Rosaria Ferrari

Increased neurodegeneration during ageing in mice lacking high-affinity nicotine receptors

We have examined neuroanatomical, biochemical and endocrine parameters and spatial learning in mice lacking the β2 subunit of the nicotinic acetylcholine receptor (nAChR) during ageing. Aged β2−/− mutant mice showed region‐specific alterations in cortical regions, including neocortical hypotrophy, loss of hippocampal pyramidal neurons, astro‐ and microgliosis and elevation of serum corticosterone levels. Whereas adult mutant and control animals performed well in the Morris maze, 22‐ to 24‐month‐old β2−/− mice were significantly impaired in spatial learning. These data show that β2 subunit‐containing nAChRs can contribute to both neuronal survival and maintenance of cognitive performance during ageing. β2−/− mice may thus serve as one possible animal model for some of the cognitive deficits and degenerative processes which take place during physiological ageing and in Alzheimers disease, particularly those associated with dysfunction of the cholinergic system.

Acute and long-term changes in the mesolimbic dopamine pathway after systemic or local single nicotine injections

We have examined several neurochemical and behavioural parameters related to the function of the mesolimbic dopamine (DA) pathway in animals treated with nicotine following three modes of drug administration, i.e. systemic intraperitoneal injection, intra‐accumbens (Acb) infusion or intraventral tegmental area (intra‐VTA) microinjection. The present modes of systemic, intra‐Acb and intra‐VTA nicotine administration elicited comparable acute increases in dialysate DA levels from the Acb. The increase in extracellular DA levels was paralleled by a significant enhancement of locomotion in a habituated environment in the case of systemic or intra‐VTA nicotine administration, whereas unilateral or bilateral intra‐Acb nicotine infusion was ineffective, showing that accumbal DA increase is not sufficient to elicit locomotion in this experimental paradigm. Intra‐VTA, but not systemic or intra‐Acb, nicotine administration caused a long‐term (at least 24‐h) increase in basal dialysate DA levels from the Acb. In addition, significant increases in tyrosine hydroxylase (TH) and GluR1 (but not dopamine transporter or NR1) mRNA levels in the VTA were detected 24 h after intra‐VTA nicotine administration. Systemic nicotine injection caused only an increase in TH mRNA levels while intra‐Acb infusion did not modify any of the mRNAs tested. The long‐term increase in basal DA levels in the Acb and TH, and GluR1 mRNA levels in the VTA upon intra‐VTA nicotine microinjection indicates that even a single nicotine injection can induce plastic changes of the mesolimbic DA pathway.

Functional striatal hypodopaminergic activity in mice lacking adenosine A(2A) receptors.

Adenosine and caffeine modulate locomotor activity and striatal gene expression, partially through the activation and blockade of striatal A2A receptors, respectively. The elucidation of the roles of these receptors benefits from the construction of A2A receptor‐deficient mice (A2A‐R−/−). These mice presented alterations in locomotor behaviour and striatal expression of genes studied so far, which are unexpected regarding the specific expression of A2A receptor by striatopallidal neurones. To clarify the functions of A2A receptors in the striatum and to identify the mechanisms leading to these unexpected modifications, we studied the basal expression of immediate early and constitutive genes as well as dopamine and glutamate neurotransmission in the striatum. Basal zif268 and arc mRNAs expression was reduced in mutant mice by 60–80%, not only in the striatum but also widespread in the cerebral cortex and hippocampus. Striatal expression of substance P and enkephalin mRNAs was reduced by about 50% and 30%, respectively, whereas the expression of GAD67 and GAD65 mRNAs was slightly increased and unaltered, respectively. In vivo microdialysis in the striatum revealed a 45% decrease in the extracellular dopamine concentration and three‐fold increase in extracellular glutamate concentration. This was associated with an up‐regulation of D1 and D2 dopamine receptors expression but not with changes in ionotropic glutamate receptors. The levels of tyrosine hydroxylase and of striatal and cortical glial glutamate transporters as well as adenosine A1 receptors expression were indistinguishable between A2A‐R−/− and wild‐type mice. Altogether these results pointed out that the lack of A2A receptors leads to a functional hypodopaminergic state and demonstrated that A2A receptors are necessary to maintain a basal level in immediate early and constitutive genes expression in the striatum and cerebral cortex, possibly via their control of dopamine pathways.

