Joel A. Maruniak

Neurogenesis persists in the subependymal layer of the adult mouse brain.

Neurogenesis of olfactory bulb granule cells is known to persist in adult rats where, in some strains, the bulbs grow throughout life. In mice, bulb growth ceases early in adulthood and here we ask if granule cell neurogenesis persists after the bulbs have stopped growing. By injecting adult mice with bromodeoxyuridine (BrdU) and allowing short and long survival times, we found that new cells form in the subependymal layer and that they migrate subsequently into the olfactory bulbs where they acquire the nuclear morphology of granule cells and express neuron-specific markers. Using [3H]thymidine, we found that most of these adult-generated granule neurons persist within the bulbs for at least 16 weeks. This shows the persistence of neurogenesis and neuronal migration in adult animals in which the olfactory bulbs have stopped growing.

Odor deprivation leads to reduced neurogenesis and reduced neuronal survival in the olfactory bulb of the adult mouse

Neurogenesis persists in the olfactory bulbs of adult mice, with new cells being generated in the proliferative subependymal layer. Our previous work has shown that unilateral odor deprivation through naris closure leads to a net loss of granule neurons in the ipsilateral (odor-deprived) olfactory bulb, while not affecting the contralateral bulb. Here we used several experimental approaches to determine if this loss of neurons results from reduced neurogenesis, reduced neuronal survival, or both. First, bromodeoxyuridine immunohistochemistry was used to determine the number of S-phase cells in the subependymal layer eight weeks after naris closure. Proliferation was reduced within and just caudal to the odor-deprived bulb compared to the open-side (control) bulb. Second, counts of pyknotic nuclei four weeks after naris closure were used to document a higher rate of cell death on the deprived side. Third, 3H-thymidine autoradiography was used to assess differences in granule cell survival on the two sides. Granule cell precursors were labeled by a single injection of 3H-thymidine eight weeks after naris closure, and the number of surviving labeled granule cells assessed four and 16 weeks later. Granule cell survival was significantly reduced within the odor-deprived bulbs. These data indicate that the loss of granule cells which follows odor deprivation is caused, at least in part, by reduced neurogenesis and reduced survival of these adult-generated neurons.

Adult naris closure profoundly reduces tyrosine hydroxylase expression in mouse olfactory bulb

Peripheral afferent innervation appears to be required for the expression of the dopamine phenotype in the rodent main olfactory bulb. Experiments utilizing neonatal naris closure as a means of sensory deprivation also suggest that odor-induced afferent activity is required for the expression of the phenotype. These experiments are confounded, however, by the significant postnatal maturation of the dopamine system. The current experiments utilized adult unilateral naris closure to address this issue. As with neonatal closure, adult deprivation produces a profound reduction in the expression of tyrosine hydroxylase (TH), the first enzyme in the dopamine biosynthetic pathway. By 4 days a small decrease is observed in TH activity and immunoreactivity. Activity reaches a nadir of 12% of control levels at about 1 month. TH mRNA is reduced similarly when analyzed at about 2 months post-closure. Glutamic acid decarboxylase protein and mRNA expression, which are co-localized with TH, remain at close to control levels indicating the continued presence of the dopamine neurons. The time-course of the loss of TH is identical to that for zinc sulphate-induced denervation of the olfactory bulb. These data support the hypothesis that odor modulated afferent activity is required for expression of the dopamine phenotype and that, if a trophic factor is involved, its release is also activity dependent.

Embryonic stem cell-derived neural progenitors incorporate into degenerating retina and enhance survival of host photoreceptors

Embryonic stem (ES) cells differentiate into all cell types of the body during development, including those of the central nervous system (CNS). After transplantation, stem cells have the potential to replace host cells lost due to injury or disease or to supply host tissues with therapeutic factors and thus provide a functional benefit. In the current study, we assessed whether mouse neuralized ES cells can incorporate into retinal tissue and prevent retinal degeneration in mnd mice. These mice have an inherited lysosomal storage disease characterized by retinal and CNS degeneration. Sixteen weeks after intravitreal transplantation into adult mice, donor cells had incorporated into most layers of the retina, where they resembled retinal neurons in terms of morphology, location in the retina, and expression of cell type–specific marker proteins. Presence of these donor cells was correlated with a reduction in the sizes and numbers of lysosomal storage bodies in host retinal cells. The presence of transplanted donor cells was also accompanied by enhanced survival of host retinal neurons, particularly photoreceptors. These results demonstrate that neuralized ES cells protect host neurons from degeneration and appear to replace at least some types of lost neurons.

Neural differentiation of mouse embryonic stem cells in vitro and after transplantation into eyes of mutant mice with rapid retinal degeneration

Embryonic stem (ES) cells can differentiate into many specialized cell types, including those of the nervous system. We evaluated the differentiation of enhanced green fluorescent protein (EGFP)-expressing B5 mouse ES cells in vitro and in vivo after transplantation into the eyes of mice with hereditary retinal degeneration. After neural induction with retinoic acid, the majority of cells in embryoid bodies expressed markers for neural progenitors as well as for immature and mature neurons and glial cells. When induced ES cells were plated in vitro, further differentiation was observed and the majority of cells expressed beta-III Tubulin, a marker for immature neurons. In addition, many plated cells expressed markers for mature neurons or glial cells. Four days after intravitreal transplantation into the eyes of rd1 mice (a model of rapid retinal degeneration), donor cells appeared attached to the vitreal surface of the retina. After 6 weeks in vivo, most transplanted cells remained adherent to the inner retinal surface, and some donor cells had integrated into the retina. Transplanted cells exhibited some properties typical of neurons, including extensive process outgrowth with numerous varicosities and expression of neuronal and synaptic markers. Therefore, after induction B5 ES cells can acquire the morphologies of neural cells and display markers for neuronal and glial cells in vitro and in vivo. Furthermore, when placed in the proper microenvironment ES-derived neural precursors can associate closely with or migrate into nervous tissue where differentiation appears to be determined by cues provided by the local environment, in this case the degenerating neural retina.

