A quantitative comparison of the efferent projections of the anterior and posterior subdivisions of the medial amygdala in female mice

Abstract

In rodents, many aspects of sociosexual behavior are mediated by chemosignals released by opposite-sex conspecifics. These chemosignals are relayed via the main (MOS) and accessory olfactory systems (AOS) to the medial amygdala (Me). The Me is subdivided into anterior (MeA) and posterior (MeP) subnuclei, and lesions targeting these regions have different effects on proceptive courtship behaviors in female mice. Differential behavioral effects of MeA vs. MeP lesions could reflect a difference in the projections of neurons located in these Me subnuclei. To examine this question, we injected female mice with the anterograde tracer, Fluoro-Ruby into either the MeA or MeP and quantified labeled puncta in 11 forebrain target sites implicated in courtship behaviors using confocal fluorescence microscopy. We found that the MeP more densely innervates the medial and intermediate regions of the posterior bed nucleus of the stria terminalis (pBNST) and the posteromedial cortical amygdala (PMCo), while the MeA more densely innervates the horizontal diagonal band of Broca (HDB) and the medial olfactory tubercle (mOT), a region that may be a component of the circuitry responsible for olfactory-mediated motivated behaviors.

Keywords: Anterograde tracing; Axon terminal; Medial amygdala; Olfactory; Sociosexual behavior.

Figure 1

A,B, Confocal image showing Fluoro-Ruby injection sites in the anterior (A) and posterior (B) medial amygdala. C,D, Schematic reconstructions of coronal sections showing the extent of individual Fluoro-Ruby injections in female mice targeting the anterior (n=6) medial amygdala. Top row: subjects 336, 340, 342; Bottom row: subjects 353, 354, 358. E,F, Schematic reconstructions of coronal sections showing the extent of individual Fluoro-Ruby injections in female mice targeting the posterior (n=6) medial amygdala. Top row: subjects 337, 341, 344; Bottom row: subjects 343, 345, 346. Sections are ordered sequentially from anterior (left) to posterior (right), with the numbers shown on the bottom right of each plate representing the distance in mm posterior to bregma. Colored regions within each plate represent individual injections, identified by the animal code. Adapted from Franklin and Paxinos (Franklin & Paxinos, 2008). MeA, anterior medial amygdala; MeP, posterior medial amygdala; opt, optic tract.

Figure 2

The posterior medial amygdala (MeP) densely innervates the posteromedial cortical amygdala (PMCo) and the posterior bed nucleus of the stria terminalis (pBNST). Epifluorescent images show dense anterograde fiber labeling in the PMCo and pBNST (A’, B’) in MeP Fluoro-Ruby-injected animals, but not MeA Fluoro-Ruby-injected animals (A, B). Retrogradely labeled cell bodies were also observed in the PMCo of subjects given Fluoro-Ruby injections into the MeP (A’), indicated by filled white arrows.

Figure 3

Overlapping anterograde fiber/puncta labeling in the posterior bed nucleus of the stria terminalis (A; pBNST) and medial olfactory tubercle (B; mOT) of a female mouse given injections of PHA-L (green) and Fluoro-Ruby (red) into the MeA and MeP, respectively. A, confocal image showing that the intermediate and medial portions of the pBNST receive input mostly from the MeP (red), while the lateral portion receives relatively equal input from both the anterior and posterior Me. A’, 60× (oil) Z plane-stacked confocal image of the boxed area shown in A. Labeled fibers with puncta originating from the MeA (green) and MeP (red) were seen throughout the posterolateral stria terminalis (STMPL), shown with DAPI nuclear counterstain (blue cell bodies). B, confocal image showing synaptic input to the mOT almost exclusively from the anterior Me (green) with very sparse labeling from the MeP (red). B’, 60× (oil) Z plane-stacked confocal image of the boxed area shown in B showing innervation of the mOT predominantly by the MeA (green fibers).

Figure 4

Z-plane stacked confocal images (60× magnification) showing anterograde fiber/puncta labeling in coronal sections of the medial portion of the posterior stria terminalis (STMPM) following Fluoro-Ruby injections into either the anterior (A, MeA) or posterior (B, MeP) medial amygdala. C, Filled white arrows point to labeled puncta, magnified from the boxed region that were included for analysis (0.8μm-1.6μm diameter); open arrows point to possible puncta that were very small (<0.8μm diameter) and therefore excluded from analysis.

Figure 5

Modified schematic from the mouse brain atlas of Franklin and Paxinos (Franklin & Paxinos, 2008) showing the location of forebrain regions in which labeled puncta (dark gray boxes; 100² μm) were counted. Counting regions included the medial olfactory tubercle (A), the shell (left) and core (right) of the nucleus accumbens (B), the horizontal diagonal band of Broca (C), the medial preoptic area (D), medial (left), intermediate (middle), and lateral (right) posterior bed nucleus of the stria terminalis (E), dorsomedial (left) and ventrolateral (right) subdivisions of the ventromedial hypothalamus (F), and posteromedial cortical amygdala (G). Numerical values represent the distance in mm from bregma for each section.

Figure 6

Quantitative estimation of labeled puncta in animals given Fluoro-Ruby injections into either the MeA or MeP. Brain regions in which puncta were counted: STMPL, STMPI, STMPM, posterolateral, intermediate, medial divisions of the bed nucleus of the stria terminalis; PMCo, posteromedial cortical amygdala; HDB, horizontal diagonal band of Broca; MPA, medial preoptic area; VMHdm, VMHvl, dorsomedial, ventrolateral portions of the ventromedial hypothalamus; mOT, medial olfactory tubercle; AcbC, AcbSh, nucleus accumbens core, shell. Data are expressed as the mean (±SEM) calculated from confocal images merged in the Z-plane. Sample sizes for each group are shown in parentheses. *P<0.040, **P<0.001 (Two-tailed Student's t-test).