Gene expression in blood is associated with risperidone response in children with autism spectrum disorders.

Children with autism spectrum disorders (ASDs) often have severe behavioral problems. Not all children with these problems respond to atypical antipsychotic medications; therefore, we investigated whether peripheral blood gene expression before treatment with risperidone, an atypical antipsychotic, was associated with improvements in severe behavioral disturbances 8 weeks following risperidone treatment in 42 ASD subjects (age 112.7±51.2 months). Exon expression levels in blood before risperidone treatment were compared with pre-post risperidone change in Aberrant Behavior Checklist-Irritability (ABC-I) scores. Expression of exons within five genes was correlated with change in ABC-I scores across all risperidone-treated subjects: GBP6, RABL5, RNF213, NFKBID and RNF40 (α<0.001). RNF40 is located at 16p11.2, a region implicated in autism and schizophrenia. Thus, these genes expressed before treatment were associated with subsequent clinical response. Future studies will be needed to confirm these results and determine whether this expression profile is associated with risperidone response in other disorders, or alternative antipsychotic response within ASD.

Corticosteroid effects on blood gene expression in Duchenne muscular dystrophy.

Though Deflazacort and prednisone improve clinical endpoints in Duchenne muscular dystrophy (DMD) patients, Deflazacort produces fewer side effects. As mechanisms of improvement and side effect differences remain unknown, we evaluated effects of corticosteroid administration on gene expression in blood of DMD patients. Whole blood was obtained from 14 children and adolescents with DMD treated with corticosteroids (DMD-STEROID) and 20 DMD children and adolescents naïve to corticosteroids (DMD). The DMD-STEROID group was further subdivided into Deflazacort and prednisone groups. Affymetrix U133 Plus 2.0 expression microarrays were used to evaluate mRNA expression. Expression of 524 probes changed with corticosteroids, including genes in iron trafficking and the chondroitin sulfate biosynthesis pathway. Deflazacort compared with prednisone yielded 508 regulated probes, including many involved in adipose metabolism. These genes and pathways help explain mechanisms of efficacy and side effects of corticosteroids, and could provide new treatment targets for DMD and other neuromuscular disorders.

Expression of c-fos protein in brain: metabolic mapping at the cellular level.

The proto-oncogene c-fos is expressed in neurons in response to direct stimulation by growth factors and neurotransmitters. In order to determine whether the c-fos protein (Fos) and Fos-related proteins can be induced in response to polysynaptic activation, rat hindlimb motor/sensory cortex was stimulated electrically and Fos expression examined immunohistochemically. Three hours after the onset of stimulation, focal nuclear Fos staining was seen in motor and sensory thalamus, pontine nuclei, globus pallidus, and cerebellum. Moreover, 24-hour water deprivation resulted in Fos expression in paraventricular and supraoptic nuclei. Fos immunohistochemistry therefore provides a cellular method to label polysynaptically activated neurons and thereby map functional pathways.

Psychosis: pathological activation of limbic thalamocortical circuits by psychomimetics and schizophrenia?

Non-competitive NMDA receptor antagonists, such as phencyclidine, ketamine and MK801, produce psychosis in humans. These drugs also produce injury to cingulate-retrosplenial cortex in adult rodents that can be prevented by GABA-receptor agonists and antipsychotics such as haloperidol and clozapine. MK801 injections into anterior thalamus reproduce limbic cortex injury, and GABA-receptor agonist injections into anterior thalamus prevent injury produced by systemic MK801. Inhibition of NMDA receptors on GABAergic thalamic reticular nucleus neurons might activate thalamocortical 'injury' circuits in animals. Pathological activation of thalamocortical circuits might also mediate the psychosis produced by NMDA-receptor antagonists in humans, and might contribute to psychosis in schizophrenia.

Induction of fos-like immunoreactivity in hypothalamic corticotropin-releasing factor neurons after adrenalectomy in the rat.