Rat trigeminal, olfactory and taste responses after capsaicin desensitization.

Experiment 1 showed that capsaicin injections severely reduced or eliminated nasal trigeminal responses to 3 odorants. Experiments 2 and 3 investigated whether desensitized animals could behaviorally detect and discriminate odors. Capsaicin treated animals had no measurable deficits in locating buried food, in odor aversion learning, or in operant odor detection and discrimination. Experiment 4 examined whether behavioral responsiveness to salty, sour and bitter tastes was affected by desensitization. Capsaicin injections did not affect responsiveness to salty or sour, but may have raised rejection thresholds for bitter. Broadly, the present results suggest that substance P-containing fibers mediate trigeminal responsiveness to odorants and irritants but that the loss of this responsiveness does not appreciably affect smell or taste, per se.

Effects of unilateral naris closure on the olfactory epithelia of adult mice

This study demonstrates, for the first time, that prolonged unilateral breathing can be harmful to the adult olfactory epithelium. Mice at least 5 months old had one naris closed by cautery and suture. These were divided into 5 groups of 10 mice which had unilateral naris closure for 1, 3, 6, 8 or 12 weeks. A control group of 10 mice was untreated. Variables that were assessed included the thicknesses and numbers of cells spanning olfactory epithelia in hematoxylin and eosin stained paraffin sections. Olfactory marker protein (OMP) immunohistochemistry was used to further visualize the differential impact of naris closure on the two sides of the nose. Unilateral naris closure for 6 weeks or longer caused dramatic losses of olfactory receptor cells in the rostral third of the open-side olfactory epithelia, but did not affect numbers of cells in caudal regions or on the closed sides. The thicknesses of the open and closed-side olfactory epithelia were significantly different for only the 8-week closure group. In most mice with unilateral naris closure for longer than 6 weeks there was little or no staining of the olfactory receptor neurons or their axon bundles for OMP in the affected regions of the open side.

Stem Cells as Vectors to Deliver HSV/tk Gene Therapy for Malignant Gliomas

The prognosis of patients diagnosed with malignant gliomas including glioblastoma multiforme (GBM) is poor and there is an urgent need to develop and translate novel therapies into the clinic. Neural stem cells display remarkable tropism toward GBMs and thus may provide a platform to deliver oncolytic agents to improve survival. First we provide a brief review of clinical trials that have used intra-tumoral herpes simplex virus thymidine kinase (HSV/tk) gene therapy to treat brain tumors. Then, we review recent evidence that neural stem cells can be used to deliver HSV/tk to GBMs in animal models. While previous clinical trials used viruses or non-migratory vector-producing cells to deliver HSV/tk, the latter approaches were not effective in humans, primarily because of satellite tumor cells that escaped surgical resection and survived due to low efficiency delivery of HSV/tk. To enhance delivery of HSV/tk to kill gliomas cells, recent animal studies have focused on the ability of neural stem cells, transduced with HSV/tk, to migrate efficiently and selectively to regions occupied by GBM cells. This approach holds the promise of targeting GBM cells that have infiltrated the brain well beyond the original site of the tumor epicenter.

Masera's organ responds to odorants

Electroolfactogram (EOG) recordings from the rat septal olfactory organ (SO) provide the first demonstration of its broad-range chemosensitivity, and clearly establish this structure as a functioning component of the mammalian intranasal chemosensory system. SO sensitivity to lower concentrations of at least one common test odorant (pentyl acetate) exceeds that at sites located on the septal portion of the main olfactory neuroepithelium. Signals from the SO, as first proposed, thus could have an alerting function and provide information relevant to odor stimulus assessment.

Quantification of the effects of long-term unilateral naris closure on the olfactory bulbs of adult mice

The effects of unilateral naris closure on the olfactory bulbs of adult mice were assessed quantitatively by measuring four parameters. Naris closures were performed when animals were at least 5 months of age and lasted for 4-8 months. The first parameter measured was mitral cell number, which revealed that there was no significant effect of closure on numbers of these cells. The next parameter measured was the area of the external plexiform layer (EPL). The area of the EPL was 41% smaller in the closed-side olfactory bulbs than in the open-side olfactory bulbs (P less than 0.01). Comparisons of the areas of the granule cell layers (GCL) showed that the closed-side GCL was 25% smaller than the open-side GCL (P less than 0.01). Finally, the number of cells in the open- and closed-side GCLs were compared. The number of cells in the closed-side GCL was 30% lower than the number of cells in the open-side GCL (P less than 0.01). These data show that the shrinkage of closed-side olfactory bulb after naris closure in adult mice is due, at least in part, to the loss of granule cells and not to the loss of the main output neurons, the mitral cells.