Short- and Long-term Changes in Striatal Neurons and Astroglia After Transient Forebrain Ischemia in Rats

BACKGROUND AND PURPOSE The striatum is one of the regions most sensitive to transient forebrain ischemia. After 30-minute ischemia, areas of massive neuronal degeneration are clearly detectable a few hours after the insult and attain their maximal extension 24 hours after the insult. However, for most cellular and neurochemical parameters it is not known whether some recovery occurs at later times. We examined certain cell populations in the caudate putamen at different times after transient ischemia. METHODS Adult male Sprague-Dawley rats were subjected to 30-minute forebrain ischemia (four-vessel occlusion model). Six experimental groups were considered: control animals and ischemic animals killed 4 hours, 1 day, 7 days, 40 days, and 8 months after reperfusion. Three striatal cell populations were examined by means of immunocytochemistry coupled to computer-assisted image analysis: vulnerable medium spiny neurons, resistant aspiny neurons, and reactive astrocytes, labeled for their content of dopamine- and cAMP-regulated phosphoprotein mr32 (DARPP-32), somatostatin and neuropeptide Y, and glial fibrillary acidic protein, respectively. RESULTS (1) The area containing DARPP-32 immunoreactive neurons was markedly decreased (15% to 20% of control caudate putamen area) at 1 day after reperfusion and partially recovered at the following times (40% to 50% at 7 days and 50% to 60% at 40 days and 8 months after reperfusion). (2) The appearance of reactive astrocytes was precocious (4 hours to 1 day after ischemia) in the medial caudate putamen, the region in which DARPP-32 recovered within 40 days after ischemia, and late (7 to 40 days after ischemia) in the lateral caudate putamen, where no DARPP-32 recovery was detected. (3) Neuropeptide Y/somatostatin-containing neurons resisted the ischemic insult and could be detected in areas devoid of DARPP-32 immunoreactive neurons as long as 8 months after reperfusion. CONCLUSIONS The present results show a marked recovery of DARPP-32-positive neurons within 40 days after 30-minute forebrain ischemia in the medial, but not the lateral, caudate putamen. Medial caudate putamen also contains a high density of reactive astrocytes on the first day after ischemia, suggesting that astrocytic support has an important role in the spontaneous recovery of ischemic neurons.

S100A1 codistributes with synapsin I in discrete brain areas and inhibits the F-actin-bundling activity of synapsin I

The Ca2+‐sensor protein S100A1 was recently shown to bind in vitro to synapsins, a family of synaptic vesicle phosphoproteins involved in the regulation of neurotransmitter release. In this paper, we analyzed the distribution of S100A1 and synapsin I in the CNS and investigated the effects of the S100A1/synapsin binding on the synapsin functional properties. Subcellular fractionation of rat brain homogenate revealed that S100A1 is present in the soluble fraction of isolated nerve endings. Confocal laser scanning microscopy and immunogold immunocytochemistry demonstrated that S100A1 and synapsin codistribute in a subpopulation (5–20%) of nerve terminals in the mouse cerebral and cerebellar cortices. By forming heterocomplexes with either dephosphorylated or phosphorylated synapsin I, S100A1 caused a dose‐ and Ca2+‐dependent inhibition of synapsin‐induced F‐actin bundling and abolished synapsin dimerization, without affecting the binding of synapsin to F‐actin, G‐actin or synaptic vesicles. These data indicate that: (i) synapsins and S100A1 can interact in the nerve terminals where they are coexpresssed; (ii) S100A1 is unable to bind to SV‐associated synapsin I and may function as a cytoplasmic store of monomeric synapsin I; and (iii) synapsin dimerization and interaction with S100A1 are mutually exclusive, suggesting an involvement of S100A1 in the Ca2+‐dependent regulation of synaptic vesicle trafficking.

The translocation of focal adhesion kinase in brain synaptosomes is regulated by phosphorylation and actin assembly.

Focal adhesion kinase (FAK) and the related proline‐rich tyrosine kinase 2 (PYK2) are non‐receptor protein tyrosine kinases that transduce extracellular signals through the activation of Src family kinases and are highly enriched in neurones. To further elucidate the regulation of FAK and PYK2 in nervous tissue, we investigated their distribution in brain subcellular fractions and analysed their translocation between membrane and cytosolic compartments. We have found that FAK and PYK2 are present in a small membrane‐associated pool and a larger cytosolic pool in various neuronal compartments including nerve terminals. In intact nerve terminals, inhibition of Src kinases inhibited the membrane association of FAK, but not of PYK2, whereas tyrosine phosphatase inhibition sharply increased the membrane association of both FAK and PYK2. Disruption of the actin cytoskeleton was followed by a decrease in the membrane‐associated pool of FAK, but not of PYK2. For both kinases, a significant correlation was found between autophosphorylation and membrane association. The data indicate that FAK and PYK2 are present in nerve terminals and that the membrane association of FAK is regulated by both phosphorylation and actin assembly, whereas that of PKY2 is primarily dependent on its phosphorylation state.

