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Effects of pentylenetetrazol-induced status epilepticus on c-Fos and HSP72 immunoreactivity in the immature rat brain
Molecular Brain Research 50 Ž1997. 79–84
Research report
Effects of pentylenetetrazol-induced status epilepticus on c-Fos and HSP72 immunoreactivity in the immature rat brain Jacques E. Motte a
a,b
, Maria Jose´ da Silva Fernandes Astrid Nehlig a,)
a,1
, Christian Marescaux a ,
INSERM U 398, Faculte´ de Medecine, 11 rue Humann, 67085 Strasbourg Cedex, France ´ ´ SerÕice de Neuropediatrie, American Memorial Hospital, 51092 Reims Cedex, France ´ ´
b
Accepted 22 April 1997
Abstract Pentylenetetrazol ŽPTZ.-induced status epilepticus ŽSE. leads to acute and long-term metabolic decreases in specific brain regions of rats at 10 ŽP10. or 21 days after birth ŽP21.. These decreases are not related to apparent neuronal damage. Therefore, to better understand the neuronal activation and stress response to PTZ in immature rats, we mapped the expression of c-Fos and of the 72 kDa heat-shock protein ŽHSP72. in the same model of severe SE induced by the repetitive i.p. injections of subconvulsive doses of PTZ. Rats were sacrificed either at 2 or 24 h after the onset of SE in order to reveal c-Fos immunoreactivity, and at 24 and 72 h for HSP72 expression. Hematoxylin-eosin staining was performed at 24, 72 and 144 h after SE. The expression of c-Fos at 2 h after SE was more marked at P21 than at P10 and was prominent at both ages in the hippocampal dentate gyrus, cerebral cortex and amygdala. Some immunoreactivity was also present in the hypothalamus, thalamus and a few brainstem and cerebellar regions at both ages. There was a good relation between the regions expressing c-Fos and those exhibiting acute metabolic decreases at P21. Conversely, PTZ seizures did not lead to any expression of c-Fos at 24 h after SE or of HSP72 at 24 or 72 h at any age. Cell density was not affected by PTZ-induced SE at any age and at any time. These results suggest that c-Fos is a useful marker of neuronal activation induced by severe and prolonged seizures in the immature brain. The lack of HSP72 and of late c-Fos expression likely reflect the absence of neuronal damage in this model of PTZ-induced SE in the immature rat. q 1997 Elsevier Science B.V. Keywords: c-Fos; Heat shock protein; Seizure; Status epilepticus; Development
1. Introduction In previous studies using pentylenetetrazol ŽPTZ.-induced status epilepticus ŽSE. in immature rats, we showed that local cerebral metabolic rates for glucose ŽLCMRglcs. and local cerebral blood flow ŽLCBF. were increased in all regions of 10-day-old rats. Conversely, in 21-day-old animals, according to the region of interest, metabolic and circulatory levels were increased, decreased or not changed compared to control levels w26,27x. Moreover, PTZ-induced SE led to long-term decreases in LCMRglcs in adult animals subjected to seizures either at 10 ŽP10. or at 21 days after birth ŽP21.. These decreases were especially )
Corresponding author. Fax: q33 Ž3. 8824-3360; E-mail: [email protected] 1 M.J.daS. Fernandes is a Brazilian postdoctoral fellow sponsored by the CNPq. 0169-328Xr97r$17.00 q 1997 Elsevier Science B.V. All rights reserved.
marked when the seizures occurred at P21 and mainly located in the regions where LCMRglcs were decreased during the acute phase of PTZ-induced SE, i.e., mainly cerebral cortex, hippocampus and sensory regions w13x. In the adult rat, metabolic decreases induced by severe and prolonged seizures coincide with neuronal damage w20,25x. Therefore, the acute and long-term metabolic and circulatory decreases related to PTZ-induced SE could be indicative of neuronal suffering in immature rats, as is the case in adult animals w20,25x. However, we have no indication of neuronal damage in this model either at P10 or at P21 w24,29x. Therefore, in order to better understand the relationship between local cerebral functional changes, cellular activation, stress and damage, in the present study, we mapped the expression of the c-Fos and 72-kDa heat shock protein ŽHSP72.. Indeed, the c-fos proto-oncogene undergoes a rapid and transient increase following a variety of external
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stimuli. The expression of its encoded protein product, Fos, appears in specific brain regions after seizures induced by various convulsants or by kindling w3,4,16,21,31,38x and allows the tracing of the specific pathways involved in brain activation. Likewise, the regional immunoreactivity of the inducible HSP72 is expressed in immature rat brain by stressed cells w8x in response to various types of insults, such as hyperthermia w17x, hypoxia-ischemia w7,8,40x and seizures w19,30,42x. The expression of HSP72 represents a sensitive and specific marker of excitation-induced stress and is known to influence the ability of cells to survive to various forms of toxicity, including seizures w18x. Thus, in the present study, we mapped the immunoreactive response of c-Fos and HSP72 to SE induced by PTZ in P10 or P21 rats.