To identify brain sites responding to the removal of corticosterone feedback by adrenalectomy (ADX), rat brains were processed for fos immunocytochemistry 1, 3, and 7 days after ADX, sham-ADX, or no surgery using a polyclonal antiserum to fos residues 132-154. Compared to SHAM, ADX rats exhibited strong fos-like immunoreactivity (FLI) only in the parvocellular neurons of the paraventricular hypothalamic nuclei (PVN) 1, 3, and 7 days after surgery. Replacement with a corticosterone pellet at the time of adrenalectomy (ADX + B) prevented this increase in PVN FLI in three of four rats at 1 day, all rats at 3 days, and two of seven rats 7 days after surgery; 100 micrograms/ml corticosterone in the drinking water for 2 days before perfusion reversed ADX-induced increases in PVN FLI in 7-day ADX rats. Providing 25 micrograms/ml corticosterone in the drinking water to ADX rats for 5 days after surgery did not prevent expression of PVN FLI, even though this dose has been shown to normalize morning basal ACTH levels in ADX rats. Virtually all parvocellular PVN neurons expressing FLI after ADX costained for CRF. Some parvocellular neurons also expressed both fos and vasopressin. In all rats, many brain regions expressed FLI that was not related to adrenalectomy. We conclude that the changes in neuronal FLI correlate with demonstrated changes in neuroendocrine activity after ADX; however, suppression of ADX-induced FLI may require higher replacement levels of corticosterone than inhibition of ADX-induced ACTH secretion.

Fluoxetine prevents PCP- and MK801-induced HSP70 expression in injured limbic cortical neurons of rats.

BACKGROUND: N-Methyl-D-aspartate (NMDA) receptor antagonists, including phencyclidine (PCP) and dizocilpine (MK801), cause schizophrenialike psychosis in humans, and produce vacuolated neurons in the cingulate and retrosplenial cortices of the rat brain. Since psychotically depressed patients and schizophrenic depressed patients may require treatment with selective serotonin reuptake inhibitors (SSRIs), it is of interest to examine the relationship between SSRIs and NMDA antagonist neurotoxicity. METHODS: The neurotoxicity of PCP and MK801 was assessed using heat shock protein (HSP70) immunocytochemistry and HSP70 Western blots because HSP70 is expressed in the injured, vacuolated neurons. Female rats were given fluoxetine (0, 5, 10, and 20 mg/kg IP) followed 1 hour later by MK801 (1 mg/kg IP) or PCP (50 mg/kg IP). RESULTS: Pretreatment with fluoxetine (20 mg/kg IP) 1 hour before MK801 prevented the induction of HSP70 by MK801 in the cingulate and retrosplenial cortices. Pretreatment with fluoxetine (10 or 20 mg/kg IP) 1 hour before PCP also prevented the HSP70 induction by PCP. CONCLUSIONS: Fluoxetine prevents the neurotoxicity of NMDA receptor antagonists in rat brain. This suggests the possibility that SSRIs could modulate psychosis, and may provide a model for examining the link between the hallucinogenic properties of PCP and lysergic acid diethylamide.

NMDA and AMPA/kainate glutamate receptors modulate dentate neurogenesis and CA3 synapsin-I in normal and ischemic hippocampus.

The effect of N-methyl-D-aspartate (NMDA) and 2-(aminomethyl)phenylacetic acid/kainate (AMPA/kainate) glutamate receptors on dentate cell proliferation and hippocampal synapsin-I induction was examined after global ischemia. Cell proliferation was assessed using BrdU labeling, and synaptic responses were assessed using synapsin-I expression. Systemic glutamate receptor antagonists (MK-801 and NBQX) increased BrdU-labeled cells in the dentate subgranular zone (SGZ) of control adult gerbils (30% to 90%, P < 0.05). After global ischemia (at 15 days after 10 minutes of ischemia), most CA1 pyramidal neurons died, whereas the numbers of BrdU-labeled cells in the SGZ increased dramatically (>1000%, P < 0.0001). Systemic injections of MK801 or NBQX, as well as intrahippocampal injections of either drug, when given at the time of ischemia completely blocked the birth of cells in the SGZ and the death of CA1 pyramidal neurons at 15 days after ischemia. Glutamate receptor antagonists had little effect on cell birth and death when administered 7 days after ischemia. The induction of synapsin-I protein in stratum moleculare of CA3 at 7 and 15 days after global ischemia was blocked by pretreatment with systemic or intrahippocampal MK-801 or NBQX. It is proposed that decreased dentate glutamate receptor activation--produced by glutamate receptor antagonists in normal animals and by chronic ischemic hippocampal injury--may trigger dentate neurogenesis and synaptogenesis. The synapsin-I induction in mossy fiber terminals most likely represents re-modeling of dentate granule cell neuron presynaptic elements in CA3 in response to the ischemia. The dentate neurogenesis and synaptogenesis that occur after ischemia may contribute to memory recovery after hippocampal injury caused by global ischemia.

