Estrous cycle impacts on dendritic spine plasticity in rat nucleus accumbens core and shell and caudate-putamen

Abstract

An important factor that can modulate neuron properties is sex-specific hormone fluctuations, including the human menstrual cycle and rat estrous cycle in adult females. Considering the striatal brain regions, the nucleus accumbens (NAc) core, NAc shell, and caudate-putamen (CPu), the estrous cycle has previously been shown to impact relevant behaviors and disorders, neuromodulator action, and medium spiny neuron (MSN) electrophysiology. Whether the estrous cycle impacts MSN dendritic spine attributes has not yet been examined, even though MSN spines and glutamatergic synapse properties are sensitive to exogenously applied estradiol. Thus, we hypothesized that MSN dendritic spine attributes would differ by estrous cycle phase. To test this hypothesis, brains from adult male rats and female rats in diestrus, proestrus AM, proestrus PM, and estrus were processed for Rapid Golgi-Cox staining. MSN dendritic spine density, size, and type were analyzed in the NAc core, NAc shell, and CPu. Overall spine size differed across estrous cycle phases in female NAc core and NAc shell, and spine length differed across estrous cycle phase in NAc shell and CPu. Consistent with previous work, dendritic spine density was increased in the NAc core compared to the NAc shell and CPu, independent of sex and estrous cycle. Spine attributes in all striatal regions did not differ by sex when estrous cycle was disregarded. These results indicate, for the first time, that estrous cycle phase impacts dendritic spine plasticity in striatal regions, providing a neuroanatomical avenue by which sex-specific hormone fluctuations can impact striatal function and disorders.

Keywords: caudate-putamen; dendritic spine; estradiol; estrous cycle; nucleus accumbens; sex differences.

Figure 1.

Location of analyzed medium spiny neuron (MSN) dendritic segments in adult rat nucleus accumbens (NAc) core, NAc shell, and caudate-putamen (CPu). (a) Males. (b) Females in diestrus. (c) Females in proestrus AM. (d) Females in proestrus PM. (e) Females in estrus. Triangles represent analyzed dendritic segment location within slice. Acronyms: AC, anterior commissure; LV, lateral ventricle; NAc, nucleus accumbens; CPu, caudate-putamen.

Figure 2.

Representative images of Golgi impregnated dendritic segments from MSNs in: (a-d) NAc core, (e-h) NAc shell, (i-l) CPu. (a) Male. (b) Female in proestrus PM. (c) Male. (d) Female in proestrus AM. (e) Female in estrus. (f) Female in diestrus. (g) Female in proestrus PM. (h) Female in diestrus. (i) Female in proestrus AM. (j) Female in diestrus. (k) Male. (l) Female in proestrus AM. Scale bar 5 μm.

Figure 3.

Schematic representing spine classification protocol, following Risher et al., 2014. Spine length, width, length/width ratio, and head number were first assessed. These attributes were then used to hierarchically and subsequently classify spines into the following types: branched (all spines exhibiting more than one head), filopodia (length > 2 um), mushroom (width > 0.6 um), long thin (length > 1 um), thin (length:width ratio > 1 um), stubby (length:width ratio ≤ 1 um).

Figure 4.

Dendritic spine density differed by striatal region independent of sex or estrous cycle phase. (a) Average spine size did not differ by striatal region. (b) Dendritic spine density is elevated in NAc core compared to NAc shell and CPu. Acronyms: LWR, length:width ratio; **p<0.01.

Figure 5.

No differences in spine size or spine density by sex independent of estrous cycle phase. (a) NAc core spine size. (b) NAc shell spine size. (c) CPu spine size. (d) NAc core spine density. (e) NAc shell spine density. (f) CPu spine density. Acronyms: LWR, length:width ratio.

Figure 6.

Overall dendritic spine size differed by estrous cycle phase in the NAc core and NAc shell, but not CPu. (a) In NAc core, average spine size was decreased in proestrus PM compared to proestrus AM. (b) In NAc shell, average spine size was increased in estrus compared to proestrus PM and diestrus. (c) CPu spine size. (d) NAc core spine density. (e) NAc shell spine density. (f) CPu spine density. Acronyms: LWR, length:width ratio; *, p<0.05; **, p<0.01.

Figure 7.

Average spine length differs by estrous cycle phase in the NAc shell and CPu, but not NAc core. (a) In NAc core, spine length trends lower in proestrus PM compared to both proestrus AM and estrus. (b) In NAc shell, spine length is elevated in estrus compared to all other phases. (c) In CPu, spine length in elevated in estrus compared to diestrus and proestrus PM. (d) NAc core spine width. (e) NAc shell spine width. (f) CPu spine width. Acronyms: ^o, 0.05<p<0.1; *, p<0.05; **, p<0.01.

Figure 8.

Visual representation of spine type distributions for males and females of each estrous cycle phase. (a) NAc core. (b) NAc shell. (c) CPu. This representation demonstrates region-specific differences in the impact of the estrous cycle upon dendritic spine type. For example, in the NAc core proestrus AM features a relatively larger proportion of long spine types, such as filopodia and long thin, while proestrus PM features a relatively larger proportion of short spine types, such as thin and stubby. Circle diameter indicates spine type proportion, with larger diameters indicating increased proportion. Circle color corresponds with spine type. Connecting lines between spine type circles indicate the presence of a statistically significant difference between proportions. Lack of a line indicates no significant difference exists in proportion between the respective spine types. Line weight represents p value, with increasing weight indicating decreasing p value. Quantitative metrics are provided in Tables 2-3. Acronyms: F, filopodia; LT, long thin; T, thin; S, stubby; M, mushroom; B, branched.