SLC25A12 expression is associated with neurite outgrowth and is upregulated in the prefrontal cortex of autistic subjects.

Autism is a neurodevelopmental disorder with a strong genetic component, probably involving several genes. Genome screens have provided evidence of linkage to chromosome 2q31-q33, which includes the SLC25A12 gene. Association between autism and single-nucleotide polymorphisms in SLC25A12 has been reported in various studies. SLC25A12 encodes the mitochondrial aspartate/glutamate carrier functionally important in neurons with high-metabolic activity. Neuropathological findings and functional abnormalities in autism have been reported for Brodmann's area (BA) 46 and the cerebellum. We found that SLC25A12 was expressed more strongly in the post-mortem brain tissues of autistic subjects than in those of controls, in the BA46 prefrontal cortex but not in cerebellar granule cells. SLC25A12 expression was not modified in brain subregions of bipolar and schizophrenic patients. SLC25A12 was expressed in developing human neuronal tissues, including neocortical regions containing excitatory neurons and neocortical progenitors and the ganglionic eminences that generate neocortical inhibitory interneurons. At mid-gestation, when gyri and sulci start to develop, SLC25A12 molecular gradients were identified in the lateral prefrontal and ventral temporal cortex. These fetal structures generate regions with abnormal activity in autism, including the dorsolateral prefrontal cortex (BA46), the pars opercularis of the inferior frontal cortex and the fusiform gyrus. SLC25A12 overexpression or silencing in mouse embryonic cortical neurons also modified dendrite length and the mobility of dendritic mitochondria. Our findings suggest that SLC25A12 overexpression may be involved in the pathophysiology of autism, modifying neuronal networks in specific subregions, such as the dorsolateral prefrontal cortex and fusiform gyrus, at both pre- and postnatal stages.

Activity-dependent regulation of genes implicated in X-linked non-specific mental retardation.

X-linked forms of non-specific mental retardation are complex disorders, for which mutations in several genes have recently been identified. These include OPHN1, GDI1, PAK3, IL1RAPL, TM4SF2, FMR2 and RSK2. To investigate the mechanisms through which alterations of these gene products could result in cognitive impairment, we analyzed their expression using quantitative PCR technique in two in vitro models of activity-dependent gene regulation: kainate-induced seizures and long-term synaptic potentiation (LTP). We found that the level of expression of four genes, PAK3, IL1RAPL, RSK2 and TM4SF2, was significantly up-regulated following kainate treatment. Furthermore we observed a significant increase in mRNA levels of PAK3 and IL1RAPL following LTP induction. These results suggest a possible role for these four genes in activity-dependent brain plasticity.

MASH-1/RET pathway involvement in development of brain stem control of respiratory frequency in newborn mice.

Respiratory abnormalities have been described in MASH-1 (mammalian achaete-scute homologous gene) and c-RET ("rearranged during transfection") mutant newborn mice. However, the neural mechanisms underlying these abnormalities have not been studied. We tested the hypothesis that the MASH-1 mutation may impair c-RET expression in brain stem neurons involved in the control of breathing. To do this, we analyzed brain stem c-RET expression and respiratory phenotype in MASH-1 +/+ wild-type, MASH-1 +/- heterozygous, and MASH-1 -/- knock-out newborn mice during the first 2 h of life. In MASH-1 -/- newborns, c-RET gene expression was absent in the noradrenergic nuclei (A2, A5, A6, A7) that contribute to modulate respiratory frequency and in scattered cells of the rostral ventrolateral medulla. The c-RET transcript levels measured by quantitative RT-PCR were lower in MASH-1 -/- and MASH-1 +/- than in MASH-1 +/+ brain stems (P = 0.001 and P = 0.003, respectively). Breath durations were shorter in MASH-1 -/- and MASH-1 +/- than in MASH-1 +/+ mice (P = 0.022) and were weakly correlated with c-RET transcript levels (P = 0.032). Taken together, these results provide evidence that MASH-1 is upstream of c-RET in noradrenergic brain stem neurons important for respiratory rhythm modulation.

Platelet arachidonate cascade of migraineurs in the interictal phase.

Morphological and functional alterations of platelets in migraineurs may be linked to the development of migraine. We examined the eicosanoid synthesis of platelets of untreated female migraineurs in a headache-free period and compared it to that of age- and blood group-matched healthy female volunteers. In the platelets of headache-free migraineurs significantly less amounts of anti-aggregatory prostaglandin D2 and prostacyclin, as well as of 12-L-hydroxy-5,8,10-heptadecatrienoic acid (a potent endogenous inducer of endothelial prostacyclin production) were produced, while the synthesis of platelet aggregatory thromboxane did not differ when compared to that of healthy women. These results suggest that the platelet eicosanoids of migraineurs in the headache-free period might promote the development of cellular, vascular and neurological events inducing headache.

Parvalbumin- and calbindin-containing neurons express c-fos protein in primary and secondary (mirror) epileptic foci of the rat neocortex.

The present experiments aimed at the description and further immunocytochemical characterization of activated neocortical neurons expressing the c-fos gene. Focal seizures were induced by the topical application of isotonic, isohydric 4-aminopyridine solution to the frontal neocortex of adult anesthetized Wistar rats. The EEG of both hemispheres was recorded from the surface of the skull. The animals were perfused with fixative, coronal plane vibratome sections were cut and stained with cocktails containing polyclonal c-fos and monoclonal calbindin or parvalbumin antibodies. The polyclonal c-fos antibody was tested with Western blotting and the diffusion of 4-aminopyridine investigated with autoradiography of [3H]4-aminopyridine. The c-fos protein was detected in every layer of the neocortex (primary focus) and in some allocortical areas of the treated hemisphere. Scattered immunostained nuclei were observed in layers II, III, IV and VI of the contralateral neocortex (mirror focus). Several parvalbumin- and calbindin-positive neurons contained the c-fos protein in both foci. The medium-sized non-pyramidal parvalbumin neurons were found in layers II-IV and VI of the neocortex and in stratum multiforme of the prepiriform cortex. The c-fos protein was colocalized with calbindin mainly in layers II and III in small and medium-sized non-pyramidal neurons. The results prove that focal epileptiform activity of the neocortex activates diverse inhibitory neuronal populations. As concluded, the inhibitory control is probably more effective in the contralateral hemisphere (mirror focus) than on the side of 4-APY treatment (primary focus).

