections

The three behavioural experiments illustrated in Supplementary Fig. 1 aimed to test the effect of functionally disconnecting either the BLA or the CeN from dopaminergic transmission in the aDLS. The rationale for using such causal manipulations is provided in Supplementary Fig. 1.

Surgery

All rats were anaesthetized with a mixture of ketamine hydrochloride (100 mg kg−1; Ketaset; Fort Dodge Animal Health Ltd, Southampton, UK) and xylazine (12 mg kg−1; Rompun; Bayer, Wuppertal, Germany) and implanted with an intravenous jugular catheter (Camcaths, Ely, UK) as described previously41,60. Rats were then immediately positioned in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA) with the incisor bar set at −3.3 mm (ref. 61).

For experiment 1, rats were given either a unilateral BLA lesion, a unilateral CeN lesion or a sham lesion (couterbalanced). BLA lesions were made using two infusions (anteroposterior (AP)−2.3/−3, mediolateral (ML)+/−4.6, dorsoventral (DV)−7.3 (ref. 62)) of quinolinic acid (0.3 μl per 2 min followed by a 2-min diffusion time). CeN lesions were made using one infusion (AP−2.3, ML+/−4.2, DV−7.7 (ref. 63)) of ibotenic acid (0.25 μl per 6 min−1 followed by a 2-min diffusion time). AP and ML coordinates were measured from bregma, DV for BLA and CeN lesions were measured from dura and skull, respectively. All rats were subsequently implanted bilaterally with 22-gauge guide cannulae (Plastics One, Roanoke, VA, USA) positioned to lie 2 mm above the aDLS (AP+1.2, ML±3, DV−3)41 infusion target.

For experiment 2, rats were implanted with four 22-gauge guide cannulae, all bilaterally targeting the aDLS (AP+1.2, ML±3, DV−3) and then either bilaterally targeting the CeN (AP−2.3, ML±4, DV−3.7) or the BLA (AP−2.6, ML±4.6, DV−3.6). AP and ML coordinates were measured from bregma, DV from skull.

For Experiment 3, rats were then implanted bilaterally with 22-gauge guide cannulae positioned to lie 2 mm above the aDLS (AP+1.2, ML±3, DV−3 (ref. 40)).

Cannulae and catheters were secured and maintained as described previously40,41. Rats were treated daily from the day before to 7 days after surgery with 10 mg kg−1 s.c. of the antibiotic, Baytril (Bayer).

Intrastriatal and intra-amygdala infusions

Intrastriatal infusions (0.5 μl per side; bilateral for control group; unilateral for BLA-lesioned and CeN-lesioned groups—see Supplementary Fig. 2) were made via 28-gauge steel hypodermic injectors (Plastics One) lowered to the injection sites 2 mm (aDLS), 4 mm (CeN) or 5 mm (BLA) ventral to the end of the guide cannulae.

α-flupenthixol, SCH23390 and raclopride were, respectively, administered at the doses of 10 or 15 μg per infusion; 0.25, 0.5 and.75 μg per side and at 0.5, 1 and 1.5 mg per side. B/M was administered at 0.3/0.03 nmol per infusion, respectively. Infusions were made over 90 s using a syringe pump (Harvard Apparatus, Holliston, MA, USA) and were followed by a 60-s period to allow diffusion of the infused drug or vehicle before injectors were removed and obturators were replaced. Test sessions (see Procedures) began 5 min later.

For all pharmacological challenges, intracranial infusions were delivered according to a Latin-square design and were separated by at least two baseline sessions to control that stable levels of cocaine seeking or cocaine self-administration were maintained throughout.

Procedures

For all the experimental groups, cocaine self-administration training sessions began 7 days following surgery (Supplementary Fig. 1). Cocaine (0.25 mg per infusion; 0.1 ml per 5 s−1) was available under a continuous reinforcement (that is, fixed-ratio 1 (FR1)) schedule. One active lever press resulted in an infusion and initiated a 20-s time out. Each cocaine infusion was accompanied by a 20-s illumination of the active lever-specific cue-light (CS). The houselight was extinguished and both levers were retracted during the time out. Pressing on the inactive lever was recorded but had no programmed consequence. The maximum number of available cocaine infusions during this stage was 30. Active and inactive lever assignment was counterbalanced. All test conditions were administered in a counterbalanced, Latin-square order of treatment, and sessions were conducted prior to, and were thus unaffected by, self-administered cocaine on that day (Fig. 1). Each test was immediate followed by the appropriate training session.

