The Absence of Caspase-8 in the Dopaminergic System Leads to Mild Autism-like Behavior.

In the last decade, new non-apoptotic roles have been ascribed to apoptotic caspases. This family of proteins plays an important role in the sculpting of the brain in the early stages of development by eliminating excessive and nonfunctional synapses and extra cells. Consequently, impairments in this process can underlie many neurological and mental illnesses. This view is particularly relevant to dopamine because it plays a pleiotropic role in motor control, motivation, and reward processing. In this study, we analyze the effects of the elimination of caspase-8 (CASP8) on the development of catecholaminergic neurons using neurochemical, ultrastructural, and behavioral tests. To do this, we selectively delete the CASP8 gene in cells that express tyrosine hydroxylase with the help of recombination through the Cre-loxP system. Our results show that the number of dopaminergic neurons increases in the substantia nigra. In the striatum, the basal extracellular level of dopamine and potassium-evoked dopamine release decreased significantly in mice lacking CASP8, clearly showing the low dopamine functioning in tissues innervated by this neurotransmitter. This view is supported by electron microscopy analysis of striatal synapses. Interestingly, behavioral analysis demonstrates that mice lacking CASP8 show changes reminiscent of autism spectrum disorders (ASD). Our research reactivates the possible role of dopamine transmission in the pathogenesis of ASD and provides a mild model of autism.

Peripheral inflammation increases the damage in animal models of nigrostriatal dopaminergic neurodegeneration: possible implication in Parkinson's disease incidence.

Inflammatory processes described in Parkinson's disease (PD) and its animal models appear to be important in the progression of the pathogenesis, or even a triggering factor. Here we review that peripheral inflammation enhances the degeneration of the nigrostriatal dopaminergic system induced by different insults; different peripheral inflammations have been used, such as IL-1ß and the ulcerative colitis model, as well as insults to the dopaminergic system such as 6-hydroxydopamine or lipopolysaccharide. In all cases, an increased loss of dopaminergic neurons was described; inflammation in the substantia nigra increased, displaying a great activation of microglia along with an increase in the production of cytokines such as IL-1ß and TNF-α. Increased permeability or disruption of the BBB, with overexpression of the ICAM-1 adhesion molecule and infiltration of circulating monocytes into the substantia nigra, is also involved, since the depletion of circulating monocytes prevents the effects of peripheral inflammation. Data are reviewed in relation to epidemiological studies of PD.

Inflammatory Animal Model for Parkinson's Disease: The Intranigral Injection of LPS Induced the Inflammatory Process along with the Selective Degeneration of Nigrostriatal Dopaminergic Neurons.

We have developed an animal model of degeneration of the nigrostriatal dopaminergic neurons, the neuronal system involved in Parkinson's disease (PD). The implication of neuroinflammation on this disease was originally established in 1988, when the presence of activated microglia in the substantia nigra (SN) of parkinsonians was reported by McGeer et al. Neuroinflammation could be involved in the progression of the disease or even has more direct implications. We injected 2 µg of the potent proinflammatory compound lipopolysaccharide (LPS) in different areas of the CNS, finding that SN displayed the highest inflammatory response and that dopaminergic (body) neurons showed a special and specific sensitivity to this process with the induction of selective dopaminergic degeneration. Neurodegeneration is induced by inflammation since it is prevented by anti-inflammatory compounds. The special sensitivity of dopaminergic neurons seems to be related to the endogenous dopaminergic content, since it is overcome by dopamine depletion. Compounds that activate microglia or induce inflammation have similar effects to LPS. This model suggest that inflammation is an important component of the degeneration of the nigrostriatal dopaminergic system, probably also in PD. Anti-inflammatory treatments could be useful to prevent or slow down the rate of dopaminergic degeneration in this disease.

Stress is critical for LPS-induced activation of microglia and damage in the rat hippocampus.

