Cynthia C. Woo

Multidimensional chemotopic responses to n-aliphatic acid odorants in the rat olfactory bulb

In an effort to understand the means by which similar chemical odorants are encoded in the mammalian brain, we exposed rats to a homologous series of n‐aliphatic acids and mapped the response of the entire olfactory bulb glomerular layer by using a high‐resolution [14C]‐2‐deoxyglucose uptake technique. We found that these similar odorants evoked spatially clustered but distinct responses in the bulb that changed systematically with carbon chain length. In addition to these chemotopic responses, different odorants within the series evoked systematic differences along two other dimensions: amount of deoxyglucose uptake and extent of the glomerular layer showing high activity. Increases along these two dimensions also were correlated with increasing carbon number. The focal glomerular responses were mirrored by responses in deeper bulb layers. Decreasing the odorant concentration decreased the deoxyglucose uptake within focal regions. The focal regions of activity occurred in pairs involving both medial and lateral representations in the bulb, perhaps reflecting the paired medial and lateral projections of olfactory sensory neurons expressing specific types of odorant feature receptor proteins. The observed spatial pattern of response also may explain both the failure of some bulb lesions to interfere with behavioral olfactory responses and the success of other lesions in blocking olfactory responses. These data support a model of parallel, distributed processing of odorants along multiple dimensions. They also support the notion that analyses of the spatial relationships among odorant responses in the olfactory bulb can demonstrate aspects of the mechanism for odor chemical coding. J. Comp. Neurol. 409:529–548, 1999.

Sensitive period for neural and behavioral response development to learned odors

Olfactory preference training early in life produces both a behavioral preference and an enhanced uptake of 2-deoxy-[14C]glucose (2-DG) in specific areas of the olfactory bulb glomerular layer. We now describe a sensitive period during the first week after birth for the development both of the enhanced neural response and the behavioral preference.

A learned odor evokes an enhanced Fos-like glomerular response in the olfactory bulb of young rats

Young rats exposed to peppermint odor and reinforcing tactile stimulation from postnatal days (PND) 1-18 increase their preference for that odor relative to controls. This early olfactory memory is accompanied by an 80% increase in the density of glomerular-layer cells displaying Fos-like immunoreactivity in response to the learned odor on PND 19. The difference is observed in midlateral portions of the olfactory bulb that align with foci of 2-deoxyglucose (2-DG) uptake in adjacent sections. Trained and control animals are not different in the Fos-like response of juxtaglomerular cells within ventrolateral 2-DG foci. Ratios of midlateral/ventrolateral response differ significantly between trained and control animals and include differences among cells of three staining intensities. These ratios are correlated with ratios of 2-DG uptake (midlateral/ventrolateral foci), which also differ significantly between trained and control rats. Juxtaglomerular cells associated with 2-DG foci also express Egr-1-like immunoreactivity. However, the midlateral Egr-1 response does not differ between trained and control rats. These results show that early memories can be associated with an increased Fos-like response in a primary sensory area of the CNS. They also suggest that only specific regions within the olfactory bulb are modified following the learning of a given odor in early life.

Expression of active caspase-3 in mitotic and postmitotic cells of the rat forebrain

Active caspase‐3 immunoreactivity was detected in the rat forebrain proliferative regions at birth and remained high in these areas for about 2 weeks, during which period labeled cells were present centroperipherally across the olfactory bulb. By the end of the third postnatal week, only a small number of immunolabeled cells remained in these forebrain structures. Active caspase‐3 immunolabeling was localized mostly to cell nuclei and co‐localized partially with TuJ1 and NeuN immunoreactivity, but not with glial fibrially acidic protein, OX‐42, γ‐aminobutyric acid, or terminal deoxynucleotidyl transferase‐mediated nick end labeling (TUNEL)‐positive labeling. Active caspase‐3 and 5‐bromo‐2′‐deoxyuridine (BrdU) double‐labeled nuclei were seen in the proliferative regions after 2 hours and in the periglomerular region of the bulb after 7 days following BrdU injections. Examination of the cells with electron microscopy confirmed that the active caspase‐3‐containing nuclei in the proliferative regions often had infoldings and appeared to be undergoing division. Some of the cells with active caspase‐3‐labeled nuclei in the bulb had synapses on their somata or dendrites. Labeled dendritic spines and a few axon terminals were also observed in the olfactory bulb. Taken together, it appears that a wave of active caspase‐3‐positive cells are dividing in the proliferative zones and then migrating to the bulb as they differentiate into neurons. Therefore, active caspase‐3 may play a role in cellular processes such as neuronal differentiation, migration, and plasticity, in addition to its role in cell death. J. Comp. Neurol. 433:4–22, 2001.

Environmental enrichment as an effective treatment for autism: A randomized controlled trial

Enriched sensorimotor environments enable rodents to compensate for a wide range of neurological challenges, including those induced in animal models of autism. Given the sensorimotor deficits in most children with autism, we attempted to translate that approach to their treatment. In a randomized controlled trial, 3-12 year-old children with autism were assigned to either a sensorimotor enrichment group, which received daily olfactory/tactile stimulation along with exercises that stimulated other paired sensory modalities, or to a control group. We administered tests of cognitive performance and autism severity to both groups at the initiation of the study and after 6 months. Severity of autism, as assessed with the Childhood Autism Rating Scale, improved significantly in the enriched group compared to controls. Indeed, 42% of the enriched group and only 7% of the control group had what we considered to be a clinically significant improvement of 5 points on that scale. Sensorimotor enrichment also produced a clear improvement in cognition, as determined by their Leiter-R Visualization and Reasoning scores. At 6 months, the change in average scores for the enriched group was 11.3 points higher than that for the control group. Finally, 69% of parents in the enriched group and 31% of parents in the control group reported improvement in their child over the 6-month study. Environmental enrichment therefore appears to be effective in ameliorating some of the symptoms of autism in children.

