Neuronal nitric oxide synthase expression in cerebellar mutant mice

Nitric oxide (NO) is a diffusible, multifunctional signaling molecule found in many areas of the brain. NO signaling is involved in a wide array of neurophysiological functions including synaptogenesis, modulation of neurotransmitter release, synaptic plasticity, central nervous system blood flow and cell death. NO synthase (NOS) activity regulates the production of NO and the cerebellum expresses high levels of nitric oxide synthase (NOS) in granule, stellate and basket cells. Cerebellar mutant mice provide excellent opportunities to study changes of NO/NOS concentrations and activities to gain a greater understanding of the roles of NO and NOS in cerebellar function. Here, we have reviewed the current understanding of the functional roles of NO and NOS in the cerebellum and present NO/NOS activities that have been described in various cerebellar mutant mice and NOS knockout mice. NO appears to exert neuroprotective effects at low to moderate concentrations, whereas NO becomes neurotoxic as the concentration increases. Excessive NO production can cause oxidative stress to neurons, ultimately impairing neuronal function and result in neuronal cell death. Based on their genetics and cerebellar histopathology, some of cerebellar mutant mice display similarities with human neurological conditions and may prove to be valuable models to study several human neurological disorders, such as autism and schizophrenia.     

Localization of ataxin-2 within the cerebellar cortex of the rat

Spinocerebellar ataxia type 2 is caused by a polyglutamine stretch in the protein ataxin-2 that is due to an expansion of a CAG repeat in the spinocerebellar ataxia-2 gene. The function of wild-type ataxin-2 has not been clarified. A widespread distribution of this protein throughout the brain has been reported. We examined the expression of ataxin-2 in cortical cerebellar cells of the adult rat. We performed a single label immunohistochemical study of ataxin-2 and a single label immunofluorescence study of ataxin-2 and zebrin on adjacent sections, to compare the distribution of the observed parasagittal band pattern. We also performed a double label immunofluorescence study of ataxin-2 and one of each parvalbumin, calbindin, and calretinin. Single label studies revealed that between 50% and 70% of the Purkinje cells express ataxin-2. The abundance of ataxin-2 was different between hemisphere and vermis, with a clear prevalence for the former. Furthermore, the distribution of ataxin-2-positive Purkinje cells showed a peculiar alternating parasagittal band pattern. Among the other cortical cerebellar cells only basket and granule cells showed ataxin-2 staining. Our dual label studies showed that about 50% of calbindin and more than 70% of parvalbumin-immunoreactive Purkinje cells were also labeled for ataxin-2. The uneven distribution of ataxin-2 expression in the Purkinje cell layer does not support the hypothesized link between ataxin-2 content and cell vulnerability. The differences in ataxin-2 expression among the cell types of cerebellar cortex, on the other hand, suggest a possible correlation between ataxin-2 content and cell function.     

X-linked transmission of the shaker mutation in rats with hereditary Purkinje cell degeneration and ataxia

This study reports on the mode of inheritance of the shaker mutation and the development of an inbred strain of the shaker rat mutation from Sprague Dawley outbred stock onto a Wistar Furth background. Neuroanatomical and behavioral expression of the affected phenotype, through seven generations of backcross and intercross breeding, has confirmed the mode of inheritance to be X-linked. Behaviorally, affected mutants present with a wide-based ataxic gait and whole body tremor. In affected mutants calbindin immunostaining for surviving cerebellar Purkinje cells revealed widespread degeneration in the anterior lobe and in limited areas of the posterior lobe. Fast Fourier transform analysis of the tremor revealed a frequency of 3–5 Hz. As predicted by X-linked inheritance, female descendants of an affected male are carriers for the genotype and the phenotype is expressed in one-half of her male offspring. There was spatially random and limited degeneration of Purkinje cells in carrier females, but they did not display overt clinical signs of ataxia and tremor. These data provide further support for using the shaker mutant rat as an animal model for studies of mechanisms underlying human heredodegenerative diseases.     

