Here we show that there was a significant increase in anxiety-like behavior in BALB/cJ mice injected with the NPY-SAP neurotoxin into the CeA relative to mice treated with the control B-SAP. Thus, CeA NPY-SAP-treated mice spent significantly less time in the open area of the elevated zero maze and made significantly less open arm entries relative to B-SAP-treated mice. Reduced open area activity was not likely related to a general reduction of motor behavior or compromised heath status as there were no significant differences between mice treated with CeA infusion of NPY-SAP or B-SAP in terms of open-field locomotor activity or in measures of consummatory behaviors. Increased anxiety-like behavior was likely the result of blunted NPY signaling in the region of the CeA, as mice treated with NPY-SAP showed a significant reduction of Y1R IR in the CeA relative to B-SAP treated animals. The Y1R IR results reinforce the conclusion that the NPY-SAP toxin successfully lesioned cells in the CeA that express Y1R. CeA-infusion of NPY-SAP site-specifically attenuated Y1R IR in the CeA, and did not significantly alter Y1R IR in nearby regions including the BLA, medial amygdala, and bed nucleus of the stria terminalis. Thus, NPY-SAP appears to be a tool that will allow very precise definition of the NPY neurocircuitry involved in modulating anxiety-like behavior. It is noteworthy that since NPY-SAP binds to all NPY receptors, the NPY-SAP treatment would have also killed Y2- and Y5-expression cells, though we did not quantify these changes in the present report. The observed increase of anxiety-like behavior following CeA injection of NPY-SAP are consistent with results obtained using other tools to blunt NPY signaling such as NPY receptor antagonist and NPY antisense [6
]. It should be noted that while NPY signaling in the CeA has been shown to modulate ethanol consumption in rodents [11
], the lack of an effect of CeA-infused NPY-SAP on ethanol intake here is likely the result of an almost complete avoidance of ethanol in the BALB/cJ mice that were used (only 1.14 to 1.21 g/kg/day).
As a control, we injected NPY-SAP into the BMH and assessed subsequent anxiety-like behavior. We chose the BMH because, to our knowledge, there is no known link to NPY signaling in this region to the modulation of anxiety-like behavior, and NPY signaling in the hypothalamus appears to be primarily involved in feeding behaviors [3
]. Unexpectedly, mice given BMH injection of NPY-SAP showed reduced anxiety-like behavior relative to B-SAP treated mice, evidenced by increased open area time and open area entries with no associated alterations of open-field locomotor activity or consummatory behaviors. While BMH infusion of NPY-SAP was associated with a significant reduction of Y1R IR in the BMH (confirming the lesioning of Y1R-expressing cells), this treatment also caused a significant increase in Y1R IR in the BLA and the CA3 region of the hippocampus when compared to B-SAP-treated mice. Increased Y1R signaling stemming from an upregulation of Y1R IR in the BLA and/or CA3 region may account for the paradoxical decrease in anxiety-like behavior in mice given NPY-SAP injection into the BMH. If it is assumed that increased Y1R IR translates into increased NPY signaling in these regions, such increased NPY signaling may have promoted anxiolysis which led to the observed reduction of anxiety-like behavior in the present work. In fact, accumulating evidence indicates that NPY signaling in the BLA and hippocampus protect against anxiety-like behaviors in rodents [17
], reinforcing the idea that an upregulation of NPY signaling in these regions accounts for the reduced anxiety-like behavior in mice given BMH infusion of NPY-SAP.
While it is unclear how destruction of Y1R-expressing cells in the BMH confers increases of Y1R IR in other brain regions, it is interesting to note that a previous study that used NPY-SAP to induce lesions of NPY receptor-expression cells in the arcuate nucleus of the hypothalamus observed a significant increase in Y1R IR in other regions including the paraventricular nucleus of the hypothalamus and the perifornical area. Analogous to the present findings, there was a paradoxical increase in feeding behavior in NPY-SAP-treated animals, hypothesized to stem from the compensatory increase in Y1R signaling on other brain regions [5
]. Similarly, area postrema lesions were found to be associated with a significant reduction of anxiety-like behavior in rats, an effect that was hypothesized to be related to compensatory increases of NPY mRNA levels in other brain regions that included the amygdala [18
]. We suggest that the observed compensatory increase in Y1R IR in the BLA and/or CA3 likely explains the reduced anxiety-like behavior in mice treated with BMH injection of NPY-SAP. Importantly, there are connections between the area of the BMH and the BLA [19
] as well as the medial hypothalamus and the hippocampus [1
], and these pathways may be involved with the compensatory increases of Y1R IR in the regions outside of the lesion site.
In conclusion, the present set of experiments suggest that the NPY-SAP neurotoxin may be a useful tool for studying the NPY neurocircuitry that modulates anxiety-like behaviors. Consistent with previous work, blunted NPY receptor signaling in the CeA following local injection of NPY-SAP was associated with increased anxiety-like behavior, reinforcing the critical role of NPY signaling in the CeA in the integration of emotional responses. The unexpected reduction of anxiety-like behavior following BMH injection of NPY-SAP may be related to the compensatory increase in Y1R IR in the BLA and/or CA3. This latter observation raises the important point that while NPY-SAP may be a useful tool, caution is necessary when drawing conclusions regarding the role of NPY signaling at the specific lesion site. Characterization of the NPY system in brain regions beyond the site of NPY-SAP injection may be necessary to gain a more accurate picture of the system involved.
- Saporin, when conjugated to NPY (NPY-SAP), kills cells expression NPY receptors.
- Injecting NPY-SAP into the central amygdala (CeA) reduces local Y1 receptor levels.
- Injecting NPY-SAP into the CeA increases anxiety-like behavior.
- NPY-SAP may be a useful tool to study the central NPY circuitry modulating anxiety.