The impact of limited sucrose intake on perineuronal nets of parvalbumin interneurons in the basolateral amygdala: A potential role in stress resilience

Abstract

Natural rewards like regular sucrose consumption can buffer physiological and behavioral stress responses, likely mediated, at least in part, by increased plasticity in parvalbumin-positive (PV+) interneurons in the basolateral amygdala (BLA). As PV+ interneuron plasticity is tightly regulated by specialized extracellular matrix structures called perineuronal nets (PNNs), this study investigated the impact of regular sucrose consumption vs. repetitive stress on the PNNs that surround PV+ interneurons in the BLA, as well as the number of glutamatergic (vGLUT1) and GABAergic (vGAT) appositions that PV+ cells receive. Male rats were given an established limited sucrose intake (LSI) feeding paradigm (vs. water-fed controls) and were co-exposed to a brief restraint stress (vs. no stress controls), daily for 14 days. Sucrose consumption increased the proportion of PV+ cells that were surrounded by PNNs, independent of stress exposure. PV+ cells with PNNs had more vGLUT1-positive and fewer vGAT-positive appositions compared to those lacking PNNs. Additionally, sucrose consumption increased the ratio of excitatory/inhibitory appositions onto PV+ cells, suggesting the possibility of elevated PV+ interneuron tone, leading to greater inhibition of the BLA's stress-excitatory output. These findings indicate that sucrose consumption influences PNN formation and structural plasticity on PV+ interneurons in the BLA, which has implications for understanding the neurological mechanisms underlying stress resilience by natural rewards.

Keywords: Basolateral amygdala; Parvalbumin; Perineuronal nets; Stress; Structural plasticity; Sucrose.

Figure 1:

(A) Experimental timeline of LSI and restraint stress protocols. (B) Representative confocal image of PV+ interneurons (magenta) and their perineuronal nets (PNNs; green) in the basolateral amygdala (BLA). PNNs were labeled using Wisteria floribunda agglutinin (WFA). White arrowheads point to PV+ cells with full PNNs while empty arrowheads point to PV+ cells with partial PNNs, both counted as PV+/WFA+ cells. Asterisks denote PV+ cells lacking a PNN (PV+/WFA−).

Figure 2:

The impact of sucrose consumption on food intake, total caloric intake, and body weight in stressed and unstressed rats. (A) Rats offered a 30% sucrose solution began to consume nearly the maximum volume provided (8 mL/day), while those offered water did not (interactive effect (p < 0.05) of DRINK X TIME). (B) Rats consuming sucrose reduced their chow intake isocalorically to compensate for the additional calories from sucrose, and repeated restraint stress reduced chow intake regardless of drink type (interactive effects (p < 0.05) of DRINK X TIME, and of STRESS X TIME). (C) Total daily caloric intake was similar between sucrose-drinking rats and water-drinking controls, whereas stress reduced total caloric intake regardless of drink type (interactive effect (p < 0.05) of STRESS X TIME). (D) Body weight was comparable between sucrose-drinking rats and water-drinking controls. However, stress reduced body weight, particularly at later experimental days (interactive effect (p < 0.05) of STRESS x TIME). All results were analyzed with repeated measures 3-way ANOVA. N = 12/group.

Figure 3:

Effect of repeated restraint stress and sucrose intake on PV+ interneurons and their PNNs in the BLA. (A) Neither sucrose nor restraint affected the (A) number of PV+ cells, or (B) proportion of PV+ cells surrounded by full PNNs. Sucrose increased the proportion of PV+ cells that were surrounded by (C) partial PNNs, or (D) any type of PNN (either full or partial), and (E) reduced the proportion of PV+ cells that were without a PNN structure. (F) As a result, sucrose increased the ratio of PV+ cells that were (vs. were not) surrounded by PNN structures. Notably, stress had neither a main nor interactive effect on any of these end points. All results were calculated with 2-way ANOVA. # indicates significant main effect of DRINK. N = 12/group.

Figure 4:

Effects of PNN presence on BLA PV+ cell properties and appositions. PV+ cells with PNNs (A) were larger (**** p < 0.0001) and (B) had higher PV+ fluorescence intensity (**** p < 0.0001) than those without PNNs. PV+ cells with PNNs also received (C) more vGLUT1+ appositions (**p < 0.01), and (D) fewer vGAT+ appositions (***p < 0.001), resulting in (E) a higher ratio of vGLUT1+ to vGAT+ appositions (***p < 0.001). All results were analyzed with paired t-tests. N = 48 rats (collapsed across treatment groups). (F) and (G) are representative confocal images of PV+ interneurons (magenta) in the BLA with and without PNNs (green), surrounded by vGLUT+ and vGAT+ appositions (red), respectively.

Figure 5:

Analysis of excitatory and inhibitory appositions onto BLA PV+ cells by stress condition and drink treatment. (A) The effect of sucrose intake on the number of vGLUT1+ appositions approached significance (p < 0.1). (B) There was no significant effect of either LSI or repeated restraint stress on the number of vGAT+ appositions onto PV+ cells in the BLA (p > 0.05). (C) Sucrose consumption increased the ratio of vGLUT1+ to vGAT+ appositions onto PV+ cells (# p < 0.05 indicates main effect of DRINK), whereas repeated restraint stress had no effect. All results were analyzed by 2-way ANOVAs. N = 12/group.

Figure 6.

Working model of a mechanism by which LSI blunts stress responses. Chronic consumption of a food reward (e.g., limited sucrose intake; LSI) increases the proportion of PV+ interneurons that have PNNs (PV+/WFA+) in the BLA – an effect that is associated with increased excitatory/inhibitory (E/I) input onto these neurons. This leads to enhanced PV+ interneuron-mediated inhibitory tone during stress, thereby dampening the overall activation of BLA principal neurons that would otherwise promote greater stress responses via direct and indirect projections to numerous other stress-regulatory brain regions. Figure created with Biorender.com