Mary Grillo

Chemical deafferentation of the olfactory bulb: Plasticity of the levels of tyrosine hydroxylase, dopamine and norepinephrine

The laminar distribution of tyrosine hydroxylase activity, dopamine and norepinephrine was determined in the dog olfactory bulb. The levels of tyrosine hydroxylase activity and dopamine were highest in the glomerular layer, whereas norepinephrine appeared to be more uniformly distributed across the layers. A similar distribution was observed within the mouse olfactory bulb. Following deafferentation of the mouse olfactory bulb, the levels of tyrosine hydroxylase activity and dopamine declined, while norepinephrine levels showed a transient increase. Subsequent to regeneration of the olfactory nerve, these levels returned to control values. The levels of tyrosine hydroxylase activity and of dopamine were very low or non-detectable in the olfactory epithelium, which contains the olfactory receptor neuron perikarya. The data obtained indicate that tyrosine hydroxylase activity and dopamine content in the bulb are more tightly coupled to each other than either is to norepinephrine content. Since the two catecholamines are in two different classes of neurons, this implies that the bulk of the tyrosine hydroxylase activity in the bulb is associated with the dopamine-containing neurons. Finally, our data are consistent with a transsynaptic control mechanism of the tyrosine hydroxylase activity and dopamine level in the olfactory bulb.

Purification and characterization of carnosine synthetase from mouse olfactory bulbs.

Carnosine synthetase was purified about 500‐fold from mouse olfactory bulb to a specific activity of approx 25 nmol/min/mg. This is an increase of 800‐fold over that previously reported for this enzyme from rat brain and 11 times higher than the most highly purified enzyme from chicken pectoral muscle. ATP was essential for activity and could not be replaced by ADP. NAD had no effect on the synthesis of carnosine. Of the β‐alanine analogues tested, the purified mouse enzyme incorporated only γ‐aminobutyric acid and β‐amino‐n‐butyric acid into peptide linkage with histidine. Synthesis of carnosine by the mouse olfactory bulb enzyme was competitively inhibited by the histidine analogues, 1‐methyl histidine and 3‐methyl histidine, with Ki values which were at least 40 times the Km value for histidine (16 μM). Ornithine and lysine were more efficient β‐alanine acceptors than 1‐methyl histidine for the mouse enzyme. Enzyme from olfactory epithelium and leg skeletal muscle of mice also showed higher Ki values for 1–methyl histidine than the Km value for histidine. In contrast, carnosine‐anserine synthetase from chicken pectoral muscle gave Km values for histidine, 1‐methyl histidine and 3‐methyl histidine, which were all in the range of 4–12 μM. The differences in substrate specificity between the enzyme from mouse and chicken implies alternate routes of anserine synthesis in these species and predicts the occurrence of certain novel peptides in mouse brain.

Transneuronal regulation of neuronal specific gene expression in the mouse olfactory bulb

Peripheral afferent denervation (deafferentation) of the rodent main olfactory bulb produces a marked decrease in tyrosine hydroxylase (TH) activity and immunoreactivity in a population of juxtaglomerular dopaminergic neurons. Preservation of activity and immunostaining for aromatic L-amino acid decarboxylase implies that these cells do not die, but change phenotype. We now report that the steady-state level of TH mRNA markedly decreases in the adult mouse olfactory bulb in response to deafferentation. This reduction is permanent following intranasal irrigation with 0.17 M zinc sulphate (ZnSO4) but reversible following deafferentation produced by intranasal irrigation with 0.7% Triton X-100. The initial declines in TH activity, protein and mRNA of dopaminergic juxtaglomerular neurons observed after Triton X-100 treatment are all reversible as the steady-state level of TH mRNA gradually returns to control levels. Steady-state levels of mRNA for olfactory marker protein (OMP), a protein found in high concentrations in olfactory receptor neurons and their processes which innervate the olfactory bulb, were also monitored following deafferentation. Following treatment with either ZnSO4 or Triton X-100, the pattern of changes in steady-state levels of OMP mRNA was similar to that observed for TH. The steady-state level of PEP19 mRNA, a peptide previously localized to granule cells in the olfactory bulb, was not altered by deafferentation. These data indicate selective and parallel regulation of TH and OMP message and protein levels following deafferentation.