A learned odor decreases the number of Fos-immunopositive granule cells in the olfactory bulb of young rats

Olfactory stimulation evokes a column of activity within the olfactory bulb extending from the glomerular layer to the granule cell layer that can be visualized with 2-deoxyglucose autoradiography, optical imaging, Fos protein immunohistochemistry and c-fos mRNA in situ hybridization. The Fos response to odors is typified by the activity of relatively few juxtaglomerular cells, which often occur in foci, and a large number of granule cells extending through much of the bulb. In this study, we characterized the granule cell response to an odor for which young rats had acquired a preference. Fos-like immunoreactive granule cells were quantified by image analysis, and densely stained cells were counted in a region previously shown to be responsive to peppermint odor. We found that odor-trained pups have about half the number of Fos-immunopositive superficial granule cells which respond to a learned odor than do control pups. We then determined whether there was a correlation between the juxtaglomerular cell response and the response of the superficial granule cells deep to those glomerular layer cells. We found a positive correlation between the number of juxtaglomerular cells and the number of granule cells demonstrating Fos immunoreactivity in both control and trained pups, a relationship that changed with early olfactory training.

Environmental Enrichment as a Therapy for Autism: A Clinical Trial Replication and Extension

Based on work done in animal models showing that autism-like symptoms are ameliorated following exposure to an enriched sensorimotor environment, we attempted to develop a comparable therapy for children with autism. In an initial randomized controlled trial, children with autism who received sensorimotor enrichment at home for 6 months had significant improvements in both their cognitive ability and the severity of their autism symptoms (Woo & Leon, 2013). We now report the outcomes of a similar randomized controlled trial in which children with autism, 3 to 6 years old, were randomly assigned to groups that received either daily sensorimotor enrichment, administered by their parents, along with standard care, or they received standard care alone. After 6 months, enriched children showed statistically significant gains in their IQ scores, a decline in their atypical sensory responses, and an improvement in their receptive language performance, compared to controls. Furthermore, after 6 months of enrichment therapy, 21% of the children who initially had been given an autism classification, using the Autism Diagnostic Observation Schedule, improved to the point that, although they remained on the autism spectrum, they no longer met the criteria for classic autism. None of the standard care controls reached an equivalent level of improvement. Finally, the outcome measures for children who received only a subset of sensory stimuli were similar to those receiving the full complement of enrichment exercises. Sensorimotor enrichment therapy therefore appears to be a cost-effective means of treating a range of symptoms for children with autism.

Synaptophysin-like immunoreactivity in the rat olfactory bulb during postnatal development and after restricted early olfactory experience

Synaptophysin is a synaptic vesicle protein that provides a marker of synaptic distribution in the brain. We used synaptophysin immunohistochemistry to detect changes in synaptic distribution in the main olfactory bulb during normal early postnatal development and following early olfactory restriction. Synaptophysin-like immunoreactivity appears early in the postnatal development of most bulbar laminae. In the first postnatal week, the entire glomerular neuropil is stained intensely, but by PND 19, the glomerular perimeter becomes more intensely stained than its core. This immunoreactivity is especially dense along that aspect of the perimeter facing the olfactory nerve layer. To determine if this developmental pattern could be affected by early olfactory experience, unilateral naris closure was performed on PND 1. In deprived bulbs at PND 19, the difference in staining between the glomerular border and the glomerular core is significantly less than in either contralateral glomeruli or glomeruli from unoperated animals. The difference between the glomerular border and the glomerular core is greater in bulbs contralateral to a closed naris than in bulbs from unoperated animals. Staining intensity measured over entire glomeruli is not altered by naris closure, nor is staining of other laminae. The changes detected after naris closure indicate that the glomerular distribution of synaptophysin may be particularly sensitive to early olfactory experience.

Early locus coeruleus lesions increase the density of β-adrenergic receptors in the main olfactory bulb of rats

Norepinephrine is supplied to both deep and superficial layers of the olfactory bulb through dense projections from the locus coeruleus.16 Beta‐adrenergic receptors are located in nearly all bulb laminae, with high‐density foci of β‐1 and β‐2‐adrenoceptors present in the glomerular layer.29 Early olfactory experiences that increase norepinephrine levels in the bulb also decrease the density of β‐1‐ and β‐2‐adrenoceptors, as well as the number of high‐density glomerular foci of β‐2‐receptors.30 Changes in bulb norepinephrine levels, therefore, may affect the density of β‐adrenoceptors in the bulb. In the current study, we test this hypothesis by performing unilateral lesions of the locus coeruleus with 6‐hydroxydopamine on postnatal day 4, and examining the density of β‐1‐ and β‐2‐adrenergic receptors in the main olfactory bulb of the rat using125I‐labeled iodopindolol receptor autoradiography on postnatal day 19. Locus coeruleus destruction resulted in a statistically significant increase in the density of adrenergic receptors in the ipsilateral bulb compared to the contralateral bulb. Both β‐1‐ and β‐2‐adrenoceptor subtypes increased in density with this manipulation, although the number of glomerular layer high‐density β‐2 foci was not significantly different between the two bulbs. These results are consistent with the hypothesis that changes in olfactory bulb norepinephrine can regulate the density of β‐adrenergic receptors in the bulb.

Granule and mitral cell densities are unchanged following early olfactory preference training

Early olfactory preference training causes both an increased number of juxtaglomerular cells and an increased number of such cells expressing Fos protein. In contrast, there are fewer cells expressing Fos protein in the granule cell layer after training. Here, we report no change in the number or size of granule and mitral cells as a consequence of these early olfactory experiences.