Ishikawa A, Ambroggi F, Nicola SM, and Fields HL Presented by: Andrew Burke

1. Dorsomedial Prefrontal Cortex Contribution to Behavioral and Nucleus Accumbens Neuronal Responses to Incentive Cues Ishikawa A, Ambroggi F, Nicola SM, and Fields HL Presented by: Andrew Burke

2. Mesocorticolimbic System Medial Prefrontal Cortex mPFC NAc VTA Nucleus Accumbens BLA Basolateral Amygdala Ventral Tegmental Area Dopamine Cells For review: Wise (1996) Neurobiol of Addiction

3. Medial Prefrontal Cortex (mPFC) Nucleus Accumbens Core &Shell Cg PrL IL Core Shell • Cg = Cingulate Cortex • PrL = Prelimbic Cortex • IL = Infralimbic Cortex Next slide

4. Medial Prefrontal Cortex (mPFC) Nucleus Accumbens Core &Shell Cg PrL IL Core Shell • Cg = Cingulate Cortex • PrL = Prelimbic Cortex • IL = Infralimbic Cortex

5. Figure 8-Recording and Injection Sites

6. Nucleus Accumbens (NAc) • “Limbic-motor interface” • Facilitates appropriate responding to reward-predictive stimuli • Subpopulations of NAc neurons • Excited/inhibited by discriminative stimuli (DSs) • Predict reward availability • Source of cue-evoked firing of the NAc • mPFC • BLA • VTA Glu NAc Glu DA

7. Background (Yun et al, 2004) • Inactivation of VTA by GABAB agonist, baclofen • Reduced behavioral response to reward associated cue • Reduced NAc firing in response to reward associated cue

8. Background (Nicola et al, 2005) • Manipulating DA activity in the nucleus accumbens (NAc) core and shell • DAT blocker (GBR12909) increased behavioral responding to a predictive stimulus • D1 receptor antagonist decreased behavioral responding to a predictive stimulus • Dopamine acts on NAc (GABA) neurons to mediate responding to a cue for reward

9. Background (2007 SFN abstracts) • Inactivation of BLA impaired behavioral responding to predictive cues • Also reduced cue-induced firing in NAc • Inactivation of dmPFC impaired behavioral responding to predicitive cues • ??

10. The Hypothesis • Inputs to the NAc contribute to cue-evoked excitations of NAc neurons include glutamergic inputs from the mPFC.

11. Animals • Adult Long-Evans rats • ~350 g (n=9) • Housed individually • 1 week of restricted food and water before training • 13 g food and 30 ml H2O during expts. • Experiments conducted in the dark (12h hours of dark per day)

12. Methods • Placed in operant chamber • White noise 65dB and orange houselight ON always • Given 2 auditory stimuli • Intermittent 6 kHz tone on 40 ms and off 50 ms (90 ms period) • Siren-frequency ramped from 4 to 8 kHz and back with 400 ms cycle period Operant Chamber White noise (65 dB) 2 stimuli (85 dB) Auditory Stimuli

13. Discriminative Stimuli Task • Auditory stimuli=tone cues were either: • Discriminative stimuli (DS) (AKA “incentive cue”) - predicted reward delivery after correct lever press during DS presentation • Nonrewarded stimulus (NS) - lever pressing did not trigger reward delivery • Active lever and DS tone were counterbalanced • Pressing the active lever during DS tone = termination of DS = 50 ml of 10% sucrose into reward receptacle • Cues presented on a variable interval schedule with average interval of 30 s • The DS (on for up to 10 s) or NS (10 s) was randomly presented at end of each interval

14. Training Procedures • Stage 1 • Introduced to chamber • Entry into reward receptacle or pressing either lever triggered delivery of 50 ml of a 10% sucrose solution • 10 s timeout was imposed after reward delivery when reward could not be delivered • Trained until they learned to obtain 100 rewards in <1 hr

15. Training Procedures • Stage 2 • 2-lever fixed ratio (FR) 1 task • Response on either lever triggered reward delivery followed by 3 s timeout. • Remained in this stage until they learned to obtain all 100 rewards in < 1 hr.

