John Y. F. Wong

Reduced BDNF mRNA Expression in the Parkinson's Disease Substantia Nigra

Brain-derived neurotrophic factor (BDNF) has potent effects on survival and morphology of dopaminergic neurons and thus its loss could contribute to death of these cells in Parkinsons disease (PD). In situ hybridization revealed that BDNF mRNA is strongly expressed by dopaminergic neurons in control substantia nigra pars compacta (SNpc). In clinically and neuropathologically typical PD, SNpc BDNF mRNA expression is reduced by 70% (P = 0.001). This reduction is due, in part, to loss of dopaminergic neurons which express BDNF. However, surviving dopaminergic neurons in the PD SNpc also expressed less BDNF mRNA (20%, P = 0.02) than their normal counterparts. Moreover, while 15% of control neurons had BDNF mRNA expression >1 SD below the control mean, twice as many (28%) of the surviving PD SNpc dopaminergic neurons had BDNF mRNA expression below this value. This 13% difference in proportions (95% CI 8-17%, P < or = 0.000001) indicates the presence of a subset of neurons in PD with particularly low BDNF mRNA expression. Moreover, both control and PD neurons displayed a direct relationship between the density of BDNF mRNA expression per square micrometer of cell surface and neuronal size (r(2) = 0.93, P </= 0.00001) which was lost only in PD neurons expressing the lowest levels of BDNF mRNA. If BDNF is an autocrine/paracrine factor for SNpc dopaminergic neurons, loss of BDNF-expressing neurons may compromise the well-being of their surviving neighbors. Moreover, neurons expressing particularly low levels of BDNF mRNA may be those at greatest risk of injury in PD and possibly the trigger for the degeneration itself.

Essential fatty acids given from conception prevent topographies of motor deficit in a transgenic model of Huntington's disease.

Transgenic R6/1 mice incorporate a human genomic fragment containing promoter elements exon 1 and a portion of intron 2 of the Huntingtin gene responsible for Huntingtons disease. They develop late-onset neurological deficits in a manner similar to the motor abnormalities of the disorder. As essential fatty acids are phospholipid components of cell membranes which may influence cell death and movement disorder phenotype, R6/1 and normal mice were randomised to receive a mixture of essential fatty acids or placebo on alternate days throughout life. Over mid-adulthood, topographical assessment of behaviour revealed R6/1 transgenics to evidence progressive shortening of stride length, with progressive reductions in locomotion, elements of rearing, sniffing, sifting and chewing, and an increase in grooming. These deficits were either not evident or materially diminished in R6/1 transgenics receiving essential fatty acids. R6/1 transgenics also showed reductions in body weight and in brain dopamine D(1)-like and D(2)-like quantitative receptor autoradiography which were unaltered by essential fatty acids.These findings indicate that early and sustained treatment with essential fatty acids are able to protect against motor deficits in R6/1 transgenic mice expressing exon 1 and a portion of intron 2 of the Huntingtin gene, and suggest that essential fatty acids may have therapeutic potential in Huntingtons disease.

Expression of brain-derived neurotrophic factor and TrkB neurotrophin receptors after striatal injury in the mouse

Brain-derived neurotrophic factor (BDNF) promotes the survival and differentiation of nigral dopaminergic neurons and supports the activity of dopaminergic cells grafted into the striatum. However, little attention has been given to the physiological role of endogenous BDNF and its receptor TrkB within the nigrostriatal dopamine system. We know that striatal injury is followed by long-term stimulation of dopaminergic activity in the striatum, could BDNF play a role in this phenomenon? One week after physical injury to the striatum of C57/Black mice, just before dopaminergic activation becomes obvious, in situ hybridization on coronal sections through mouse striatum reveals that BDNF mRNA expression increases significantly before returning to basal levels within 1 month. Expression of mRNA for TrkB follows a very different pattern. No change of expression of the full-length and catalytically competent TrkBTK+ receptor is seen. However, expression of the truncated form of the receptor TrkTK-, which lacks the catalytic tyrosine kinase domain, does increase and stays elevated for at least 2 months after injury. When combined with observations of dopaminergic activation after striatal injury and the neuroprotective effects of BDNF introduced into the striatum, our findings suggest that BDNF and TrkBTK- do indeed play a role in dopaminergic regeneration and repair.