Nitric oxide synthase inhibitor facilitates focal seizures induced by aminopyridine in rat.

The effects of N-nitro-L-arginine (NA), a nitric oxide (NO) synthase inhibitor, were investigated on the focal ictal-like seizure induced by 3-aminopyridine in rat neocortex in vivo. Intraperitoneal and intracerebroventricular (i.c.v.) injections of NA markedly facilitated propagation of epileptiform events. In addition, NA injected i.c.v. increased the number/hour of individual ictal periods while decreasing their duration. In the presence of NA and an N-methyl-D-aspartate (NMDA) receptor antagonist D(-)2-amino-5-phosphonovaleric acid (APV) the number of ictal periods increased while their duration synergically decreased. APV by itself did not change the number of ictal episodes but decreased their duration. Our results suggest that NO inhibits the induction and propagation of seizure activity. We cannot distinguish the proportion of neuronal and/or vascular NO involved in our experimental conditions, but these effects seem to be independent of the NMDA receptors.

Anticonvulsive effect of urethane on aminopyridine-induced epileptiform activity.

The effects of urethane anaesthesia on the development and spread of ictal-like epileptiform activity induced by 3-aminopyridine, was investigated on the somatosensory cortex of rats. Under urethane anaesthesia in most animals only one abortive ictal period appeared with dose-dependent latency. Urethane completely abolished high frequency afterdischarges (5-10 Hz) and prevented the development of recurrent seizures in both primary and mirror foci. Urethane also eliminated ongoing epileptiform activity induced previously by 3-aminopyridine under nembutal anaesthesia. The antiepileptic effects of urethane on ictal-like activity may be the consequence of a presynaptic action through reduction of glutamate-mediated excitation together with a depressant effect on responsiveness of cortical neurones. The afterdischarges of high frequency could be essential to development and stabilization of repetitive tonic events in the neocortex.

Cellular mechanisms of neocortical secondary epileptogenesis.

Firing activity, membrane parameters and postsynaptic responses were studied by recording intracellularly from different types of neurons during the development of a secondary neocortical epileptiform focus (mirror focus, Mf) contralateral to the site of an aminopyridine-induced focus (primary focus, Pf) in anesthetized rats. Three different stages in the development of secondary epileptogenesis were observed. (i) in the Pf stage epileptiform discharges appeared only in the ECoG recorded from the Pf, but neurons in the Mf showed reduced firing activity; (ii) in the Pf + Mf stage, synchronous ictal epileptiform activity occurred in the Pf and Mf. Changes in the balance between inhibition and excitation, appearance of novel electrophysiological phenomena (e.g. antidromic like action potentials, PDS (paroxysmal depolarization shift) potentials, rebound bursts), enhanced intrinsic bursting, and a transition from regular spiking to bursting were observed at the cellular level; (iii) in the Pf/Mf stage in 10% of the animals, the surface epileptic discharges were in synchrony with cellular activity in the Mf but were temporally independent of Pf activity, suggesting that during secondary epileptogenesis the Pf and the Mf can have underlying epileptogenic mechanisms which are different in origin.

Stimulation of substantia nigra pars reticulata suppresses neocortical seizures.

The effects of unilateral electrical stimulation of the substantia nigra pars reticulata (SNpr) on the electrocorticographic (ECoG) manifestations of seizures were studied in anesthetized rats. Epileptiform activity was provoked in the primary focus (Pf) by unilateral, local application of 3-aminopyridine which induced secondary focus in the homologous area of the contralateral cortex (mirror focus, Mf). The position of the electrode for stimulation of SNpr was contralateral to the Pf. The results showed a strong suppressive nigral effect on cortical seizure propagation and on seizure susceptibility in both hemispheres. Stimulation of the SNpr prevented the manifestation of sustained epileptiform events, decreased the rate of seizure appearance in the Mf, delayed the onset of paroxysmal activity and markedly reduced the amplitude and duration of ictal episodes at both foci. Seizure potentials of lower frequencies disappeared, while the relative proportion of those of higher frequency increased in SNpr-stimulated animals. SNpr stimulation had no significant effect on fully developed seizures. Our observations support the idea that SNpr might be involved in the control of cortical seizure susceptibility, regulating other structures which are possibly involved in the generation and propagation of seizure.

Study of electrical activity in the area of the hypothalamic ventromedial nucleus and the lateral hypothalamus of rats fed on a high protein and high fat diet.

The authors studied bioelectric potentials in the area of the hypothalamic ventromedial nucleus and the lateral hypothalamus of rats fed on a standard, a high protein and a high fat diet. On the first 3--6 days after changing from the standard to the high fat and high protein diets, a decrease in the amplitude of electrical activity was recorded in both the areas in question. It was also found that the frequency of electrical activity in the hypothalamic ventromedial nucleus or the lateral hypothalamus rose, after 3 days administration of the high fat or the high protein diet, in correlation to the type of diet, and that, in the frequency spectrum, a change occurred in the proportion of basic frequency in relation to superimposed frequencies distorting it. It was further found that there was a permanent difference between the amplitude of electrical activity in the lateral hypothalamus and the hypothalamic ventromedial nucleus.