Naris Closure Research Articles

In the Piriform Cortex, the Primary Impetus for Information Encoding through Synaptic Plasticity Is Provided by Descending Rather than Ascending Olfactory Inputs.

Information encoding by means of persistent changes in synaptic strength supports long-term information storage and memory in structures such as the hippocampus. In the piriform cortex (PC), that engages in the processing of associative memory, only short-term synaptic plasticity has been described to date, both in vitro and in anesthetized rodents in vivo. Whether the PC maintains changes in synaptic strength for longer periods of time is unknown: Such a property would indicate that it can serve as a repository for long-term memories. Here, we report that in freely behaving animals, frequency-dependent synaptic plasticity does not occur in the anterior PC (aPC) following patterned stimulation of the olfactory bulb (OB). Naris closure changed action potential properties of aPC neurons and enabled expression of long-term potentiation (LTP) by OB stimulation, indicating that an intrinsic ability to express synaptic plasticity is present. Odor discrimination and categorization in the aPC is supported by descending inputs from the orbitofrontal cortex (OFC). Here, OFC stimulation resulted in LTP (>4 h), suggesting that this structure plays an important role in promoting information encoding through synaptic plasticity in the aPC. These persistent changes in synaptic strength are likely to comprise a means through which long-term memories are encoded and/or retained in the PC.

Anatomical and molecular consequences of Unilateral Naris Closure on two populations of olfactory sensory neurons expressing defined odorant receptors

Mammalian olfactory sensory neurons (OSNs), the primary elements of the olfactory system, are located in the olfactory epithelium lining the nasal cavity. Exposed to the environment, their lifespan is short. Consequently, OSNs are regularly regenerated and several reports show that activity strongly modulates their development and regeneration: the peripheral olfactory system can adjust to the amount of stimulus through compensatory mechanisms. Unilateral naris occlusion (UNO) was frequently used to investigate this mechanism at the entire epithelium level. However, there is little data regarding the effects of UNO at the cellular level, especially on individual neuronal populations expressing a defined odorant receptor. Here, using UNO during the first three postnatal weeks, we analyzed the anatomical and molecular consequences of sensory deprivation in OSNs populations expressing the MOR23 and M71 receptors. The density of MOR23-expressing neurons is decreased in the closed side while UNO does not affect the density of M71-expressing neurons. Using Real Time qPCR on isolated neurons, we observed that UNO modulates the transcript levels for transduction pathway proteins (odorant receptors, CNGA2, PDE1c). The transcripts modulated by UNO will differ between populations depending on the receptor expressed. These results suggest that sensory deprivation will have different effects on different OSNs’ populations. As a consequence, early experience will shape the functional properties of OSNs differently depending on the type of odorant receptor they express.

ORA1, a Zebrafish Olfactory Receptor Ancestral to All Mammalian V1R Genes, Recognizes 4-Hydroxyphenylacetic Acid, a Putative Reproductive Pheromone

The teleost v1r-related ora genes are a small, highly conserved olfactory receptor gene family of only six genes, whose direct orthologues can be identified in lineages as far as that of cartilaginous fish. However, no ligands for fish olfactory receptor class A related genes (ORA) had been uncovered so far. Here we have deorphanized the ORA1 receptor using heterologous expression and calcium imaging. We report that zebrafish ORA1 recognizes with high specificity and sensitivity 4-hydroxyphenylacetic acid. The carboxyl group of this compound is required in a particular distance from the aromatic ring, whereas the hydroxyl group in the para-position is not essential, but strongly enhances the binding efficacy. Low concentrations of 4-hydroxyphenylacetic acid elicit increases in oviposition frequency in zebrafish mating pairs. This effect is abolished by naris closure. We hypothesize that 4-hydroxyphenylacetic acid might function as a pheromone for reproductive behavior in zebrafish. ORA1 is ancestral to mammalian V1Rs, and its putative function as pheromone receptor is reminiscent of the role of several mammalian V1Rs as pheromone receptors.