16. Training Procedures • Stage 3 • One-lever FR task • Pressing active lever during cue presentation triggered reward delivery with 10 s timeout • Cue presented at end of timeout and remained on until an active lever press • Lever pressing in absence of the cue was not rewarded • Timeout increased from 10 s to 20 s then to 30 s when rats obtained >100 rewards during the session • Stayed in stage 3 until latency to press lever was <15 s after DS cue presentation

17. Training Procedures • Stage 4 • Trained on DS task until the • Correct response to the DS occurred >90% of the time • Incorrect responses to the NS only occurred about 20% of the time • When this criteria was met rats were rewarded with brain surgery

18. Surgery • Anesthetized with isoflurane • Implanted with bilateral guide cannula (27ga) directed at the dmPFC • Bilateral recording electrode arrays (consisting of 8 electrodes) were implanted into the Nac core • Rats allowed to recover from surgery for 1 week before experiment began

19. Electrophysiology and Microinjections • Retrained on DS task for >6 days with recording cables connected • Disabling the dmPFC • Performed DS task for 60 min to obtain a baseline for recording • Removed from behavior/recording chamber • Infused saline or M/B (mixed solution containing 25ng of muscimol (GABAA agonist) and 50ng baclofen (GABAB agonist) into dmPFC slowly (4 min total) • Returned to chamber immediately for a 2 h post injection session for subsequent recording

20. Microinjection confusion? • “All 9 rats received bilateral injection of all drugs” • “unilateral injections of saline, 25 or 50 ng M/B were given to eight, nine, and six rats, respectively” • 8 of the 9 received Saline, 9=25ng, 6=50ng? • In 4 rats bilateral injection was first • In 2 rats unilateral injection was first • The remaining 3 rats were given injections in random order • Doses were always given in random order • Two animals were implanted with movable electrodes that could be deepened by 150 µm

21. Data Analysis • Behavior analyzed • DS response latency • Rate of uncued responding on active levers • Rate of responding in the absence of DS and NS • DS and NS response ratios • Proportion of these cues during which the animal made a response on active lever • Effects of bilateral inj. analyzed with 1-way repeated measures ANOVA • Effect of unilateral inj. analyzed with 1-way ANOVA • Post hoc test: Fisher’s PLSD

22. Electrophysiology Data Analysis • Raster plots were also used to visualize firing of individual neurons • Incentive cue, operant and receptacle exit related responses illustrated with a perievent time histogram (PETH) (AKA, peristimulus time histogram) • Mean firing increases or decreases and pre-DS baselines were compared across neurons between pre and post injection conditions using the paired t test

23. Figure 1-inactivation of the dmPFC reduces behavioral responding to DSs Results indicate that the dmPFC is required for behavioral responding to reward-predicative cues during the DS task.

24. Figure 2

25. Figure 2-Inactivation of the dmPFC reduces the DS-evoked excitation of Nac neurons

26. Figure 2-Inactivation of the dmPFC reduces the DS-evoked excitation of NAc neurons Averaged PETHs (0.5 s bin width) of all recorded cue-excited neurons before (black) and after (red) 25 ng M/B, 50 ng of M/B, and saline injections.

27. Figure 2-Inactivation of the dmPFC reduces the DS-evoked excitation of NAc neurons Comparisons of DS-evoked excitations before and after each drug injection.

28. Figure 3-Inactivation of the dmPFC reduces the DS-evoked inhibition of NAc neurons

29. Figure 3-Inactivation of the dmPFC reduces the DS-evoked inhibition of NAc neurons

30. Results Figures 2 and 3 • Both excitatory and inhibitory neuronal profiles in response to the reward associated cue were reduced when dmPFC is inactivated • Previous study (Nicola, et al 2004) suggests that incentive cue excitations AND inhibitions are reduced when the animals fails to make the appropriate response (press the lever)

31. Figure 4-Inactivation of the dmPFC reduces incentive cue excitation and inhibition on trials when the animal makes appropriate response to the incentive cue (DS). Excitatory Reduction of incentive cue excitation/inhibition after dmPFC inactivation is at least in part responsible for the reduction in behavioral responding to the DS. Inhibitory

32. Figure 5 - Inactivation of the dmPFC does NOT affect operant, reward-related and receptacle exit neuronal firing except for a small effect of 25 ng of M/B on operant excitations.