For Experiment 1 (Supplementary Fig. 1 top panel), following 10 training sessions under FR1, the effects of aDLS-dopamine receptor blockade on early-stage cocaine seeking were tested. Infusions (bilateral for control group, unilateral and contralateral to the lesion in BLA and CeN groups) of α-flupenthixol (0 and 10 μg per infusion) were made into the aDLS. During each 15-min test session, every active lever press resulted in a 1-s light CS presentation, and cocaine was only delivered on the first lever press following the 15-min interval (that is, FI15 (FR1:S)). Thus, the early-performance tests were conducted prior to, and were thus unaffected by, self-administered cocaine on that day (Fig. 1). Each test session was immediately followed by a FR1 cocaine self-administration training session (that is, up to 30 reinforcers in a 2-h session).

Following the early-performance cocaine-seeking tests, the response requirement was increased across the daily training sessions from FR1 to FR3, FR5(FR2:S), FR10(FR2:S) and ultimately FR10(FR4:S)41. Under each intermediate second-order schedule, completion of the unit schedule (given within parentheses) resulted in a 1-s CS light presentation; cocaine infusion and the 20-s time out were given only on completion of the overall schedule. Therefore, for the intermediate stage assessments, rats had been trained under conditions in which contingent presentations of the cocaine-associated CS occurred after 4 responses (FR4:S); cocaine was delivered on completion of the tenth set of 4 lever presses, that is, 40 lever presses. After reaching the FR10(FR4:S) schedule, rats began the transition-stage cocaine-seeking tests. During each 15-min test session with aDLS α-flupenthixol infusions (0 and 10 μg per infusion), every four active lever presses resulted in a 1-s light CS presentation, and cocaine was only delivered on the fourth lever press following the 15-min interval (that is, FI15(FR4:S)). Thus, the transition-stage performance tests were again conducted prior to, and were unaffected by, daily self-administered cocaine. Each test session was immediately followed an FR10(FR4:S) cocaine self-administration training session (30 reinforcers over 2 h).

The response requirements were then again increased through daily training sessions across the following reinforcement SOCR:FR10(FR6:S), FR10(FR10:S) and finally to an overall schedule of FI15(FR10:S)13,40,41 (Supplementary Fig. 1) during which responding was maintained by contingent presentation of the cocaine-associated Cs every tenth lever press (FR10:S), and each of the five daily cocaine infusions was delivered after the tenth lever press following completion of each 15-min fixed interval. Following 15 sessions, the habitual-stage tests were conducted in which the effects of aDLS α-flupenthixol infusions (0, 5, 10 and 15 μg per infusion) were assessed. The first 15-min interval of the schedule provides a time period in which no cocaine has been administered, yet rats are actively seeking cocaine.

Finally, on completion of contralateral testing rats were assessed for effects of ipsilateral aDLS-dopamine receptor blockade (0 and 10 μg per infusion) (Supplementary Fig. 5). Rats in the BLA and CeN groups were given infusions in the aDLS on the same side of the brain as their unilateral amygdala lesion; rats in the control group were given unilateral aDLS infusions.

For Experiment 2 (Supplementary Fig. 1 middle panel), following five training sessions under FR1 the effects of amygdala inactivation on early-stage cocaine seeking were tested. Bilateral infusions into the amygdala (either the CeN or the BLA depending on the group) of B/M22 or PBS (counterbalanced) were made. Dopamine receptor blockade in the aDLS was not assessed at this stage as it has already been established that it has no effect on cocaine-seeking at this point40,41.

The response requirement was then increased across the daily training sessions as described for Experiment 1 all the way to FI15(FR10:S). Following 15 sessions of training under this schedule, habitual-stage tests were conducted. Rats received bilateral inactivation (via B/M) of the amygdala (CeN or BLA), bilateral dopamine receptor blockade (α-flupenthixol, 10 μg per side) in the aDLS, a disconnection procedure involving unilateral amygdala inactivation with contralateral aDLS-dopamine receptor blockade and an ipsilateral procedure in which both the amygdala and aDLS were treated in the same hemisphere. For each of these tests, PBS control infusions were intermixed.

