Sensitivity to neurotoxic stress is not increased in progranulin-deficient mice

Loss-of-function mutations in the progranulin (GRN) gene are a common cause of autosomal dominant frontotemporal lobar degeneration, a fatal and progressive neurodegenerative disorder common in people less than 65 years of age. In the brain, progranulin is expressed in multiple regions at varying levels, and has been hypothesized to play a neuroprotective or neurotrophic role. Four neurotoxic agents were injected in vivo into constitutive progranulin knockout (Grn^−/−) mice and their wild-type (Grn^+/+) counterparts to assess neuronal sensitivity to toxic stress. Administration of 3-nitropropionic acid, quinolinic acid, kainic acid, and pilocarpine induced robust and measurable neuronal cell death in affected brain regions, but no differential cell death was observed between Grn^+/+ and Grn^−/− mice. Thus, constitutive progranulin knockout mice do not have increased sensitivity to neuronal cell death induced by the acute chemical models of neuronal injury used in this study.     

Impact of the lesion procedure on the profiles of motor impairment and molecular responsiveness to L-DOPA in the 6-hydroxydopamine mouse model of Parkinson's disease

6-Hydroxydopamine (6-OHDA) lesions are being used in the mouse for basic research on Parkinson's disease and L-DOPA-induced dyskinesia. We set out to compare unilateral lesion models produced by intrastriatal or intramesencephalic injections of a fixed 6-OHDA concentration (3.2 μg/μl) in C57BL/6 mice. In the first experiment, toxin injections were performed either at two striatal coordinates (1 or 2 μl per site, termed "striatum(2 × 1 μl)" and "striatum(2 × 2 μl)" models), in the medial forebrain bundle (MFB), or in the substantia nigra pars compacta (SN) (1 μl per site). All the four lesion models produced significant forelimb use asymmetry, but spontaneous turning asymmetry only occurred in the MFB and striatum(2 × 2 μl) models. After the behavioral studies, the induction of phosphorylated extracellular signal-regulated kinases 1 and 2 (pERK1/2) by acute L-DOPA (30 mg/kg) was used as a marker of post-synaptic supersensitivity. Striatal pERK1/2 expression was sparse in the SN and striatum(2 × 1 μl) groups, but pronounced in the striatum(2 × 2 μl) and MFB-lesioned mice. In further experiments, mice with MFB and striatal(2 × 2 μl) lesions were used to compare behavioral and molecular responses to chronic L-DOPA treatment (12 days at 3 and 6 mg/kg/day). Maximally severe abnormal involuntary movements (AIMs) occurred in all MFB-lesioned mice, whereas only 35% of the mice with striatal lesions developed dyskinesia. Striatal tissue levels of dopamine were significantly lower in the dyskinetic animals (both MFB and striatum(2 × 2 μl) groups) in comparison with the non-dyskinetic ones. Noradrenaline levels were significantly reduced only in MFB lesioned animals and did not differ among the dyskinetic and non-dyskinetic cases with striatal lesions. In all groups, the L-DOPA-induced AIM scores correlated closely with the number of cells immunoreactive for tyrosine hydroxylase or FosB/?FosB in the striatum. In conclusion, among the four lesion procedures examined here, only the MFB and striatum(2 × 2 μl) models yielded a degree of dopamine denervation sufficient to produce spontaneous postural asymmetry and molecular supersensitivity to L-DOPA. Both lesion models are suitable to reproduce L-DOPA-induced dyskinesia, although only MFB lesions yield a pronounced and widespread expression of post-synaptic supersensitivity markers in the striatum.     

Blood harmane (1-methyl-9H-pyrido[3,4-b]indole) concentration in dystonia cases vs. controls

BackgroundHarmane (1-methyl-9H-pyrido[3,4-b]indole) (HA) is a potent neurotoxin that has been linked to two neurological diseases, essential tremor and Parkinson's disease. Blood harmane concentrations [HA] are elevated in patients with both diseases. An important question is whether HA is specifically linked with these diseases or alternatively, is a non-specific marker of neurological illness.ObjectivesWe assessed whether blood [HA] was elevated in patients with a third neurological disease, dystonia, comparing them to controls.MethodsBlood [HA] was quantified by high performance liquid chromatography. Subjects comprised 104 dystonia cases and 107 controls.ResultsMean log blood [HA] in dystonia cases was similar to that of controls (0.41 ± 0.51 g⁻¹⁰/ml vs. 0.38 ± 0.61 g⁻¹⁰/ml, t = 0.42, p = 0.68). In unadjusted and adjusted logistic regression analyses, log blood [HA] was not associated with the outcome (diagnosis of dystonia vs. control): odds ratio (OR)_unadjusted = 1.11, 95% confidence interval (CI) = 0.69–1.79, p = 0.68; OR_adjusted = 1.07, 95% CI = 0.58–1.97, p = 0.84.ConclusionsIn contrast to the elevated blood [HA] that has been reported in patients with essential tremor and Parkinson's disease, our data demonstrate that blood [HA] was similar in patients with dystonia and controls. These findings provide the first support for the notion that an elevated blood [HA] is not a broad feature of neurological disease, and may be a specific feature of certain tremor disorders.     

