Davor Stanic

Axonal sprouting following lesions of the rat substantia nigra

Parkinsons disease is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Symptoms do not appear until most nigral neurons are lost, implying that compensatory mechanisms are present. Sprouting has been proposed as one of these mechanisms. This study quantified the extent of compensatory axonal sprouting following injury of dopaminergic neurons within the substantia nigra pars compacta. Specifically, the extent of the axonal arbour and axonal varicosity morphology was measured after partial destruction (with 6-hydroxydopamine) of the substantia nigra of the adult male rat. Four months later, the substantia nigra was injected with the anterograde neuronal tracer dextran-biotin to trace the full extent of individual axons. An unbiased estimate of neuron number was performed in each animal. This demonstrated nigral neuronal loss ranging from 10 to 90% on the side that received the injection whilst a 7% reduction was observed in the side contralateral to the lesion. Coincident with this loss, some nigral neurons lose tyrosine hydroxylase expression. Vigorous axonal sprouting was observed in the terminal arbours of lesioned animals and was associated with an increased axonal varicosity size. Axonal varicosities and branching points were primarily confined to the dorsal 1.5mm of the caudate-putamen, an area predominantly innervated by nigral neurons. It appears that dopaminergic neurons were responsible for this sprouting because the density of dopamine transporter immunoreactive varicosities in the caudate-putamen was maintained until about a 70% loss of neurons. It was concluded that substantial compensation in the form of sprouting and new dopaminergic synapse formation occurs following lesions of the substantia nigra pars compacta.

Timecourse of striatal re-innervation following lesions of dopaminergic SNpc neurons of the rat

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.

Characterization of neuropeptide Y2 receptor protein expression in the mouse brain. I. Distribution in cell bodies and nerve terminals.

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. J. Comp. Neurol. 499:357–390, 2006.

Ethosuximide reduces epileptogenesis and behavioral comorbidity in the GAERS model of genetic generalized epilepsy

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 antiepileptogenic activity in the WAG/Rij rat model of genetic generalized epilepsy (GGE) with absence seizures. Here we examined whether chronic treatment with ESX (1) possesses antiepileptogenic effects in the genetic absence epilepsy rats from Strasbourg (GAERS) model of GGE, (2) is associated with a mitigation of behavioral comorbidities, and (3) influences gene expression in the somatosensory cortex region where seizures are thought to originate.

Neuropeptide Y2 receptor protein is present in peptidergic and nonpeptidergic primary sensory neurons of the mouse.

The localization of the neuropeptide tyrosine (NPY) Y2 receptor (Y2R) protein was studied in mouse dorsal root ganglia (DRGs) and spinal cord, by using a recently developed rabbit anti‐Y2R antibody and a sensitive immunohistochemical method. Y2R‐like immunoreactivity (‐LI) was observed in about 10% of the small/medium‐sized lumbar DRG neurons. Among these, about 44% were calcitonin gene‐related peptide‐immunoreactive, and about 38% bound isolectin B4. In the dorsal horn of the spinal cord, an intense Y2R‐LI was seen in the most superficial layers, mostly restricted to laminae I–II. This immunoreactivity was completely abolished by dorsal rhizotomy. Y2R‐L1 was also detected on the skin, more abundantly in hairy than glabrous skin. Specificity experiments showed complete disappearance of the Y2R‐LI described above after incubation with antibody preadsorbed with the immunogenic peptide. Furthermore, Y2R‐LI was also absent in a Y2R knockout mouse. These results demonstrate that the NPY Y2R is associated mainly with both peptidergic and nonpeptidergic small, presumably nociceptive, neurons projecting to the superficial layers of the dorsal horn. The results also support a role for this receptor and NPY in pain mechanisms. J. Comp. Neurol. 489:328–348, 2005.

Effects of long-term treatment with dopamine receptor agonists and antagonists on terminal arbor size

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 Parkinsons disease and in controlling plasticity following injury, loss or transplantation of DA neurons.

