Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization.

Here we describe a fluorescence in situ hybridization protocol that allows for the detection of two mRNA species in fresh frozen brain tissue sections. This protocol entails the simultaneous and specific hybridization of hapten-labeled riboprobes to complementary mRNAs of interest, followed by probe detection via immunohistochemical procedures and peroxidase-mediated precipitation of tyramide-linked fluorophores. In this protocol we describe riboprobes labeled with digoxigenin and biotin, though the steps can be adapted to labeling with other haptens. We have used this approach to establish the neurochemical identity of sensory-driven neurons and the co-induction of experience-regulated genes in the songbird brain. However, this procedure can be used to detect virtually any combination of two mRNA populations at single-cell resolution in the brain, and possibly other tissues. Required controls, representative results and troubleshooting of important steps of this procedure are presented. After tissue sections are obtained, the total length of the procedure is 2-3 d.

Molecular characterization of the injury-induced aromatase transcript in the adult zebra finch brain.

In the zebra finch (Taeniopygia guttata), the aromatase gene is transcribed from one of two promoters resulting in two transcripts constitutively expressed in brain or ovary. These transcripts differ only in Exon 1 which lies in the 5' un-translated region (UTR). An inducible form of aromatase is expressed following brain injury in glia. Towards characterizing this transcript, we (a) examined the up-regulation of amplicons within the aromatase transcript using quantitative PCR (qPCR), (b) performed 5' and 3' rapid amplification of cDNA ends (RACE) on injured brain RNA and (c) sequenced the injury-induced aromatase transcript. qPCR suggested that inducible aromatase may contain a novel 3'UTR. However, neither 3' nor 5' RACE revealed novel UTRs in the injured telencephalon. We then sequenced aromatase from injured entopallium, a region that lacks detectable constitutive aromatase. Inducible aromatase was identical in sequence to the known neural aromatase transcript. These data suggest that injury-induced aromatase differs from ovarian, but is indistinguishable from neuronal aromatase. We suggest that an injury-specific signal in glia may modulate aromatase transcription. Alternatively, injury-induced aromatase transcription may be silenced under constitutive conditions. To the best of our knowledge, this is the first report that documents the sequence of inducible aromatase in any vertebrate.

Inhibition of injury-induced glial aromatase reveals a wave of secondary degeneration in the songbird brain.

Mechanical or anoxic/ischemic brain insult results in reactive gliosis and a pronounced wave of apoptotic secondary degeneration (WSD). Reactive glia express aromatase (estrogen synthase) and glial estrogen synthesis decreases apoptosis and the volume of degeneration. Whether aromatization by glia affects gliosis itself or the initiation/maintenance of the WSD remains unknown. Adult male zebra finches (Taeniopygia guttata) were injured with a needle that contained the aromatase inhibitor fadrozole or vehicle into contralateral hemispheres. Birds were killed at 0, 2, 6, 24, 72h, 2 or 6 weeks postinjury. Gliosis and degeneration were measured with vimentin- and Fluoro-Jade B-expression, respectively. Reactive gliosis was detectable at 6 h, reached asymptote at 72 h, and continued until 6 weeks postinsult. Gliosis extended further around fadrozole-injury than vehicle, an effect driven by a larger area of gliosis around fadrozole- relative to vehicle-injury at 72 h postinsult. Glial aromatase was inhibited for about 2 weeks postinjury since aromatase relative optical density was higher around fadrozole-injury relative to vehicle-injury until this time-point. Degeneration around vehicle-injury reached asymptote at 2 h postinsult, but that around fadrozole-injury peaked 24-72 h postinjury and decreased thereafter. Thus, the injury-induced WSD as described in mammals is detectable in zebra finches only following glial aromatase inhibition. In the zebra finch, injury-induced estrogen provision may decrease reactive gliosis and severely dampen the WSD, suggesting that songbirds are powerful models for understanding the role of glial aromatization in secondary brain damage.

The excitatory thalamo-"cortical" projection within the song control system of zebra finches is formed by calbindin-expressing neurons.

The learning and production of vocalizations in songbirds are controlled by a system of interconnected brain nuclei organized into a direct vocal motor pathway and an anterior forebrain (pallium-basal ganglia-thalamo-pallial) loop. Here we show that the thalamo-pallial ("thalamo-cortical") projection (from the medial part of the dorsolateral thalamic nucleus to the lateral magnocellular nucleus of the anterior nidopallium--DLM to LMAN) within the anterior forebrain loop is composed of cells positive for the calcium-binding protein calbindin. We show that the vast majority of cells within DLM express calbindin, based both on immunocytochemistry (ICC) for calbindin protein and in situ hybridization for calb mRNA. Using a combination of tract-tracing and ICC we show that the neurons that participate in the DLM-to-LMAN projection are calbindin-positive. We also demonstrate that DLM is devoid of cells expressing mRNA for the GABAergic marker zGAD65. This observation confirms that the calbindin-expressing cells in DLM are not GABAergic, in accordance with previous electrophysiological data indicating that the DLM-to-LMAN projection is excitatory. Furthermore, we use ICC to determine the trajectory of the fibers within the DLM-to-LMAN projection, and to demonstrate a sex difference in calbindin expression levels in the fibers of the DLM-to-LMAN projection. Our findings provide a clear-cut neurochemical signature for a critical projection in the songbird vocal control pathways that enable song learning.

Estrogen provision by reactive glia decreases apoptosis in the zebra finch (Taeniopygia guttata).

Upregulation of aromatase (estrogen synthase) in glia around the site of neural injury may limit neural degeneration. Systemic administration of estrogen limits neural damage, but the specific role of local estrogen provision in this effect is unclear. In male zebra finches, we tested the effect of local aromatase inhibition and estrogen replacement on type of cellular degeneration and the distance of this degeneration from the source of insult. Subjects received injections of the aromatase inhibitor fadrozole into one telencephalic lobe and fadrozole and estradiol into the contralateral lobe. Seventy-two hours later, we used Fluoro-Jade B and TUNEL to label dying and apoptotic cells, respectively. Since each subject was its own control, we were able to assess the influence of local estrogen replacement in relative distinction from circulating steroids and constitutive aromatization. Cellular degeneration around the lesion was measured with Fluoro-Jade B, TUNEL, and indirectly with aromatase expression. Additionally, the glial nature of aromatase-positive cells around the injury was queried by co-localization with vimentin. The estrogen replaced injury had fewer apoptotic cells clustered more closely around the injury compared to the hemisphere injected with fadrozole alone. Since Fluoro-Jade B and TUNEL labeled similar numbers of cells, and the distance of these cells from the injection was identical, we suggest that estrogen replacement functions primarily to restrict apoptosis in the current paradigm. Lastly, aromatase-positive cells around injuries co-localize vimentin, establishing their glial nature. Thus, glial estrogen provision at sites of neural insult may be critical in limiting the cellular degeneration caused by injury via an inhibition of apoptosis.