Entopeduncular Nucleus Projections to the Lateral Habenula Contribute to Cocaine Avoidance

Abstract

The aversive properties associated with drugs of abuse influence both the development of addiction and relapse. Cocaine produces strong aversive effects after rewarding effects wear off, accompanied by increased firing in the lateral habenula (LHb) that contributes to downstream activation of the rostromedial tegmental nucleus (RMTg). However, the sources of this LHb activation are unknown, as the LHb receives many excitatory inputs whose contributions to cocaine aversion remain uncharacterized. Using cFos activation and in vivo electrophysiology in male rats, we demonstrated that the rostral entopeduncular nucleus (rEPN) was the most responsive region to cocaine among LHb afferents examined and that single cocaine infusions induced biphasic responses in rEPN neurons, with inhibition during cocaine's initial rewarding phase transitioning to excitation during cocaine's delayed aversive phase. Furthermore, rEPN lesions reduced cocaine-induced cFos activation by 2-fold in the LHb and by a smaller proportion in the RMTg, while inactivation of the rEPN or the rEPN-LHb pathway attenuated cocaine avoidance behaviors measured by an operant runway task and by conditioned place aversion (CPA). These data show an essential but not exclusive role of rEPN and its projections to the LHb in processing the aversive effects of cocaine, which could serve as a novel target for addiction vulnerability.SIGNIFICANCE STATEMENT Cocaine produces well-known rewarding effects but also strong aversive effects that influence addiction propensity, but whose mechanisms are poorly understood. We had previously reported that the lateral habenula (LHb) is activated by cocaine and contributes to cocaine's aversive effects, and the current findings show that the rostral entopeduncular nucleus (rEPN) is a major contributor to this LHb activation and to conditioned avoidance of cocaine. These findings show a critical, though not exclusive, rEPN role in cocaine's aversive effects, and shed light on the development of addiction.

Keywords: addiction; avoidance; cocaine; entopeduncular nucleus; lateral habenula; rostromedial tegmental nucleus.

Figure 1.

LHb afferents are activated by acute cocaine infusion. A, Schematics of experimental procedure and cocaine cFos induction, and representative photomicrograph of immunostaining of CTb injecton site in the LHb. B, The LH showed the highest proportion of CTb-labeled neurons among these LHb afferent. C, Proportions of CTb-labeled neurons expressing cFos in the VP, LPO, LH, and rEPN, showing the largest cocaine-induced increases in LHb-projecting neurons in the rEPN. D, Representative photomicrographs of immunostaning of CTb and cFos in the rEPN, LH, and VP. Black arrows indicate neurons immunoreactive for both CTb and cFos, red arrows indicate single labeled CTb neurons. Scale bars: 100 µm (A, D).

Figure 2.

rEPN neuron activation during cocaine's delayed aversive phase. A, Schematic of recording experiments in rEPN (upper panel) and recording sites in rEPN (lower panel), showing that recording sites were located in the LHb-projecting region. B, Schematic of recording paradigm. Animals performed a discrimination task containing of a block of 100 reward (food), neutral, and shock trials, followed by a single dose of cocaine or saline intravenous infusion. C, Behavioral outcomes during reward training. Animals were first trained with reward and neutral trials for 8 d before adding the shock trials for discrimination task. Trials were successful if animals entered food port within 2 s after reward cue onset, or withheld entry for 2 s after neutral or shock cues. D, Behavioral outcomes for individual rats during each recording session of discrimination task. Each dot in reward, neutral, and shock trials represents one individual rat, and lines connect sessions from the same rats. E, Average responses of rEPN neurons to reward, neutral, and shock trials in the discrimination task. Black bars indicate the analysis windows used in the adjacent panel. Reward cue responses were averaged in a 200- to 400-ms time window after cue onset, while neutral and shock cue response were averaged during a 0- to 100-ms time window. F, Among these neurons, 10/20 showed biphasic responses to cocaine with inhibition 0–10 min and excitation 15–30 min after injections (red trace), while 4/20 showed only inhibition (blue trace) and 6/20 showed excitation or no response to cocaine (black trace). These neurons did not respond to saline infusion (dashed trace). Responses of biphasic and inhibited neurons 0–10 min postcocaine were significantly decreased compared with saline, and response of biphasic neurons 15–30 min postcocaine were significantly increased. G, A majority (8 out of 10) of biphasic neurons also exhibited monotonic encoding of valence cues. H–J, Individual neuron responses 0–10 and 15–30 min after cocaine infusions positively correlated with responses to reward cues and shock cues/shocks during 200- to 400- and 0- to 100-ms poststimulus time windows.

Figure 3.

rEPN lesion deminished cocaine-induced cFos in the LHb and RMTg. A, Experimental procedure and representative photomicrographs of the ipsilateral (lesioned) and contralateral (intact) rEPN for the neuronal marker NeuN. Scale bar: 100 µm. B, Illustration of the extent of the lesion for four lesioned animals. C, Representative photomicrographs of immunostaining of LHb and RMTg cFos in the lesion side and the intact side. Scale bar: 100 µm. D, rEPN lesion greatly reduced cocaine-induced cFos in the LHb, while slightly diminishing cFos in the RMTg.

Figure 4.

The rEPN and its projections to the LHb mediate cocaine avoidance behaviors. A, Cocaine runway paradigm. In each session, animals started at the start box and received a single cocaine infusion uponreaching the goal box, or timed out after 900 s and was manually placed into goal box and given cocaine. B, Muscimol (red dots) and vehicle (blue dots) infusion sites in the rEPN. rEPN muscimol infusions blocked development of cocaine avoidance (C) and reduced run direction reversals (D) during sessions 1–7. E, Locomotion averaged over sessions 1–7 was not different between groups. F, Behavioral paradigm of CPA. Animals received CNO infusion into the LHb 10 min after cocaine infusion and were placed into paired chamber 5 min after CNO infusion for 15 min. G, Photomicrographs of immunostaining of mCherry in the rEPN and LHb. H, CNO (red dots) and vehicle (blue dots) infusion sites in the LHb. I, Illustration of the extent of the virus expression for eight hM4Di animals. J, K, Inactivation of rEPN-LHb pathway blocked CPA induced by acute cocaine withdrawal. Preference sores are calculated by the difference in time spent and number of entries to the cocaine-paired chamber before and after the conditioning.