Axoplasmic transport of carnosine (β-alanyl-L-histidine) in the mouse olfactory pathway.

Carnosine in the chemoreceptor neurons of the olfactory epithelium can be labeled in vivo by intranasal irrigation with either14C-β-alanine or14C-L-histidine. This newly synthesized carnosine (but not the precursor amino acids) is translocated to the olfactory bulb, where the olfactory chemoreceptor axons synapse with the dendrites of mitral cells and other second-order neurons. Labeled carnosine arrives in the bulb several hours after intranasal administration of precursor. Similar arrival time is seen for macromolecules after intranasal administration of [3H]L-fucose, [14C]L-proline, or [14C]L-histidine. Macromolecules labeled with [3H]uridine take much longer to reach the bulb. Carnosine is also labeled after [3H]uridine administration. No labeling of macromolecules is observed after administration of 1-[14C]-β-alanine. Oral administration of the same dose of [14C]-β-alanine gives almost no labeled carnosine in bulb or epithelium. This method has permitted us to estimate that the half-life of labeled carnosine in both the bulb and epithelium is about 20 h. This method provides a means of selectively prelabeling the olfactory chemoreceptor neurons in the olfactory epithelium and their synapses in the olfactory bulb prior to cellular and subcellular separation procedures, and may also enable us to monitor the influences of olfactory stimulation on synthesis and transport of carnosine.

Enzymatic and immunological evidence for two forms of carnosinase in the mouse

Carnosinase (aminoacyl-L-histidine hydrolase, EC 3.4.13.3) hydrolyzes the dipeptide carnosine (beta-alanyl-L-histidine), which is thought to play a role in cerebral and skeletal muscular function and has been implicated as a neuroaffector in the olfactory bulb. Carnosinase activity is present in many tissues of the mouse including heart, liver and lung, but it is most active in kidney, uterus and nasal olfactory mucosa. Kinetic measurements with 1H-NMR spectroscopy indicate that the enzyme is stereospecific and can hydrolyze L-but not D-carnosine. Anserine is a poorer substrate, while homocarnosine is essentially a non-substrate. However, these two dipeptides are effective inhibitors of the hydrolysis of L-carnosine. Carnosinase activity is unaffected when assayed in 2H2O at 99% isotopic purity. From considerations of the effect of Mn2+ on (1) substrate concentration velocity curves; (2) thermostability, and (3) inhibitor behavior, tissues with carnosinase can be divided into two groups. Kidney, uterus and olfactory mucosa represent one group, while central nervous system, muscle, spleen, etc. represent the second. The validity of this classification is confirmed by immunological evidence. Antiserum prepared against carnosinase purified from kidney cross-reacts with and inhibits the activity of olfactory mucosa, kidney and uterus but not that from central nervous system, heart or liver.

Proximal regions of the olfactory marker protein gene promoter direct olfactory neuron-specific expression in transgenic mice.

Olfactory marker protein (OMP) expression is highly restricted to mature olfactory neurons (ON). Less than 0.3 kb of upstream 5′ flanking sequence of the OMP gene directs lacZ expression preferentially to ON in several independently derived lines of transgenic mice. A larger transgene with 0.8 kb of upstream flanking sequence also gave lacZ expression in ON and in a few ectopic sites in the central nervous system (CNS). In addition to the main olfactory epithelium, endogenous OMP is also expressed in chemosensory neurons of the vomeronasal and septal organs, and lacZ expression was detected in neurons of these sites as well. This confirmed the presence of regulatory sequences in the proximal portion of the OMP gene. Endogenous OMP expression in ON was normal in all transgenic lines. Strikingly, in several transgenic lines lacZ expression was restricted to subsets of ON. In one such line, ON axons were intensely stained for lacZ and projected to a subset of olfactory bulb glomeruli. Although identifiable subsets of ON and their termination fields have been described previously, this is the first demonstration of this phenomenon in transgenic mice. These lines of transgenic mice thus provide in vivo models for characterization of genetic elements regulating developmental and functional organization of the olfactory neuroepithelium.