Activity-Dependent Modulation of Odorant Receptor Gene Expression in the Mouse Olfactory Epithelium

Activity plays critical roles in development and maintenance of the olfactory system, which undergoes considerable neurogenesis throughout life. In the mouse olfactory epithelium, each olfactory sensory neuron (OSN) stably expresses a single odorant receptor (OR) type out of a repertoire of ∼1200 and the OSNs with the same OR identity are distributed within one of the few broadly-defined zones. However, it remains elusive whether and how activity modulates such OR expression patterns. Here we addressed this question by investigating OR gene expression via in situ hybridization when sensory experience or neuronal excitability is manipulated. We first examined the expression patterns of fifteen OR genes in mice which underwent neonatal, unilateral naris closure. After four-week occlusion, the cell density in the closed (sensory-deprived) side was significantly lower (for four ORs), similar (for three ORs), or significantly higher (for eight ORs) as compared to that in the open (over-stimulated) side, suggesting that sensory inputs have differential effects on OSNs expressing different OR genes. We next examined the expression patterns of seven OR genes in transgenic mice in which mature OSNs had reduced neuronal excitability. Neuronal silencing led to a significant reduction in the cell density for most OR genes tested and thinner olfactory epithelium with an increased density of apoptotic cells. These results suggest that sensory experience plays important roles in shaping OR gene expression patterns and the neuronal activity is critical for survival of OSNs.

Postnatal experience modulates functional properties of mouse olfactory sensory neurons

Early experience considerably modulates the organization and function of all sensory systems. In the mammalian olfactory system, deprivation of the sensory inputs via neonatal, unilateral naris closure has been shown to induce structural, molecular and functional changes from the olfactory epithelium to the olfactory bulb and cortex. However, it remains unknown how early experience shapes the functional properties of individual olfactory sensory neurons (OSNs), the primary odor detectors in the nose. To address this question, we examined the odorant response properties of mouse OSNs in both the closed and open nostril after 4 weeks of unilateral naris closure, with age-matched untreated animals as control. Using a patch-clamp technique on genetically tagged OSNs with defined odorant receptors (ORs), we found that sensory deprivation increased the sensitivity of MOR23 neurons in the closed side, whereas overexposure caused the opposite effect in the open side. We next analyzed the response properties, including rise time, decay time, and adaptation, induced by repeated stimulation in MOR23 and M71 neurons. Even though these two types of neuron showed distinct properties with regard to dynamic range and response kinetics, sensory deprivation significantly slowed down the decay phase of odorant-induced transduction events in both types. Using western blotting and antibody staining, we confirmed the upregulation of several signaling proteins in the closed side as compared with the open side. This study suggests that early experience modulates the functional properties of OSNs, probably by modifying the signal transduction cascade.

Continuous Neural Plasticity in the Olfactory Intrabulbar Circuitry

In the mammalian brain each olfactory bulb contains two mirror-symmetric glomerular maps linked through a set of reciprocal intrabulbar projections. These projections connect isofunctional odor columns through synapses in the internal plexiform layer (IPL) to produce an intrabulbar map. Developmental studies show that initially intrabulbar projections broadly target the IPL on the opposite side of the bulb and refine postnatally to their adult precision by 7 weeks of age in an activity-dependent manner (Marks et al., 2006). In this study, we sought to determine the capacity of intrabulbar map to recover its precision after disruption. Using reversible naris closure in both juvenile and adult mice, we distorted the intrabulbar map and then removed the blocks for varying survival periods. Our results reveal that returning normal olfactory experience can indeed drive the re-refinement of intrabulbar projections but requires 9 weeks. Since activity also affects olfactory sensory neurons (OSNs) (Suh et al., 2006), we further examined the consequence of activity deprivation on P2-expressing OSNs and their associated glomeruli. Our findings indicate that while naris closure caused a marked decrease in P2-OSN number and P2-glomerular volume, axonal convergence was not lost and both were quickly restored within 3 weeks. By contrast, synaptic contacts within the IPL also decreased with sensory deprivation but required at least 6 weeks to recover. Thus, we conclude that recovery of the glomerular map precedes and likely drives the refinement of the intrabulbar map while IPL contacts recover gradually, possibly setting the pace for intrabulbar circuit restoration.