Catecholamine neurons with Alzheimer's neurofibrillary changes and alteration of tyrosine hydroxylase

Summary Immunohistochemistry with antisera against tyrosine hydroxylase was performed on neurons with Alzheimer's neurofibrillary changes in the substantia nigra and locus ceruleus. These specimens were obtained from brains with Alzheimer's disease, Pick's disease, progressive supranuclear palsy, Alzheimer's type parkinsonism, parkinsonism-dementia complex on Guam, and normal aging. Under these neurologic conditions the affected cate-cholamine neurons with Alzheimer's neurofibrillary changes were stained positively with antisera against tyrosine hydroxylase. The results suggested that in these neurons, Alzheimer's neurofibrillary changes seemed to develop independently before the reduction of tyrosine hydroxylase protein synthesis.     

Development of glutamic acid decarboxylase-immunoreactive elements in the cerebellar cortex of normal and lurcher mutant mice.

The development of glutamic acid decarboxylase-immunoreactivity (GAD-IR) in cells, fibers, and varicosities of the cerebellar cortex has been examined by light microscopy in normal and lurcher mutant mice between postnatal day 3 and 30 (P3-P30). Purkinje cell morphology was demonstrated in adjacent sections by using an antiserum to the 28Kd vitamin D-dependent calcium binding protein (CaBP). In early postnatal lurcher mice, but not in normal littermates, GAD-IR fibers, presumably Purkinje cell pseudopodia, invade the external granular layer. The plexus of CaBP-IR axons in the internal granular layer is much less complex in lurcher mice than in normal littermates, even before the onset of lurcher Purkinje cell degeneration at P8. In normal mice, GAD-IR fibers encapsulate Purkinje cell somata by P15. Lurcher Purkinje cells, in contrast, receive scattered contacts by GAD-IR puncta and possess a "cap" of such elements surrounding the primary dendrite and apical soma. Pinceau formations, visible as a knot of GAD-IR puncta hanging from the base of Purkinje cells in normal P15 mice, are not present in lurcher littermates. "Empty baskets" or collapsed pinceau formations in regions devoid of Purkinje cells are not revealed by anti-GAD immunohistochemistry in the P17-P30 lurcher cerebellar cortex.     

Reduced tyrosine hydroxylase and GTP cyclohydrolase mRNA expression, tyrosine hydroxylase activity, and associated neurochemical alterations in Nurr1-null heterozygous mice

The nuclear receptor Nurr1 is essential for the development of midbrain dopamine neurons and appears to be an important regulator of dopamine levels as adult Nurr1-null heterozygous (+/-) mice have reduced mesolimbic/mesocortical dopamine levels. The mechanism(s) through which reduced Nurr1 expression affects dopamine levels has not been determined. Quantitative real-time PCR revealed a significant reduction in tyrosine hydroxylase (TH) and GTP cyclohydrolase (GTPCH) mRNA in ventral midbrain of +/- mice as compared to wild-type mice (+/+). The effect on TH expression was only observed at birth, while reduced GTP cyclohydrolase was also observed in the adult ventral tegemental area. No differences in dopamine transporter, vesicular monoamine transporter, dopamine D2 receptor or aromatic amino acid decarboxylase were observed. Since TH and GTPCH are both involved in dopamine synthesis, regulation of in vivo TH activity was measured in these mice. In vivo TH activity was reduced in nucleus accumbens and striatum of the +/- mice (24.7% and 15.7% reduction, respectively). In the striatum, gamma-butyrolactone exacerbated differences on +/- striatal TH activity (29.8% reduction) while haloperidol equalized TH activity between the +/+ and +/-. TH activity in the nucleus accumbens was significantly reduced in all conditions measured. Furthermore, dopamine levels in the striatum of +/- mice were significantly reduced after inhibition of dopamine synthesis or after haloperidol treatment but not under basal conditions while dopamine levels in the nucleus accumbens were reduced under basal conditions. Based on these data the +/- genotype results in changes in gene expression and impairs dopamine synthesis which can affect the maintenance of dopamine levels, although with differential effects between mesolimbic/mesocortical and nigrostriatal dopamine neurons. Together, these data suggest that Nurr1 may function to modify TH and GTPCH expression and dopamine synthesis.     

