Histone deacetylase inhibitors de-repress tyrosine hydroxylase expression in the olfactory bulb and rostral migratory stream

Abstract

Most olfactory bulb (OB) interneurons are derived from neural stem cells in the subventricular zone (SVZ) and migrate to the OB via the rostral migratory stream (RMS). Mature dopaminergic interneurons in the OB glomerular layer are readily identified by their synaptic activity-dependent expression of tyrosine hydroxylase (TH). Paradoxically, TH is not expressed in neural progenitors migrating in the RMS, even though ambient GABA and glutamate depolarize these progenitors. In forebrain slice cultures prepared from transgenic mice containing a GFP reporter gene under the control of the Th 9kb upstream regulatory region, treatment with histone deacetylase (HDAC) inhibitors (either sodium butyrate, Trichostatin A or Scriptaid) induced Th-GFP expression specifically in the RMS independently of depolarizing conditions in the culture media. Th-GFP expression in the glomerular layer was also increased in slices treated with Trichostatin A, but this increased expression was dependent on depolarizing concentrations of KCl in the culture media. Th-GFP expression was also induced in the RMS in vivo by intra-peritoneal injections with either sodium butyrate or valproic acid. Quantitative RT-PCR analysis of neurosphere cultures confirmed that HDAC inhibitors de-repressed Th expression in SVZ-derived neural progenitors. Together, these findings suggest that HDAC function is critical for regulating Th expression levels in both neural progenitors and mature OB dopaminergic neurons. However, the differential responses to the combinatorial exposure of HDAC inhibitors and depolarizing culture conditions indicate that Th expression in mature OB neurons and neural progenitors in the RMS are regulated by distinct HDAC-mediated mechanisms.

Figure 1

Laminar organization of TH expression in the perinatal mouse olfactory bulb. A, Th in situ hybridization (ISH) reveals that Th message is expressed in the glomerular (gl), mitral (m) and superficial granule cell (sgc) layers. However, Th is not expressed in the rostral migratory stream (RMS). B, TH immunohistochemistry (IHC) reveals that TH protein in limited almost exclusively to the glomerular layer, and is also absent from the RMS. The absence of TH protein from the mitral or superficial granule cells layers indicates that TH is post-transcriptionally regulated in these regions. Sagittal sections are from mouse pups approximately 4 days old. Other abbreviations: AOB, accessory olfactory bulb. Bar = 250 μm.

Figure 2

Induction of Th-GFP expression in the RMS and OB of forebrain slice cultures by TSA. A, weak basal Th-GFP expression in the glomerular and superficial granule cells layers, but not in the RMS (arrow), was detected in slices cultured in non-depolarizing media (supplemented with NaCl). B, Th-GFP expression was induced in the glomerular and superficial granule cell layers, but not in the RMS (arrow), in slices cultured with depolarizing media (supplemented with KCl). C, Th-GFP was induced in the RMS (arrow) of slices cultured in non-depolarizing media with TSA. D, Th-GFP expression was induced in the glomerular and superficial granule cell layers, as well as the RMS (arrow), in slices cultured with depolarizing media and TSA. E, relative fluorescence intensity ratios of GFP expression in the glomerular layer revealed TSA increased Th-GFP expression in the glomerular layer only when the slices were cultured in depolarizing media. F, depolarizing concentrations of KCl in the culture media did not affect the Th-GFP fluorescence intensity in the RMS. Bar = 250 μm.

Figure 3

In vivo induction of Th-GFP expression in the RMS by sodium butyrate (NaB). A, the RMS of a mouse treated with NaB contained Th-GFP expression in the RMS (outlined with dashed line). B, high magnification image of boxed region in A revealed leading and trailing processes of GFP expressing cells in the RMS. C, in contrast, Th-GFP was not observed in the RMS of saline injected control littermates. Bar = 125 μm for A and C, and 25 μm for B.

Figure 4

Induction of Th expression in SVZ-derived neurosphere cultures by HDAC inhibitors. Quantitative RT-PCR analysis showed that Th expression levels were increased in neurospheres treated for 48 hours with either TSA, NaB or VPA.