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Improved in situ hybridization methods for mRNA detection in tissues have been developed based on the hybridization chain reaction (HCR). We show that in situ HCR methods can be used for the detection of microRNAs in tissue sections from mouse retinas. In situ HCR can be used for the detection of two microRNAs simultaneously or for the combined detection of microRNA and mRNA. In addition, miRNA in situ HCR can be combined with immunodetection of proteins. We use these methods to characterize cells expressing specific microRNAs in the mouse retina. We find that miR-181a is expressed in amacrine cells during development and in adult retinas, and it is present in both GABAergic and glycinergic amacrine cells. The detection of microRNAs with in situ HCR should facilitate studies of microRNA function and gene regulation in the retina and other tissues.

The mammalian retina contains a complex mixture of different types of neurons. We find that microRNA miR-216b is preferentially expressed in postmitotic retinal amacrine cells in the mouse retina, and expression of miR-216a/b and miR-217 in retina depend in part on Ptf1a, a transcription factor required for amacrine cell differentiation. Surprisingly, ectopic expression of miR-216b directed the formation of additional amacrine cells and reduced bipolar neurons in the developing retina. We identify the Foxn3 mRNA as a retinal target of miR-216b by Argonaute PAR-CLIP and reporter analysis. Inhibition of Foxn3, a transcription factor, in the postnatal developing retina by RNAi increased the formation of amacrine cells and reduced bipolar cell formation. Foxn3 disruption by CRISPR in embryonic retinal explants also increased amacrine cell formation, whereas Foxn3 overexpression inhibited amacrine cell formation prior to Ptf1a expression. Co-expression of Foxn3 partially reversed the effects of ectopic miR-216b on retinal cell formation. Our results identify Foxn3 as a novel regulator of interneuron formation in the developing retina and suggest that miR-216b likely regulates Foxn3 and other genes in amacrine cells.

The mammalian retina contains a complex mixture of different types of neurons. We find that the microRNA miR-216b is preferentially expressed in postmitotic retinal amacrine cells in the mouse retina, and expression of miR-216a/b and miR-217 in the retina depend in part on Ptf1a, a transcription factor required for amacrine cell differentiation. Surprisingly, ectopic expression of miR-216b, or the related miR-216a, can direct the formation of additional amacrine cells in the developing retina. In addition, we observe the loss of bipolar neurons in the retina after miR-216b expression. We identify the mRNA for the transcriptional regulator Foxn3 as a retinal target of miR-216b by Argonaute PAR-CLIP and reporter analysis. Inhibition of Foxn3 in the postnatal developing retina by RNAi also increases the formation of amacrine cells and reduces bipolar cell formation, while overexpression of Foxn3 inhibits amacrine cell formation prior to the expression of Ptf1a. Disruption of Foxn3 by CRISPR in embryonic retinal explants also reduces amacrine cell formation. Co-expression of Foxn3 can partially reverse the effects of ectopic miR-216b on retinal cell type formation. Our results identify Foxn3 as a novel regulator of interneuron formation in the developing retina and suggest that miR-216b likely regulates expression of Foxn3 and other genes in amacrine cells.

The cellular mechanisms underlying the exceptional vulnerability of the basal forebrain (BF) cholinergic neurons during pathological aging have remained elusive. Here we employed an adeno-associated viral vector-based RNA interference (AAV-RNAi) strategy to suppress the expression of tropomyosin-related kinase A (trkA) receptors by cholinergic neurons in the nucleus basalis of Meynert/substantia innominata (nMB/SI) of adult and aged rats. Suppression of trkA receptor expression impaired attentional performance selectively in aged rats. Performance correlated with trkA levels in the nMB/SI. trkA knockdown neither affected nMB/SI cholinergic cell counts nor the decrease in cholinergic cell size observed in aged rats. However, trkA suppression augmented an age-related decrease in the density of cortical cholinergic processes and attenuated the capacity of cholinergic neurons to release acetylcholine (ACh). The capacity of cortical synapses to release ACh in vivo was also lower in aged/trkA-AAV-infused rats than in aged or young controls, and it correlated with their attentional performance. Furthermore, age-related increases in cortical proNGF and p75 receptor levels interacted with the vector-induced loss of trkA receptors to shift NGF signaling toward p75-mediated suppression of the cholinergic phenotype, thereby attenuating cholinergic function and impairing attentional performance. These effects model the abnormal trophic regulation of cholinergic neurons and cognitive impairments in patients with early Alzheimer's disease. This rat model is useful for identifying the mechanisms rendering aging cholinergic neurons vulnerable as well as for studying the neuropathological mechanisms that are triggered by disrupted trophic signaling.