Subunit and region-specific decreases in nicotinic acetylcholine receptor mRNA in the aged rat brain

We have investigated possible changes in the mRNA levels for several alpha and beta subunits of the nicotinic acetylcholine receptor (nAChR) and the level of binding for nicotinic ligands in 7- to 32-month-old rats. Alpha4 and beta2, and to a lesser extent alpha6 and beta3, mRNA levels showed decreases between 20 and 30% at 29 months of age which in some areas reached 50% at 32 months of age. Alpha7 showed a small increase from 7 to 14 months and then a progressive decrease from 14 to 32 months down to the 7-month levels. 3H-epibatidine binding did not significantly change from 7 to 32 months of age in rat tel- and diencephalon. Binding in the substantia nigra was exceptional in that it showed a significant decrease starting from 23 months of age. 125I-alpha-bungarotoxin binding showed a pattern of change which roughly paralleled that of alpha7 mRNA. These findings show that an alteration in some steps of nAChR biosynthesis takes place during aging, which may be related to functional changes in nicotinic transmission.

Loss of high-affinity nicotinic receptors increases the vulnerability to excitotoxic lesion and decreases the positive effects of an enriched environment

Pharmacological activation of nicotinic acetylcholine receptors (nAChRs) exerts neuroprotective effects in cultured neurons and the intact animal. Much less is known about a physiological protective role of nAChRs. To understand whether endogenous activation of β2* nAChRs contributes to the maintenance of the functional and morphological integrity of neural tissue, adult β2‐/‐ mice were subjected to in vivo challenges that cause neurodegeneration and cognitive impairment (intrahippocampal injection of the excitotoxin quinolinic acid), or neuroprotection and cognitive potentiation (2‐month exposure to an enriched environment). The excitotoxic insult caused an increased deficit in the Morris water maze learning curve and increased loss of hippocampal pyramidal cells in β2 ‐/‐ mice. Exposure to an enriched environment improved performance in contextual and cued fear conditioning and object recognition tests in β2+/+, whereas the improvement was absent in β2‐/‐ mice. In addition, β2+/+, but not β2‐/‐, mice exposed to an enriched environment showed a significant hypertrophy of the CA1/3 regions. Thus, lack of β2* nAChRs increased susceptibility to an excitotoxic insult and diminished the positive effects of an enriched environment. These results may be relevant to understanding the pathophysiological consequences of the marked decrease in nAChRs that occurs in neurodegenerative diseases such as Alzheimers disease and Parkinsons disease.— Zanardi, A., Ferrari, R., Leo, G., Maskos, U., Changeux, J.‐P., Zoli, M. Loss of high affinity nicotinic receptors increases the vulnerability to excitotoxic lesion and decreases the positive effects of an enriched environment. FASEB J. 21, 4028–4037 (2007)

Neuron-Glia Cross Talk in Rat Striatum after Transient Forebrain Ischemia

Striatum is highly vulnerable to transient forebrain ischemia induced by the 4 vessel occlusion (4V0) method (Brierley 1976. Pulsinelli et al. 1982, Zini et al. 1990a). Massive degeneration and loss of Nissl-stained neurons occur within 24 hr from an ischemia of long duration (30 min) (Pulsinelli et al. 1982). Neuronal loss is mainly restricted to the lateral part of caudate-putamen (Pulsinelli et al. 1982, Zini et al. 1990a). Cellular alterations include loss of medium-size spiny projection neurons (Pulsinelli et al. 1982, Francis and Pulsinelli 1982), largely corresponding to dopaminoceptive neurons (Benfenati et al. 1989, Zoli et al. 1989), and increase in reactive astrocytes (Pulsinelli et al. 1982, Grimaldi et al. 1990) and microglia (Gehrmann et al. 1982). On the other hand, large cholinergie (Francis and Pulsinelli 1982) and medium-size aspiny somatostatin (SS)/neuropeptide Y (NPY)-containing interneurons are resistant to the ischemic insult (Pulsinelli et al. 1982, Grimaldi et al. 1990). In a few instances, such as in the case of SS and NPY immunoreactivity (IR), the initial loss is followed by full recovery within 7 (SS) or 40 (NPY) days post-ischemia (Grimaldi et al. 1990). However, it is not known whether some kind of recovery is present for the bulk of medium-size spiny projections neurons after the first days post-ischemia.