Harriet Baker

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.

Olfactory afferent regulation of the dopamine phenotype in the fetal rat olfactory system

Recent studies strongly suggest that functional olfactory receptor cell innervation is necessary for the maintenance of the dopamine phenotype in the adult rat olfactory bulb. To determine whether afferent innervation is required for the initial expression of the dopaminergic phenotype during development, the current studies investigated the association between afferent innervation and phenotypic expression using both in vivo and in vitro systems. Ontogeny of the dopamine phenotype in the rat main olfactory bulb was assessed by the appearance of immunoreactivity for tyrosine hydroxylase, the first enzyme in the dopamine biosynthetic pathway. Development of receptor afferent innervation of the bulb was demonstrated with olfactory marker protein immunoreactivity. Tyrosine hydroxylase-immunoreactive cells occurred only in regions of the olfactory bulb receiving afferent innervation. However, the appearance of afferent fibers in the olfactory bulb preceded tyrosine hydroxylase expression by three to four days (gestational days 14-15 versus 18, respectively). In explant cultures, significant numbers of tyrosine hydroxylase-containing cells were observed only in en bloc co-cultures of presumptive olfactory bulb and epithelium. Explant cultures of presumptive olfactory bulb alone contained few, if any, tyrosine hydroxylase-immunoreactive cells. Similarly, explants produced by recombining previously separated presumptive olfactory bulb and epithelium exhibited very few tyrosine hydroxylase-immunostained cells. These data suggest that expression of the dopamine phenotype, as indicated by the presence of tyrosine hydroxylase, depends on a critical level of afferent innervation. The results also support previous studies which indicated that neuronal activity or an activity-dependent process may be required for induction of tyrosine hydroxylase expression.

Unilateral, neonatal olfactory deprivation alters tyrosine hydroxylase expression but not aromatic amino acid decarboxylase or GABA immunoreactivity

Recent publications have demonstrated an important role for olfactory afferent innervation in maintenance of the dopamine phenotype of olfactory bulb target neurons. The mechanisms underlying the control of phenotypic expression in this system are not known. These studies employed the model of unilateral neonatal olfactory deprivation to investigate the effects of lack of odorant stimulation on dopamine expression in the rat. Immunoreactivity of tyrosine hydroxylase, the first and rate-limiting enzyme in dopamine biosynthesis, used as a marker of the dopamine system, exhibited a large decrease both 40 and 70 days following olfactory deprivation. The losses were region specific suggesting that the deprivation was not complete. The number of immunoreactive GABAergic neurons was not reduced. The number of neurons containing aromatic L-amino acid decarboxylase (the second enzyme in the dopamine biosynthetic pathway) was also not decreased. Olfactory marker protein immunoreactivity in the glomeruli, a marker for afferent innervation, was not significantly altered indicating that the olfactory bulb was not denervated. These data demonstrate that neonatal deprivation, and the resulting lack of odorant stimulation, produces a transneuronal alteration in dopamine expression without neuronal loss. The studies also suggest that neuronal activity or the activity-dependent release of a trophic factor is necessary for the expression of the dopamine phenotype.

Olfactory bulb dopamine neurons survive deafferentation-induced loss of tyrosine hydroxylase

Peripheral deafferentation of the rodent olfactory bulb results in loss of dopamine content, tyrosine hydroxylase activity and immunocytochemical staining for tyrosine hydroxylase in juxtaglomerular dopamine neurons. Reinnervation of the bulb by afferent neurons results in the return of all parameters to control levels suggesting that the dopamine neurons did not degenerate but that the expression of tyrosine hydroxylase enzyme was transneuronally regulated in a static population of juxtaglomerular cells. To evaluate this possibility, we determined the activity and immunocytochemical localization of the second enzyme in the dopamine biosynthetic pathway, DOPA decarboxylase. At a time when tyrosine hydroxylase activity was reduced to 25% of control values, DOPA decarboxylase activity in the lesioned bulb was maintained at about 65% of that in the unlesioned bulb. Immunocytochemical staining with antibodies to both enzymes, performed sequentially in the same sections, demonstrated that in the unlesioned bulb tyrosine hydroxylase and DOPA decarboxylase are co-localized in the same population of juxtaglomerular neurons. Similar results were obtained in adjacent sections each stained with one of the two antibodies. In contrast, in the deafferented bulb, about three times as many neurons were stained with DOPA decarboxylase as with tyrosine hydroxylase antibodies. The DOPA decarboxylase activity measurements and immunocytochemistry argue for the continued presence, in the lesioned olfactory bulb, of a population of tyrosine hydroxylase deficient dopamine neurons. The data suggest that olfactory receptor cell innervation transneuronally regulates the expression of tyrosine hydroxylase by mechanisms separate from those controlling the levels of DOPA decarboxylase.

