Synaptic Connections of Aromatase Circuits in the Medial Amygdala Are Sex Specific

Ex vivo characterization of the intrinsic electrophysiological properties of arom⁺ and arom⁻ neurons in males and females. A, Schematic of whole-cell patch recording strategy from the MeA of transgenic mice. B, Arom⁺ neurons were distinguished from arom⁻ neurons based on tdTomato expression in a cross between a transgenic mouse line expressing Cre in arom⁺ neurons (Yao et al., 2017) and a tdTomato reporter line (Madisen et al., 2010). C, Left, Aromatase-expressing neurons are shown in red. Right, Two recorded neurons filled with neurobiotin-streptavidin are shown in cyan. Middle, Merged channels showing one recorded neuron was arom⁺ (arrow) and the other was arom⁻ (arrowhead). Scale bar, 250 μm. D, Firing pattern of a single arom⁺ neuron (top) and arom⁻ neuron (bottom) in response to current steps ranging from 20 to 220 pA. E, Average number of spikes are shown for male (blue) and female (red) MeA neurons, with arom ⁺ neurons (top) and arom⁻ neurons (bottom). Shaded areas indicate SEM.

Strategy for circuit mapping from arom⁺ neurons in the MeA. A, Conditional AAV vectors were used to express TVA, RG, and mCherry in arom⁺ neurons of the MeA (Materials and Methods). After 10 d, G-deleted rabies (GFP) was injected at the same stereotaxic coordinates to infect arom⁺ starter neurons, after which 7 d were allowed for rabies to be transported from arom⁺ neurons in the MeA to the AOB. B, Horizontal image of MeA showing arom⁺ neurons (red) and arom⁺ →arom⁺) in cyan (arrowhead). Colabeled neurons were interpreted as starter neurons (arrow). C, The AOB was rendered transparent and imaged intact. A maximum intensity projection of the entire AOB showing →arom⁺ neurons monosynaptically labeled from the ipsilateral MeA. Arrow, Labeled mitral cell; arrowhead, labeled dendritic arbor in glomerulus; linea alba, thin straight line. D, 3D representation of all →arom⁺ neurons in the AOB in a single animal (cyan, cell bodies; blue, glomeruli). Note the strong anterior bias of both cell bodies and glomeruli. Scale bars, 250 μm.

Quantitative analysis of AOB input to arom⁺ neurons in the MeA. A, Percentage of →arom⁺ neuron cell bodies in the anterior versus posterior AOB. B, Percentage of →arom⁺ glomeruli in the anterior versus posterior AOB. C, Neuroanatomical reconstruction of the all →arom⁺ in the MeA of a single female mouse. Two neurons are highlighted: the first receives input only from anterior glomeruli, while the second, with a markedly smaller dendritic arbor, receives input from only posterior glomeruli (linea alba, thin straight line). D, The fraction of →arom⁺ neurons that receive input from only the anterior AOB glomeruli (black), from only the posterior AOB glomeruli (gray), or from both the anterior and posterior AOB glomeruli (white). Error bars indicate SEM.

Classical retrograde tracing from the MeA to the AOB. A, Fluoro-Gold was iontophoretically injected into the MeA, and 10 d were allowed for retrograde transport of Fluoro-Gold from the MeA to the AOB. B, A Fluoro-Gold injection site demonstrates labeling in both the dorsal and ventral MeA. C, Retrogradely labeled projection neurons in the AOB mitral cell layer showing dense labeling in both the anterior and posterior AOB. D, Percentage of neurons located in the anterior versus posterior AOB. Scale bars, 250 μm.

Sex differences in the AOB projection to arom⁺ MeA neurons. A, The average number of arom⁺ projecting AOB neurons in female (red) or male (blue) mice. B, Average number of arom⁺ projecting AOB neurons in female (red) or male (blue) mice after normalization by the number of starter neurons in the MeA. C, Relationship between the numbers of arom⁺ starter neurons in the MeA and →arom⁺ neurons in the AOB of male (blue squares) and female (red circles) mice. Dashed lines indicate the best linear fit to the data, and the shaded regions indicate the 95% confidence interval on each fit. Error bars indicate SEM.