Flunarizine induces a transient loss of tyrosine hydroxylase immunoreactivity in nigrostriatal neurons

Neurotoxic effects of flunarizine (Fz), a selective calcium channel blocker, on the nigrostriatal dopamine system was investigated. Systemic injections of Fz to mice resulted in a transient loss of tyrosine hydroxylase (TH) immunoreactive nigrostriatal neurons without cell loss. TH immunoreactivity in these neurons was greatly reduced as rapidly as one day after drug administration (regardless of dosage used) and thereafter recovered in both dose- and time-dependent manners. Such a novel neurotoxic action of Fz may constitute a morphological substrate for reversible drug-induced parkinsonian signs described in recent clinical case reports.     

A small proportion of cholinergic neurones in the nucleus basalis magnocellularis of ferret appear to stain positively for tyrosine hydroxylase

The immunofluorescence and peroxidase-antiperoxidase methods for immunohistochemistry were used to show that a small number of neurones in the nucleus basalis magnocellularis of ferret stain positively for what appears to be tyrosine hydroxylase, a marker for catecholaminergic neurones. With a double immunocytochemical staining method, it was confirmed that the tyrosine hydroxylase-like immunoreactivity is localized in neurones that also stain strongly for choline acetyltransferase.     

Inferior olivary-induced expression of Fos-like immunoreactivity in the cerebellar nuclei of wild-type and Lurcher mice

Earlier behavioural studies have shown that the expression of the immediate-early gene c-fos, as visualized by the immunohistochemical detection of Fos, in the inferior olive (IO) correlated closely with expression in related areas of the cerebellar nuclei. It has been speculated that the expression of c-fos within the cerebellar nuclei may be induced by enhanced spiking activity of the immunopositive neurons in the inferior olive. Two potential mechanisms may play a role in this process: a direct induction by way of the collaterals of the olivary climbing fibres to the cerebellar nuclei, or indirectly, by climbing fibre activity-induced depression of mossy fibre-parallel fibre-induced simple spike frequency of the Purkinje cells resulting in a subsequent disinhibition of the related parts of the cerebellar nuclei. In an attempt to distinguish between these possible mechanisms, we analysed Fos immunoreactivity in the olivocerebellar system of wild-type mice and in the mutant mouse Lurcher which lacks Purkinje cells. The tremorgenic agent harmaline, which is known to induce enhanced and rhythmic firing of olivary neurons was given intraperitoneally to anaesthetized mice of both genotypes. Harmaline application coincides with the induction of Fos-immunoreactive neurons in most areas of the IO in both wild-type and Lurcher mice. Both types of mice also showed enhanced expression in the larger neurons of the cerebellar nuclei. However, in the smaller, GABAergic nucleo-olivary neurons, increased c-fos expression was only observed in the wild-type mice. We conclude that: (i) increased olivary activity indeed may result in increased c-Fos expression in related areas of the cerebellar nuclei; (ii) because the indirect mode of induction is not operative in Lurcher mice, the olivary collateral innervation of the cerebellar nuclei is sufficient for c-fos induction in the larger nucleobulbar neurons in Lurcher and potentially also in wild-type mice; however (iii) for the nucleo-olivary cells an intact cerebellar cortical input is necessary to evoke increased expression of c-fos following harmaline application.     