For Experiment 3 (Supplementary Fig. 1 bottom panel), following 10 sessions under FR1 rats were trained under fixed interval SOCR with daily increases in the interval duration, from 1 to 15 min as previously described13. Rats were then trained for 20 sessions under the FI15(FR10:S) schedule prior to being challenged with bilateral intra-aDLS infusions of vehicle, SCH23390 or raclopride.

Histology

Histology was conducted as described previously40,41.

Electrophysiological study

Despite the different time course in the functional control over the recruitment of aDLS-dependent cue-controlled cocaine cocaine-seeking behaviour between the BLA and the CeN and their dissociable neural targets, we wanted to identify better the neural pathway whereby the BLA controls aDLS function. Such remote functional connectivity having never been tested before, we decided to use extracellular electrophysiological recordings of aDLS medium spiny neurons to measure the nature and the characteristics of the influence of BLA stimulation over the spike probability of these neurons.

Animals

Male Sprague–Dawley rats (Charles River, France) weighing 300 g at experiment start were housed two per cage under conditions similar to the behavioural experiments.

Surgery

Rats were anesthetized by a single i.p. injection of urethane (1.7 g kg−1, in distilled water). Rats were positioned in a stereotaxic frame (m2e-Unimecanique, France) and maintained at 37 °C with a homeothermic blanket. Burr holes were drilled in the cleaned skull above the electrode targets, and the overlying dura was carefully resected. The coordinates are relative to bregma in the right hemisphere.

Stimulating electrodes

A concentric electrode (SNEX-100, Phymep) was implanted in M1 (AP+4.15; ML+3 ; DV−2.5). A current pulse (0.5-ms long) was applied by an isolated stimulator (DS3 digitimer) triggered by a 1401 Plus system (Cambridge Electronic Design, Cambridge, UK). Because the target aDLS MSNs are silent, cortical stimulation was continuously applied at 0.3 Hz. A bipolar electrode (SNEX-200, Phymep) was lowered into the BLA (AP−2.5; ML+5; DV−7.3). The two poles of the electrodes are oriented in the same sagittal plan.

Electrophysiological recordings and neuron selection

The recording electrode was lowered to the aDLS (AP+1.8; ML+3, from DV−3.3 to DV−4.4) ipsilateral to the stimulating electrodes. The recording electrodes were pulled from borosilicate glass capillaries (GC150F, Harvard Apparatus, UK) using a P97 micropipette puller (Sutter instrument, CA, USA). The tip was broken back under microscopic control to achieve an impedance of 10–15 MΩ as measured in situ with an axoclamp2B (Axon instruments, Foster City, CA) by bridge balance. The electrodes were filled with 0.4 M NaCl. Through the electrode, the extracellular potential was recorded with an axoclamp2B amplifier in the bridge mode versus a reference electrode maintained in contact with the skull skin by a sponge moistened with 0.9% NaCl. The signal was amplified 10 × by the axoclamp2B, further amplified 100 × and band pass filtered (low-pass filter at 300 Hz and high-pass filter at 20 kHz) via a differential AC amplifier (model 1700; AM systems, Carlsborg, WA). Spike occurrence was continuously recorded by a 1401 Plus Cambridge Electronic Design system running Spike 2.

The neurons of interest were isolated by slowly lowering the recording electrode in the aDLS while a single electrical stimulation pulse was delivered to M1 at an overall rate of 0.3 Hz. A total of ∼50 striatal neurons that were excited by stimulation of M1 were recorded. Because MSNs compose ∼95% of striatal neurons64,65 and because putative interneurons were not tested, almost all of the neurons recorded in this study were likely to be the medium spiny subtype identified after stimulation of the primary glutamatergic input to the DLS, namely M1. Neurons were considered as pMSNs based on their electrophysiological properties: a spike duration above 1 ms over 20 trials and the shape of their action potential, as previously described51,66,67 (Supplementary Fig. 6A).

Neurons with sustained spontaneous firing, which produced more than two spikes following a single electrical stimulation, with a spike duration <1 ms were considered to be putative interneurons, and were not analysed.