33. Figure 5 - Receptacle entry

34. Figure 5 - Receptacle Exit

35. Table 1

36. Figure 6 - Correlation of DS responding behavior with neuronal firing to DSs. A, B: DS response ratio was significantly correlated with the magnitude of DS excitation but not DS inhibition. C: negative correlation between DS excitation and behavioral response latency Suggest that reduced excitation of NAc core after dmPFC inactivation may be the cause of impaired behavioral responses to the cue.

37. Figure 7-Unilateral dmPFC inactivation reduces DS responding and both ipsilateral DS excitation and contralateral DS inhibition

38. Figure 7 - Unilateral dmPFC inactivation on NAc neuronal firing • Ipsilateral excitatory projection from the dmPFC to the NAc is essential for appropriate cue responding (E). • Could be a contribution to incentive cue responding for the contralateral inhibitory effect (H)

39. Conclusions • M/B inactivation of the dmPFC • Reduced operant behaviors in response to incentive cues during this DS task • Minimal effects on consumption behaviors • Reduced magnitude of excitatory neuronal responses in NAc Core • Reduced the magnitude of inhibitory neuronal responses in NAc Core • Minimal effects on baseline firing in NAc Core

40. Conclusions - Dopamine • Dopamine alone does not directly excite NAc neurons (Nicola, et al 2000, 2004) • Dopmaine manipulations altered responding for cue (Nicola, et al 2005) • The enhanced neuronal and behavioral responding after DA release onto NAc and PFC may be caused by increasing PFC glutamate excitatory afferents to the NAc GABA neurons

41. Extrapolation to drug reinstatement • dmPFCNAc core projection is implicated in the reinstatement of drug seeking (Kalivas) • Authors suggest a parallel between present findings and those of drug reward • Is the incentive salience of sucrose reward and drug the same? • Suggested that other areas, excluding mesolimbic DA system, mediates incentive value of food rewards, also learning new associations between unconditioned stimuli and reward (ie, TPP, NAcopiod system) • Are the same pathways and mechanisms involved in both drug reinstatement and palatable reward in food deprived animals? • “Dopamine systems are only implicated in reward when individuals are in a state of physiological deprivation” • Discussed in Berridge & Robinson (1998)

42. Tonic or Phasic Activity • Inactivating VTA reduced cue excitatory & inhibitory (phasic) and baseline (tonic) NAc activity, & reduced beh response (Yun, et al 2004) • Reduced DA in mPFC after VTA inactivation inactivates the mPFC and NAc • Here, mPFC blockade is sufficient to reduce behavioral response and blocks mostly phasic activity • It is the phasic responses, not tonic, that drive this incentive cue behavior

43. Cue-evoked inhibitions • mPFC Glu projection to NAc is excitatory and should cause increased firing rate • A direct ipsilateral projection is likely responsible for the excitatory NAc response to the cue • Perhaps the projections responsible for inhibitory responses in NAc derive from polysynaptic connections • i.e., PFC(GLU+)NAc?(GABA-)NAc(GABA-)

44. References • Ambroggi F, Ishikawa A, Seroussi A, Fields HL, Nicola SM (2007) Evidence that dopamine enhance nucleus accumbens responses to incentive cues by gating an excitatory input from the basolateralamygdala. Soc NeurosciAbstr 33:310.8. • Berridge K, Rominson TE (1998) What is the role of dopamine in reward: hedonic impact, reward learning, or incentive salience? Brain Research Rev 28:309-369. • Ishikawa A, Ambroggi F, Nicola SM, Fields HL (2007) Contrasting contributions of the prefrontal cortex and amygdala to cue-evoked reward seeking behavior. Soc NeurosciAbstr 33:310.10. • McFarland K, Kalivas PW (2001) The circuitry mediating cocaine-induced reinstatement of drug-seeking behavior. J Neurosci 21:8655– 8663. • Nicola SM, Surmeier J, Malenka RC (2000) Dopaminergic modulation of neuronal excitability in the striatum and nucleus accumbens. Annu Rev Neurosci 23:185–215. • Nicola SM, Taha SA, Kim SW, Fields HL (2005) Nucleus accumbens dopamine release is necessary and sufficient to promote the behavioral response to reward-predictive cues. Neuroscience 135:1025–1033. • Yun IA, Wakabayashi KT, Fields HL, Nicola SM (2004b) The ventral tegmental area is required for the behavioral and nucleus accumbens neuronal firing responses to incentive cues. J Neurosci 24:2923–2933.