Somatotopic organization of tyrosine hydroxylase expression in the rat locus coeruleus: long term effect of RU24722

Tyrosine hydroxylase (TH) tissue concentration was determined by immunostaining of tissue sections directly transferred onto nitrocellulose membranes in the restricted region of the noradrenergic perikarya of the locus coeruleus (LC) along its postero-anterior axis. TH containing cells were systematically counted on adjacent post fixed sections stained by immunohistochemistry. The absolute quantity of TH was estimated in each section and was found to be linearly related to the number of TH immuno-positive cells found in the adjacent section. The ratio between these two parameters was thus used as an index of the cellular concentration of TH in noradrenergic cells. In the LC of control rats, the TH cellular concentration was lower (-39%) in the anterior than in the posterior half of the structure. Three days after an injection of 20 mg/kg of RU24722, an eburnamine derivative known to increase the quantity of TH in the LC, increases in quantities of TH were found in both portions of the LC. Moreover in the posterior LC the increase in the amount of TH resulted from a significant increase in the number of TH-immunopositive cells. In the anterior part, however, it was primarily the result of a significant increase in TH cellular concentration. Throughout the LC there was an increase in the cellular concentration of TH which was inversely proportional to the concentrations found in control animals. TH mRNA content was measured by a quantitative in situ hybridization in sections of both the posterior and anterior LC one day after a single injection of RU24722 at the same dose. The quantity of TH mRNA was significantly increased in both parts. The number of TH mRNA-expressing neurons also increased, especially in the anterior LC. Thus the effects at the level of TH protein and TH mRNA were strikingly parallel though increase in TH protein occurred later than the increase in the TH mRNA. These results suggest that in the rat LC: (1) there is a significant population of 'sleeping cells' in which TH expression is either inactivated or, at a low level of activation; (2) TH cellular concentration could exert a retrocontrol on its own expression in cells of the LC that contained TH and (3) TH expression appears to be regulated by different selective mechanisms in these two different subpopulations of noradrenergic cells within the LC.     

Cellular density in the cerebellar molecular layer in essential tremor,spinocerebellar ataxia,and controls

BackgroundIt would be useful to identify additional postmortem markers of Purkinje cell loss in essential tremor (ET). In hereditary cerebellar ataxia, Purkinje cell loss has been reported to result in a secondary increase in the density of the remaining cell populations in the cerebellar molecular layer. However, this phenomenon has not been studied in ET. We quantified cerebellar molecular layer cellular density in 15 ET cases, 15 controls, and 7 spinocerebellar ataxia (SCA) cases (2:2:1 ratio).MethodsA standard neocerebellar tissue block was stained with Luxol fast blue Hematoxylin & Eosin. Within 5 selected fields, cell soma (e.g., stellate, basket, and glial cell bodies) were counted. Cellular density was the number of cells/cm².ResultsThe Purkinje cell count differed across the three groups (p < 0.001), with the highest counts in controls, intermediate counts in ET cases and lowest counts in SCA cases. ET cases and controls had similar molecular layer cellular density (p = 0.79) but SCA cases had higher values than both groups (p < 0.01). A robust inverse correlation between Purkinje cell count and molecular layer cellular density (i.e., brains with more Purkinje cell loss had higher molecular layer cellular density), observed in SCA and controls (r = −0.55, p = 0.008), was not observed in ET cases.DiscussionAlthough Purkinje cell counts were reduced in ET cases compared to controls, an increase in molecular layer cellular density was not evident in ET. The increase in molecular layer cellular density, observed in SCA cases, may require a more marked loss of PCs than occurs in ET.     

Reduced nicotinamide nucleotides prevent nitration of tyrosine hydroxylase by peroxynitrite

Tyrosine hydroxylase (TH) is the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter dopamine (DA). TH activity is inhibited by peroxynitrite (ONOO(-)) by a mechanism that involves nitration of tyrosine residues and oxidation of cysteine residues in the enzyme. Reduced forms of the nicotinamide adenine dinucleotide cofactors, NADH and NADPH, protect TH from inhibition by ONOO(-) and prevent nitration of tyrosine residues. NAD, the oxidized form of the cofactors, neither protects TH from ONOO(-)-induced inhibition nor prevents the nitration of tyrosine residues in the enzyme. These results suggest that the redox status of the nicotinamide nucleotide cofactors could influence the ability of ONOO(-) to modify proteins that are important to the function of DA neurons.     

