We recently demonstrated that brain-generated E2 controls the gain of central auditory neurons, in real-time, by directly regulating fast neurotransmission 
. To determine whether estrogen-associated circuits may influence visual processing, we first assessed whether the cellular components required for estrogen production and sensitivity are available in the visual cortex, more specifically in the mouse V1. To this end, we first cloned the genes encoding aromatase (ARO; a.k.a., estrogen-synthase), and each of the classic estrogen receptors (ERα and ERβ), from a mouse cDNA library via PCR. We then used these cDNAs to generate antisense riboprobes and carried out a detailed analysis by fluorescence in-situ hybridization to determine whether or not estrogen-associated circuits are found in V1. In addition, given that this method enabled us to specifically identify estrogen-producing and estrogen-sensitive cells at single cell resolution, it was possible to quantitatively study these neuronal populations with stringent, unbiased stereological methods.
V1 is Highly Enriched with Estrogen-Producing and Estrogen-Sensitive Neurons
Remarkably, we found that V1 is highly enriched in estrogen-producing (ARO-positive) and estrogen-responsive neurons (ER-positive) (). More specifically, we found that ARO-positive neurons are expressed at high levels in cortical layers II to VI (). Within and across cortical layers, the distribution of cells positive for ARO mRNA was largely homogenous. Stereological quantification revealed that the supragranular (II/III), granular (IV) and infragranular (V/VI) layers of V1 contained 25.1±0.9 (mean ± S.E.), 25.2±0.8 and 24.1±0.7×103 neurons/mm3 that were positive for ARO, respectively ().
The primary visual cortex (V1) contains a large population of estrogen-producing and estrogen-producing neurons.
Large populations of neurons in the adult V1 putatively produce and are sensitive to estrogens.
V1 neurons also express both estrogen receptors abundantly. We found that ERα and ERβ are expressed in all cortical layers, with the exception of layer I (). Although ERα is expressed at significant levels, expression of ERβ is more robust, as revealed by unbiased quantification. In particular, we determined that 15.4±0.6, 16.2±0.6 and 17.1±0.7×103 neurons/mm3 were positive for ERα mRNA in the supragranular, granular and infragranular layers of V1, respectively (). Quantification of the population of ERβ-positive neurons revealed 19.8±0.7, 19.9±0.8 and 20.9±0.7×103 neurons/mm3 in layers II/III, IV and V/VI, respectively (). When considering all cortical layers combined, our results showed that 63.0%±0.4, 41.3%±0.5 and 51.5%±0.8 of the overall neuronal population in V1 expresses ARO, ERα and ERβ, respectively, indicating that V1 is a major site associated with estrogenic circuits.
Estrogen-Associated Networks in V1 Are Activated by Visual Experience
We next tested if estrogen-associated circuits in V1 are engaged by visual stimulation. To this end, we housed animals overnight in a dark-room and subsequently stimulated mice with ambient light for 30 min. The V1 was then processed for a stringent double-fluorescence in-situ hybridization method that we developed and described in detail previously, where it is possible to identify two mRNAs in the same brain sections, at single-cell resolution 
. We used the expression of the activity-dependent transcription factor egr-1
to identify visually-driven neurons in V1, and riboprobes directed against ARO or each of the ERs, to identify estrogen-producing and -responsive neurons (). The expression of egr-1
has been used by a large contingent of research groups, including our own, to reliably identify visually-driven neurons, and the 30 min time-point was chosen as it corresponds to peak egr-1
mRNA accumulation following stimulus onset (for reviews, see 
Visual experience activates estrogen-associated networks in V1.
Quantitative analyses revealed that 21.8±0.8, 20.5±0.7 and 22.4±0.9×103 neurons/mm3 in V1 co-localize egr-1 and ARO mRNAs in supragranular, granular and infragranular layers, respectively. When considering all layers together, these results indicate that 87.3%±3.4 of visually-driven neurons are estrogen-producing cells. In contrast, we found that 4.7±0.7, 5.1±0.6 and 4.4±0.8×103 neurons/mm3 co-localize egr-1 and ERα mRNAs, and 12.1±0.8, 11.7±0.8 and 10.5±0.9×103 neurons/mm3 co-express egr-1 and ERβ in supragranular, granular and infragranular layers of V1, respectively. These results further revealed that 29.2%±1.8 and 56.6%±3.4 of the overall neuronal population of V1 neurons expressing ERα and ERβ are engaged by visual experience. These findings suggest that although both estrogen-producing and estrogen-sensitive neurons in V1 are significantly driven by visual input, sensory experience predominantly affects the population of estrogen-producing cells.
Estrogen-Associated Circuits in V1 Are Neurochemically Heterogeneous
We next set out to determine the neurochemical identity of estrogen-producing and estrogen-sensitive neurons in V1. To this end, we carried out double-FISH experiments combining riboprobes directed at estrogen-associated networks (ARO, ERα or ERβ) and classic markers for excitatory or inhibitory neurons – the vesicular glutamate transporter 2 (vGlut2) and the 65 kDa glutamic acid decarboxylase (GAD65), respectively ().
Neurochemical identity and heterogeneity of estrogen-associated circuits in V1.
We found that estrogen-producing (ARO-positive) cells are largely composed of excitatory neurons. More specifically, quantitative analysis showed that the supragranular, granular and infragranular layers of V1 displayed 18.8±0.6, 19.1±0.7 and 18.6±0.7×103 neurons/mm3 that co-expressed ARO and vGlut2 mRNAs, respectively. In contrast, a significantly smaller fraction of ARO neurons were positive for GAD65 (6.7±0.6, 5.9±0.9 and 6.2±0.8×103 neurons/mm3 in supragranular, granular and infragranular layers, respectively; (). When all cortical layers were considered together, our results revealed that 76.1%±0.5 and 25.4%±1.1 of the estrogen-producing neurons in V1 (ARO-positive) were excitatory and inhibitory, respectively ().