Global ischemia induces immediate-early genes encoding zinc finger transcription factors.

Ischemia induces immediate-early genes (IEGs) in brain. Since prolonged expression of some IEGs may precede neuronal death, some researchers have suggested that these IEGs mediate neuronal death. We therefore examined the effect of 5 and 10 min of global ischemia on the expression of the IEGs NGFI-A, NGFI-B, NGFI-C, egr-2, egr-3, and Nurr1 in gerbil brain. All of the IEGs were induced after 30 min of reperfusion in the hippocampus. Most of them were induced in several other regions as well, including cortex, hypothalamus, thalamus, and amygdala. The acute IEG induction decreased in most brain areas by 2-6 h. However, at 24 h following 5 min of ischemia NGFI-A continued to be expressed in the CA1 region and dentate gyrus. In the dentate gyrus, NGFI-C continued to be expressed for 24 h and egr-3 for as long as 72 h. In other brain areas, all of the IEGs returned to control levels by 72 h except in CA1, where most messenger RNA (mRNA) levels were decreased; this decrease correlated with marked neuronal loss. The persistent expression of NGFI-A in CA1 neurons destined to die and the persistent expression of NGFI-A, NGFI-C, and egr-3 genes in dentate granule cell neurons that survive may indicate that some transcription factors modulate cell death whereas others support cell survival when expressed for prolonged periods. The protein products of several transcription factors, including c-fos, are known to downregulate their own expression. The persistent expression of NGFI-A in the CA1 neurons destined to die could therefore be due to ischemia-induced transcriptional activation caused by, e.g., increased intracellular calcium levels plus a lack of negative feedback caused by the blockade of the translation of NGFI-A mRNA into protein.

Focal increases of white matter glucose utilization produced by electrical stimulation of rat motor cortex.

The right motor cortex was electrically stimulated in adult, awake rats for 45 min. Local cerebral glucose utilization (LCGU) was measured in white matter pathways with the (14C)-2-deoxyglucose method. Stimulation increased LCGU in focal regions of the right internal capsule to 51.3 mumol/100 g/min, compared to 39.8 on the control left side. Stimulation also increased LCGU in the right, medial pontine pyramidal tract to 36.2 mumol/100 g/min, compared with 27.3 on the control left side. The data demonstrate that electrical stimulation of motor cortex neurons increases LCGU 30 to 40% in the efferent myelinated axons of those neurons.

Thiamine deficiency limits glucose utilization and glial proliferation in brain lesions of symptomatic rats.

The effects of thiamine (B1) deficiency on local CMRglu (LMCRglu) in the vestibular nuclei were studied with the 14C-2-deoxyglucose autoradiographic method in awake asymptomatic and symptomatic rats. Animals on the B1-deficient diet for 98 days developed symptoms of ataxia and opisthotonos. The results show that B1 deficiency produces: (1) bilateral vestibular nuclei lesions in symptomatic animals; (2) very low LCMRglu rates in these lesions; and (3) limitation of glial proliferation in the lesions. Giving B1 to B1-deficient symptomatic animals produced a cellular proliferation response consisting mostly of microglia in the lesioned areas of the vestibular nuclei and a high LCMRglu rate in the regions of microglial proliferation.

Sequential metabolic changes in rat brain following middle cerebral artery occlusion: a 2-deoxyglucose study.

The distribution and time course of altered cerebral metabolism following permanent focal ischemia was studied in rat using the 2-deoxyglucose (2DG) technique. Increased 2DG uptake preceded decreased 2DG uptake and infarction in the caudate putamen and cortex. Decreased 2DG uptake without infarction was observed for 72 h in thalamus and for 24 h in hippocampus (areas remote from the ischemic zones). This study supports the concept of cell excitation as a pathophysiologic process in permanent focal ischemia. The time course of increased metabolism may demarcate the time window of opportunity for the previously demonstrated attenuation of stroke size with inhibition of cell excitation by pharmacologic blockade of excitatory amino acid neurotransmission.

