Peri-infarct depolarizations during focal ischemia in the awake Spontaneously Hypertensive Rat. Minimizing anesthesia confounds in experimental stroke.

Anesthesia profoundly impacts peri-infarct depolarizations (PIDs), but only one prior report has described their monitoring during experimental stroke in awake animals. Since temporal patterns of PID occurrence are model specific, the current study examined PID incidence during focal ischemia in the awake Spontaneously Hypertensive Rat (SHR), and documented the impact of both prior and concurrent isoflurane anesthesia. For awake recordings, electrodes were implanted under isoflurane anesthesia 1day to 5weeks prior to occlusion surgery. Rats were then subjected to permanent or transient (2h) tandem occlusion of the middle cerebral and ipsilateral common carotid arteries, followed by PID monitoring for up to 3days. Comparison perfusion imaging studies evaluated PID-associated hyperemic transients during permanent ischemia under anesthesia at varied intervals following prior isoflurane exposure. Prior anesthesia attenuated PID number at intervals up to 1week, establishing 2weeks as a practical recovery duration following surgical preparation to avoid isoflurane preconditioning effects. PIDs in awake SHR were limited to the first 4h after permanent occlusions. Maintaining anesthesia during this interval reduced PID number, and prolonged their occurrence through several hours following anesthesia termination. Although PID number otherwise correlated with infarct size, PID suppression by anesthesia was not protective in the absence of reperfusion. PIDs persisted up to 36h after transient occlusions. These results differ markedly from the one previous report of such monitoring in awake Sprague-Dawley rats, which found an extended biphasic PID time course during 24h after both permanent and transient filament occlusions. PID occurrence closely reflects the time course of infarct progression in the respective models, and may be more useful than absolute PID number as an index of ongoing pathology.

Glial elements contribute to stress-induced torsinA expression in the CNS and peripheral nervous system.

DYT1 dystonia is caused by a single GAG deletion in exon 5 of TOR1A, the gene encoding torsinA, a putative chaperone protein. In this study, central and peripheral nervous system perturbations (transient forebrain ischemia and sciatic nerve transection, respectively) were used to examine the systems biology of torsinA in rats. After forebrain ischemia, quantitative real-time reverse transcriptase-polymerase chain reaction identified increased torsinA transcript levels in hippocampus, cerebral cortex, thalamus, striatum, and cerebellum at 24 h and 7 days. Expression declined toward sham values by 14 days in striatum, thalamus and cortex, and by 21 days in cerebellum and hippocampus. TorsinA transcripts were localized to dentate granule cells and pyramidal neurons in control hippocampus and were moderately elevated in these cell populations at 24 h after ischemia, after which CA1 expression was reduced, consistent with the loss of this vulnerable neuronal population. Increased in situ hybridization signal in CA1 stratum radiatum, stratum lacunosum-moleculare, and stratum oriens at 7 days after ischemia was correlated with the detection of torsinA immunoreactivity in interneurons and reactive astrocytes at 7 and 14 days. Sciatic nerve transection increased torsinA transcript levels between 24 h and 7 days in both ipsilateral and contralateral dorsal root ganglia (DRG). However, increased torsinA immunoreactivity was localized to both ganglion cells and satellite cells in ipsilateral DRG but was restricted to satellite cells contralaterally. These results suggest that torsinA participates in the response of neural tissue to central and peripheral insults and its sustained up-regulation indicates that torsinA may contribute to remodeling of neuronal circuitry. The striking induction of torsinA in astrocytes and satellite cells points to the potential involvement of glial elements in the pathobiology of DYT1 dystonia.

Metabolic effects of 1-methyl-4-phenylpyridinium (MPP(+)) in primary neuron cultures.

