Delay of punishment highlights differential vulnerability to developing addiction-like behavior toward sweet food

Drug addiction involves excessive motivation to consume drugs and continued use despite negative consequences. While motivation and reward mechanisms have been extensively studied, mechanisms underlying persistence of behavior despite adverse outcomes have only recently become a major focus. In animal models, sensitivity to punishment—typically footshock paired with reward-seeking actions—reduces seeking and taking behavior and serves as a marker of the transition to compulsive, addiction-like behavior. Temporal discounting reduces the perceived impact of future events; individuals who discount more steeply are considered more impulsive and at higher risk for addiction. Negative consequences of addictive behaviors are often delayed; thus, individuals who heavily discount delayed punishment may be especially vulnerable. Punishment, like reward, is discounted with delay in humans and animals, making it important to study the behavioral impact of delays between reward-seeking actions and punishment. The authors previously developed a self-adjusting progressive shock strength (PSS) procedure that quantifies individual resistance to punishment while minimizing exposure to highly aversive shocks. In the present study, they used the PSS procedure to test how introducing delays (0–12 s) between lever presses/food delivery and footshock affects resistance to punishment and to characterize individual differences, including relationships with motivation (progressive ratio), anxiety-like behavior, and pain sensitivity.

Prior work shows that contingent footshock punishment reduces operant seeking and taking, and individual differences in punishment sensitivity are linked to addiction-like behavior. Temporal discounting literature indicates that both rewards and punishers lose impact with delay; humans and animals accept more negative consequences when punishment is delayed. Individual differences in delay discounting are associated with impulsivity and addiction risk. Earlier PSS work demonstrated that resistance to punishment is influenced by motivational states and shows trait-like stability, while reducing exposure to excessively aversive shocks compared to fixed-intensity punishment procedures. Studies also suggest that pavlovian (fear/conditioned suppression) and instrumental punishment processes can sometimes be dissociated, and that prior shock history can sensitize or produce tolerance to future punishment. Collectively, these findings motivate assessing how delayed punishment modulates resistance to punishment and whether individual differences emerge under delayed contingencies.

Subjects: Forty-eight male Sprague-Dawley rats (8–9 weeks; Janvier Labs), experimentally naïve, housed under standard conditions. Two cohorts (n=24 each) were used with different delay orders. Procedures followed EU directive 2010/63/EU; approved by COMETHEA. Food restriction: From operant training onward, rats received 20 g/day chow given 1 h after sessions; water ad libitum. Design: After 9 daily FR1 training sessions (45 min each; each active lever press delivered one 45 mg sucrose pellet), rats were tested once per week either in PSS sessions (delayed punishment) or, during the first three PSS weeks on alternate weeks, in a progressive ratio (PR) session. Thereafter, only PSS sessions were interleaved with FR1 training. Delays between lever press/food delivery and footshock were fixed within a session and tested 2–4 times each. Tested delays: 0, 3, 6, and 12 s. Cohort 1 delay order: 0 s (×3), 3 s (×2), 12 s (×3), 6 s (×3). Cohort 2: 6 s (×2), 3 s (×2), 0 s (×2), 12 s (×2). Behavior across cohorts did not differ; data combined. 24–72 h after the last operant sessions, anxiety-like behavior (open field) and pain sensitivity (hot plate) were assessed. Apparatus: MedAssociates operant chambers within sound-attenuating boxes; two levers on right wall, recessed food tray; floor grid connected to shockers (ENV-414SA) delivering footshock at fixed 0.45 mA. A diode above each lever; another diode on opposite wall flashed to indicate punishment contingencies. Events controlled with Med-PC IV. Training (FR1): Each active lever press yielded one pellet; food delivery accompanied by 2-s lever light; followed by 18-s timeout with houselight on. Inactive lever presses recorded but not reinforced. PSS procedure: Self-adjusting progressive shock strength as in Desmercieres et al. (2022). Active lever presses delivered a pellet and a footshock of fixed intensity (0.45 mA) with variable duration. Shock duration escalated across steps if the rat completed 2 trials at the current step. Duration steps (s): 0, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.12, 0.13, 0.15, 0.18, 0.20, 0.23, 0.27, 0.31, 0.35, 0.41, 0.47, 0.54, 0.62, 0.71. If no response occurred for 5 min, duration reset to 0 and progression reinitialized. Shock strength quantified as electrical charge (mC) = current (0.45 mA) × shock duration (s). The PSS breakpoint was the total electrical charge sustained during the session, incorporating both tolerated charge and willingness to resume responding after punishment-induced pauses. PR schedule: Response requirement increased after each pellet per the exponential progression ratio = round((5×e^{0.2×n})−5); step values: 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, 40, 50, 62, 77, 95, 118, 145, 178, 219, 268, 328, 402, 492, 603, 737. Session ended after 10 min without completing a step (≤1 h typical). Open field (anxiety-like behavior): White plexiglass arena 50×50×40 cm; animals habituated 30 min to room; tested 30 min. Video tracking (Viewpoint) computed percent time in center (25×25 cm square) as anxiety-like measure (more center time = less anxiety-like behavior). Hot plate (pain sensitivity): Ugo Basile DS37 at 48 °C. After 10-min habituation, rats placed on plate within 25 cm diameter cylinder; latency to escape/jump recorded; 120 s cutoff. Statistical analysis: Med-PC data converted via Python tool Med_to_csv. Normality assessed (Shapiro–Wilk). Due to non-normal distributions with delays, nonparametric Friedman tests analyzed delay effects in all rats. Median split of 0-s PSS breakpoint (total charge) within each cohort classified rats as shock-sensitive vs shock-resistant (robustness checked with alternative delays/combined cohorts). Mann–Whitney tests compared initial vs final FR1 baselines and pain sensitivity between groups. For delay effects within sensitivity groups, two-way repeated-measures ANOVA (Geisser–Greenhouse correction when needed) with Sidak post hoc tests. Significance at p<0.05.