Carnosine, homocarnosine and anserine in vertebrate retinas.

The dipeptides carnosine, homocarnosine and anserine are differentially distributed among the retinas of several vertebrate species. Retinas of birds are rich in anserine while those of frogs have primarily carnosine. Several mammalian species contain only very low levels of homocarnosine. The biological function of these dipeptides is unknown but their presence and synthesis in retina may confound studies of uptake, metabolism and cellular localization of their component amino acids ?-alanine, gamma-aminobutyric acid and histidine.

Monoclonal Antibodies to Mammalian Carnosine Synthetase

Abstract: A set of mouse monoclonal antibodies has been generated against rabbit muscle camosine synthetase. The immunoreactivity of these antibodies has been characterized using an immunoassay that permits the separation and direct measurement of the synthetase activity on a second antibody bead complex. Four IgG monoclonal antibodies bind the carnosine synthetase activity from muscle of all mammals tested (mouse, rat, rabbit, cow, dog, and monkey) but not that from chicken muscle. This indicates the mammalian enzymes share epitopes that are absent from the avian enzyme. In addition, relative tissue levels of synthetase activity can be quantified with this immunoassay. Thus, high levels of carnosine synthetase activity are immunoprecipitated from the olfactory tissues of both rat and rabbit. Synthetase activity is generally lower in other tissues (muscle, brain, heart, liver, and gut). Nevertheless, the cross‐reactivity of the synthetase from several tissues (olfactory mucosa, muscle, brain, gut, heart, and liver) of a single species indicates the enzyme protein contains similar epitopes in these tissues. Immunoaffinity purification of this low‐abundance, unstable enzyme should now be possible for subsequent studies of structure and regulation.

LacZ andOMP are co-expressed during ontogeny and regeneration in olfactory receptor neurons of omp promoter-lacZ transgenic mice

The ontogeny and cellular specificity of expression of β‐galactosidase activity and olfactory marker protein (OMP) are compared in olfactory tissue of the H‐OMP‐lacZ‐3 line of transgenic mice. In this line the expression oflacZ is driven by a 0.3 kb fragment of the rat OMP promoter. During fetal development,lacZ expression is detectable in olfactory receptor neurons (ORNs) shortly after the initial appearance of endogenous OMP. The β‐galactosidase marker was observed only in mature olfactory receptor neurons where it co‐localized with endogenous OMP. It was absent from immature neurons that express the growth associated phosphoprotein B50/GAP43. Lesion of the peripheral olfactory pathway by intranasal irrigation with Triton X‐100 eliminated expression of both OMP andIacZ in the olfactory neuroepithelium. Subsequent regeneration of the full complement of olfactory receptor neurons was associated with co‐expression of both OMP and β‐galactosidase activity. Neither OMP nor β‐galactosidase activity was induced in any other cell type of the regenerating olfactory mucosa. Thus, as little as 0.3 kb of the OMP promoter has the ability to targetlacZ expression to olfactory receptor neurons in a temporally and spatially defined manner. We discuss the potential utility of this transgenic line for future studies of the olfactory system.

Expression of catfish amino acid taste receptors inXenopus oocytes

We demonstrate that poly (A+)RNA isolated from catfish barbels directs the expression of functional amino acid taste receptors in theXenopus oocyte. The activity of these receptors is monitored in ovo by the two electrode voltage clamp technique. Specific conductance changes recorded in response to amino acid stimulation are analogous to those recorded electrophysiologically from intact catfish barbels. These responses exhibit specificity, reproducibility, rapid onset and termination, and desensitization to repetitive stimulation. A functional assay system that encompasses the full complement of transduction events from the ligand-receptor interaction to subsequent conductance changes is necessary to identify molecular components responsible for these events. Our results demonstrate that theXenopus oocyte can be used to characterize and identify clones coding for amino acid taste receptors analogous to its use in studying receptor molecules for other neuroactive compounds.