Neuropeptide Y Impairs Retrieval of Extinguished Fear and Modulates Excitability of Neurons in the Infralimbic Prefrontal Cortex

Abstract

Neuropeptide Y (NPY), a 36 aa peptide, regulates stress and emotional behaviors. Preclinical and clinical studies support an association of NPY with trauma-evoked syndromes such as posttraumatic stress disorder (PTSD), although the exact contribution of NPY is not clear. In the current study, we examined functional attributes of NPY in the infralimbic (IL) cortex, an area that regulates fear memories and is reported to be hypoactive in PTSD. Carriers of NPY gene polymorphism rs16147 have been reported to have elevated prefrontal NPY expression. Infusion of NPY into the IL cortex in rats significantly impaired fear extinction memory without affecting conditioned fear expression or acquisition of extinction. Neuroendocrine stress response, depression-like behavior, and working memory performance were not affected by NPY infusion into the IL. The NPY Y1 receptor antagonist BIBO3304 completely abolished NPY effects on fear extinction retrieval. Y1 receptor expression was localized on CaMKII-positive pyramidal projection neurons and GAD67-positive interneurons in the IL. Patch-clamp recordings revealed increased inhibitory synaptic transmission onto IL projection neurons in the presence of NPY. Thus, NPY dampens excitability of IL projection neurons and impairs retrieval of extinction memory by inhibiting consolidation of extinction. Of relevance to PTSD, elevation of prefrontal NPY attributable to the genetic polymorphism rs16147 may contribute to IL hypoactivity, resulting in impaired extinction memory and susceptibility to the disorder.

Significance statement: Neuropeptide Y (NPY), a stress modulatory transmitter, is associated with posttraumatic stress disorder (PTSD). Contribution of NPY to PTSD symptomology is unclear. PTSD patients have reduced activity in the infralimbic (IL) subdivision of the medial prefrontal cortex (mPFC), associated with compromised extinction memory. No information exists on fear modulation by NPY in the IL cortex, although NPY and NPY receptors are abundant in these areas. This study shows that IL NPY inhibits consolidation of extinction, resulting in impaired retrieval of extinction memory and modulates excitability of IL projection neurons. In addition to providing a novel perspective on extinction memory modulation by NPY, our findings suggest that elevated mPFC NPY in gene polymorphism rs16147 carriers or after chronic stress could increase susceptibility to PTSD.

Keywords: Neuropeptide Y; Y1; extinction; fear; infralimbic; prefrontal.

Figure 1.

NPY infusion into the IL cortex impairs retrieval of extinction. A, Representative illustration showing location of cannula placements in the IL cortex based on histological assessment. Coronal sections represent coordinates +3.2 mm (top) and +2.7 mm (bottom) relative to bregma. B, Image showing the spread of fluorescent labeled NPY (FAM-NPY) within the IL subdivision of the mPFC. C, Timeline of the experiment showing infusion of NPY before extinction training on Day 2. D, Freezing response to tone for rats infused with aCSF (n = 12) or NPY (3, 10, and 30 pmol/0.2 μl; n = 6–8). Rats infused with 10 or 30 pmol NPY showed significantly higher freezing compared with aCSF rats during extinction retrieval on Day 3, as noted by higher levels of freezing (right). No significant difference in percentage freezing was noted during conditioning (left) or extinction training (middle). E, Schematic for post-extinction training infusion of NPY. F, Infusion of NPY (10 pmol) into the IL post-extinction acquisition significantly impaired retrieval of extinguished fear compared with the aCSF-infused group (n = 7 per group). Data shown are mean ± SEM; *p < 0.05 versus aCSF and 3 pmol NPY (Fig. 1D) or aCSF (Fig. 1F) group. For conditioning, data on post-shock freezing for shocks 1–6 are shown. For extinction and retrieval, data from tones 1–9 are shown. Magnitude of freezing plateaued at these time points, and no additional changes in freezing were observed in subsequent trials. PL, Prelimbic cortex; CC, corpus callosum. BL represents baseline freezing during 2 min before the delivery of conditioning, extinction, or retrieval trials.

