**Background:** Impulsivity and novelty-seeking are well-established risk factors for the development of substance use disorder and behavioral addictions. Dysfunction in the medial prefrontal cortex (mPFC) is central to these disorders, but the specific contributions of distinct neuronal populations within the mPFC remain poorly understood. Vasoactive intestinal peptide (VIP)-expressing interneurons in the mPFC receive convergent glutamatergic, serotonergic, and cholinergic inputs from extra-cortical regions, positioning them as potential mediators between long-range inputs and local cortical processing. VIP interneurons provide inhibitory input onto somatostatin (SST) interneurons, which in turn innervate pyramidal neurons, creating a disinhibitory circuit. The infralimbic cortex (IL), a subregion of the mPFC, has been specifically implicated in behavioral inhibition and response control. This study hypothesized that selective ablation of VIP interneurons in the IL would modulate impulsive responding and novelty-seeking behavior.
**Methods:** The authors generated heterozygous VIP::ZsGreen mice by crossing VIP-IRES-Cre mice with Ai6 reporter mice, resulting in ZsGreen expression localized to VIP-expressing neurons. Selective ablation of VIP interneurons was achieved via bilateral stereotaxic injection of 400 nL of a cre-dependent caspase-3 AAV (pAAV-flex-taCasp3-TEVp) into the border of the IL and dorsal peduncular cortex (coordinates: +1.54 mm from bregma, ±0.3 mm lateral, 3.3 mm ventral). Sham animals received identical injections of sterile saline. After 14 days for viral expression and recovery, mice underwent behavioral testing in the following order: (1) three-choice serial reaction time task (3CSRTT) to measure motor impulsivity, (2) open field test for spatial anxiety-like behavior and locomotion, (3) social interaction test for novelty-seeking, and (4) binge-like eating assay for hedonic food intake. In the 3CSRTT, mice were trained to respond to illuminated nose poke holes within 5 seconds. During testing, the intertrial interval (ITI) was randomized between 5 s, 7.5 s, and 12.5 s across 250 trials. Premature responses (poking during the ITI) served as the primary measure of impulsive action. Ablation was confirmed post-mortem by quantifying ZsGreen-expressing neurons in the IL region between +1.10 mm and +1.98 mm rostral of bregma.
**Key Results:** Caspase-3 injection successfully ablated VIP interneurons in the IL, confirmed by significant loss of ZsGreen-expressing neurons (unpaired t-test, p = 0.0164). In the 3CSRTT, there was no statistically significant difference in premature, correct, or incorrect responding between sham and ablated animals when all ITIs were pooled, though a trend toward increased premature responses was observed (unpaired t-test, p = 0.093). However, when trials were separated by ITI duration, ablated animals showed a significant increase in premature responses exclusively during the longest ITI (12.5 s; p = 0.004). A two-way ANOVA revealed a significant interaction between treatment and ITI-dependent response type (F(8,206) = 7.056, p < 0.0001). This effect was observed in both sexes with no significant sex differences. In the social interaction assay, VIP-ablated mice did not spend significantly more time exploring a novel mouse (unpaired t-test, p = 0.5532, 95% CI = [-24.79, 13.59]) and did not show significantly different latency to first contact (unpaired t-test, p = 0.5929, 95% CI = [-43.73, 25.52]). In the open field test, ablation did not affect time spent in the center (unpaired t-test, p = 0.2851, 95% CI = [-26.19, 8.036]) or total locomotion (unpaired t-test, p = 0.7447, 95% CI = [-5751, 4166]). In the binge-like eating assay, ablation did not significantly alter high-fat diet consumption over 30 minutes (unpaired t-test, p = 0.8128, 95% CI = [-0.2789, 0.3523]).
**Clinical Implications:** This study provides the first behavioral characterization of IL VIP interneuron ablation, demonstrating that these neurons are specifically required for controlling impulsive responding during periods of high expectation (long-delay trials) without affecting anxiety, novelty-seeking, or hedonic feeding. The findings suggest a circuit-level mechanism in which VIP interneurons function as an adaptive disinhibitory gate that becomes active specifically during prolonged waiting periods. When this gate is absent (via ablation), impulsive responding increases. This mechanism aligns with the known role of the mPFC in appropriately timed reactions and response inhibition. The specificity of the effect—increased impulsivity without broader behavioral changes—highlights VIP interneurons as a potential therapeutic target for disorders characterized by impulsivity, such as substance use disorder and behavioral addictions. The study also notes that while no sex-specific differences reached statistical significance, the study may lack sufficient power to detect such effects (p_female = 0.112, p_male = 0.173 for long-delay premature responses). Limitations include the use of a global ablation approach rather than temporally precise manipulation, and the possibility that compensatory mechanisms from parvalbumin and somatostatin interneurons may mask additional behavioral effects.