Sexual differences in locus coeruleus neurons and related behavior in C57BL/6J mice

Abstract

Background: In addition to social and cultural factors, sex differences in the central nervous system have a critical influence on behavior, although the neurobiology underlying these differences remains unclear. Interestingly, the Locus Coeruleus (LC), a noradrenergic nucleus that exhibits sexual dimorphism, integrates signals that are related to diverse activities, including emotions, cognition and pain. Therefore, we set-out to evaluate sex differences in behaviors related to LC nucleus, and subsequently, to assess the sex differences in LC morphology and function.

Methods: Female and male C57BL/6J mice were studied to explore the role of the LC in anxiety, depressive-like behavior, well-being, pain, and learning and memory. We also explored the number of noradrenergic LC cells, their somatodendritic volume, as well as the electrophysiological properties of LC neurons in each sex.

Results: While both male and female mice displayed similar depressive-like behavior, female mice exhibited more anxiety-related behaviors. Interestingly, females outperformed males in memory tasks that involved distinguishing objects with small differences and they also showed greater thermal pain sensitivity. Immunohistological analysis revealed that females had fewer noradrenergic cells yet they showed a larger dendritic volume than males. Patch clamp electrophysiology studies demonstrated that LC neurons in female mice had a lower capacitance and that they were more excitable than male LC neurons, albeit with similar action potential properties.

Conclusions: Overall, this study provides new insights into the sex differences related to LC nucleus and associated behaviors, which may explain the heightened emotional arousal response observed in females.

Keywords: Anxiety; Depression; Electrophysiology; Female; Learning and memory; Locus coeruleus; Noradrenaline; Pain; Patch clamp; Sex.

Fig. 1

Evaluation of anxiety, well-being and depressive-like behavior in male and female mice. The results of anxiety-like behavior expressed as a the relative time spent in the open arms of the EPM by male and female mice. b Percentage of time spent in the lit compartment in the light–dark test. c Percentage of time spent in the central zone in the OFT, along with representative heatmaps of activity. d Graph depicting the material burrowed (in grams) and e the AUC of the material burrowed over time. f Time spent grooming in the splash test. Evaluation of the depressive-like behavior expressed as g immobility time in the TST and h FST. The data are presented as the mean ± SEM of n = 7–10 mice per group: *p < 0.05, **p < 0.01 vs male. From b to h females were in proestrus and estrus (P/E) stages. AUC, area under the curve

Fig. 2

Evaluation of the NOR paradigm and the sensorial assessment of male and female mice. a Schematic representation of the NOR experimental design to assess short-term (STM, learning index) and long-term memory (LTM, memory index), using two different objects. Graphs depicting b the total distance traveled in the habituation phase of the NOR paradigm and c its representation in 1 min intervals. Graphs showing d the percentage of preference exploring identical objects (represented as A) and e the number of interactions during the 15-min training phase of the test. f Graph representing the discrimination index (DI) between objects following the STM and LTM protocols when using a novel object (represented as B in STM protocol and C in LTM protocol) that differed drastically from the familiar object. g Schematic representation of the NOR experimental design to assess STM using objects with minimal differences between them. h Graph depicting the DI following the STM protocol and representative heatmaps showing activity around the objects, when the novel object (represented as D cross out) presented strong similarity with the familiar one (represented as D). Evaluation of the i mechanical response in the von Frey test using calibrated filaments from 0.16 to 10 g and j the paw withdrawal (in seconds) in the plantar test. The data are presented as the mean ± SEM of n = 10 mice per group: *p < 0.05, **p < 0.01 vs male; #p < 0.05 vs the first minute. Females were in proestrus and estrus (P/E) stages. DI, discrimination index; AU, arbitrary units

Fig. 3

Quantification of TH⁺ cells and the LC volume in male and female mice. a Representative images of the LC in male and female mice stained for TH by DAB, and b quantification of the number of TH⁺ cells in both groups. c Representative confocal images of the area occupied by the soma (white line) and/or dendrites (yellow line) outlined for volume estimation. d Quantification of the number of TH⁺ cells detected by immunofluorescence in the entire LC. e Graph depicting the distribution of TH⁺ cells along the rostrocaudal axis of the LC and f its representation as the AUC. g The volume occupied by the soma, h somatodendrites and i dendrites of the entire LC. j Graph depicting the distribution of TH⁺ dendrites along the rostrocaudal axis of the LC and k its representation as the AUC. The data are presented as the mean ± SEM of n = 5 animals per group for DAB and another 5 animals per group for inmunofluorescence: *p < 0.05, **p < 0.01 vs male. Scale bars = 100 μm. IV, fourth ventricle; TH, Tyrosine Hydroxylase; DAB, 3,3′-diaminobenzidine tetrahydrochloride; AUC, area under curve

Fig. 4

Evaluation of the passive properties and excitability of LC neurons in male and female mice. Population graphs depicting differences in a the membrane capacitance, b a slight difference in the membrane resistance and c similar membrane resting potentials. d Representative example and graph of the IRK currents. e Representative example of the action potential (AP) and corresponding parameters, such as the amplitude, half-width, threshold and after hyperpolarization potential (AHP). f Representative examples of the voltage responses of identified LC neurons to current injection of + 100 and − 100 pA, respectively. g Rheobase. h Graph showing the activity driven in response to the injection of positive currents (+ 25 pA steps). i Graph showing the voltage deflections in response to the injection of negative currents (− 25 pA steps). The data are presented as the mean ± SEM of n = 5 mice per group: *p < 0.05, **p < 0.01 ***p < 0.001 vs male. ANOVA (sex factor): ^#p < 0.05, ^###p < 0.001