Differential Regulation of Dopaminergic Gene Expression byEr81

A recent study proposed that differentiation of dopaminergic neurons requires a conserved "dopamine motif" (DA-motif) that functions as a binding site for ETS DNA binding domain transcription factors. In the mammalian olfactory bulb (OB), the expression of a set of five genes [including tyrosine hydroxylase (Th)] that are necessary for differentiation of dopaminergic neurons was suggested to be regulated by the ETS-domain transcription factor ER81 via the DA-motif. To investigate this putative regulatory role of ER81, expression levels of these five genes were compared in both olfactory bulbs of adult wild-type mice subjected to unilateral naris closure and the olfactory bulbs of neonatal Er81 wild-type and mutant mice. These studies found that ER81 was necessary only for Th expression and not the other cassette genes. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSA) experiments showed that ER81 bound directly to a consensus binding site/DA-motif in the rodent Th proximal promoter. However, the ER81 binding site/DA-motif in the Th proximal promoter is poorly conserved in other mammals. Both ChIP assays with canine OB tissue and EMSA experiments with the human Th proximal promoter did not detect ER81 binding to the Th DA-motif from these species. These results suggest that regulation of Th expression by the direct binding of ER81 to the Th promoter is a species-specific mechanism. These findings indicate that ER81 is not necessary for expression of the OB dopaminergic gene cassette and that the DA-motif is not involved in differentiation of the mammalian OB dopaminergic phenotype.

Expression of EGR-1 in a subset of olfactory bulb dopaminergic cells

In the adrenal medulla, binding of the immediate early gene (IEG) proteins, EGR-1 (ZIF-268/KROX-24/NGFI-A) and AP-1, to the tyrosine hydroxylase (Th) proximal promoter mediate inducible Th expression. The current study investigated the potential role of EGR-1 in inducible Th expression in the olfactory bulb (OB) since IEGs bound to the AP-1 site in the Th proximal promoter are also necessary for activity-dependent OB TH expression. Immunohistochemical analysis of a naris-occluded mouse model of odor deprivation revealed weak EGR-1 expression levels in the OB glomerular layer that were activity-dependent. Immunofluorescence analysis indicated that a majority of glomerular cells expressing EGR-1 also co-expressed TH, but only small subset of TH-expressing cells contained EGR-1. By contrast, granule cells, which lack TH, exhibited EGR-1 expression levels that were unchanged by naris closure. Together, these finding suggest that EGR-1 mediates activity-dependent TH expression in a subset of OB dopaminergic neurons, and that there is differential regulation of EGR-1 in periglomerular and granule cells.

Secreted TARSH regulates olfactory mitral cell dendritic complexity

Olfactory sensory neurons synapse with mitral cells to form stereotyped connections in the olfactory bulb (OB). Mitral cell apical dendrites receive input from olfactory sensory neurons expressing the same odorant receptor. During development, this restricted dendritic targeting of mitral cells is achieved through eliminating elaborated dendritic trees to a single apical dendrite. Through a genome-wide microarray screen, we identified TARSH (Target of NESH SH3) as a transiently expressed molecule in mitral cells during the dendritic refinement period. TARSH expression is restricted to pyramidal neurons along the main olfactory pathway, including the anterior olfactory nucleus and piriform cortex. The dynamic TARSH expression is not altered when odor-evoked activity is blocked by naris closure or in AC3 knockout mice. We also demonstrate that TARSH is a secreted protein. In dissociated OB cultures, secreted TARSH promotes the reduction of mitral cell dendritic complexity and restricts dendritic branching and outgrowth of interneurons. Dendritic morphological changes were also observed in mitral cells overexpressing TARSH themselves. We propose that TARSH is part of the genetic program that regulates mitral cell dendritic refinement.