Disruption of mitochondrial function has been proposed as an action of 1-methyl-4-phenylpyridinium (MPP(+)) that is responsible for its toxicity. In order to characterize effects of MPP(+) on energy metabolism in primary culture neurons, we monitored levels of several metabolites in cultured rat cerebellar granule cells exposed to MPP(+). The toxin produced a rapid concentration-dependent reduction in intracellular phosphocreatine (PCr), amounting to a 50-80% decrease within 30-60 min at 50 microM, that was maintained through the 1 week exposure interval examined. In contrast, ATP levels remained comparable to those of untreated neurons for approximately 4 days, at that time a 50% reduction in ATP was observed in association with a decrease in cell viability. Acute decreases in PCr were accompanied by increases in creatine such that the total creatine levels were maintained. Lactate levels in the culture medium were significantly increased (from 4.5 to 6.0 mM) within 6 hr after addition of MPP(+), with a concentration dependence similar to that observed for the reduction in PCr. Increased lactate production in the presence of MPP(+) coincided with a more rapid depletion of glucose in the culture medium. MPP(+) induced a rapid and sustained decrease in intracellular pH calculated from the creatine kinase equilibrium, and this acidification is considered primarily responsible for the observed decrease in PCr. These studies provide direct evidence that toxic concentrations of MPP(+) have acute effects on energy metabolism in primary culture neurons, consistent with an increased dependence on glycolysis to meet metabolic demand, but indicate that toxicity is not associated with overt, immediate failure to maintain cellular ATP.

Postischemic temperature as a modulator of the stress response in brain: dissociation of heat shock protein 72 induction from ischemic tolerance after bilateral carotid artery occlusion in the gerbil.

Brief ischemia induces tolerance to subsequent more severe insults, and induction of the 70 kDa heat shock/stress protein, hsp72, has been suggested to play a role. This study tested the requirement for hsp72 expression in a gerbil tolerance model in which postischemic temperature was varied to modulate the level of hsp72 induction. Gerbils were subjected to 2 min bilateral common carotid artery occlusion and kept under halothane anesthesia for 90 min, during which rectal temperature was either maintained at 37 degrees C (normothermic, NT) or elevated to 39.5 degrees C (hyperthermic, HT) during 15-60 min recirculation. Hsp72 mRNA expression was determined by in situ hybridization with a (35)S-labeled oligonucleotide probe at 3, 24 and 48 h. Separate groups were subjected to a test challenge of 5 min ischemia 48 h after the priming insult, and CA1 neuron counts were obtained at 1 week. Significant protection was observed in both NT and HT groups. However, while 90% of hippocampi from NT animals showed detectable protection of CA1 neurons, less than half showed detectable hsp72 mRNA induction. These results indicate that, within the limits of experimental detection, hsp72 expression is not required for induction of ischemic tolerance.

Transient global ischemia in rats yields striatal projection neuron and interneuron loss resembling that in Huntington's disease.

The various types of striatal projection neurons and interneurons show a differential pattern of loss in Huntington's disease (HD). Since striatal injury has been suggested to involve similar mechanisms in transient global brain ischemia and HD, we examined the possibility that the patterns of survival for striatal neurons after transient global ischemic damage to the striatum in rats resemble that in HD. The perikarya of specific types of striatal interneurons were identified by histochemical or immunohistochemical labeling while projection neuron abundance was assessed by cresyl violet staining. Projectionneuron survival was assessed by neurotransmitter immunolabeling of their efferent fibers in striatal target areas. The relative survival of neuron types was determined quantitatively within the region of ischemic damage, and the degree of fiber loss in striatal target areas was quantified by computer-assisted image analysis. We found that NADPHd(+) and cholinergic interneurons were largely unaffected, even in the striatal area of maximal damage. Parvalbumin interneurons, however, were as vulnerable as projection neurons. Among immunolabeled striatal projection systems, striatoentopeduncular fibers survived global ischemia better than did striatopallidal or striatonigral fibers. The order of vulnerability observed in this study among the striatal projection systems, and the resistance to damage shown by NADPHd(+) and cholinergic interneurons, is similar to that reported in HD. The high vulnerability of projection neurons and parvalbumin interneurons to global ischemia also resembles that seen in HD. Our results thus indicate that global ischemic damage to striatum in rat closely mimics HD in its neuronal selectivity, which supports the notion that the mechanisms of injury may be similar in both.