3H]Ethynylbicycloorthobenzoate ([3H]EBOB) binding in recombinant GABAA receptors

Ethynylbicycloorthobenzoate (EBOB) is a recently developed ligand that binds to the convulsant site of the GABAA receptor. While a few studies have examined the binding of [3H]EBOB in vertebrate brain tissue and insect preparations, none have examined [3H]EBOB binding in preparations that express known configurations of the GABAA receptor. We have thus examined [3H]EBOB binding in HEK293 cells stably expressing human alpha1beta2gamma2 and alpha2beta2gamma2 GABAA receptors, and the effects of CNS convulsants on its binding. The ability of the CNS convulsants to displace the prototypical convulsant site ligand, [35S]TBPS, was also assessed. Saturation analysis revealed [3H]EBOB binding at a single site, with a K(d) of approximately 9 nM in alpha1beta2gamma2 and alpha2beta2gamma2 receptors. Binding of both [3H]EBOB and [35S]TBPS was inhibited by dieldrin, lindane, tert-butylbicycloorthobenzoate (TBOB), PTX, TBPS, and pentylenetetrazol (PTZ) at one site in a concentration-dependent fashion. Affinities were in the high nM to low microM range for all compounds except PTZ (low mM range), and the rank order of potency for these convulsants to displace [3H]EBOB and [35S]TBPS was the same. Low [GABA] stimulated [3H]EBOB binding, while higher [GABA] (greater than 10 microM) inhibited [3H]EBOB binding. Overall, our data demonstrate that [3H]EBOB binds to a single, high affinity site in alpha1beta2gamma2 and alpha2beta2gamma2 GABAA receptors, and modulation of its binding is similar to that seen with [35S]TBPS. [3H]EBOB has a number of desirable traits that may make it preferable to [35S]TBPS for analysis of the convulsant site of the GABAA receptor.     

Absence of neurotoxic effects in leopard sharks, Triakis semifasciata, following domoic acid exposure.

Domoic acid (DA), a potent neurotoxin produced by select species of algae and diatoms, kills neurons bearing kainic acid-type glutamate receptors. Studies have shown that DA bioaccumulates in invertebrates and fish that consume the diatoms. In every vertebrate species tested or observed in the wild, dietary or systemic DA causes neuronal damage or clinical signs of neurotoxicity. Sharks, like marine birds and mammals, are exposed to DA through their diet; however, no research has demonstrated the effect of DA on shark behavior or physiology. In this study, juvenile leopard sharks (Triakis semifasciata) were given DA by intracoelomic injection at doses of 0, 1, 3, 9, and 27 mg/kg and observed for 7 days. The sharks failed to demonstrate behavioral or histological changes in response to the toxin. We identified putative brain glutamate receptors by probing western blots with an antibody specific for kainic acid-type glutamate receptors and demonstrated receptor localization in the cerebellum with immunohistochemistry. Blood levels of DA in three sharks dosed at 9 mg/kg fell rapidly within 1.5h of injection. We show that leopard sharks possess the molecular target for DA but are resistant to doses of DA known to be toxic to other vertebrates.     

Evaluation of neurodegeneration in scrapie-infected animals by selective methods that detect cellular degeneration

Scrapie is a fatal neurodegenerative disease of sheep and goats. The precise details of neuronal and neurite degeneration in scrapie-infected animals remain unknown. Using specific silver staining methods, we compared the neurodegeneration caused by treatment of rats with kainic acid (KA) or ibogaine (IBO) to the neuropathology observed in mice infected with the C602 strain of scrapie. As reported previously, KA resulted in extensive silver labeling of neurons, especially in the cortex, putamen and hippocampus. IBO silver labeling was observed only in small clusters of Purkinje neurons in the paravermal region of the cerebellum. However, in scrapie-infected mice, a few silver stained neurons (differing from the dark degenerating neurons observed following neurotoxic exposure) were found in layer II of cortex, cingulate cortex, zona incerta, thalamus and hypothalamus. Some silver grains were observed in glial-like cells, especially those in the paraventricular region. Degenerating axons were positive for silver staining and were found in the cortex, cingulate cortex, corpus callosum, habenulae, septum, fornix, thalamus, caudate putamen and a few in fasciculus retroflexus and substantia nigra. Our results suggest that the limbic system is one of the important loci for the neurodegenerative effect of at least some scrapie strains.     