Effect of Chronic Angiotensin‐Converting Enzyme Inhibition on Striatal Dopamine Content in the MPTP‐Treated Mouse

Abstract: We have previously shown that chronic treatment with the angiotensin‐converting enzyme inhibitor perindopril increased striatal dopamine levels by 2.5‐fold in normal Sprague—Dawley rats, possibly via modulation of the striatal opioid or tachykinin levels. In the present study, we investigated if this effect of perindopril persists in an animal model of Parkinson’s disease, the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐treated mouse. C57BL/6 mice were treated with the neurotoxin (30 mg/kg/day intraperitoneally) for 4 days and then left for 3 weeks to allow the degeneration of striatal dopaminergic terminals. At this time, the mice exhibited a 40% decrease in striatal dopamine content and an accompanying 46% increase in dopamine D2 receptor levels compared with control untreated mice. The dopamine content returned to control levels, and the increase in dopamine D2 receptor levels was attenuated in mice treated with perindopril (5 mg/kg/day orally for 7 days) 2 weeks after the last dose of MPTP. When the angiotensin‐converting enzyme inhibitor was administered (5 mg/kg/day for 7 days) immediately after the cessation of the MPTP treatment, there was no reversal of the effect of the neurotoxin in decreasing striatal dopamine content. Our results demonstrate that perindopril is an effective agent in increasing striatal dopamine content in an animal model of Parkinson’s disease.

Expression of glial cell line-derived neurotrophic factor (GDNF) mRNA following mechanical injury to mouse striatum.

ALTHOUGH glial cell line-derived neurotrophic factor (GDNF) expression is low in the adult brain, its administration protects dopaminergic neurons against a range of insults, leading to the suggestion of a role in dopaminergic regeneration. If locally produced GDNF is to fulfil a role in dopaminergic regeneration after injury, it seems reasonable to hypothesize that its expression will increase after mechanical trauma. We have demonstrated that GDNF mRNA expression increases within 6 h of using a wire knife to injure adult mouse striatum. Expression doubles after 1 week and remains elevated for at least 1 month. Most GDNF expression is associated with haemosiderin-containing cells, indicating production by brain macrophages. GDNF production by macrophages may be essential for neural regeneration following CNS trauma.

Electroencephalographic characterisation of pentylenetetrazole-induced seizures in mice lacking the α4 subunit of the neuronal nicotinic receptor

Autosomal Dominant Nocturnal Frontal Lobe Epilepsy (ADNFLE) is associated in some kindreds with mutations in the genes encoding the alpha 4 or beta 2 subunits of the neuronal nicotinic acetylcholine receptor (nAChR). Functional characterisation of the described ADNFLE mutations in oocyte preparations has produced conflicting results, with some studies suggesting hypofunction but others showing increased ligand sensitivity or delayed desensitisation. Knockout mice were studied to investigate extreme hypofunction of alpha 4 nAChRs in vivo. Mutant (Mt) and control mice underwent epidural electroencephalographic (EEG) recording for 2 h in the untreated state and for 1 h following administration of the gamma-amino butyric acid (GABA) antagonist, pentylenetetrazole (PTZ, 80 mg/kg). No spontaneous seizures occurred and no EEG differences were observed between the genotypes in drug naïve mice. Following PTZ, however, Mt mice showed markedly increased mortality compared to controls (85 vs 30%, P<0.001). Mts also had a greater number of generalised clonic seizures in the first 40 min following injection. In the same period, the EEGs of Mt mice showed an excess of spikes (P=0.033), multi-spike complexes (P=0.002) and continuous fast activity (P=0.017) compared to controls. These findings demonstrate that intact alpha 4 nAChR subunits provide significant in vivo protection against the proconvulsant effects of GABA antagonism.

Sprouting of Dopaminergic Axons after Striatal Injury: Confirmation by Markers Not Dependent on Dopamine Metabolism

Striatal injury increases dopamine metabolism in the nigrostriatal system but it is unclear whether this response is due to increased synthesis and activation of tyrosine hydroxylase within existing dopamine terminals and/or branching and sprouting of new terminals. While monitoring the density of tyrosine hydroxylase immunoreactive fibers suggests that sprouting occurs, this technique alone cannot adequately answer this question since the intensity of staining and thus the visibility of individual fibers are intimately linked to dopaminergic activity. However, by examining axons and their branches using markers that are independent of dopamine metabolism it is possible to determine whether dopaminergic sprouting does in fact take place. One month after using a Scouten wire knife to create a small lesion in the left striatum of normal C57/bl-6 mice, silver staining revealed an increase in the total number of neuronal fibers throughout the injured striatum. This was accompanied by intense staining of tyrosine hydroxylase-positive fibers around the wound and an increased density of striatal fibers labeled with dextran-biotin after injection of this neuronal tracer into the substantia nigra 1 month after striatal surgery and 5 days prior to sacrifice. The increase in tyrosine hydroxylase immunoreactivity confirms previous observations of increased dopaminergic activity after striatal injury. The increases in silver staining and dextran-biotin transport provide independent evidence that this increase in dopaminergic activity occurs because of sprouting of new fibers originating in the substantia nigra.