Estrogen-associated circuits in V1 are neurochemically distinct.
Remarkably, our dFISH studies also revealed that the population of estrogen-sensitive neurons (ER-positive) is neurochemically heterogeneous. Specifically, we found that 3.3±0.7, 2.5±0.8 and 3.0±0.6×103 neurons/mm3 co-expressed ERα and vGlut2, and 12.8±0.9, 14.1±0.8 and 13.9±0.8×103 neurons/mm3 were double-labeled for ERα and GAD65 mRNAs in the supragranular, granular and infragranular layers of V1, respectively (). In stark contrast, quantitative analysis revealed that whereas 19.1±0.7, 17.9±0.7 and 18.8±0.9×103 neurons/mm3 were positive for ERβ and vGlut2 mRNAs, 2.1±0.9, 2.0±1.0 and 1.9±0.8×103 neurons/mm3 co-expressed ERβ and GAD65 in V1's supragranular, granular and infragranular layers, respectively (). Quantitatively, the populations of ERα- and ERβ-positive cells that were excitatory or inhibitory were significantly different from each other (p<0.001 for all layers). When considering all cortical laminae together, these findings showed that 18.2%±1.7 and 84.2%±1.8 of the ERα-expressing neurons in V1 exhibit excitatory and inhibitory phenotypes, respectively (). Conversely, 92.0%±2.1 and 10.1%±0.4 of the ERβ-positive neurons are composed of excitatory and inhibitory cells (). These findings provide direct evidence of a marked dichotomy on the neurochemical identity of ERα- versus ERβ-positive cells within V1.
Populations of ARO- and ERβ-Positive Neurons Exhibit a Moderate Degree of Overlap
The findings above demonstrate that ERα-positive neurons are predominantly GABAergic and, therefore, represent a different neuronal population relative to ARO and ERβ-positive neurons, which are chiefly excitatory. It is unclear, however, whether or not ARO-and ERβ-positive neurons are expressed in the same cells, or different neurons. Such configurations would support models by which estrogen acts through autocrine-like and paracrine fashions, respectively. To address this issue we performed double-FISH studies for ARO and ERβ. Quantitative analysis with unbiased stereological methods revealed that 14.3±0.5, 14.8±0.8 and 16.3±0.6×103 neurons/mm3 co-express ARO and ERβ mRNAs in the supragranular, granular and infragranular layers of V1, respectively. These observations reveal that, when considering all cortical layers together, 61.4%+5.6 of all ARO-positive neurons in V1 co-express ERβ. Overall these findings suggest that ARO and ERβ-positive neurons overlap to a moderate degree and suggest that locally-generated estrogen is positioned to affect V1 neuronal physiology through autocrine and/or paracrine fashions.
Estrogen-Associated Networks Are Highly Stable in Response to Acute Visual Experience
To investigate if acute epochs of visual experience affect the density of estrogen-associated circuits, we subjected different groups of animals to 30 min, 1 h and 2 h of visual stimulation following overnight dark-rearing. Stereological quantification of the numerical densities of neurons positive for ARO, ERα and ERβ mRNA was carried out in brains processed for FISH.
We found that acute visual experience did not affect the population of ARO-positive cells in V1 (; ). Likewise, acute visual stimulation did not impact the population of cells expressing either estrogen receptor across cortical layers of V1 (; and ). Overall, the findings above indicate that acute visual experience does not affect the numerical densities of ARO- ERα- and ERβ-positive neurons and suggest that estrogen-associated circuits in V1 are highly stable to short epochs of visual stimulation.
The numerical densities of ARO, ERα or ERß-positive neurons in V1 are not affected by acute visual stimulation.
Acute sensory stimulation does not affect the numerical densities of ARO-positive cells across cortical layers of V1.
Acute sensory stimulation does not affect the numerical densities of ERα-positive cells across cortical layers of V1.
Acute sensory stimulation does not affect the numerical densities of ERβ-positive cells across cortical layers of V1.
Estrogen-Associated Circuits Are Not Affected by Chronic Visual Deprivation
Although acute visual stimulation does not influence the density of estrogen-producing and estrogen-responsive neurons, it is possible that chronic (long-term) changes in visual experience affect the constitution of estrogen-associated circuits. To directly investigate this question, we raised a group of mice from birth to adulthood in complete darkness. We then quantitatively compared the distribution of ARO, ERα and ERβ-positive cells in the V1 of these chronically light-deprived mice against that of age-matched, normally-raised controls.
Surprisingly, our results showed that chronic visual deprivation does not affect the density of ARO-positive neurons in V1 (; ). Likewise, animals that were dark-reared from birth exhibited numerical densities of ERα and ERβ-positive cells across cortical layers of V1 that were not significantly different from those observed in normally-reared controls (; and , respectively).
Chronic visual deprivation, from birth to adulthood, does not affect the density of estrogen-associated circuits in V1.
Chronic visual deprivation does not affect the numerical densities of ARO-positive cells across cortical layers of V1.
Chronic visual deprivation does not affect the numerical densities of ERα-positive cells across cortical layers of V1.
Chronic visual deprivation does not affect the numerical densities of ERβ-positive cells across cortical layers of V1.
Our results show that chronic visual deprivation throughout post-natal development does not affect the numerical densities of estrogen-associated circuits. These findings suggest that visual experience is not required for the adequate implementation and maintenance of estrogen-sensitive or estrogen-responsive circuits in V1.