Visualization of spatiotemporal differentiation of dopaminergic interneurons in adult mouse olfactory bulb using transgenic mice

Olfactory bulb (OB) interneurons, predominantly periglomerular (PG) and granule (GR) cells, are derived from neural stem cells in the subventricular zone (SVZ) throughout life and migrate to the OB in the rostral migratory stream (RMS). In the adult superficial GR layer transgene expression was found, either enhanced green fluorescent protein or LacZ reporter driven by a 9 kb tyrosine hydroxylase (TH) promoter, that marked the dopamine (DA) phenotype. To demonstrate that the reporters and endogenous TH were similarly regulated expression of both parameters was shown to decline in the OB PG cells ipsilateral to odor deprivation produced by adult unilateral naris closure. The present findings suggested that DAergic differentiation might begin prior to progenitors reaching a PG position despite evidence that TH protein expression occurred only after PG cells received olfactory afferent stimulation. Of many genes previously hypothesized to regulate OB DA expression, regulated expression of the orphan receptor, Nurr1, but not the homeobox-containing genes, Dlx-1 and -2, was consistent with a role in regulation of the DA phenotype in adult mice OB. The studies show that transgenic lines are useful for analyzing spatiotemporal regulation by both intrinsic (programmed) and environmental factors in the neurogenesis of adult mouse OB DAergic interneurons.

Activity plays a role in eliminating olfactory sensory neurons expressing multiple odorant receptors in the mouse septal organ

A fundamental belief in the field of olfaction is that each olfactory sensory neuron (OSN) expresses only one odorant receptor (OR) type. Here we report that coexpression of multiple receptors in single neurons does occur at a low frequency. This was tested by double in situ hybridization in the septal organ in which greater than 90% of the sensory neurons express one of nine identified ORs. Notably, the coexpression frequency is nearly ten times higher in newborn than in young adult mice, suggesting a reduction of the sensory neurons with multiple ORs during postnatal development. In addition, such reduction is prevented by four-week sensory deprivation or impaired apoptosis. Furthermore, the high coexpression frequency is restored following four-week naris closure performed in young adult mice. The results indicate that activity induced by sensory inputs plays a role in ensuring the one cell–one receptor rule in a subset of olfactory sensory neurons.

Naris occlusion alters the electro-olfactogram: Evidence for compensatory plasticity in the olfactory system

Unilateral naris occlusion (NO) has been widely used as a method of olfactory deprivation to study the role of stimulus-driven activity in olfactory development [P.C. Brunjes, Unilateral naris closure and olfactory system development, Brain Res. Rev. 19 (1994) 146-160]. Recent immunochemical studies of the olfactory epithelium (OE) following NO provide evidence for a previously unknown compensatory response to deprivation [D.M. Coppola, A. Waguespack, M. Reems, M.L. Butman, J. Cherry, Naris occlusion alters transductory protein immunoreactivity in olfactory epithelium, Histol. Histopathol. 21 (2006) 487-501; A. Waguespack, M. Reems, M.L. Butman, J. Cherry, D.M. Coppola, Olfactory receptor neurons have enhanced olfactory marker protein immunoreactivity in the nasal cavity ipsilateral to naris occlusion, Brain Res. 1044 (2005) 1-7]. To further investigate this phenomenon we measured electro-olfactograms (EOG) from the open and occluded OE of adult mice that had undergone NO as newborns. EOG waveforms from the open side OE of NO mice were indistinguishable from those obtained from untreated animals. However, amplitudes of EOGs from the occluded OE of NO mice were greater, on average, than those recorded at matched locations from the open side. This result was consistent across turbinates, odors, and all but the highest odor concentration. In addition, EOGs recorded from the occluded OE had significantly slower onset and recovery kinetics. Responses in a double-pulse protocol confirmed that the kinetics of the cellular or population processes that underlie the EOG are slowed by NO. These results provide the most direct support, to date, for compensatory plasticity in the olfactory system.