The hippocampus is insensitive to strong inflammatory stimulus under normal conditions and one of the most severely affected areas in Alzheimer's disease. We have analyzed the effect of chronic stress for 9 days in the hippocampus unilaterally injected with LPS. In non-stressed rats, LPS injection failed to activate microglia although a subset of degenerating cells in the CA1 area was evident. This effect was not accompanied by loss of Neu-N positive neurons in the CA1 area. In stressed rats, LPS injection had a dramatic effect in activating microglia along with astrogliosis and BDNF mRNA induction. NeuN immunostaining demonstrated a loss of about 50% of CA1 pyramidal neurons under these conditions. Fluoro jade B histochemistry demonstrated the presence of degenerating cells in most of CA1 area. Mechanistically, combination of chronic stress and LPS resulted in prominent activation of MAPKs including JNK, p38 and ERK clearly different from LPS injection in controls. Further, LPS+stress induced a dramatic decrease in phosphorylated levels of both Akt and CREB, which fully supports a consistent deleterious state in the hippocampal system under these conditions. Treatment with RU486, a potent inhibitor of glucocorticoid receptor activation, significantly protected animals against the deleterious effects observed in LPS-stressed animals.

[The prevalence of infection by the hepatitis B, C and human immunodeficiency viruses in drug users]. / Prevalencia de la infección por los virus de la hepatitis B, C e inmunodeficiencia humana en usuarios de drogas.

OBJECTIVE: Estimate the prevalence of the hepatitis B (HBV) infection, hepatitis C (HCV) and human immunodeficiency virus (HIV) and its coexistence in intravenous drug users, in order to start afterwards a vaccination and sanitary training programmes. PATIENTS AND METHODS: Intravenous drug users attended in a health centre and in the drugs addition deshabitation centre of reference located in a marginal urban quarter. Patients were detected from the health centre. During one year (June 1995-1996) facts were collected. The age, sex, consumption, type, administration mechanism and also the described serologies were analysed. It has been carried out descriptive statistics and applied the chi-square [correction of square-ji] test. RESULTS: A study of 355 patients, 295 (83.1%) males and 60 (16.9%) females was carried out. The average age was 28.6 years (SD = 6.5). All serologies in 113 (31.8%) were available. The positive serologies for HIV, 64.6% for HBV and 64.4% had 71.1% for HCV. The three of them coexisted in a 35.4% between HIV, 39.1% of them were VHB and 88% VHC. 49.1% were VHB and VHC. The infection from any of the three virus was related with intravenous administration mechanism, but not with sex or drug type. CONCLUSION: The infection caused from the virus above mentioned is frequent in drug users. A not negligible percentage of patients could benefit from the hepatitis B vaccine administration (67.6%) or other preventive measures.

Differences in the stimulation of the phosphoinositide cycle by amine neurotransmitters in cultured rat forebrain neurones and astrocytes.

In this study, we compared the stimulation by carbachol (CCh), noradrenaline (NA), and histamine (HA) of phosphoinositide hydrolysis in rat forebrain neuronal and glial cultures. When Ca2+ was omitted from the stimulation buffer (low microM extracellular Ca2+), amine-induced [3H]inositol phosphate accumulation was reduced to a higher extent in astrocytes (70-80% for CCh and NA and 100% for HA) than in neurones (around 50-60% for all the amines). Furthermore, guanosine 5'-[gamma-thio]trisphosphate (GTP[S]) stimulation of phosphoinositidase C (PIC) in membranes was 5-fold higher in neurones than in astrocytes. These results indicate differences in the mechanism of PIC stimulation in the two cell types. After 30 min stimulation in the presence of 10 mM Li+, a higher accumulation of [3H]inositol 4-monophosphate and [3H]inositol 1,4-bisphosphate than of [3H]inositol 1/3-monophosphate occurred for all agonists in neurones, whereas the opposite was observed in astrocytes. Moreover, in these cells stimulation for 5 min in the absence of Li+ produced a 2-3-fold accumulation of all metabolites of the 3-kinase pathway of inositol-1,4,5-trisphosphate metabolism but not of those of the 5-phosphatase pathway. Thus, regardless of the amine receptor stimulated, the 3-kinase route appeared to prevail in astrocytes and the 5-phosphatase pathway in neurones. The histamine response in neurones differed from that of the other agonists in that it rapidly declined. Taken together these results indicate that the heterogeneity in amine stimulation of the phosphoinositide cycle previously observed in brain slices could arise to a great extent from the cellular diversity of this preparation and be related to the differential contribution of the amine receptors located in neurones and astrocytes.