min. Sections were then sequentially incubated twice in PBS, once in 0.6% hydrogen peroxide in PBS and twice in PBS containing 0.4% normal goat serum ŽVector Laboratories, Burlingame, CA, USA. for Fos or horse serum ŽVector. for HSP, 0.25% Triton-X100 and 1.5% bovine serum albumin ŽBSA.. Sections were then incubated overnight at 208C with the primary antibody, a rabbit
2. Materials and methods 2.1. Animals and pharmacological treatment Adult Sprague–Dawley rats ŽIffa–Credo Breeding Laboratories, L’Arbresle, France., one male and two females by cage were housed together in mating groups for 5 days and constantly maintained under standard laboratory conditions on a 12r12 h lightrdark cycle Žlights on at 06.00 h.. After delivery, litter sizes were reduced to 10 pups for homogeneity Žday of birth was considered as day 0.. Experiments were performed on P10 or P21 rats. To reach a SE of controlled intensity and quite long duration, the animals received repeated i.p. injections of PTZ Ž10 mgrml in saline., as previously described w10x. At P10 and P21, the rats were first injected with 40 mgrkg PTZ, followed by 20 mgrkg 10 min later and then, every 10 min, additional i.p. administrations of 10 mgrkg PTZ until SE was reached. This injection procedure induced EEG and behavioral changes characteristic of each age group, P10 or P21 w6,24x. Control animals received the same number of saline injections as their paired PTZ-exposed congeners. For the study of HSP72 expression, the animals were put back with their mother in their normal environment at about 6–8 h after the onset of SE. 2.2. Fos and HSP72 immunohistochemistry The immunohistochemical detection of c-Fos protein was performed in P10 and P21 rats at 2 Ž6–8 animals at each age. and 24 h Ž3 animals at each age. while the expression of HSP72 was measured at 24 Ž6–8 animals at each age. and 72 h Ž3 animals at each age. after the onset of SE. The expression of both proteins was revealed simultaneously in 2 matched control animals at each age. Brains were removed, immediately frozen in isopentane and stored at y808C. Serial coronal sections, 20 m m thick, were cut in a cryostat. Brain sections were fixed in 4% paraformaldehyde dissolved in phosphate buffer saline ŽPBS. at pH 7.4 for 7
Fig. 1. Expression of c-Fos in the hippocampus of P21 control rats ŽA., and of rats subjected to PTZ-induced SE at P10 ŽB. or P21 ŽC.. Note the total absence of labelling in the control hippocampus as well as marked labelling in the granular cell layer of the dentate gyrus in rats exposed to PTZ at both ages. Immunoreactivity of c-Fos is moderate in area CA3 and rather weak in areas CA2 and CA1 of the hippocampus and shows also an overall stronger expression at P21 than at P10. Scale bar s100 m m.
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Fig. 2. Expression of c-Fos in the piriform cortex of control P10 rats ŽA. or of P10 ŽB. and P21 rats ŽC. subjected to PTZ-induced SE, and in the entorhinal cortex of P10 ŽD. and P21 rats ŽE. subjected to PTZ-induced SE. Note the lack of immunoreactivity in the control piriform cortex and the quite marked labelling in the piriform and entorhinal cortex of seizing animals which exhibits a similar distribution at both ages that is stronger in the most superficial layer of rats seizing at P21 compared to those seizing at P10. Scale bar s 100 m m.