Selective glial vulnerability following transient global ischemia in rat brain.

Global cerebral ischemia selectively damages neurons, but its contribution to glial cell death is uncertain. Accordingly, adult male rats were sacrificed by perfusion fixation at 1, 2, 3, 5, and 14 days following 10 minutes of global ischemia. This insult produces CA1 hippocampal neuronal death at post-ischemic (PI) day 3, but minor or no damage to neurons in other regions. In situ end labeling (ISEL) and immunohistochemistry identified fragmented DNA of dead or dying glia and distinguished glial subtypes. Rare ISEL-positive oligodendroglia, astrocytes, and microglia were present in control brain. Apoptotic bodies and ISEL-positive glia significantly increased at PI day 1 in cortex and thalamus (p < 0.05), but were similar to controls in other regions and at other PI intervals. Most were oligodendroglia, although ISEL-positive microglia and astrocytes were also observed. These results show that oligodendroglia die rapidly after brief global ischemia and are more sensitive than neurons in certain brain regions. Their selective vulnerability to ischemia may be responsible for the delayed white matter damage following anoxia or CO poisoning or that associated with white matter arteriopathies. Glial apoptosis could contribute to the DNA ladders of apoptotic oligonucleosomes that have been found in post-ischemic brain.

Cryptic expression of the 70-kDa heat shock protein, hsp72, in gerbil hippocampus after transient ischemia.

The 70 kDa heat shock protein, hsp72, is known to be induced following transient global ischemia in brain, as detected by immunocytochemistry and in situ hybridization techniques. However, while hsp72 mRNA is expressed rapidly following postischemic recirculation, immunocytochemistry fails to detect hsp72 protein for many hours after such insults, even in cell populations that readily express Fos and other proteins encoded by ischemia-induced mRNAs. In the present study, hsp72 expression in gerbil hippocampus was compared by immunocytochemistry and immunoblot methods at several intervals following 10 min ischemia. As established in previous studies, hsp72 immunoreactivity remained undetectable in postischemic neurons at 6 h following such insults. In contrast, immunoblots of dissected gerbil hippocampus demonstrated nearly maximal accumulation of hsp72 at this time point. These results indicate that the protein is present, but cryptic to detection in perfusion-fixed sections, during early recirculation. The constitutively expressed heat shock cognate protein, hsc70, did not show significant changes in level or distribution by either method, except for a decrease in CA1 staining at 48 h. These results confirm that hsp72 rapidly accumulates to high levels in postischemic hippocampus, and suggest that further studies of its subcellular localization during this interval may offer insight into its functional role as a component of the stress response in neurons after such insults.

Postischemic hyperthermia increases expression of hsp72 mRNA after brief ischemia in the gerbil.

Brain temperature during ischemia critically determines insult severity, and temperature changes during recirculation may also affect subsequent injury. We have examined the impact of postischemic temperature on induction of the 70 kDa stress protein, hsp72, after brief ischemia in the gerbil. Animals were subjected to 2 min ischemia after which they were maintained under continuous halothane anesthesia during 3 h recirculation, and were either kept normothermic or subjected to hyperthermia comparable to that which occurs spontaneously in gerbils released from anesthesia immediately after the occlusion. Quantitative in situ hybridization showed striking dependence of hsp72 induction on postischemic hyperthermia. This result establishes that delayed temperature-sensitive signals mediate this injury-associated transcriptional response, and demonstrates that postischemic temperature must be carefully monitored in studies of gene expression and induced tolerance employing brief ischemic insults.

Species heterogeneity between gerbils and rats: quinolinate production by microglia and astrocytes and accumulations in response to ischemic brain injury and systemic immune activation.

