Epigenetic control of neurotransmitter expression in olfactory bulb interneurons

Abstract

Defining the molecular mechanisms that underlie development and maintenance of neuronal phenotypic diversity in the CNS is a fundamental challenge in developmental neurobiology. The vast majority of olfactory bulb (OB) interneurons are GABAergic and this neurotransmitter phenotype is specified in migrating neuroblasts by transcription of either or both glutamic acid decarboxylase 1 (Gad1) and Gad2. A subset of OB interneurons also co-express dopamine, but transcriptional repression of tyrosine hydroxylase (Th) suppresses the dopaminergic phenotype until these neurons terminally differentiate. In mature OB interneurons, GABA and dopamine levels are modulated by odorant-induced synaptic activity-dependent regulation of Gad1 and Th transcription. The molecular mechanisms that specify and maintain the GABAergic and dopaminergic phenotypes in the OB are not clearly delineated. In this report, we review previous studies and present novel findings that provide insight into the contribution of epigenetic regulatory mechanisms for controlling expression of these neurotransmitter phenotypes in the OB. We show that HDAC enzymes suppress the dopaminergic phenotype in migrating neuroblasts by repressing Th transcription. In the mature interneurons, both Th and Gad1 transcription levels are modulated by synaptic activity-dependent recruitment of acetylated Histone H3 on both the Th and Gad1 proximal promoters. We also show that HDAC2 has the opposite transcriptional response to odorant-induced synaptic activity when compared to Th and Gad1. These findings suggest that HDAC2 mediates, in part, the activity-dependent chromatin remodeling of the Th and Gad1 proximal promoters in mature OB interneurons.

Keywords: Adult neurogenesis; ChIP; Dopamine; GABA; GAD; Glutamic acid decarboxylase; HDAC; NRSE; OB; Olfactory bulb; RMS; SVZ; TH; TSA; Tyrosine hydroxylase; acH3; acetylated Histone H3; chromatin immunoprecipitation; gamma aminobutyric acid; glutamic acid decarboxylase; histone deacetylase; neural restrictive silencer elements; olfactory bulb; qPCR; quantitative polymerase chain reaction; rostral migratory stream; subventricular zone; trichostatin A; tyrosine hydroxylase.

Figure 1

Laminar organization and circuit diagram for the olfactory bulb. A, a cartoon summary of the key synaptic connections in the main olfactory bulb based on studies in mice. Olfactory receptor neuronal axons terminate in the glomerular layer where they form axo- and dendro-dendritic connections with both the mitral and tufted cells and inhibitory periglomerular layer interneurons. The mitral/tufted cells are the primary output cells and their axons project to other cortical regions. The transmission of odorant sensory information for higher level processing in the cortex is modulated by inhibitory neurons in the granule cell layer. B, a cartoon of the mouse forebrain showing the position of the individual laminae of the main olfactory bulb (OB), rostral migratory stream (RMS) and subventricular zone (SVZ).

Figure 2

Odorant-mediated regulation of Gad1 transcription in the olfactory bulb. A, a mouse subjected to odorant deprivation by unilateral naris occlusion. The open and closed nares are indicated by the arrow and arrowhead, respectively. B, qRT-PCR analysis reveals Gad1 mRNA levels are reduced in the olfactory bulb that is ipsi-lateral to the closed nares (closed) when compared to the bulb corresponding to the unobstructed contra-lateral nares (open). C, chromatin immunoprecipitation (ChIP) shows that occupancy of pan- acetylated Histone H3 (acH3) on the Gad1 proximal promoter is dramatically reduced in the closed bulb relative to the open bulb. For comparison, negative control ChIP experiments with antibodies to rabbit IgG are also shown. D, immunofluorescence analysis for acH3 in the olfactory bulbs of mice subjected to unilateral naris occlusion shows that the reduced stimulation produced by odor deprivation does not diminish total levels of acH3 in the bulb ipsi-lateral to the naris closure (closed). Rather, there appears to be a slight increase in the acH3 intensity in this bulb. Thus, the changes in acH3 occupancy on the Gad1 proximal promoter observed in C are the result of mechanisms that specifically target the Gad1 promoter.

Figure 3

Histone deacetylase activity suppresses Th transcription in the rostral migratory stream (RMS) and olfactory bulb (OB). A, in situ hybridization in the adult OB shows strong levels of Th transcription in the glomerular layer (GL) as well as weak expression levels in the mitral and superficial granule cell layers (MCL and GCL, respectively). There is scattered transcription in the external plexiform layer (EPL), but no expression in the internal plexiform layer (IPL). In contrast to the in situ hybridization studies, immunohistochemical analysis with antibodies to TH protein reveal that protein expression is limited to only the glomerular layer. B and C, GFP expression in neonatal forebrain slice cultures from Th-GFP mice after 24 hour treatment with either vehicle (control) or 1.2 μM trichostatin A (TSA), respectively. The presence of the HDAC inhibitor, TSA, permits GFP expression within migrating progenitors in the RMS. The slices in B and C were cultured with depolarizing conditions (25mM KCl), which induces GFP expression in the glomerular layer. The presence of TSA, however, substantially increases GFP expression levels in the glomerular layer relative to the control slices. Insets show higher magnifications images of boxed areas. D and E, primary neural progenitor cultures from Th-GFP mice 24 hours after treatment with either vehicle (control) or 1.2 μM trichostatin A (TSA), respectively. The neuronal progenitor marker, β-tubulin III (red) is expressed by several cells in both sets of cultures. By contrast, only TSA-treated cultures show Th-GFP co-expressed in a subset of neuronal progenitors (yellow cells). F, in mice subjected to unilateral naris occlusion, chromatin immunoprecipitation (ChIP) experiments show that occupancy of pan-acetylated Histone H3 (acH3) on the Th proximal promoter is drastically reduced in the bulb ipsi-lateral to nares closure (closed) relative to the bulbs that are contra-lateral (open). For comparison, negative control ChIP experiments with antibodies to rabbit IgG are also shown.

Figure 4

HDAC2 expression in olfactory bulb dopaminergic neurons. A-C, in the OB of adult Th-GFP mice, immunofluorescence analysis reveals that nearly all GFP-expressing dopaminergic neurons (green) also co-express HDAC2 (red). Regions with Th-GFP and HDAC2 co-expression appear as yellow. D, in mice subjected to unilateral naris occlusion, qRT-PCR analysis shows Th and HDAC2 transcription levels have opposite responses to synaptic activity. In bulbs contra-lateral to naris closure (open), where odorant-mediated synaptic activity is highest, Th expression is greatest and HDAC2 is depressed. By contrast, Th expression is depressed and HDAC2 is maximal in the olfactory bulb ipsi-lateral to naris closure (closed) where synaptic activity is lowest.