Heat shock protein hsp72 induction in cortical and striatal astrocytes and neurons following infarction.

Transient global and transient focal ischemia induced the 72 kDa heat shock protein (hsp72) in neurons in cortex, striatum, and other regions known to be injured during transient ischemia. A novel finding was the induction of hsp72 in islands (cylinders in three dimensions) of cells composed of astrocytes around the perimeter and neurons in the interior. Since histology showed pale staining in these regions, it is proposed that these islands represent areas of focal infarction in the distribution of small cortical and lenticulostriate arteries. Although the factors responsible for hsp72 induction during ischemia and infarction are unknown, these results suggest differences in mechanisms of hsp72 induction in astrocytes compared to neurons.

DNA fragmentation and HSP70 protein induction in hippocampus and cortex occurs in separate neurons following permanent middle cerebral artery occlusions.

DNA nick end-labeling (TUNEL) and heat shock protein (HSP)70 immunocytochemistry were performed on the same brain sections 1 (n = 6), 3 (n = 12), and 7 (n = 7) days following permanent middle cerebral artery (MCA) occlusions produced in adult rats using the endovascular carotid suture method. In the cortex at 1 and 3 days following MCA occlusions, HSP70 immunoreactive neurons were located outside areas of infarction and showed little evidence of DNA fragmentation. HSP70-stained cortical neurons were intermingled with TUNEL cells near the infarct, but extended for greater distances away from the infarct. DNA fragmentation occurred in CA1 hippocampal neurons in 39% of the animals at 1 and 3 days following ipsilateral MCA occlusion. Bilateral DNA fragmentation occurred in CA1 neurons in one subject. HSP70 protein was expressed in CA1 hippocampal neurons in nine of 18 (50%) animals at 1 and 3 days following MCA occlusions, including all animals that exhibited hippocampal DNA fragmentation. Three animals had bilateral expression of HSP70 in CA1 neurons. Cells that stained for either HSP70 protein or DNA fragmentation existed in close proximity to one another. Approximately 5-7% of HSP70-stained cells were TUNEL stained and 3% of TUNEL-positive cells also stained for HSP70. There was no HSP70 staining or DNA fragmentation in the brains of sham-operated controls (n = 4) or in the brains of animals 7 days following MCA occlusions. These data suggest that ischemic cells capable of translating HSP70 protein generally do not undergo DNA fragmentation. These data support the concept that most HSP70 protein-containing neurons in the cortical "penumbra" and hippocampus survive ischemic injury and are "reversibly injured." It is shown that CA1 hippocampal pyramidal neurons die or are reversibly injured in approximately 50% of animals following permanent MCA occlusions. Although the mechanism of this hippocampal injury is unknown, it could relate to transynaptic activation of N-methyl-D-aspartate (NMDA) receptors that mediate induction of early genes in hippocampus.

Microglia/macrophages proliferate in striatum and neocortex but not in hippocampus after brief global ischemia that produces ischemic tolerance in gerbil brain.

The current study determined whether short durations of ischemia that produce ischemia-induced tolerance stimulate glial proliferation in brain. Adult male gerbils were injected with BrdU (50 mg/kg) and dividing cells were detected using immunocytochemistry after sham operations, 2.5 or 5 minutes of global ischemia, or ischemia-induced tolerance. The 2.5-minute ischemia and the ischemia-induced tolerance did not kill hippocampal CA1 pyramidal neurons, whereas the 5-minute ischemia did kill the neurons. At 4 days after 2.5-minute global ischemia, when cell proliferation was maximal, BrdU-labeled cells increased in striatum and in neocortex, but not in hippocampus. The majority of the BrdU-labeled cells were double-labeled with isolectin B4, showing that these dividing cells were primarily microglia or macrophages, or both. Similarly, BrdU-labeled microglia/macrophages were found in striatum and neocortex but not in hippocampus of most animals 4 days after ischemia-induced tolerance (2.5 minutes of global ischemia followed 3 days later by 5 minutes of global ischemia). No detectable neuronal cell death existed in striatal and cortical regions where the microglia/macrophage proliferation occurred. Though 3 of 7 animals subjected to 2.5 minutes of ischemia showed decreased myelin-associated glycoprotein (MAG) immunostaining and increased numbers of adenomatous polyposis coli-stained oligodendrocytes in lateral striatum, this did not explain the microglia/macrophage proliferation. Data show that ischemia-induced tolerance in the gerbil is associated with proliferation of microglia/macrophages in striatum and cortex but not in hippocampus. Because there is no apparent neuronal death, it is postulated that the microglia/macrophage proliferation occurs in response to an unknown nonlethal injury to neurons or glia and may be beneficial.

