J. N. Armstrong

The application of antisense oligonucleotide technology to the brain: some pitfalls.

Summary1. Amphetamine-induced c-fos andegr-1 expression in the striatum was used as a model in which to study the effects of antisense oligodeoxynucleotides (ODNs) directed at c-fos. Using direct infusions of ODNs into the striata of animals we have demonstrated that c-fos antisense ODNs retain most of their biological activity with 2- or 3-base substitutions. The c-fos antisense and mismatch ODNs attenuated Fos immunoreactivity but had little effect on Egr-1 immunoreactivity.2. In another group of studies examining the role of c-fos in amygdala kindling, we have demonstrated that ODNs cause neurotoxic damage following repeated daily infusions into the amygdala. The damage observed was greatly diminished when the time interval between infusions was extended.

Expression of the 27,000 mol. wt heat shock protein following kainic acid-induced status epilepticus in the rat

Western analysis and immunohistochemistry were used to determine the time-course and the distribution of the 27,000 mol. wt heat shock protein, Hsp27, in rat brain following systemic administration of kainic acid. No Hsp27 immunoreactivity was detected in naive control animals or in rats that failed to develop status epilepticus. Hsp27 immunoreactivity was detected as early as 12 h in the parietal cortex, piriform cortex and the hippocampus of rats that developed status epilepticus. The number of cells expressing Hsp27 and the intensity of Hsp27 immunoreactivity were increased 24 h after kainic acid administration. Hsp27 immunoreactivity was still observed seven days post-kainic acid injection. The morphology of the Hsp27-positive cells and double immunofluorescence against Hsp27 and glial fibrillary acidic protein revealed that Hsp27-positive cells were astrocytes. In addition, the distribution of Hsp27 suggested that astrocytic Hsp27 was dependent on excitation-induced metabolic stress rather than the direct effect of kainic acid on astrocytes.

The inducible 70,000 molecular/weight heat shock protein is expressed in the degenerating dentate hilus and piriform cortex after systemic administration of kainic acid in the rat.

Using both immunohistochemistry and in situ hybridization, we examined the rat brain for the expression of the inducible 70,000 mol. wt heat shock protein, Hsp70, at 3,6,12 and 24 h after systemic administration of kainic acid. In contrast to previous reports, the present study demonstrates that neurons in the regions most susceptible to seizure-induced cell death accumulate both Hsp70 messenger RNA and protein. Neurons in the denate hilus and piriform cortex contained Hsp70 messenger RNA at 6 h and protein at 12 h. These neutrons contained little or no Hsp70 messenger RNA or protein at 24 h when the majority of cells in these area were pyknotic. Injured neurons in areas such as the parietal cortex, which are less susceptible to seizure-induced cell death, expressed and maintained high levels of Hsp70 messenger RNA and protein at 12 and 24 h. This work suggest that Hsp70 messenger RNA and protein are rapidly and transiently expressed in dying neurons, and contradicts the notion that Hsp70 only accumulates in injured neurons that survive.

Cell specific expression of Hsp70 in neurons and glia of the rat hippocampus after hyperthermia and kainic acid-induced seizure activity

In this study we investigated the time course, cell-type and stress-specific expression of hsp70 mRNA and Hsp70 protein in glial cells and neurons in the rat brain following heat shock treatment and kainic acid-induced status epilepticus. Transcripts for hsp70 were detected in hippocampal homogenates from 1.5 to 6 h following hyperthermia and from 3 to 24 h following kainic acid-induced seizures. In situ hybridization revealed hsp70 mRNA to be region specific and time-dependent following hyperthermia and kainic acid-induced seizures. Western analysis indicated that Hsp70 reached maximal levels at 3 h after hyperthermia and 12 h after kainic acid-induced seizures. Immunohistochemistry revealed low level expression of Hsp70 protein in dentate granule cells at 1.5 and 3 h after hyperthermia. No Hsp70 protein was detected in neurons of the pyramidal cell layer or dentate hilus at any time following hyperthermia. Small Hsp70-immunoreactive cells were detected throughout the hippocampus following hyperthermia that, based on cell size, distribution, and double-labeling with vimentin, were considered to be glia. In contrast, high levels of Hsp70 protein were detected in neurons of the pyramidal cell layer and dentate hilus at 24 h after seizure-inducing kainic acid injection. These results suggest that expression of Hsp70 protein is cell-specific depending on the stressor. In addition, finding high levels of Hsp70 mRNA in the dentate granule cells after hyperthermia, but little or no Hsp70 protein, suggests that the synthesis of the protein is also regulated at the post-transcriptional level following hyperthermia.