Quinolinic acid is an excitotoxic kynurenine pathway metabolite, the concentration of which increases in human brain during immune activation. The present study compared quinolinate responses to systemic and brain immune activation in gerbils and rats. Global cerebral ischemia in gerbils, but not rats, increased hippocampus indoleamine-2,3-dioxygenase activity and quinolinate levels 4 days postinjury. In a rat focal ischemia model, small increases in quinolinate concentrations occurred in infarcted regions on days 1, 3, and 7, although concentrations remained below serum values. In gerbils, systemic immune activation by an intraperitoneal injection of endotoxin (1 mg/kg of body weight) increased quinolinate levels in brain, blood, lung, liver, and spleen, with proportional increases in lung indoleamine-2,3-dioxygenase activity at 24 h postinjection. In rats, however, no significant quinolinate content changes occurred, whereas lung indoleamine-2,3-dioxygenase activity increased slightly. Gerbil, but not rat, brain microglia and peritoneal monocytes produced large quantities of [13C(6)]-quinolinate from L-[13C(6)]tryptophan. Gerbil astrocytes produced relatively small quantities of quinolinate, whereas rat astrocytes produced no detectable amounts. These results demonstrate that the limited capacity of rats to replicate elevations in brain and blood quinolinic acid levels in response to immune activation is attributable to blunted increases in local indoleamine-2,3-dioxygenase activity and a low capacity of microglia, astrocytes, and macrophages to convert L-tryptophan to quinolinate.

DNA fragmentation follows delayed neuronal death in CA1 neurons exposed to transient global ischemia in the rat.

Apoptosis is an active, gene-directed process of cell death in which early fragmentation of nuclear DNA precedes morphological changes in the nucleus and, later, in the cytoplasm. In ischemia, biochemical studies have detected oligonucleosomes of apoptosis whereas sequential morphological studies show changes consistent with necrosis rather than apoptosis. To resolve this apparent discrepancy, we subjected rats to 10 minutes of transient forebrain ischemia followed by 1 to 14 days of reperfusion. Parameters evaluated in the CA1 region of the hippocampus included morphology, in situ end labeling (ISEL) of fragmented DNA, and expression of p53. Neurons were indistinguishable from controls at postischemic day 1 but displayed cytoplasmic basophilia or focal condensations at day 2; some neurons were slightly swollen and a few appeared normal. In situ end labeling was absent. At days 3 and 5, approximately 40 to 60% of CA1 neurons had shrunken eosinophilic cytoplasm and pyknotic nuclei, but only half of these were ISEL. By day 14, many of the necrotic neurons had been removed by phagocytes; those remaining retained mild ISEL. Neither p53 protein nor mRNA were identified in control or postischemic brain by in situ hybridization with riboprobes or by northern blot analysis. These results show that DNA fragmentation occurs after the development of delayed neuronal death in CA1 neurons subjected to 10 minutes of global ischemia. They suggest that mechanisms other than apoptosis may mediate the irreversible changes in the CA1 neurons in this model.

Heat-shock protein and C-fos expression in focal microvascular brain damage.