affinity-purified polyclonal antibody ŽSanta Cruz Biotechnology, Santa Cruz, CA, USA, dilution 1:500 in PBS containing goat serum. for Fos or a monoclonal anti-72 Kd HSP ŽAmersham, Les Ulis, France, dilution 1:200 in PBS containing horse serum. for the detection of HSP72. The sections were rinsed twice in PBS containing the appropriate serum and incubated for 1 h at 208C with the secondary antibody, biotinylated goat anti-rabbit antibody, dilution 1:400 for Fos ŽVector. and biotinylated anti-mouse antibody, dilution 1:50 for HSP72 ŽVector. in the corresponding serumrTriton-X100rBSArPBS mixture. Sections were rinsed twice in the latter medium and covered with the ABC reagent ŽVectastain Kit, Vector. for 1 h at 208C. Sections were rinsed twice in PBS and incubated for 5–8 min in a mixture of 0.025% diaminobenzidine, 0.01% nickel chloride and 0.05% hydrogen peroxide in PBS. Then, sections were dehydrated in ethanol and coverslipped. In addition to control animals exposed only to saline, immunocytochemical control sections from 2 animals subjected to SE Ž2 for Fos and 2 for HSP72. underwent the procedures described above except for the exposure to the primary antibody which eliminated all staining.
The distribution of positive neurons was recorded from forebrain to cerebellum. Direct visual counting of Fos or HSP72 expressing neurons was performed using a 4-point grading scale of 0–3, with 0 corresponding to the absence of reactive cells and 1, 2 and 3 to low, moderate and high density labelled cells, respectively. 2.3. Rating of cell surÕiÕal Cell density was measured on adjacent sections from P10 and P21 rats taken at 24, 72 and 144 h after SE. Sections were stained by the hematoxylin-eosin method for cell counting that was performed under light microscopy. 3. Results 3.1. Expression of c-Fos In control P10 and P21 rats, all cerebral regions were devoid of c-Fos immunoreactivity ŽFigs. 1 and 2.. At both ages and at 2 h after the onset of SE, the expression of c-Fos induced by PTZ was prominent in the cerebral
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cortex and the dentate gyrus ŽFigs. 1 and 2.. Within the cerebral cortex, the piriform, entorhinal and parietal areas were strongly labelled at both ages and c-Fos expression was also high in the prefrontal cortex at P21. Conversely, c-Fos immunoreactivity was weak in the cingulate cortex, especially at P21. Within forebrain regions, labelling was weak in the septum and the caudate nucleus in which region it was absent at P21. c-Fos expression was moderate in the amygdala at both ages. Within the hippocampus, labelling was virtually absent in the CA1 area, weak in the CA3 region and strong in the dentate gyrus, already at P10 ŽFigs. 1 and 2.. The ventromedian hypothalamus and the medial thalamus were weakly marked at both ages while the paraventricular hypothalamus and the lateral thalamus expressed only c-Fos at P21 and P10, respectively. Within brainstem regions, weak c-Fos labelling appeared only at P21 in the medial geniculate body and substantia nigra, was moderate in the other regions and only strong in the cerebellar nuclei after PTZ-induced SE at P21. Conversely, at 24 h after the onset of SE, no c-Fos immunoreactivity could be detected either at P10 or at P21. 3.2. Expression of HSP72 PTZ-induced SE did not lead to any expression of HSP72 in any brain area and at any time, 24 or 72 h after SE, either at P10 or at P21. The lack of expression of HSP72 in the present model was not related to a technical problem since the same method allowed us to map HSP72 immunoreactivity in adult rats subjected to lithium-pilocarpine-induced SE w23x. 3.3. Cell density The analysis of hematoxylin-eosin stained sections did not reveal any difference in cell density at 24, 72 or 144 h after PTZ-induced SE in P10 or P21 rats. Very few scattered pycnotic nuclei could be seen at both ages at 72 h together with less well delineated cell borders but there was absolutely no visible difference in cell shape and density between control and PTZ-exposed P10 or P21 rats at 144 h. 4. Discussion The present data show that PTZ-induced SE lasting for 60 min leads to an early widespread expression of c-Fos that is quite strong in the cerebral cortex and the dentate gyrus of both P10 and P21 rats. Conversely, there is no late expression of c-Fos or induction of HSP72 by PTZ seizures in the immature rat brain. 4.1. Early expression of c-Fos during seizures in the immature rat brain Our data are in good accordance with previous studies concerning the induction of c-Fos following the adminis-