The expression of the neuronal intermediate filament protein peripherin in the rat embryo.

The expression of the neuronal type III intermediate filament protein peripherin was examined in the rat embryo during and following neuronogenesis in the spinal cord and the peripheral nervous system. In situ hybridization analysis reveals that peripherin mRNA is found in the mid-gestational rat embryo in ventral and lateral motoneurons in the spinal cord, and in neurons of all peripheral ganglia examined, including spinal, sympathetic, and enteric ganglia. Peripherin mRNA is seen only in post-migratory motoneurons or neuronal cells in aggregating ganglia, indicating that precursor cells do not express peripherin. To examine the expression of the protein, an affinity-purified antibody (anti-per) specific for a bacterially produced peripherin fusion protein was generated. Anti-per specifically recognizes a 58 kDa, cytoskeletal-enriched, nerve growth factor (NGF)-inducible protein of the expected tissue distribution. Immunocytodetection with anti-per shows that the initiation of peripherin protein synthesis is coincident with the morphological differentiation of neurons. In development, peripherin is one constituent of a program of gene expression activated at terminal neuronal differentiation.

Thiamine deficiency induces oxidative stress and exacerbates the plaque pathology in Alzheimer’s mouse model

Mitochondrial dysfunction, oxidative stress and reductions in thiamine-dependent enzymes have been implicated in multiple neurological disorders including Alzheimers disease (AD). Experimental thiamine deficiency (TD) is an established model for reducing the activities of thiamine-dependent enzymes in brain. TD diminishes thiamine-dependent enzymes throughout the brain, but produces a time-dependent selective neuronal loss, glial activation, inflammation, abnormalities in oxidative metabolism and clusters of degenerating neurites in only specific thalamic regions. The present studies tested how TD alters brain pathology in Tg19959 transgenic mice over expressing a double mutant form of the amyloid precursor protein (APP). TD exacerbated amyloid plaque pathology in transgenic mice and enlarged the area occupied by plaques in cortex, hippocampus and thalamus by 50%, 200% and 200%, respectively. TD increased Abeta(1-42) levels by about three fold, beta-CTF (C99) levels by 33% and beta-secretase (BACE1) protein levels by 43%. TD-induced inflammation in areas of plaque formation. Thus, the induction of mild impairment of oxidative metabolism, oxidative stress and inflammation induced by TD alters metabolism of APP and/or Abeta and promotes accumulation of plaques independent of neuron loss or neuritic clusters.

Rapid down‐regulation of tyrosine hydroxylase expression in the olfactory bulb of naris‐occluded adult rats

In most sensory systems, afferent innervation regulates morphological and biochemical characteristics of target cells for a limited time during development. Sensory deprivation experiments in adult rats also have suggested a critical period for afferent influences on olfactory bulb structure and function. Previous odorant deprivation studies that employed unilateral naris closure in neonatal rats demonstrated down‐regulation of the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH) in dopamine neurons intrinsic to the olfactory bulb. Accompanying the altered biochemical parameters was a decrease in bulb size. To distinguish between deprivation‐induced alterations in TH expression secondary to developmental sequelae and those occurring in mature neurons, the consequences of unilateral naris closure were assessed in young adult rats. In agreement with previous studies significant postnatal increases occurred in TH expression and total protein, an indication of bulb size. At 30 days post‐closure, total protein was unaltered in the ipsilateral olfactory bulb but showed a small (12.9%), significant decline at 60 days. In contrast to the limited morphological consequences of odor deprivation, profound reductions occurred in TH expression. TH activity ipsilateral to the closure decreased significantly by 14 days post‐closure and remained depressed for up to 6 months. In parallel with enzyme activity, TH immunoreactivity did not decline in the first few days post‐closure. In situ hybridization revealed that TH mRNA levels decreased rapidly, i.e., by 2 days post‐closure, reached a nadir at 1 month, and remained depressed for at least 6 months. The capacity of odor deprivation in the adult rat olfactory system to down‐regulate TH expression suggests that this phenotypic alteration occurs independently of a presumed critical period.

Neurons of the superior nucleus of the medial habenula and ependymal cells express IL-18 in rat CNS.