BLA stimulation protocol

Once a pMSN was found, the effect of the pre-stimulation of the BLA on the M1-evoked spike probability was tested. M1 stimulation current intensity was adjusted to produce about 50% evoked spike probability (the current intensity was adjusted for each neuron). To obtain a stable baseline spiking probability, M1 stimulation was delivered for 20 trials at a rate of 0.3 Hz.

To test the effect of the BLA stimulation alone on the spike probability of the selected pMSNs, M1 stimulation was turned off and BLA stimulation was applied alone for at least 40 trials. Following these 20 trials, a single BLA stimulus pulse (.5 ms, .5 mA) was delivered before the M1 stimulation pulse for 20 trials with IsI ranging from 1 to 1,000 ms (IsI tested: 1, 5, 50, 100, 200, 300, 500, 1,000 ms). These IsI, which were applied following a Latin-square design, were chosen to test mono- and poly-synaptic delays.

AcbC glutamate receptor blockade

AcbC glutamatergic receptors were blocked by an infusion of a mixture of the AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid) and NMDA (N-methyl-d-aspartate) receptor antagonists—CNQX and AP568,69. An injection electrode filled with the CNQX/APV solution (4 mg ml−1 and 8 mg ml−1, respectively) was lowered into the AcbC (AP+1.6; ML+1.4; DV−7) ipsilateral to the stimulating and recording electrodes. The injection electrode was pulled from borosilicate glass capillaries (GC100F, Harvard Apparatus) using a P97 micropipette puller. A scale system on the pipette allowed us to accurately measure each injected volume by visual observation of the meniscus. After the injection, the absence of meniscus movement showed that no spontaneous leakage occurred. The pulled tip of the injection pipette was broken under a microscopic control at an external diameter of ∼30–40 μm. A volume of 0.5 μl of CNQX/AP5 was injected into the AcbC11 with a picoinjector (UC-8 controller, Bioscience tools). BLA–M1 co-stimulations were thus applied and we measured the effect of an infusion of the CNQX/APV mixture into the AcbC on the influence exerted by BLA pre-stimulation over spike probability of aDLS pMSNs in response to M1 stimulation for the specific 100–200–300 ms IsI, those at which the recorded neurons in the aDLS had been shown to be sensitive to BLA stimulation prior to M1 stimulation.

Data and statistical analyses

Data are presented as mean±s.e.m. except when individual points are displayed (Fig. 2b). Analyses were performed using Statsoft STATISTICA 10 software.

Assumptions about normality of distribution and homogeneity of variance were assessed using the Kolmogorov–Smirnov and Levene tests, respectively. In case of violation of at least one of these assumptions, data sets were log-transformed (Fig. 2b,c).

For behavioural studies, lever presses during each 15-min drug-seeking tests were analysed using two-way analyses of variance (ANOVAs) with lever, dose and training level as within-subject factors.

For electrophysiological studies, spike probability evoked by M1 stimulation was calculated as the ratio between the number of evoked APs and the number of stimulation cycles. Change in probability of spike discharge evoked by BLA stimulation was calculated by comparing the probability of spike discharge in M1 stimulation-only situation and the spike probability on each pre-stimulation condition.

A cluster analysis on average spike probability of pMSNs, obtained for the BLA–M1 IsI of 100, 200 and 300 ms, identified three neuronal populations according to the nature of BLA pre-stimulation influence on the spike probability of discharge evoked by the M1 stimulation.

Latency to AP and AP duration were calculated as previously described66. Briefly, the AP duration was measured as the time between start of depolarization and the depolarization of each AP evoked by M1 stimulation. AP waveforms were averaged using the spike 2 software and extracted as the shape of the APs of the recorded neuron.

When no AP was recorded in some downregulated neurons (on only four occasions), provided there was no effect of the stimulation on the AP duration and latency to first AP overall, the value was computed as the average of the other values (50, 100, 200 or 300 ms IsI). Variations in spike probability, latency to first AP and AP duration were analysed by ANOVA with neuronal type as between-subject factor and stimulation condition (including IsI) as within-subject factors.

Significance was set at α=0.05. Significant interactions were analysed further using the Dunnet post hoc or Newman–Keuls post hoc tests where appropriate. Tests used are stated in either the main text or figure legends. Effect sizes are reported using partial η2 values70.