tration of PTZ. Indeed, a major expression of the protein is present in the cerebral cortex, the dentate gyrus of the hippocampus, the amygdala and some thalamic and hypothalamic nuclei, and virtually absent from most midbrain and brainstem areas, as previously shown with PTZ in adult w3,5,14,21,34,37x or immature rodents w14,34x. However, in previous studies with immature animals, the induction of c-Fos either was not apparent before P20 in the mouse w34x or occurred only at 4 h after a prolonged seizure episode in the P10 rat w14x. However, in the other studies on immature animals, seizures were induced by the injection of a unique dose of the convulsant w14,34x while, in the present study, we injected several subconvulsive doses of PTZ in order to reach a 60–80 min SE. With this injection schedule, there is a time lag of 40–70 min before the onset of SE during which alterations of the EEG and isolated seizures occur w6,12,28x. After the onset of SE, there was no return to basal EEG activity for 30–50 min and numerous spontaneous seizures occurred thereafter w12,28x. Therefore, the expression of c-Fos in P10 rats at 2 h after the onset of SE in the present study could be related to the episodic seizure activity preceding SE in addition to the severe and sustained convulsive activity induced by our injection procedure. The basal expression of the c-fos gene during postnatal development is low until P10 in the cerebral cortex and P13 in the cerebellum and then doubles in both structures at P20 w10x. Likewise, the expression of c-fos mRNA induced by kainate administration is close to basal levels until P7 and doubles in hippocampus between P7 and P13 w35x. The marked expression of the c-Fos protein after PTZ prolonged seizures at P10 ww14x, the present studyx is probably not related to the immaturity of the transcriptional apparatus that facilitates stimulus-transcription coupling since the c-fos gene is inducible in the brain of the P7–P8 rat after hypoxia ischemia w9x and in the brain of the P3 mouse after brain tissue disruption w33x. In the present study, the early c-Fos immunoreactivity at P21 presents many similarities with that recorded in the adult rat after PTZ seizures w37x. Indeed, c-Fos expression is moderate to high in the cerebral cortex, the amygdala, the dentate gyrus, the hypothalamic paraventricular nucleus and the medial thalamus. There is also a slight labelling in the substantia nigra in P21 and in adult rats w37x. 4.2. Relationship between early c-Fos immunoreactiÕity and local cerebral glucose utilization during seizures There is some mismatch between the spatial distribution of early c-Fos immunoreactivity and the changes in LCMRglcs and LCBF induced by acute PTZ seizures in P10 or P21 rats w26,27x. Indeed, in P10 rats, LCMRgls and rates of LCBF largely increased over control values in all brain areas but hippocampus and inferior colliculus where they were similar to control values. In P21 rats, LCMRglcs and
J.E. Motte et al.r Molecular Brain Research 50 (1997) 79–84
LCBF rates remained higher than control values in most brainstem areas while they decreased in hippocampus, cerebral cortex, white matter and some thalamic and motor areas w26,27x. However, c-Fos protein is expressed in almost the same regions at both ages, mainly in cerebral cortex, hippocampus and amygdala. Thus, there is no direct correlation between the areas of metabolic activation and those expressing c-Fos but it is interesting to notice that c-Fos is mainly expressed in the areas where LCMRglcs are decreased during acute PTZ seizures at P21 w26,27x or in the long term w13x, i.e., cerebral cortex, amygdala and hippocampus. However, these functional changes occur without any change in cell density, as reported in the present study and could rather reflect a functional reorganization induced by seizures and related cellular activation. Moreover, the lack of expression of c-Fos at 24 h after PTZ-induced seizures recorded at both P10 and P21 is in accordance with the absence of lesions recorded in this model of seizures at both ages. Indeed, it has been shown that the second wave of c-Fos labelling usually occurs 24–48 h after acute seizures in cells destined to die w22x. The anatomical discrepancy between changes in LCMRglcs and regional c-Fos immunoreactivity can be explained by the fact that c-Fos immunohistochemistry reveals gene activation within specific cell nuclei at the site of activation while glucose utilization reflects changes in the activity of the whole cell body and can reflect either the activity at the site of action, along the whole neuronal pathway