Cortical brain damage was produced in rats by a focal pulse from a Nd-YAG laser, and evolution of the lesion was evaluated at 30 min, and 2, 8, and 24 h with respect to microvascular perfusion, blood-brain barrier (BBB) permeability, and expression of both the heat-shock/stress protein, hsp72, and the c-fos proto-oncogene transcription factor. A double-labeling fluorescence technique employing intravenously injected Evans blue albumin (EBA) and fluorescein-labeled dextran was used to map and measure BBB damage and microvascular perfusion in fresh frozen brain sections. Hsp72 and c-fos mRNAs were localized by in situ hybridization, and the respective proteins were identified by immunocytochemistry. Parallel sections were stained for glial fibrillary acidic protein and for routine histologic examination. Striking hsp72 mRNA expression was evident by 2 h in an approximately 300 microns wide rim surrounding an area of expanding BBB damage. Increased hsp72 mRNA was observed only in regions of preserved microcirculation, where the hsp72 protein was subsequently localized exclusively in the vasculature at 24 h after the insult. Hsp72-positive endothelial cells spanned the narrow margin between the lesion and histologically normal, glial fibrillary acidic protein (GFAP)-positive cortical tissue. There was no hsp72 expression in the area of subcortically migrating edema fluid. Inductions of c-fos mRNA and Fos protein were not strikingly evident around the focal brain lesion, but were observed transiently throughout the injured hemisphere at 30 min and 2.5 h, respectively, indicating that spreading depression was triggered by the focal injury. These results are in striking contrast to those previously obtained from studies of models of focal ischemic or traumatic brain injury, which are characterized by a complex pattern of glial and neuronal hsp72 expression in the periphery of an infarct, and which suggest that the tightly demarcated lesion produced by the Nd-YAG laser lacks these components of graded injury that are evident following other types of focal brain damage.

Gene expression and induced ischemic tolerance following brief insults.

Striking changes in gene expression occur after transient ischemic insults, including the induction of heat shock proteins that are believed to play a central role in cellular defense mechanisms, and proto-oncogene (immediate-early gene) transcription factors that regulate the expression of diverse target genes. Such effects potentially contribute to a wide range of pathophysiological responses. A number of studies have characterized models of induced ischemic tolerance in which brief priming insults are demonstrated to result in reduced vulnerability to subsequent challenges. In the present study we have optimized a model of induced ischemic tolerance in the gerbil by carefully monitoring the duration of ischemic depolarization during each insult. Using this model we demonstrate that the threshold depolarization required to induce tolerance is comparable to those for induction of several transcription factor mRNAs, while mRNA encoding the heat shock protein, hsp72, is strongly induced only after more severe insults that approach the threshold for ischemic neuronal injury.

Distribution of phosphodiester and phosphorothioate oligonucleotides in rat brain after intraventricular and intrahippocampal administration determined by in situ hybridization.

The distribution and stability of exogenously administered oligonucleotides (oligos) are important variables determining the potential utility of antisense oligos as agents for modifying gene expression within a given brain region in vivo. In the present study, phosphodiester (PO) and phosphorothioate (PS) oligos antisense with respect to a recently cloned rat hsp70 sequence were localized in rat brain following intraventricular and intrahippocampal administration using an in situ hybridization detection method. Unlabeled PO and PS oligos were dissolved in artificial cerebrospinal fluid and infused under stereotaxic control using a syringe pump. At various intervals after administration frozen brain sections were collected on gelatin-coated slides and hybridized with 35S-labeled probe consisting of the corresponding phosphodiester sense sequence. After intraventricular administration the unmodified PO oligo exhibited a limited and strictly periventricular distribution. In contrast the PS oligo showed significant penetration into and accumulation within brain, with extensive uptake in ipsilateral striatum and dorsal hippocampus, as well as in midline periventricular structures. Both oligos remained detectable for at least two days after administration. Following intrahippocampal injection the PO oligo was rapidly lost from the injection site, with detectable signal persisting only along the hippocampal fissure at 24 h. The PS oligo exhibited a more diffuse initial distribution as well as greater stability. While there was no indication of specific accumulation in the major hippocampal neuron layers through 24 h, there was some indication of selective localization in neuronal soma by 48 h. These results confirm that the relative instability of unmodified oligos may severely limit their utility as antisense reagents in brain in vivo. While PS oligos show more widespread distribution than PO oligos after intraventricular infusion, even these do not detectably accumulate in cortex and other structures without immediate access to the ventricular space under the dosing conditions employed here. The hybridization approach used in these studies should prove to be of general use in verifying the targeting of specific brain structures with antisense oligos by various routes of administration.

Preparative methods for brain slices: a discussion.

