Umeå University's logo

It is well established that environmental influences play a key role in sculpting neuronal connectivity in the brain. One example is the olfactory sensory map of topographic axonal connectivity. While intrinsic odorant receptor signaling in olfactory sensory neurons (OSN) determines anterior-posterior counter gradients of the axonal guidance receptors Neuropilin-1 and Plexin-A1, little is known about stimulus-dependent gradients of protein expression, which correlates with the functional organization of the olfactory sensory map along its dorsomedial (DM)-ventrolateral (VL) axis. Deficiency of the Alzheimer's β-secretase BACE1, which is expressed in a DM(low)-VL(high) gradient, results in OSN axon targeting errors in a DM > VL and gene dose-dependent manner. We show that expression of BACE1 and the all-trans retinoic acid (RA)-degrading enzyme Cyp26B1 form DM-VL counter gradients in the olfactory epithelium. Analyses of mRNA and protein levels in OSNs after naris occlusion, in mice deficient in the olfactory cyclic nucleotide-gated channel and in relation to onset of respiration, show that BACE1 and Cyp26B1 expression in OSNs inversely depend on neuronal activity. Overexpression of a Cyp26B1 or presence of a dominant negative RA receptor transgene selectively in OSNs, inhibit BACE1 expression while leaving the DM(low)-VL(high) gradient of the axonal guidance protein Neuropilin-2 intact. We conclude that stimulus-dependent neuronal activity can control the expression of the RA catabolic enzyme Cyp26B1 and downstream genes such as BACE1. This result is pertinent to an understanding of the mechanisms by which a topographic pattern of connectivity is achieved and modified as a consequence of graded gene expression and sensory experience.

Stimulus-dependent expression of the retinoic acid-inactivating enzyme Cyp26B1 in olfactory sensory neurons (OSNs) forms a dorsomedial (DM)-ventrolateral (VL) gradient in the mouse olfactory epithelium. The gradient correlates spatially with different rates of OSN turnover, as well as the functional organization of the olfactory sensory map, into overlapping zones of OSNs that express different odorant receptors (ORs). Here, we analyze transgenic mice that, instead of a stimulus-dependent Cyp26B1 gradient, have constitutive Cyp26B1 levels in all OSNs. Starting postnatally, OSN differentiation is decreased and progenitor proliferation is increased. Initially, these effects are selective to the VL-most zone and correlate with reduced ATF5 expression and accumulation of OSNs that do not express ORs. Transcription factor ATF5 is known to stabilize OR gene choice via onset of the stimulus-transducing enzyme adenylyl cyclase type 3. During further postnatal development of Cyp26B1 mice, an anomalous DMhigh-VLlow expression gradient of adenylyl cyclase type 3 appears, which coincides with altered OR frequencies and OR zones. All OR zones expand ventrolaterally except for the VL-most zone, which contracts. The expansion results in an increased zonal overlap that is also evident in the innervation pattern of OSN axon terminals in olfactory bulbs. These findings together identify a mechanism by which postnatal sensory-stimulated vitamin A metabolism modifies the generation of spatially specified neurons and their precise topographic connectivity. The distributed patterns of vitamin A-metabolizing enzymes in the nervous system suggest the possibility that the mechanism may also regulate neuroplasticity in circuits other than the olfactory sensory map.

The aim of the studies included in the thesis is to better understand the interplay between neuronal activity-dependent gene regulation and the bioactive vitamin A metabolite all-trans-retinoic acid (RA) during postnatal development, refinement and maintenance of precise neuronal connectivity using the olfactory sensory neuron (OSN) in the olfactory epithelium (OE) of genetically modified mice as a model. We show that:

Inhibition of RA receptor (RAR)-mediated transcription in OSNs reduces expression of the olfactory cyclic nucleotide-gated (CNG) ion channel, which is required for odorant receptor (OR)-mediated stimulus transduction. This, results in increased OSN death and errors in precise connectivity. The increased cell death may be a consequence of reduced intrinsic excitability and/or reduced influx of Ca²⁺ ions while the errors in connectivity may be due to altered OR-dependent expression of axonal guidance proteins, such as Kirrel-2 and Neuropilin-1.

Expression of the RA catabolic enzyme Cyp26B1 in OSNs is positively regulated by RAR-mediated transcription as well as sensory stimulation in a CNG channel-dependent manner. This shows that neuronal activity and local vitamin A metabolism are parts of novel regulatory feedback loop controlling precise connectivity and neuronal survival. The feedback loop may be a form of homeostatic plasticity in response to global changes in neuronal activity.

BACE1, an enzyme is implicated in Alzheimer´s disease, and Cyp26B1 are inversely regulated by CNG channel-dependent sensory stimulation. Cyp26B1 expression is switched on at birth, forms a topographic expression gradient in OE and inhibits BACE1 expression into an inverse counter gradient. Taken together these results reveal a novel neuronal activity-dependent mechanism by which sensory stimuli can shape spatial gene expression via altered RA bioavailability.

Increased Cyp26B1 expression stimulates turnover of OSNs during adult neurogenesis by a non-cell-autonomous mechanism. The gradient of Cyp26B1 expression correlates with spatially-regulated diversification of OSNs into subpopulations that express different subsets of OR genes. Cyp26B1 expression influences spatial OR diversification of OSNs by two different mechanisms. In the ventrolateral OE, Cyp26B1 inhibits OR expression by blocking OSN differentiation at a stage that may be associated with the cell intrinsic mechanism regulating OR gene choice. In the dorsomedial OE the expression frequency of some ORs is unaltered while other increases, presumably as a consequence of neuronal activity-dependent competition. A probable function of graded and activity-dependent Cyp26B1 expression is to form a topographic partitioning of the olfactory sensory map into functional domains, which gradually differ from each other with regard to experience-driven plasticity and neurogenic potential along the dorsomedial-ventrolateral axis of OE.

Little is known about the identities and functions of extracellular signaling molecules that work in concert with neuronal activity to regulate refinement and maintenance of the mouse olfactory sensory map. We show that expression of a dominant negative retinoic acid receptor (RAR) in olfactory sensory neurons (OSNs) increased the number of glomeruli that incorrectly contained OSN axons expressing different odorant receptors. This phenotype became apparent postnatally, coincided with increased cell death, and was preceded by increased Neuropilin-1 and reduced Kirrel-2 expressions. Kirrel-2-mediated cell adhesion influences odorant receptor-specific axonal convergence and is regulated by odorant receptor signaling via the olfactory cyclic nucleotide-gated (CNG) ion channel. Accordingly, we found that inhibited RAR function correlated with reduced CNG channel expression. Naris occlusion experiments and analysis of CNG channel-deficient mice further indicated that RAR-regulated CNG channel levels influenced the intrinsic neuronal activity required for cell survival in the absence of odor stimulation. Finally, we showed that CNG channel activity regulated expression of the retinoic acid-degrading enzyme Cyp26B1. Combined, these results identify a novel homeostatic feedback mechanism involving retinoic acid metabolism and CNG channel activity, which influences glomerular homogeneity and maintenance of precisely connected OSNs.