Histological changes in rat nasal epithelia after unilateral neonatal naris occlusion

The authors studied the extent of the different epithelia lining the nasal fossae of the albino rat after neonatal closure of one naris. Newborn pups were anesthetized by hypothermia and the external opening of their right naris cauterized, while littermates served as controls. Animals were sacrificed at 3 months, and the occluded (OCF) and nonoccluded (NOF) fossae of experimental animals as well as both fossae of control animals (CTF) were histologically studied. In both control and experimental animals, nasal fossae were lined by five different types of epithelia: squamous stratified, transitional, metaplastic, respiratory, and olfactory epithelia. It was found that closure of one naris provokes reorganization of the epithelial lining in both the occluded and nonoccluded side. In CTF airflow, physical conditions as well as pollutants and biological agents irritate the epithelial lining, causing squamous metaplasia as well as metaplastic epithelium showing inflammation in rostral levels. In CTF caudal levels, the metaplastic epithelium appears to a lesser degree and the respiratory epithelium prevails, except for the most caudal level where the olfactory epithelium is prevalent. In OCF, the protected environment created prevents the occurrence of metaplastic epithelium, the transitional, respiratory, and olfactory epithelia developing in the corresponding area instead. In NOF, where the airflow is double, the same pattern occurs as in CTF, although metaplastic epithelium values are approximately double, suggesting a clear linear effect. An outstanding feature observed was the increased extent of the olfactory epithelium in OCF regarding NOF, although changes in its morphological structure were not found. Airflow properties, including pressure, coldness, velocity, and turbulence, as well as biological and chemical hazards present in inflow, cause histological reorganization of the nasal epithelium lining during postnatal development. Results prove the need to consider airflow changes in nasal fossae surgery and point to the protective value of naris closure in ENT clinics, supporting it as a treatment of atrophic rhinitis.

Activation of noradrenergic transmission by α 2-adrenoceptor antagonists counteracts deafferentation-induced neuronal death and cell proliferation in the adult mouse olfactory bulb

The olfactory bulb is the target of neural progenitor cells that are generated in the subventricular zone of the lateral ventricle in the adult brain. This permanent neurogenesis is likely influenced by olfactory input to the bulb since previous studies have shown that cell proliferation and/or apoptotic death are stimulated by naris closure or surgical transection of the olfactory nerve. Since the olfactory bulb is densely innervated by noradrenergic afferents originating in the locus coeruleus, we have studied the impact of pharmacologically activating this noradrenergic system on cell death and proliferation following unilateral olfactory axotomy in the adult mouse olfactory bulb. We found that noradrenaline release in the olfactory bulb was significantly increased by intraperitoneal injections of the selective α 2-adrenoceptor antagonists, dexefaroxan (0.63 mg/kg) and 5-fluoro-methoxyidazoxan (F 14413; 0.16 mg/kg). A chronic treatment with either compound for 7 days following olfactory axotomy significantly reduced neuronal death, glial activation and cell proliferation in the deafferented olfactory bulb. These data (1) confirm that α 2-adrenoceptor antagonists, presumably by facilitating central noradrenergic transmission, afford neuroprotection in vivo, as previously shown in models of cerebral ischemia, excitotoxicity and devascularization-induced neurodegeneration, and (2) support a role of the locus coeruleus noradrenergic system in promoting survival of neurons in areas of the brain where neurogenesis persists in the adult.