Limiting ischemic injury by inhibition of excitatory amino acid release.

Excitatory amino acids (EAAs) are important mediators of ischemic injury in stroke. N-Methyl-D-aspartate (NMDA) receptor antagonists have been shown to be very effective neuroprotective agents in animal models of stroke, but may have unacceptable toxicity for human use. An alternative approach is to inhibit the release of EAAs during stroke. BW1003C87 [5-(2,3,5-trichlorophenyl)-2,4-diaminopyrimidine], a drug that inhibits veratrine-induced release of the EAA glutamate in vitro, was tested in a rat model of proximal middle cerebral artery (MCA) occlusion. BW1003C87 significantly decreased ischemia-induced glutamate release in brain when given either 5 min before or 15 min following permanent MCA occlusion. Pretreated and posttreated rats had smaller infarct volumes and preserved glucose metabolism in the ischemic cortex at 24 h after MCA occlusion. BW1003C87 did not induce heat shock protein in the cingulate or retrosplenial cortex, suggesting that it does not injure neurons in these regions as do NMDA antagonists. These results demonstrate that drugs that inhibit glutamate release in ischemia may be nontoxic and show promise for the treatment of stroke.

Multiple molecular penumbras after focal cerebral ischemia.

Though the ischemic penumbra has been classically described on the basis of blood flow and physiologic parameters, a variety of ischemic penumbras can be described in molecular terms. Apoptosis-related genes induced after focal ischemia may contribute to cell death in the core and the selective cell death adjacent to an infarct. The HSP70 heat shock protein is induced in glia at the edges of an infarct and in neurons often at some distance from the infarct. HSP70 proteins are induced in cells in response to denatured proteins that occur as a result of temporary energy failure. Hypoxia-inducible factor (HIF) is also induced after focal ischemia in regions that can extend beyond the HSP70 induction. The region of HIF induction is proposed to represent the areas of decreased cerebral blood flow and decreased oxygen delivery. Immediate early genes are induced in cortex, hippocampus, thalamus, and other brain regions. These distant changes in gene expression occur because of ischemia-induced spreading depression or depolarization and could contribute to plastic changes in brain after stroke.

Induction of 70-kDa heat shock protein and hsp70 mRNA following transient focal cerebral ischemia in the rat.

Induction of the 70-kDa heat shock protein (HSP70) was demonstrated immunocytochemically in adult rats 4 h to 7 days following temporary middle cerebral artery (MCA) occlusions lasting 30, 60, or 90 min. Maximal HSP70 induction occurred approximately 24 h following ischemia. Thirty minutes of ischemia induced HSP70 in neurons throughout the cortex in the MCA distribution, whereas 90 min of ischemia induced HSP70 in neurons in the penumbra. HSP70 protein was induced in endothelial cells in infarcted neocortex following 60-90 min of MCA occlusion, and HSP70 was induced in endothelial cells in infarcted regions of lateral striatum following 30-90 min of MCA occlusion. hsp70 mRNA was induced in the MCA distribution in cortex and to a lesser extent in striatum at 2 h to 3 days following 60 min of ischemia. It is proposed that brief ischemia induces hsp70 mRNA and HSP70 protein in the cells most vulnerable to ischemia--the neurons. HSP70 protein is not induced in most neurons and glia following 60-90 min of ischemia in areas destined to infarct, whereas it is induced in vascular endothelial cells.

Induction of c-fos, junB, c-jun, and hsp70 mRNA in cortex, thalamus, basal ganglia, and hippocampus following middle cerebral artery occlusion.