Levels of biogenic amines, their metabolites, and tyrosine hydroxylase activity in the human epileptic temporal cortex

The levels of serotonin (5-HT), 5 hydroxyindoleacetic acid (5-HIAA), dopamine (DA), homovanillic acid (HVA), norepinephrine (NE), and tyrosine hydroxylase (TH) activity were measured in the focus (spiking) and nonfocus (nonspiking) regions of the temporal neocortex of 20 patients with intractable complex partial seizures. The levels of 5-HT, DA, 5-HIAA, and HVA were higher in the focus when compared to the nonfocus. Values for NE and TH activity were not different when focus and nonfocus were compared. The ratios of metabolite to precursor for 5-HT and DA were not significantly different between the focus and the nonfocus, suggesting that the changes observed were the result of a modification in the synthesis and release of these amines. Such changes in the epileptic focus could be caused by altered transsynaptic regulatory processes, which occur as a result of neuronal loss, gliosis, or neuronal sprouting.     

Abnormal expression of tyrosine hydroxylase immunoreactivity in Purkinje cells precedes the onset of ataxia in dilute-lethal mice.

Expression of tyrosine hydroxylase (TH) immunostaining in the cerebellum was examined in dilute-lethal mice (DL) prior to and following the onset of ataxia. DL walked normally on postnatal days 7 and 8. Falling over when walking was exhibited by about 20% of DL on day 9 and by all DL by day 10. TH-positive Purkinje cells in lobules IX and X of the vermis of either ataxic or non-ataxic DL were clearly observed on day 9 when compared to control mice, and had drastically increased by day 10. These results revealed that abnormal TH expression occurred in some Purkinje cells of DL cerebella, preceding the onset of ataxia.     

Cytological investigations on the cerebellar cortex of sudden infant death victims

Summary The study was based on the hypothesis that cerebellar hypoxia may play a role in sudden infant death syndrome resulting in morphological changes of the cerebellar cortex, especially with respect to Purkinje cell density. In the morphological evaluation of the Purkinje cell layer, special consideration was additionally given to secondary alterations (i.e., macrophage and/or astrocyte reaction). A total of 12 sudden infant death syndrome cases were compared with an age- and sex-matched control group. The Purkinje cell density was evaluated by determining the number of these cells per suface unit on parasagittal sections in both hemispheres. The myelomonocytic and glial reaction was demonstrated by immunohistochemical methods using lysozyme as leukocyte and macrophage markers and glial fibrillary acidic protein as an astrocyte marker. Qualitatively, no alterations resembling a macrophage or glial cell reaction were detected in sudden infant death syndrome. No differences between the right and left cerebellar hemisphere could be established in the victims of sudden infant death syndrome nor in the controls. The number of Purkinje cells per 0.352 mm² cortex was higher in the younger victims of sudden infant death (younger than 45 weeks of gestation) than in all matched controls. A statistically significant difference in Purkinje cell density, however, could not be established, and, especially, no indications of hypoxia were observed in the cerebellar cortex.     

Spontaneous alternation and habituation in Purkinje cell degeneration mutant mice

Purkinje cell degeneration (pcd) mutant mice were found to be as active as normal mice in a T-maze. The pcd mutants, contrary to normal mice, did not alternate spontaneously in either a 2-trial or a 4-trial test. Results are discussed in terms of a role for the cerebellum in behavioral inhibition and visuo-spatial organization.     