Essential conservation of D1 mutant phenotype at the level of individual topographies of behaviour in mice lacking both D1 and D3 dopamine receptors

RationaleIn the absence of agonists and antagonists evidencing appropriate selectivities, individual and interactive properties of D1 and D3 dopamine receptors would be illuminated most powerfully by their co-deletion.ObjectivesTo define and contrast the behavioural phenotype of D1/D3 double knockout mice in comparison with wild types, and with individual D1 and D3 mutants.MethodsBehavioural phenotype was characterised using an ethologically based topographical technique.ResultsOn comparison with wild types, D1/D3 double mutants were characterised topographically as follows: increases in sniffing and locomotion, which evidenced delayed habituation; reductions in rearing free, rearing seated, grooming, chewing and stillness. Though the D1/D3 double mutant ethogram comprised elements of both single mutant D1 and D3 lines, this phenotype was largely reflective of the D1 mutant component.ConclusionsDistinct patterns of initial exploratory behaviour and of temporal change over subsequent habituation were evident across the three genotypes, with particular conservation of the D1 phenotype in D1/D3 double mutants. Under the present conditions, there was little systematic evidence for D1:D3 interactions in the regulation of these aspects of behaviour.

Late direct and transneuronal effects in mice with targeted expression of a toxin gene to D1 dopamine receptor neurons

Detailed analysis of a novel transgenic model of basal ganglia disease has been undertaken. In this model the expression of an attenuated form of the diphtheria toxin gene was tightly controlled by D1 dopamine receptor regulatory domains. The behavioral and both direct toxin-mediated and transneuronal effects observed in pups in the first postnatal week have been described. Although younger pups are bradykinetic, older pups have a hyperkinetic syndrome with gait abnormality, postural instability and myoclonic jerks typical of human basal ganglia diseases such as Huntingtons disease. As expected, striatal D1 dopamine receptor, dynorphin and substance P transcripts were not detected by in situ hybridization but there was a 27% increase in striatal D2 dopamine receptor messenger RNA and a 65% increase in enkephalin messenger RNA expression. Receptor autoradiographic studies confirmed the lack of D1-class binding in the mutant striatum and in contrast to young pups, a substantial increase in striatal D2-class binding. Autoradiographic quantitation also showed a 30% increase in striatal dopamine transporter binding. In addition to the changes described in the striatopallidal and nigrostriatal pathways, up-regulated dynorphin and substance P messenger RNA expression was also seen in the cortex. The capacity of the developing brain for neurochemical adaptation following injury is dramatic. The results show that primary loss of D1 dopamine receptor-positive striatonigral pathway neurons is sufficient to generate a hyperkinetic phenotype.

Proconvulsant-induced seizures in α4 nicotinic acetylcholine receptor subunit knockout mice

Abstract The genetic basis of a number of epilepsy syndromes has been identified but the precise mechanism whereby these mutations produce seizures is unknown. Three mutations of the α4 subunit of the neuronal nicotinic acetylcholine receptor (nAChR) have been identified in autosomal dominant nocturnal frontal lobe epilepsy. In vitro studies of two mutations suggest an alteration of receptor function resulting in decreased ion channel current flow. We investigated the response of α4 nAChR subunit knockout mice to the γ-aminobutyric acid (GABA) receptor antagonists; pentylenetetrazole (PTZ) and bicuculline (BIC), the glutamate receptor agonist kainic acid (KA), the glycine receptor antagonist strychnine and the K+ channel blocker 4-aminopyridine (4-AP). Mutant (Mt) mice had a greater sensitivity to PTZ and BIC, with an increase in major motor seizures and seizure-related deaths. Furthermore, Mt mice were more sensitive to KA and strychnine, but the effects were much smaller compared to those seen with the GABA receptor antagonists. Paradoxically, Mt mice appeared to be relatively protected from 4-AP-induced major motor seizures and death. The results show that a functional deletion of the α4 nAChR subunit in vivo is associated with a major increase in sensitivity to GABA receptor blockers.