Changes in function and ultrastructure of striatal dopaminergic terminals that regenerate following partial lesions of the SNpc

Following partial substantia nigra lesions, remaining dopaminergic neurones sprout, returning terminal density in the dorsal striatum to normal by 16 weeks. This suggests regeneration and maintenance of terminal density is regulated to release appropriate levels of dopamine. This study examined the structure and function of these reinnervated terminals, defining characteristics of dopamine uptake and release, density and affinity of the dopamine transporter (DAT) and ultrastructural morphology of dopamine terminals in the reinnervated dorsal striatum. Finally, rotational behaviour of animals in response to amphetamine was examined 4 and 16 weeks after substantia nigra pars compacta (SNpc) lesions. Dopamine transport was markedly reduced 16 weeks after lesioning along with reduced density and affinity of DAT. Rate of dopamine release and peak concentration, measured electrochemically, was similar in lesioned and control animals, while clearance was prolonged after lesioning. Ultrastructurally, terminals after lesioning were morphologically distinct, having increased bouton size, vesicle number and mitochondria, and more proximal contacts on post-synaptic cells. After 4 weeks, tendency to rotate in response to amphetamine was proportional to lesion size. By 16 weeks, rotational behaviour returned to near normal in animals where lesions were less than 70%, although some animals demonstrated unusual rotational patterns at the beginning and end of the amphetamine effect. Together, these changes indicate that sprouted terminals are well compensated for dopamine release but that transport mechanisms are functionally impaired. We discuss these results in terms of implications for dyskinesia and other behavioural states.

An Ancient Duplication of Exon 5 in the Snap25 Gene Is Required for Complex Neuronal Development/Function

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.

Characterization of NPY Y2 receptor protein expression in the mouse brain. II. Coexistence with NPY, the Y1 receptor, and other neurotransmitter-related molecules

Neuropeptide Y (NPY) is widely expressed in the brain and its biological effects are mediated through a variety of receptors. We examined, using immunohistochemistry, expression of the Y2 receptor (R) protein in the adult mouse brain and its association with NPY and the Y1R, as well as a range of additional neurotransmitters and signaling‐related molecules, which previously have not been defined. Our main focus was on the hippocampal formation (HiFo), amygdaloid complex, and hypothalamus, considering the known functions of NPY and the wide expression of NPY, Y1R, and Y2R in these regions. Y2R‐like immunoreactivity (‐LI) was distributed in nerve fibers/terminal endings throughout the brain axis, without apparent colocalization with NPY or the Y1R. Occasional coexistence between NPY‐ and Y1R‐LI was found in the HiFo. Following colchicine treatment, Y2R‐LI accumulated in cell bodies that coexpressed γ‐aminobutyric acid (GABA) in a population of cells in the amygdaloid complex and lateral septal nucleus, but not in the HiFo. Instead, Y2R‐positive nerve terminals appeared to surround GABA‐immunoreactive (ir) cells in the HiFo and other neuronal populations, e.g., NPY‐ir cells in HiFo and tyrosine hydroxylase‐ir cells in the hypothalamus. In the HiFo, Y2R‐ir mossy fibers coexpressed GABA, glutamic acid decarboxylase 67 and calbindin, and Y2R‐LI was found in the same fibers that contained the presynaptic metabotropic glutamate receptor 2, but not together with any of the three vesicular glutamate transporters. Our findings provide further support that Y2R is mostly presynaptic, and that Y2Rs thus have a modulatory role in mediating presynaptic neurotransmitter release. J. Comp. Neurol. 519:1219–1257, 2011.

Developmental changes in frequency of the ciliary somatostatin receptor 3 protein

Primary cilia extend from the surface of most vertebrate cells and display several signaling molecules, including the somatostatin receptor 3 (SSTR3), enabling cilia to play essential roles as chemical, osmotic and mechanical sensors. The SSTR3 is widely distributed in the adult rat brain, and also influences cell proliferation and apoptosis. To establish whether the SSTR3 is positioned to influence these developmental processes, we examined, using immunohistochemistry, the embryonic and postnatal development of SSTR3 expression in the rat hippocampal formation, and its association with newly born and mature neurons in adult rats. Elongated SSTR3-immunoreactive (-ir) cilia first appeared in the hippocampal formation CA3 region of postnatal day (P) 0 animals, and their density increased to high levels by P2, remained at high levels through to P30, but were at low levels in 5-month old rats. A similar developmental pattern was observed in the CA1 region, where SSTR3-ir ciliated structures were first detected on P2. In contrast, density levels in the granular cell layer of the dentate gyrus were very high by P30, and remained elevated in adult rats. SSTR3-ir cilia did not colocalize with neuroblasts in the hippocampal formation or olfactory bulb, but appeared to be localized to more mature cells in these regions. A few SSTR3-ir neurons were also observed in the hippocampal formation. These findings support the hypothesis that the ciliary SSTR3 is well positioned to influence the cell cycle and apoptotic processes during postnatal development, and in neurogenic regions of the adult rat brain.