Hyperthermic induction of the 27-kDa heat shock protein (Hsp27) in neuroglia and neurons of the rat central nervous system

The 27‐kDa heat shock protein (Hsp27) is constitutively expressed in many neurons of the brainstem and spinal cord, is strongly induced in glial cells in response to ischemia, seizures, or spreading depression, and is selectively induced in neurons after axotomy. Here, the expression of Hsp27 was examined in brains of adult rats from 1.5 hours to 6 days after brief hyperthermic stress (core body temperature of 42°C for 15 minutes). Twenty‐four hours following hyperthermia, Western blot analysis showed that Hsp27 was elevated in the cerebral cortex, hippocampus, cerebellum, and brainstem. Immunohistochemistry for Hsp27 revealed a time‐dependent, but transient, increase in the level of Hsp27 immunoreactivity (Hsp27 IR) in neuroglia and neurons. Hsp27 IR was detected in astrocytes throughout the brain and in Bergmann glia of the cerebellum from 3 hours to 6 days following heat shock. Peak levels were apparent at 24 hours, gradually declining thereafter. In addition, increases in Hsp27 IR were detected in the ependyma and choroid plexus . Hyperthermia induced Hsp27 IR in neurons of the subfornical organ and the area postrema within 3 hours and reached a maximum by 24 hours with a return to control levels 4–6 days after hyperthermia. Specific populations of hypothalamic neurons also showed Hsp27 IR after hyperthermia. These results demonstrate that hyperthermia induces transient expression of Hsp27 in several types of neuroglia and specific populations of neurons. The pattern of induced Hsp27 IR suggests that some of the activated cells are involved in physiological responses related to body fluid homeostasis and temperature regulation. J. Comp. Neurol. 428:495–510, 2000.

Acute administration of cocaine, but not amphetamine, increases the level of synaptotagmin IV mRNA in the dorsal striatum of rat

Synaptotagmin IV (Syt IV) is an inducible member of a multi-gene family of synaptic vesicle proteins that participate in Ca2+-dependent and Ca2+-independent interactions during membrane trafficking. We have examined the pattern of expression of Syt IV mRNA following the administration of cocaine and amphetamine. A single acute dose of cocaine, but not amphetamine, resulted in a transient increase, as determined by in situ hybridization, in the steady-state level of Syt IV mRNA in the dorsal striatum of rats 1 h after the administration of the drug. No change in the hybridization pattern of the Syt IV-specific probe to other regions of the rat brain were observed following cocaine or amphetamine administration at the time points examined (1, 3, 6, 12 and 24 h). The pattern of synaptotagmin I-(Syt I) specific hybridization remained constant, relative to controls, for both the cocaine- and amphetamine-treated animals. Northern hybridization analysis of mRNA isolated from striatal tissue using oligonucleotide probes specific to Syt I and Syt IV demonstrated that the probes hybridized exclusively to transcripts of the sizes previously reported for these two synaptotagmins and confirmed that the relative level of Syt IV to Syt I mRNA increased following the administration of cocaine but not amphetamine. These results indicate that these drugs have different effects on altering the levels of Syt IV mRNA. This work, in conjunction with earlier work that demonstrated that cocaine and amphetamine have different effects on the expression of immediate early genes such as c-Fos, supports the hypothesis that these psychotropic agents evoke different patterns of gene expression which may lead to alteration in synaptic efficacy.

Differential expression of c-fos, Hsp70 and Hsp27 after photothrombotic injury in the rat brain

In situ hybridization and immunohistochemistry were used to examine the expression of c-fos, Hsp70 and Hsp27 following photothrombotic injury in the right fronto-parietal cortex of the rat. C-fos mRNA and protein were detected in the entire cerebral cortex on the lesioned side. Hsp70 mRNA accumulation was observed only adjacent and peripheral to the site of the lesion. At 1 h after photothrombotic injury, Hsp70 expression delineates the area of necrosis at 24 h after photothrombotic injury. Hsp27 protein was observed in the ipsilateral cerebral cortex with the exception of the deep layers of the cingulate cortex. In addition, while c-Fos immunoreactivity was localized in cell nuclei, Hsp27 immunoreactivity was detected in the cytoplasm of astrocytes. These results demonstrate that unilateral cortical injury induces changes in gene expression that vary according to cell type and brain region.

The effects of hypoxia-ischemia on expression of c-Fos, c-Jun and Hsp70 in the young rat hippocampus

The expression of c-Fos, c-Jun and Hsp70 was examined in the hippocampus at 6, 12, 24, 48, 72 h, 4, 7 and 42 days following a combination of unilateral common carotid artery ligation and 60 min of systemic hypoxia (8% oxygen, 92% nitrogen) in 25-day-old male rats. While pyknotic cells were not visible in the hippocampus of control animals, pyknosis was evident in the ipsilateral, but not the contralateral hippocampus, of hypoxic-ischemic animals beginning at 24 h post-hypoxia. Immunohistochemical analysis revealed no c-Fos-, c-Jun- or Hsp70-immunoreactivity (IR) in any control animals. However, at 6 h post-hypoxia, Fos- and Jun-IR was evident throughout the injured ipsilateral hippocampus and later appeared throughout the contralateral hippocampus, which never showed signs of pyknosis. In contrast, Hsp70-IR was first observed at 24 h post-hypoxia and was restricted to the injured ipsilateral hippocampus. Hsp70-IR was not, however, limited to dying neurons. H-I/seizure animals did not express these proteins at any time point. These results suggest that, even in irreversibly injured neurons, Fos, Jun and Hsp70 appear to be involved in the aftermath of ischemia but probably do not play a pivotal role in the outcome of H-I compromised cells. Furthermore, compounded injury (H-I/seizure) appears to block the synthesis these proteins.