The habenular-interpeduncular pathway is involved in the modulation of several functions including neuroendocrine and stress responses. Interleukin-18 (IL-18) is a pro-inflammatory cytokine predominantly studied as a modulator of immune functions and also produced in the adrenal cortex following activation of the hypothalamic-pituitary-adrenal axis. In the central nervous system, IL-18 was demonstrated to induce sleep and to influence long-term potentiation and was proposed to mediate local inflammatory reactions. The present study investigated the localization of IL-18 and its expression following either acute or chronic restraint stress in the brain of adult male Wistar rats. Using immunocytochemistry and in situ hybridization we report the unprecedented localization of IL-18 in the neurons of the superior part of the medial habenula (MHbS), their projections to the interpenducular nucleus and its expression in the ependymal cells surrounding the third and the lateral ventricles. In addition, acute (2 h) or chronic (6 h/day for 3 weeks) restraint stress induced a strong elevation of IL-18 immunostaining in the MHbS but not in ependymal cells. The present data suggest that IL-18 may participate in the modulation of stress responses in the MHbS. They also suggest that ependymal cells may be the source of IL-18 previously reported in the cerebrospinal fluid (CSF). The role of IL-18 in the ependyma and the CSF remains to be elucidated.

Inducible cAMP Early Repressor Can Modulate Tyrosine Hydroxylase Gene Expression after Stimulation of cAMP Synthesis

Members of the CREB/CREM/ATF family of transcription factors either enhance or repress transcription after binding to the cAMP response elements (CREs) of numerous genes. The rat gene for tyrosine hydroxylase (TH) bears a canonical CRE, at base pairs −38 through −45 from the transcription initiation site, that is essential for basal and cAMP-stimulated transcription (Kim, K.-S., Lee, M. K., Carroll, J., and Joh, T. H. (1993) J. Biol. Chem. 268, 15689-15695; Lazaroff, M., Patankar, S., Yoon, S. O., and Chikaraishi, D. M. (1995) J. Biol. Chem. 270, 21579-21589). The current study identifies CRE-binding proteins induced in pharmacological paradigms characterized by TH activation. PC12- and rat adrenal gland-derived nuclear proteins retarded a TH-CRE oligonucleotide in gel mobility shift assays with virtually identical patterns. These differed substantially from patterns exhibited by extracts from locus ceruleus or from neuroblastoma (SK-N-BE(2)C) and locus ceruleus-derived (CATH.a) cell lines. Forskolin stimulation of PC12 cells and reserpine treatment of rats increased, in nuclear extracts derived from cells and adrenal glands, respectively, the amount of a fast moving CRE/protein complex that was supershifted by an anti-CREM antibody. Subsequent Western, Northern, and polymerase chain reaction analyses indicated that a specific member of the CREM family, the inducible cAMP early repressor (ICER), was strongly induced in both systems. Cotransfection of PC12 cells with TH2400CAT plasmid and the expression vector pCMV-ICER-Ib demonstrated that ICER efficiently represses the transcriptional activity of the TH gene promoter. In addition, PKA-stimulated transcriptional activity of the promoter was effectively suppressed by ICER. These results suggest that ICER can modulate cAMP-stimulated transcription of the TH gene and provide a model accounting for rapid reversal of increased TH transcription following elevations in cAMP.

Decrease in tyrosine hydroxylase, but not aromatic l-amino acid decarboxylase, messenger RNA in rat olfactory bulb following neonatal, unilateral odor deprivation

Unilateral naris cauterization in rats results in occlusion of the affected naris and blockade of odorant access to ipsilateral olfactory receptor cells in the olfactory epithelium. These receptor cells project exclusively to the olfactory bulb (OB) and appear to regulate expression of the dopaminergic phenotype in a population of OB juxtaglomerular neurons. Unilateral odor deprivation results in a loss of normal stimulatory input to the OB and a marked and specific decrease in ipsilateral OB tyrosine hydroxylase (TH) expression. The expression of co-localized aromatic L-amino acid decarboxylase (AADC) is not similarly affected. We have used this procedure in neonatal rats to examine the effect of stimulus deprivation on OB TH and AADC mRNA levels. Both Northern blot and in situ hybridization analyses revealed a pronounced decrease in ipsilateral as compared to contralateral OB TH mRNA levels 40 days after naris closure. In contrast, the levels of OB AADC mRNA were unaltered by naris closure. By in situ hybridization histochemistry, both TH and AADC mRNAs were localized to OB juxtaglomerular neurons. Odor deprivation was associated with an apparent region-specific reduction in TH mRNA within the ipsilateral OB glomerular layer. By densitometric analysis, the loss of TH-specific message was quantitatively consistent with the decrease in TH activity, suggesting that the observed plasticity of OB dopaminergic neurons following functional deafferentation can be attributed to a selective, transneuronally-mediated down regulation of TH gene transcription.