Deletion mutagenesis of rat PC12 tyrosine hydroxylase regulatory and catalytic domains

The functional organization of rat tyrosine hydroxylase was investigated by deletion mutagenesis of the regulatory and catalytic domains. A series of tyrosine hydroxylase cDNA deletion mutants were amplified by PCR, cloned into the pET3C prokaryotic expression vector, and the mutant proteins were partially purified fromE. coli. The results show that the deletion of up to 157 N-terminal amino acids activated the enzyme, but further deletion to position 184 completely destroyed catalytic activity. On the carboxyl end, the removal of 43 amino acids decreased but did not eliminate activity, suggesting that this region may play a different role in the regulation of the enzyme. These findings place the amino end of the catalytic domain between residues 158 and 184 and the carboxyl end at or prior to position 455. Deletions within the first 157 amino acids in the N-terminus caused an increase in hydroxylating activity, a decrease in the apparentK _m for tyrosine and phenylalanine substrates, and a substantial increase in theK _i for dopamine inhibition. The results define this region of the N-terminus as the regulatory domain of tyrosine hydroxylase, whose primary functions are to restrict the binding of amino acid substrates and to facilitate catecholamine inhibition. The results also suggest that the well-established role of the regulatory domain in restricting cofactor binding may be secondary to an increase in catecholamine binding, which in turn lowers the affinity for the cofactor. These findings provide new insight into the functional organization and mechanisms of regulation of tyrosine hydroxylase.     

Purkinje cell loss and the noradrenergic system in the cerebellum of pcd mutant mice

Purkinje cells in the cerebellum receive inhibitory noradrenergic input from the locus coeruleus. In pcd mutant mice all Purkinje cells degenerate by 45 days of age. The purpose of the present studies was to determine if the loss of these cerebellar neurons affects the amounts of norepinephrine in the cerebellum of mice 25–280 days of age. No significant changes in norepinephrine content were detected during or after Purkinje cell degeneration. However, since degeneration led to a reduction in cerebellar weight, the norepinephrine concentration was increased in pcd mutants. These results indicate that despite the loss of a major postsynaptic target (Purkinje cells), the cerebellar noradrenergic input remains stable.     

A new group of tyrosine hydroxylase-immunoreactive neurons in the cat thalamus

A new cell group composed of a large number of neurons immunoreactive to tyrosine hydroxylase (TH) was demonstrated in the paraventricular nucleus and midline nuclei of the cat thalamus, using four different anti-TH sera after colchicine treatment. However, in these regions, we did not detect any cell bodies containing other catecholamine synthesizing enzymes nor dopamine.     

Central noradrenaline metabolism in cerebellar ataxic mice

Central noradrenaline (NA) metabolism was investigated in 4 types of ataxic mutant mice, weaver, reeler, staggerer and rolling mouse Nagoya (RMN) and hypocerebellar mice experimentally produced by neonatal treatment with cytosine arabinoside (ara-C). As neurochemical markers for synthesis, steady-state level and turnover of NA, we measured tyrosine hydroxylase (TH) activity, NA and total (free + conjugated) 3-methoxy-4-hydroxyphenylethyleneglycol (total MHPG) concentrations in the cerebellum, cerebral cortex and spinal cord. In the weaver and staggerer whose cerebellar weight loss was severe (40 and 21% of controls, respectively), the 3 neurochemical markers were all increased in the cerebellum, and a similar increase was observed in other regions. In the reeler, the 3 markers were also markedly increased in the cerebellum, but no similar pattern of increase was observed in other regions. In the RMN whose cerebellum showed the least weight loss (76% of control), only TH activity and MHPG concentration were increased in the cerebellum and spinal cord but not in the cerebral cortex. In ara-C-treated mice, the neurochemical markers were also increased in the cerebellum, but to a lesser extent in other regions. These results suggest that, although total contents and total activity of these markers per whole cerebellum were significantly decreased, the central NA metabolism was basically enhanced throughout the CNS of the cerebellar ataxic mice irrespective of their cause. The degree and extent of this enhancement appeared to be correlated with the degree of the cerebellar weight loss.