• Baseline FR1 responding remained stable across the experiment (initial baseline 121.74 ± 1.53 vs final baseline 122.29 ± 2.45 responses/session; Mann–Whitney p=0.0643).
• Delayed punishment reduced the effectiveness of shock as a punisher. Increasing delay produced higher responding and higher tolerated shock: active responses increased from ~22 at 0 s to ~48 at 6 s and ~51 at 12 s; total electrical charge sustained increased from 0.97 mC (0 s) to a maximum of 8.17 mC (12 s). Friedman tests: number of responses p<0.0001; electrical charge p<0.0001.
• PSS breakpoints were highly intercorrelated across all delays (Spearman R^2 > 0.65, p<0.0001), indicating stable individual differences despite delay manipulations.
• PSS and motivation (PR breakpoint) were uncorrelated (Spearman R^2 = 0.00025, p=0.91), indicating distinct processes.
• Classification by median split at 0 s identified shock-sensitive and shock-resistant subgroups. Resistant rats had significantly higher PSS breakpoints than sensitive rats at all delays and showed increases at delays as short as 3 s; sensitive rats showed significant increases only at 12 s. Two-way RM ANOVA: • Sensitivity main effect: Responses F(1,46)=124.4, p<0.0001; Electrical charge F(1,46)=42.24, p<0.0001. • Delay main effect: Responses F(2.328,107.1)=43.10, p<0.0001 (GG ε=0.78); Electrical charge F(1.999,91.95)=31.32, p<0.0001 (GG ε=0.67). • Sensitivity × Delay interaction: Responses F(3,138)=13.01, p<0.0001; Electrical charge F(3,138)=16.13, p<0.0001.
• Conditioned suppression: Shock-sensitive rats showed significant reductions in responding on the session following PSS compared to the day before, at all delays; resistant rats showed no suppression. Two-way RM ANOVA: sensitivity main effect F(1,46)=44.92, p<0.0001; no delay effect; no interaction.
• Pain sensitivity (hot plate): No group difference (Mann–Whitney U=258.5, p=0.54) and no correlation with PSS (Spearman R^2=0.010, p=0.49).
• Anxiety-like behavior (open field): Sensitive rats showed higher anxiety-like behavior (less center time) than resistant rats (Mann–Whitney U=164.5, p<0.05) and modest correlation with PSS (Spearman R^2=0.10, p=0.027).
• Day-to-day behavior indicated that sensitive rats developed conditioned suppression even in non-punished FR1 sessions, returning to baseline only after several shock-free sessions; PR performance was similar between groups.

Introducing a delay between action/food delivery and footshock reduced the efficacy of punishment, consistent with temporal discounting of aversive outcomes. Critically, delay highlighted two phenotypes: a shock-resistant subgroup that tolerated higher punishment and showed steeper discounting of delayed punishment, and a shock-sensitive subgroup that remained sensitive to punishment even when delayed, with minimal discounting. These patterns model clinically relevant vulnerabilities: excessive discounting of negative future consequences can promote persistent pursuit of rewards despite delayed harms, a hallmark of addiction-like behavior. Conversely, shock-sensitive animals exhibited conditioned suppression and elevated anxiety-like behavior, suggesting vulnerability to opportunity loss due to fear/avoidance even when immediate threat is absent. Resistance to punishment did not reflect greater appetitive motivation (no correlation with PR) or reduced pain sensitivity, indicating distinct underlying processes. The PSS procedure, which does not separate pavlovian from instrumental components, likely promotes strong conditioned suppression in sensitive rats; nevertheless, individual rankings of punishment resistance were stable across months of testing and across delays, supporting trait-like consistency. These findings underscore the importance of temporal contingencies in punishment when modeling compulsive behavior and suggest that delayed punishment in PSS can identify individuals at dual risk: low aversive sensitivity and steep temporal discounting of negative outcomes.

The study demonstrates that delaying punishment reduces its suppressive effect on food-seeking and reveals robust individual differences: a shock-resistant subgroup that discounts delayed punishment steeply and a shock-sensitive subgroup that remains highly suppressible and develops conditioned suppression and anxiety-like behavior. The PSS procedure provides a refined, self-adjusting measure of resistance to punishment and, with delayed contingencies, can identify phenotypes potentially vulnerable to addiction-like behavior or to opportunity loss driven by anxiety. Future research should: (1) test delayed punishment in females; (2) further dissociate pavlovian fear and instrumental punishment contributions; (3) evaluate whether delayed-punishment PSS predicts susceptibility to drug addiction; and (4) refine sensory-specific assessments of pain to better parse contributions of nociception versus aversive learning.

• Sex limitation: Only male rats were studied; prior work suggests sex differences in discounting and probabilistic punishment.
• Pain assessment mismatch: Pain sensitivity measured with thermal nociception (hot plate) whereas punishment used electrical footshock; this provides a coarse measure and may miss modality-specific differences.
• Timing of anxiety assessment: Anxiety-like behavior was measured at the end of prolonged PSS training and may reflect consequences of prior punishment experience rather than pre-existing traits.
• Pavlovian-operant confound: The PSS procedure does not isolate pavlovian conditioned fear from instrumental punishment processes, potentially contributing to conditioned suppression in sensitive rats.
• Prior shock exposure effects: Although PSS minimizes exposure to highly aversive shocks, prior shock experience can induce tolerance or sensitization, which could influence later behavior.