affected, or only at the target area w39x. Moreover, as pointed out by others, c-Fos immunoreactivity is unlikely to represent all the structures involved in the seizure events. Indeed, some structures like the substantia nigra, the cerebellar cortex and the reticular formation do not seem to be able to express the c-Fos protein. The lack of immunoreactivity in those regions has been shown in seizures induced by PTZ w2,3,21x, kainic acid w16,31x or lithium-pilocarpine w23x and could be related to the fact that the changes in neuronal activity associated with these drug-induced seizures do not provide the critical stimulus for the expression of c-Fos immunoreactivity w36,37x. 4.3. Lack of expression of HSP72 after PTZ-induced SE in immature rats The lack of expression of HSP72 at 24 and 72 h after PTZ-induced SE in the brain of immature rats is not related to the immaturity of the transcriptional apparatus that facilitates stimulus-transcription coupling since HSP70 and HSP72 genes and proteins are inducible in the brain of the P7 rat following hypoxic-ischemic injury w1,7,15x. However, to our knowledge there is no evidence of the expression of HSP72 after seizures in the immature brain, except in P21 rats after lithium-pilocarpine-induced SE wMotte et al., unpublished datax. But, compared to PTZ seizures that do not induce any damage in the immature rat
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brain w24,29x, lithium-pilocarpine SE leads to extensive neuronal damage in P21 and adult rats w11,32,41x. Thus, the lack of HSP72 expression after PTZ-induced SE in immature rats recorded in the present study could be related to the fact that the changes in neuronal activity induced by these seizures do not provide the critical stimulus for the expression of HSP72, as previously shown for c-Fos immunoreactivity w36,37x. Indeed, in the lithium-pilocarpine model of seizures in the adult rat, HSP72 immunoreactivity never appears before 50 min of severe seizure activity at which time only a very limited number of neurons are labelled while 2–3 h of status epilepticus are necessary to induce a more widespread and strong response of HSP72 w23x. Likewise, in the adult rat brain, PTZ-induced seizures do not lead to the expression of HSP70 mRNA w30x. The lack of expression of HSP after PTZ-induced seizures could relate to the lack of neuronal damage in this model of seizures w24,29x, as recorded in the present study at 24, 72 and 144 h after the induction of SE. In conclusion, the data of the present study show that PTZ-induced SE lasting for 60–80 min induce the early expression of c-Fos immunoreactivity mainly in the dentate gyrus, amygdala and cerebral cortex of P10 and P21 rats while there is no late expression of c-Fos or induction of HSP72 at any age. Thus, c-Fos immunoreactivity can be considered as a good marker of neuronal activation while the lack of late c-Fos and HSP72 expression could reflect the fact that cellular activation does not reach the critical level for induction of the stress protein and of neuronal damage in the present model of PTZ-induced SE in the immature rat. References w1x K.S. Blumenfeld, F.A. Welsh, V.A. Harris, M.A. Pesenson, Regional expression of c-fos and heat shock protein-70 mRNA following hypoxia-ischemia in the immature rat brain, J. Cereb. Blood Flow Metab. 12 Ž1992. 987–995. w2x M. Dragunow, R. Faull, The use of c-fos as a metabolic marker in neuronal pathway tracing, J. Neurosci. Methods 29 Ž1989. 261–265. w3x M. Dragunow, H.A. Robertson, Generalized seizures induce c-fos proteinŽs. in mammalian neurons, Neurosci. Lett. 82 Ž1987. 157–161. w4x M. Dragunow, H.A. Robertson, Kindling stimulation induces c-fos proteinŽs. in granule cells of the rat dentate gyrus, Nature 329 Ž1987. 441–442. w5x M. Dragunow, H.A. Robertson, Localization and induction of c-fos protein-like immunoreactive material in the nuclei of adult mammalian neurons, Brain Res. 440 Ž1988. 252–260. w6x G. El Hamdi, A. Pereira de Vasconcelos, P. Vert, A. Nehlig, An experimental model of generalized seizures for the measurement of local cerebral glucose utilization in the immature rat I. Behavioral characterization and determination of lumped constant, Dev. Brain Res. 69 Ž1992. 231–241. w7x D.M. Ferriero, H.Q. Soberano, R.P. Simon, F.R. Sharp, Hypoxiaischemia induces heat shock protein-like ŽHSP72. immunoreactivity in neonatal rat brain, Dev. Brain Res. 53 Ž1990. 145–150. w8x M.F. Gonzalez, K. Shiraishi, K. Hisanaga, S.M. Sagar, M. Mandabach, F.R. Sharp, Heat shock proteins as markers of neuronal injury, Mol. Brain Res. 93 Ž1989. 93–100.