Criteria for slice health and factors that affect slice health were discussed by many of the participants in the conference. In addition to the standard parameters of slice health (energy metabolism, morphology, electrophysiological responsiveness) more subtle but possibly equally important manifestations of slice health were discussed. These included protein synthesis, and more subtle changes, of which we are becoming increasingly aware. The latter include synthesis of stress-related proteins, altered levels of phosphorylation, altered levels of proteolysis. These last were only touched on, but it is becoming apparent they do in fact constitute important manifestations of differences between the slice preparation and the in vivo tissue. They may well lead to quite different responses in slices from those that occur in vivo. While many ways of optimizing slice wellness were discussed, there was a fair consensus that certain adjustments will optimize the most widely measured aspects of cell function. These include the following, wherever possible. Use of young animals, use of the interface chamber, preparing slices with the vibratome, pre-treating animals with ice-cold cardiac perfusion before sacrificing, using pre-incubation media which reduce NMDA receptor activation, free radical formation and cell swelling. When possible these treatments should perhaps be continued into the normal incubation. This being said, many viewpoints were actually expressed in the discussion, and it should be read to get a feel for the usefulness of the different approaches.

Immunocytochemical and in situ hybridization approaches to the optimization of brain slice preparations.

Methods are described for determining the expression of specific mRNAs and proteins in brain slices, in order to elucidate changes in gene expression during preparation of vibratome slices from hippocampus of adult rats. In situ hybridization with 35S-labeled oligonucleotides was used to evaluate the level and distribution of c-fos and hsp72 mRNAs in 15-microns frozen sections prepared from these slices. Commercially available antibodies were used to examine the distribution of induced Fos and Jun proto-oncogenes as well as expression of the neuronal cytoskeletal protein, microtubule-associated protein 2 (MAP2), in 50-microns vibratome sections from immersion-fixed slices. These studies confirm the induction of c-fos and hsp72 mRNAs during routine incubation, as previously observed in hippocampal slices obtained with a tissue chopper and incubated under somewhat different conditions, indicating that such responses are likely to be common features of many slice preparations. Accumulation of Fos and Jun immunoreactivities in neurons and glia was generally consistent with the distribution of c-fos mRNA induction observed in slices, and the neuronal component of this response was comparable to the expression of these proteins observed after transient ischemia in vivo. MAP2 immunoreactivity detected in the dendritic processes of neurons tended to show an increase in staining intensity during slice incubation, although loss of dendritic staining in specific regions was occasionally observed in association with the absence of Fos and Jun expression and histological evidence of neuron damage. These results support the use of MAP2 immunoreactivity as a sensitive indicator of neuronal integrity in slices.(ABSTRACT TRUNCATED AT 250 WORDS)

Reexpression of developmentally regulated MAP2c mRNA after ischemia: colocalization with hsp72 mRNA in vulnerable neurons.

Levels of mRNAs encoding the microtubule-associated proteins MAP2b and MAP2c as well as the 70-kDa stress protein [72-kDa heat shock protein (hsp72)] were evaluated in postischemic rat brain by in situ hybridization with oligonucleotide probes corresponding to the known rat sequences. Rats were subjected to 10-min cardiac arrest, produced by compression of major thoracic vessels, followed by resuscitation. The normally expressed MAP2b mRNA showed transient twofold elevations in all hippocampal neuron populations at 6-h recirculation, followed by a return to control levels by 24 h. MAP2b hybridization was progressively lost thereafter from the vulnerable CA1 and outer cortical layers, preceding both the fall in immunoreactive MAP2b and the eventual cell loss in these regions. The depletion of MAP2b mRNA coincided with an increase in the alternatively spliced MAP2c in vulnerable regions during 12-48 h of recirculation, precisely overlapping the late component of hsp72 expression that persisted in these cell populations. Previous studies have suggested that the initial induction of hsp72 provides an index of potential postischemic injury in neuron populations that may or may not be injured, while lasting hsp72 mRNA expression is associated with cell damage. In contrast, the present results demonstrate that MAP2c expression under these conditions occurs uniquely in neuron populations subject to injury. Available evidence suggests that MAP2c expression represents a plastic response in subpopulations of neurons that will survive in these regions, although it remains to be explicitly determined whether it may also be transiently expressed in dying cells. In any case, these observations demonstrate that reexpression of developmentally regulated MAP2c mRNA is a relatively late postischemic response in vulnerable cell populations, indicating that pathways regulating MAP2 splicing may be closely associated with mechanisms of neuron injury and/or recovery.