Middle cerebral artery (MCA) occlusion in halothane-anesthetized rats induced c-fos, junB, and c-jun immediate early gene mRNAs and hsp70 heat shock gene mRNA in brain. In situ hybridization studies showed that c-fos and junB were induced throughout all of the cortex at 1 and 4 h following MCA occlusion. hsp70 was induced in the core and margins of the MCA ischemia. By 24 h, there was little expression of c-fos, junB, c-jun, and hsp70 in the core of the MCA infarct; there was modest induction of hsp70 at the margins of the infarct; and there was diffuse induction of c-fos, junB, and c-jun in all of the cortex outside the infarct. MCA occlusion also induced these genes in subcortical structures. c-fos, junB, and hsp70 were induced in ipsilateral medial striatum, most of thalamus including medial and lateral geniculate nuclei, substantia nigra, and hippocampus. Most of these structures, except for the striatum, are not supplied by the MCA. These data show that changes in gene expression can occur in regions remote from an infarction.

Prolonged expression of hsp70 mRNA following transient focal cerebral ischemia in transgenic mice overexpressing CuZn-superoxide dismutase.

The distribution of heat shock protein hsp70 mRNA after 10 min of middle cerebral artery (MCA) occlusion was investigated through in situ hybridization in transgenic (Tg) mice overexpressing CuZn-superoxide dismutase (CuZn-SOD) and in control nontransgenic (nTg) littermates. In the ischemic cortex of nTg mice, hsp70 mRNA was detected 1 h after reperfusion and was observed for up to 6 h. In Tg mice, however, it was still detectable within the cortex even at 24 h. In the caudate putamen, hsp70 mRNA appeared at 1 h and was present for up to 6 h in both nTg and Tg mice. Although hsp70 mRNA was detected in the thalamus only at 1 h in nTg mice, it was observed for up to 6 h in Tg mice. Similarly, hsp70 mRNA was detected in the hippocampus of nTg mice only at 1 h, whereas it was detected in Tg mice at 1 h and continued up to 24 h, with high intensity in the CA1 subfield. Despite the significant amounts of hsp70 mRNA in both Tg and nTg mice following ischemia, there was no observable neuronal necrosis (as assessed using hematoxylin and eosin staining) for up to 7 days. Cortical cerebral blood flow (CBF), measured by laser-Doppler flowmetry, did not differ between nTg and Tg mice during ischemia and reperfusion, despite exhibiting hyperemia following hypoperfusion. These results suggest that oxidative stress affects the expression of hsp70 following temporary focal ischemia. An alteration in oxidation stress, which resulted from reduced levels of superoxide radicals in the presence of the CuZn-SOD transgenes, may permit the prolonged expression of hsp70.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA fragmentation and Prolonged expression of c-fos, c-jun, and hsp70 in kainic acid-induced neuronal cell death in transgenic mice overexpressing human CuZn-superoxide dismutase.

Kainic acid (KA) neurotoxicity was examined in transgenic (Tg) mice overexpressing human CuZn-superoxide dismutase (SOD-1). The doses of KA required to produce seizures, the severity of the seizures, and the regions damaged were similar in SOD-1 Tg and non-transgenic wild-type mice. Intraperitoneal KA injection induced seizure-related neuronal damage in the CA3 and CA1 regions of the hippocampus and in other regions of the brain in both SOD-1 Tg and wild-type mice. These damaged neurons were labeled with the terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL) technique up to 72 h, although no significant difference in the number of TUNEL-positive neurons was observed between SOD-1 Tg and wild-type mice. In situ hybridization showed that c-fos, c-jun, and hsp70 genes were expressed in the hippocampus, cortex, and other regions of the brain after KA treatment. The expression of these genes was maximal 1 to 4 h following KA treatment but persisted longer in the hippocampus and other regions in SOD-1 Tg compared with wild-type mice; however, cell death in the hippocampus, assessed using cresyl violet staining, was similar in SOD-1 Tg and wild-type mice. The data show that superoxide radicals modulate both immediate early gene and heat shock gene expression after KA-induced seizures. The prolonged expression of c-fos, c-jun, and hsp70 in SOD-1 Tg compared with wild-type mice may indicate that hippocampal neurons survive longer in SOD-1 Tg than in wild-type animals; however, cell death as well as the seizure threshold, seizure severity and the pattern of regional vulnerability were not affected substantially by increased levels of SOD in the brain.