No abstract

Previously we described the extent of sprouting that axons of the rat substantia nigra pars compacta (SNpc) undergo to grow new synapses and re-innervate the dorsal striatum 16 weeks after partial lesions. Here we provide insights into the timing of events related to the re-innervation of the dorsal striatum by regenerating dopaminergic nigrostriatal axons over a 104-week period after partial SNpc lesioning. Density of dopamine transporter and tyrosine hydroxylase immunoreactive axonal varicosities (terminals) decreased up to 80% 4 weeks after lesioning but returned to normal by 16 weeks, unless SNpc lesions were greater than 75%. Neuronal tracer injections into the SNpc revealed a 119% increase in axon fibres (4 mm rostral to the SNpc) along the medial forebrain bundle 4 weeks after lesioning. SNpc cells underwent phenotypic changes. Four weeks after lesioning the proportion of SNpc neurons that expressed tyrosine hydroxylase fell from 90% to 38% but returned to 78% by 32 weeks. We discuss these phenotype changes in the context of neurogenesis. Significant reductions in dopamine levels in rats with medium (30-75%) lesions returned to normal by 16 weeks whereas recovery was not observed if lesions were larger than 75%. Finally, rotational behaviour of animals in response to amphetamine was examined. The clear rightward turning bias observed after 2 weeks recovered by 16 weeks in animals with medium (30-75%) lesions but was still present when lesions were larger. These studies provide insights into the processes that regulate sprouting responses in the central nervous system following injury.

Neuropeptide Y (NPY), a 36-amino-acid peptide, mediates biological effects by activating Y1, Y2, Y5, and y6 receptors. NPY neurons innervate many brain regions, including the hypothalamus, where NPY is involved in regulation of a broad range of homeostatic functions. We examined, by immunohistochemistry with tyramide signal amplification, the expression of the NPY Y2 receptor (Y2R) in the mouse brain with a newly developed rabbit polyclonal antibody. Y2R immunoreactivity was specific with its absence in Y2R knockout (KO) mice and in adjacent sections following preadsorption with the immunogenic peptide (10(-5) M). Y2R-positive processes were located in many brain regions, including the olfactory bulb, some cortical areas, septum, basal forebrain, nucleus accumbens, amygdala, hippocampus, hypothalamus, substantia nigra compacta, locus coeruleus, and solitary tract nucleus. However, colchicine treatment was needed to detect Y2R-like immunoreactivity in cell bodies in many, but not all, areas. The densest distributions of cell bodies were located in the septum basal forebrain, including the bed nucleus, and amygdala, with lower density in the anterior olfactory nucleus, nucleus accumbens, caudal striatum, CA1, CA2, and CA3 hippocampal fields, preoptic nuclei lateral hypothalamus, and A13 DA cells. The widespread distribution of Y2R-positive cell bodies and fibers suggests that NPY signaling through the Y2R is common in the mouse brain. Localization of the Y2R suggests that it is mostly presynaptic, a view supported by its frequent absence in cell bodies in the normal mouse and its dramatic increase in cell bodies of colchicine-treated mice.

Summary Purpose Ethosuximide (ESX) is a drug of choice for the symptomatic treatment of absence seizures. Chronic treatment with ESX has been reported to have disease-modifying anti-epileptogenic activity in the WAG/Rij rat model of genetic generalised epilepsy (GGE) with absence seizures. Here we examined whether chronic treatment with ESX (i) possesses anti-epileptogenic effects in the GAERS model of GGE, (ii) is associated with a mitigation of behavioural comorbidities, and (iii) influences gene expression in the somatosensory cortex region where seizures are thought to originate. Methods GAERS and Non-Epileptic Control (NEC) rats were chronically treated with ESX (in drinking water) or control (tap water) from 3 to 22 weeks of age. Subsequently, all animals received tap water only for another 12 weeks to assess enduring effects of treatment. Seizure frequency and anxiety-like behaviours were serially assessed throughout the experimental paradigm. Treatment effects on the expression of key components of the epigenetic molecular machinery, the DNA methyltransferase enzymes, were assessed using qPCR. Key findings ESX treatment significantly reduced seizures in GAERS during the treatment phase, and this effect was maintained during the 12 weeks post-treatment phase (p<0.05). Further, the anxiety-like behaviours present in GAERS were reduced by ESX treatment (p<0.05). Molecular analysis revealed that ESX treatment was associated with increased expression of DNA methyltransferase enzyme mRNA in cortex. Significance Chronic ESX treatment has disease-modifying effects in the GAERS model of GGE, with anti-epileptogenic effects against absence seizures and mitigation of behavioural comorbidities. The cellular mechanism for these effects may involve epigenetic modifications.