Effects of unilateral naris closure on the nasal and maxillary sinus mucosa in rabbit

In nasal septal deviation, the deviated narrow side is more frequently combined with sinusitis due to the mechanical obstruction of ostiomeatal unit. The purpose of this study was to determine whether reduced airflow has a harmful effect on nasal inflammation and the development of rhinosinusitis. Twenty-three healthy New Zealand white rabbit were used. One side of the nostril was closed in 15 rabbits and 5 rabbits served as control. Rabbits were sacrificed at 4, 8 and 12 weeks. The anterior maxillary and septal mucosa were obtained for EM and LM study. Mucociliary clearance rate was measured using the India ink method. At 4 weeks, the opened side mucosa showed ciliary losses and ulcerations in the mucosa, but at 8 and 12 weeks, the closed side showed more severe ulceration and ciliary loss than open side. The mucociliary clearance rate was 12.3 mm/min in the control group, but the open side was 8.7 and 7.1 mm/min and the closed side was 4.7 and 6.4 mm/min at 4 and 8 weeks, respectively. Electron microscopy showed many abnormal cilia at 8 weeks in both sides. These observations suggest that inadequate airflow is an important causative factor of nasal and sinus infection, and that patients with septal deviation have a similar chance of developing sinusitis in either side.

Differentiation of the dopaminergic phenotype in the olfactory system of neonatal and adult mice

Olfactory bulb (OB) interneurons are derived primarily postnatally from progenitors in the anterior subventricular zone (SVZa) and migrate to the OB in the rostral migratory stream (RMS). Progenitors differentiate into phenotypically diverse granule and periglomerular cells by as yet undefined mechanisms. To visualize spatiotemporal aspects of periglomerular dopamine (DA) neuron differentiation, two independently derived transgenic mouse lines were analyzed with a 9-kb tyrosine hydroxylase (TH) promoter to drive either a LacZ or an enhanced green fluorescent protein (EGFP) reporter gene. Both reporters showed similar neonatal expression that varied from low levels in RMS, to moderate in the superficial granule cell layer, to strong in relatively large cells, possibly external tufted cells, in the periglomerular region. TH mRNA and protein were not detected in the RMS but were colocalized with the transgenes in neonatal superficial granule and periglomerular cells. By comparison, TH protein in adults was further limited to periglomerular cells. To demonstrate that transcriptional regulation was the same for EGFP and TH, expression was shown to decline similarly in the OB ipsilateral to odor deprivation produced by adult unilateral naris closure. Of two genes previously hypothesized to regulate OB DA expression, only regulated expression of the orphan receptor Nurr1, but not the homeobox-containing genes Dlx-1 and -2, was consistent with a role in regulation of the DA phenotype. These data demonstrate for the first time that DA phenotypic differentiation in neonates begins with low-level transcription in migrating progenitors in the RMS and culminates with activity-dependent protein expression in periglomerular cells innervated by olfactory receptor cells.

Postnatal refinement of peripheral olfactory projections.

Axonal projections from the olfactory epithelium to the olfactory bulb are organized into glomeruli according to the expressed odorant receptor. Using gene-targeted mice, we show that glomerular maturation proceeds along different time courses for two similar receptors and requires sensory input during distinct sensitive periods. During early development, some glomeruli are innervated by axons of neurons that do not express the same receptor. These heterogeneous glomeruli normally disappear with age, but they persist in adults deprived of sensory input by unilateral and permanent naris closure. Persistence may be due, in part, to prolonged survival of olfactory sensory neurons.