Loss of parvalbumin immunoreactivity defines selectively vulnerable thalamic reticular nucleus neurons following cardiac arrest in the rat.

The thalamic reticular nucleus (NRT) is one of the most vulnerable structures to selective neuronal damage both in human cardiac arrest patients and in experimental rodent global cerebral ischemia models. The detailed distribution of neuronal injury within the NRT was examined following 10-min cardiac arrest in the rat with conventional Nissl staining, 45Ca autoradiography and immunocytochemistry of the calcium binding proteins parvalbumin (PV) and calretinin (CR). While Nissl staining was almost unable to show the exact boundary of the nucleus and of the lesion, immunocytochemistry of PV proved to be the most useful index of the exact location and extent of neuronal loss in the NRT after ischemia. Calcium autoradiography was a sensitive method for detecting the lesion, and showed a similar distribution to the loss of PV staining, but did not give optimal spatial resolution. Quantitative analysis of PV staining at 7 days of recirculation demonstrated cell loss restricted to the lateral aspect of the middle segment of the NRT, identical with the distribution of large fusiform neurons in the somatosensory component of the nucleus. CR-positive neurons in the NRT were completely spared, although not all surviving neurons contained CR. These studies provide the first detailed characterization of the distribution of vulnerable neurons within the NRT after experimental ischemia and suggest that immunocytochemistry of PV is a useful tool for quantitative analysis of the lesion for use in further experiments to elucidate the mechanisms of selective vulnerability of the NRT.

Coexpression of c-fos and hsp70 mRNAs in gerbil brain after ischemia: induction threshold, distribution and time course evaluated by in situ hybridization.

Levels of mRNAs encoding the proto-oncogene, c-fos, and the 70 kDa stress protein, hsp70, were evaluated in gerbil brain following transient cerebral ischemia of varied duration by in situ and blot hybridization techniques. Blots of total hippocampal RNA obtained after 5 min ischemic insults confirmed a characteristic, transient time course of c-fos expression with a striking elevation within 1 h and a return to control levels by 3 h recirculation. Hsp70 hybridization was significant at 1 h and continued to increase until 3-6 h after the insult. Striking accumulation of c-fos mRNA was detected within 15 min recirculation in dentate granule cells, persisting through 1 h, and a weaker signal was evident in CA1 and CA3 pyramidal neurons of hippocampus, as well as in prepiriform/entorhinal cortex and neocortical regions, during the same interval. Hsp70 hybridization showed an identical distribution at 1 h recirculation. Ischemic insults of 1 min duration resulted in no detectable increase of either mRNA, while 2 min ischemia resulted in changes comparable to those seen after 5 min insults. This common threshold corresponds to the ischemic interval required for energy depletion and resultant failure of intracellular ion homeostasis. In contrast, expression of hsp70 mRNA was not observed under conditions of brief depolarization accompanying cortical or hippocampal spreading depression that were shown to induce c-fos. A delayed component of c-fos mRNA expression was not detected in this model, while persistent hsp70 hybridization, restricted to hippocampal CA1 neurons, was evident at 48 h after either 2 min or 5 min ischemic insults. The parallels in c-fos and hsp70 mRNA expression during early recirculation suggest that overlapping mechanisms triggered following postischemic depolarization contribute to their induction after transient ischemia.