Alternative splicing is an evolutionary innovation to create functionally diverse proteins from a limited number of genes. SNAP-25 plays a central role in neuroexocytosis by bridging synaptic vesicles to the plasma membrane during regulated exocytosis. The SNAP-25 polypeptide is encoded by a single copy gene, but in higher vertebrates a duplication of exon 5 has resulted in two mutually exclusive splice variants, SNAP-25a and SNAP-25b. To address a potential physiological difference between the two SNAP-25 proteins, we generated gene targeted SNAP-25b deficient mouse mutants by replacing the SNAP-25b specific exon with a second SNAP-25a equivalent. Elimination of SNAP-25b expression resulted in developmental defects, spontaneous seizures, and impaired short-term synaptic plasticity. In adult mutants, morphological changes in hippocampus and drastically altered neuropeptide expression were accompanied by severe impairment of spatial learning. We conclude that the ancient exon duplication in the Snap25 gene provides additional SNAP-25-function required for complex neuronal processes in higher eukaryotes.

Aromatase catalyses the last step of oestrogen synthesis. There is growing evidence that local oestrogens influence many brain regions to modulate brain development and behaviour. We examined, by immunohistochemistry, the expression of aromatase in the adult male and female mouse brain, using mice in which enhanced green fluorescent protein (EGFP) is transcribed following the physiological activation of the Cyp19A1 gene. EGFP-immunoreactive processes were distributed in many brain regions, including the bed nucleus of the stria terminalis, olfactory tubercle, medial amygdaloid nucleus and medial preoptic area, with the densest distributions of EGFP-positive cell bodies in the bed nucleus and medial amygdala. Differences between male and female mice were apparent, with the density of EGFP-positive cell bodies and fibres being lower in some brain regions of female mice, including the bed nucleus and medial amygdala. EGFP-positive cell bodies in the bed nucleus, lateral septum, medial amygdala and hypothalamus co-expressed oestrogen receptor (ER) α and β, or the androgen receptor (AR), although single-labelled EGFP-positive cells were also identified. Additionally, single-labelled ERα−, ERβ- or AR-positive cell bodies often appeared to be surrounded by EGFP-immunoreactive nerve fibres/terminals. The widespread distribution of EGFP-positive cell bodies and fibres suggests that aromatase signalling is common in the mouse brain, and that locally synthesised brain oestrogens could mediate biological effects by activating pre- and post-synaptic oestrogen α and β receptors, and androgen receptors. The higher number of EGFP-positive cells in male mice may indicate that the autocrine and paracrine effects of oestrogens are more prominent in males than females.

This study demonstrates that pharmacological manipulation of the dopamine (DA) receptors can modulate the size of the axonal tree of substantia nigra pars compacta (SNpc) neurons in mice. Pharmacological blockade or genetic ablation of the D2 receptor (D2R) resulted in sprouting of DA SNpc neurons whereas treatment with a D2 agonist resulted in pruning of the terminal arbor of these neurons. Agents such as cocaine, that indirectly stimulate D2R, also resulted in reduced terminal arbor. Specific D1 agonists or antagonists had no effect on the density of DA terminals in the striatum. We conclude that the D2 receptor has a central role in regulating the size of the terminal arbor of nigrostriatal neurons. These findings have implications relating to the use of dopaminergic agonists in the management of Parkinson's disease and in controlling plasticity following injury, loss or transplantation of DA neurons.

Failed storage capacity, leading to pulsatile delivery of dopamine (DA) in the striatum, is used to explain the emergence of 'wearing off' and dyskinaesia in Parkinson's disease. In this study, we show that surviving DA neurons in 6-OHDA lesioned rats sprout to re-innervate the striatum, and maintain terminal density until approximately 60% of neurons are lost. We demonstrate that DA terminal density correlates with baseline striatal DA concentration ([DA]). Electrochemical and synaptosome studies in 6-OHDA lesioned rats and primates suggest that impaired striatal DA re-uptake and increased DA release from medial forebrain bundle fibres contribute to maintaining striatal DA levels. In lesioned rats where terminal density fell by 60% or more, L-DOPA administration increased striatal DA levels markedly. The striatal [DA] produced by L-DOPA directly correlated with the extent of dyskinaesia, suggesting that dyskinaesia was related to high striatal [DA]. While sprouting and decreased dopamine uptake transporter function would be expected to contribute to the marked increase in L-DOPA induced [DA], the increased [DA] was most marked when DAergic fibres were >60% denervated, suggesting that other release sites, such as serotonergic fibres might be contributing. In conclusion, the extent of dyskinaesia was directly proportional to the extent of DA terminal denervation and levels of extra-synaptic striatal DA. We propose that sprouting of DA terminals and decreased dopamine uptake transporter function prevent the appearance of Parkinsonian symptoms until about 60% loss of nigral neurons, but also contribute to dysregulated striatal DA release that is responsible for the emergence of dyskinaesia and 'wearing off'.