Trans-neuronal regulation of cortical apoptosis in the adult rat olfactory system

Previous work has identified a population of neurons within the anterior piriform cortex that undergo rapid apoptosis following de-afferentation by olfactory bulbectomy in adult rats. The specific initiation signal for apoptosis in this paradigm is unknown, but may include an activity-dependent trans-neuronal cascade. The present report examined the effect of adult-onset unilateral naris occlusion, which reduces olfactory bulb afferent excitation of piriform cortex, on apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling [TUNEL]) in the rat anterior piriform cortex. Adult Long-Evans hooded rats received unilateral naris occlusion or a control manipulation and were sacrificed after 1, 5, 7, 10 or 20 days later. For comparison, a second group of rats received a unilateral bulbectomy and were sacrificed 24 h later. Counts of TUNEL-stained cell profiles were performed for layers I/II and layer III of the anterior piriform cortex ipsilateral and contralateral to the manipulation. The results confirmed that unilateral bulbectomy produced a dramatic increase in TUNEL labeling in layers I/II of the ipsilateral piriform cortex 24 h after bulbectomy. Unilateral naris closure also produced enhanced TUNEL labeling, although the magnitude of the effect was less than that produced by bulbectomy, and enhanced TUNEL labeling was apparent both ipsilateral and contralateral to the sealed naris compared to controls. Deprivation-induced TUNEL labeling was detectable by 24 h post-closure, peaked at 5 days and was no different from controls by 20 days post-closure. Neither bulbectomy nor naris closure affected TUNEL labeling in layer III. Together, these results suggest that there is a population of superficial cells in piriform cortex whose survival is tightly regulated by sensory input.

Dlx-1 and Dlx-2 expression in the adult mouse brain: relationship to dopaminergic phenotypic regulation.

Expression of the homeodomain-containing transcription factors Dlx-1 and Dlx-2 in the lateral (LGE) and medial (MGE) ganglionic eminences, subpallial embryonic structures, is required for generation of telencephalic interneurons. LGE- and MGE-derived progenitors migrate and populate a number of forebrain structures, including the cortex, hippocampus, and olfactory bulb (OB). Previous reports focusing on embryogenesis of telencephalic neurons in Dlx-1 and Dlx-2 null mice suggested a specific role for these genes in expression of the OB dopamine (DA) phenotype. We have investigated whether these genes also are expressed in adult brain, especially in those pallial derivatives, such as the OB, hippocampus, and possibly cortex, where neurogenesis continues in adults. With a highly sensitive, nonradioactive in situ hybridization technique and both DLX-2 and pan DLX antisera, widespread expression of both genes was found in adult mouse fore- but not mid- or hindbrain. The adult unilateral naris closure paradigm was employed to establish a causative role for Dlx in regulating tyrosine hydroxylase (TH) expression; TH is the first enzyme in DA biosynthesis. TH mRNA, but not Dlx expression, was significantly down-regulated in the OB ipsilateral to closure. These findings suggest that Dlx-1 and -2 do not play a direct role in DA phenotypic differentiation and TH gene regulation in adult OB. The widespread expression of Dlx mRNA and protein in the adult brain suggests that these genes may have additional roles in mature animals.

Changes in Immunoreactivity to Calcium-Binding Proteins in the Anterior Olfactory Nucleus of the Rat after Neonatal Olfactory Deprivation

The effects of olfactory deprivation on the density of neuronal populations expressing the calcium-binding proteins calbindin D-28k, calretinin, and parvalbumin in the anterior olfactory nucleus of the rat were studied immunohistochemically in 60-day-old rats subjected to unilateral naris closure on the day of birth. The neuronal populations were characterized morphologically and topologically, and the density of each cell type was calculated in each subdivision of the anterior olfactory nucleus at seven rostrocaudal levels. Data were gathered into three groups: data from either the ipsilateral or contralateral anterior olfactory nucleus of experimental animals and data from control animals. Statistical analysis indicated that disruption of the normal afferent activity to one olfactory bulb affects the expression of the calcium-binding proteins investigated in the anterior olfactory nucleus, as revealed by variations in the density of certain neuronal populations. The observed effects were very heterogeneous and could not be related to any specific neuronal type, location, or to the expression of a given calcium-binding protein. Nevertheless, as a general rule the most affected neuronal populations were those expressing calbindin D-28k located in the rostral subdivisions. These subdivisions are the latest to develop in mammals and are those that receive the largest amount of inputs from the olfactory bulb.