Localization of radioactive125I-Labelled botulinus toxin at the neuromuscular junction of mouse diaphragm

Summary Autoradiography at the light microscopic level demonstrated that the¹²⁵I-labelled neurotoxin fromClostridium botulinum type A crystalline toxin binds specifically to the neuromuscular junction of the mice diaphragm.     

Metabolism of dietary ODAP in humans may be responsible for the low incidence of neurolathyrism

OBJECTIVES: The reasons for the very low incidence of the disease neurolathyrism in humans even after excessive consumption of the pulse, Lathyrus sativus, under severe drought and famine conditions, and its continued consumption by large populations during normal periods without any deleterious effects have been examined in the context of a possible metabolism or detoxification of beta-N-oxalyl-L-alpha, beta-diaminopropionic acid (ODAP), the major neurotoxic amino acid of L. sativus. DESIGN AND METHODS: ODAP in urine samples from 54 subjects habitually consuming the pulse and in three volunteers on an L. sativus diet was determined by the OPT method following clean up of the samples on an alumina column. Urinary oxalate was also determined in these individuals. RESULTS: Twenty-five subjects showed no excretion of ODAP and it was only less than 0.7% of the dietary intake in the remaining 29 subjects. Urinary excretion of ODAP in three volunteers was also less than 1% with a peak excretion in the 4-h sample. The 4-h blood sample from one volunteer had a maximum ODAP concentration of 177 microM. The urinary oxalate content in the volunteers was nearly 3-fold higher compared to controls. CONCLUSIONS: The low excretion of dietary ingested ODAP in humans is in sharp contrast to that seen in animals and indicates a metabolism or detoxification of ODAP which may be unique to humans and may explain the low incidence of neurolathyrism.     

Vulnerabilities of ventral mesencephalic neurons projecting to the nucleus accumbens following infusions of 6-hydroxydopamine into the medial forebrain bundle in the rat

The terminal arbors of dopaminergic projections in the nucleus accumbens (Acb) core degenerate more rapidly, completely and permanently in a variety of neurotoxic circumstances than do those in the medial shell. It is unknown if this always reflects purely losses of the distal parts of axons from the core (as proposed in methamphetamine intoxication), or whether, in some circumstances, the disproportionate loss of core axons may also stem from an intrinsic vulnerability to degeneration of core-projecting neuronal perikarya. Experiments described here addressed this issue in the following manner. Three days after Fluoro-Gold (FG), a retrogradely transported tracer, had been iontophoresed selectively into the core or medial shell of male Sprague-Dawley rats, each received an infusion of saline vehicle containing or lacking 6-hydroxydopamine (6-OHDA) in the ipsilateral medial forebrain bundle (MFB). Twenty-one days later the brains were processed to exhibit ventral mesencephalic neurons containing FG. Application of an unbiased sampling method revealed substantially greater losses of FG labeled neurons relative to controls in rats that had received 6-OHDA lesions and deposition of FG in the Acb core as compared to the medial shell. Of the few core-projecting neurons that remained in the ventral mesencephalon after these lesions, 54% did not co-localize tyrosine hydroxylase immunoreactivity (TH-ir) and, thus, were not expected to degenerate. The capacity to selectively remove core-projecting dopaminergic neurons may be useful in the determination of molecular correlates of vulnerability and resistance to neurotoxicity and to possibly test the role of the core in reinforcement paradigms.     

Effects of 6-OHDA lesions of the central medial nucleus accumbens on rat intravenous morphine self-administration

The function of dopaminergic innervations of the central medial nucleus accumbens in the processes maintaining intravenous morphine self-administration was assessed by lesioning with 6-OHDA and comparing drug intake with sham-vehicle treated littermates. Localized bilateral lesions of this structure resulted in significant increases in morphine intake shifting the dose-effect relationship to the right with twice the dose necessary to maintain prelesion rates of self-administration. Content of dopamine and dihydroxyphenylacetic acid was decreased in the nucleus accumbens after the lesion, but unchanged in the adjacent pyriform cortex and anterior caudate nucleus-putamen, while serotonin was significantly decreased in the pyriform cortex. High affinity uptake measurements also suggested nucleus accumbens dopaminergic and pyriform cortex serotonergic innervations to be affected by the lesion. The shift to the right in the dose effect relationship after the lesion suggests these neuronal systems to be excitatory to the processes mediating self-administration.