Cyclic GMP inhibition of stimulated phosphoinositide hydrolysis in neuronal cultures.

We used a radioreceptor binding assay to analyse the effect of dibutyryl cyclic GMP (dbcGMP) and the nitric oxide (NO) donor sodium nitroprusside (SNP) on inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) mass accumulation in response to the muscarinic receptor agonist carbachol and K(+)-induced depolarization in neuronal primary cultures. In forebrain neurones carbachol induced a transient rise in Ins(1,4,5)P3 with a maximal increase of about 2.5-fold at 5 s. At this time 30 mM K+ induced a somewhat lower stimulation. Both carbachol and high K+ stimulations were significantly inhibited by dbcGMP and by SNP. The effect of SNP was blocked by haemoglobin. dbcGMP inhibition of the carbachol effect was also observed in cerebellar granule cell cultures. These results show for the first time that the NO/cGMP generating system can modulate phosphoinositide turnover in CNS neurones in a manner similar to that previously reported to occur in peripheral neurones and non-neuronal tissues.

Effect of zinc, copper and glucocorticoids on metallothionein levels of cultured neurons and astrocytes from rat brain.

The knowledge of brain metallothionein (MT) regulation and especially of MT presence in specific cell types is scarce. Therefore, the effect of several well-known MT inducers, measured by radioimmunoassays using antibodies that cross-react with MT-I and MT-II or specific for MT-I and which do not cross-react with human growth inhibitory factor (GIF or MT-III), has been studied in primary cultures of neurons or astrocytes obtained from rat cerebrum. MT-I levels in glial cells were about ten times higher than those in neuronal cells (538 +/- 194 vs. 49 +/- 16 pg MT-I/micrograms protein, mean +/- S.D. from three separate cell preparations). Increasing the concentration of Zn in the bovine serum albumin (BSA)-containing culture medium up to 50 microM significantly increased MT-I levels by up to 3.5-fold in neurons and 2.5-fold in astrocytes. In contrast, Cu up to 50 microM increased MT-I levels in a saturable manner in both neurons (up to 5-fold) and astrocytes (up to 1.5-fold), the maximum effect occurring at 5 microM Cu. In general, the combination of Zn and Cu further increased MT-I levels. The effect of the metals on MT-I appeared to reflect metal uptake, since MT-I induction was less marked when the BSA concentration in the medium was increased from 2 to 10 mg/ml. Dexamethasone increased MT-I levels in both neurons and astrocytes in vitro in a concentration-dependent manner. Endotoxin, IL-1 and IL-6 did not have a significant effect on glial MT levels at the concentrations studied. The administration of dexamethasone to rats increased MT-I levels in non-frontal cortex, cerebellum, pons+medulla, midbrain and hippocampus, but not in hypothalamus, frontal cortex and striatum. Endotoxin increased liver but not brain MT-I levels. Immunocytochemical studies in adult rat brain preparations with a polyclonal antibody that cross-reacts with MT-I and MT-II indicated that immunostaining was always nuclear in glial cells, whereas in neurons it was nuclear in the cerebral cortex, hippocampus and the granular layer of the cerebellum, and nuclear plus cytoplasmic in Purkinje cells in the cerebellum, hypothalamic nuclei and gigantocellular reticular nucleus in the brain stem. Meninges, choroidal plexus, ependymal and endothelial cells were also MT-immunoreactive.