Figure 2.

NPY Y₁R subtype mediates effects of NPY on extinction memory. A, Timeline of experiment. Y₁R antagonist BIBO was infused 10 min before NPY infusion 30 min before extinction training on Day 2. B, Freezing response of rats infused with aCSF (n = 7), NPY (10 pmol; n = 9), BIBO (200 pmol; n = 8), and BIBO and NPY (BIBO-NPY; n = 10). NPY-evoked impairment of extinction retrieval (increased freezing; right) was attenuated significantly in rats infused with BIBO to levels observed in the aCSF treatment group. Rats infused with BIBO by itself did not elicit any significant differences in freezing during retrieval of extinction compared with aCSF cohort. No significant differences in conditioning (left) or conditioned fear expression and extinction learning (middle) were observed between groups. C, Mean percentage freezing during trials 1–4 showed significantly increased freezing in the NPY treatment group compared with aCSF, BIBO-NPY, and BIBO-alone groups. Data shown are mean ± SEM. * p < 0.05 versus other groups; ^#p < 0.05 versus the BIBO-NPY group. For conditioning, data on post-shock freezing for shocks 1–6 is shown. For extinction and retrieval, data from tones 1–9 is shown. Magnitude of freezing plateaued at these time points, and no additional changes in freezing were observed for subsequent trials. BL represents baseline freezing during 2 min before the delivery of conditioning, extinction, or retrieval trials.

Figure 3.

Infusion of NPY into the IL cortex does not affect postrestraint neuroendocrine stress response, depression-like behavior in the FST, or working memory performance in the RAM. A, HPA response to restraint stress. Blood was collected from the tail vein at 15, 30, 60, and 120 min from the time the animals were exposed to the stressor (aCSF, n = 10; NPY, n = 9). B, FST behavior test. Immobility scores over 5 min for aCSF-infused (n = 12) and NPY-infused (n = 11) rats in the FST. Scores during the initial 5 min are reported because of high immobility in both groups after this period. C, D, RAM memory test. Mean across-phase (C) and within-phase (D) errors during retrieval testing are reported (n = 10 per group). No significant difference was noted between the NPY- and aCSF-infused animals in HPA response, immobility in the FST, or mean across-phase errors or within-phase errors in the RAM test compared with aCSF-infused rats. Data are mean ± SEM (p > 0.05).

Figure 4.

NPY potentiates GABAergic inhibition onto layer V pyramidal neurons in the IL-PFC. A, Examples of IPSCs in a layer V IL-PFC neuron after focal electrical stimulation in layers II–III, before (Baseline) and after the administration of NPY (1 μm). B, NPY significantly enhanced IPSC amplitude in these neurons (aCSF, n = 5; NPY, n = 11; p < 0.0001; two-way ANOVA). C, Representative examples of AMPAR-mediated EPSCs in a layer V neuron either before (Baseline) or after bath application of NPY (1 μm). D, Plot of normalized EPSC amplitude as a function of time demonstrating a slight but significant reduction in EPSC amplitudes after NPY exposure (n = 12; p = 0.028; two-way ANOVA) compared with control conditions (i.e., continued perfusion with aCSF; n = 5).

Figure 5.

Colocalization of Y₁R (A–F) and NPY (G–I) with CaMKII-IR or GAD67-IR cells in the IL cortex. Y₁R-immunopositive cells (A) showed colocalization with CaMKII-IR cells (B) as seen in merged image (C). A few Y₁R-positive cells also colocalized with GAD67-IR cells (E) as seen in merged image (F). NPY-IR cells (G) showed colocalization with GAD67-IR cells (H; see I for merged image). Double-labeled cells (arrows) and single-labeled CaMKII or GAD67 cells (arrowheads) are visible. Scale bar, 20 μm.