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Research report
Effects of pentylenetetrazol-induced status epilepticus on c-Fos and HSP72 immunoreactivity in the immature rat brain Jacques E. Motte a
a,b
, Maria Jose´ da Silva Fernandes Astrid Nehlig a,)
a,1
, Christian Marescaux a ,
INSERM U 398, Faculte´ de Medecine, 11 rue Humann, 67085 Strasbourg Cedex, France ´ ´ SerÕice de Neuropediatrie, American Memorial Hospital, 51092 Reims Cedex, France ´ ´
b
Accepted 22 April 1997
Abstract Pentylenetetrazol ŽPTZ.-induced status epilepticus ŽSE. leads to acute and long-term metabolic decreases in specific brain regions of rats at 10 ŽP10. or 21 days after birth ŽP21.. These decreases are not related to apparent neuronal damage. Therefore, to better understand the neuronal activation and stress response to PTZ in immature rats, we mapped the expression of c-Fos and of the 72 kDa heat-shock protein ŽHSP72. in the same model of severe SE induced by the repetitive i.p. injections of subconvulsive doses of PTZ. Rats were sacrificed either at 2 or 24 h after the onset of SE in order to reveal c-Fos immunoreactivity, and at 24 and 72 h for HSP72 expression. Hematoxylin-eosin staining was performed at 24, 72 and 144 h after SE. The expression of c-Fos at 2 h after SE was more marked at P21 than at P10 and was prominent at both ages in the hippocampal dentate gyrus, cerebral cortex and amygdala. Some immunoreactivity was also present in the hypothalamus, thalamus and a few brainstem and cerebellar regions at both ages. There was a good relation between the regions expressing c-Fos and those exhibiting acute metabolic decreases at P21. Conversely, PTZ seizures did not lead to any expression of c-Fos at 24 h after SE or of HSP72 at 24 or 72 h at any age. Cell density was not affected by PTZ-induced SE at any age and at any time. These results suggest that c-Fos is a useful marker of neuronal activation induced by severe and prolonged seizures in the immature brain. The lack of HSP72 and of late c-Fos expression likely reflect the absence of neuronal damage in this model of PTZ-induced SE in the immature rat. q 1997 Elsevier Science B.V. Keywords: c-Fos; Heat shock protein; Seizure; Status epilepticus; Development
1. Introduction In previous studies using pentylenetetrazol ŽPTZ.-induced status epilepticus ŽSE. in immature rats, we showed that local cerebral metabolic rates for glucose ŽLCMRglcs. and local cerebral blood flow ŽLCBF. were increased in all regions of 10-day-old rats. Conversely, in 21-day-old animals, according to the region of interest, metabolic and circulatory levels were increased, decreased or not changed compared to control levels w26,27x. Moreover, PTZ-induced SE led to long-term decreases in LCMRglcs in adult animals subjected to seizures either at 10 ŽP10. or at 21 days after birth ŽP21.. These decreases were especially )
Corresponding author. Fax: q33 Ž3. 8824-3360; E-mail: [email protected] 1 M.J.daS. Fernandes is a Brazilian postdoctoral fellow sponsored by the CNPq. 0169-328Xr97r$17.00 q 1997 Elsevier Science B.V. All rights reserved.
marked when the seizures occurred at P21 and mainly located in the regions where LCMRglcs were decreased during the acute phase of PTZ-induced SE, i.e., mainly cerebral cortex, hippocampus and sensory regions w13x. In the adult rat, metabolic decreases induced by severe and prolonged seizures coincide with neuronal damage w20,25x. Therefore, the acute and long-term metabolic and circulatory decreases related to PTZ-induced SE could be indicative of neuronal suffering in immature rats, as is the case in adult animals w20,25x. However, we have no indication of neuronal damage in this model either at P10 or at P21 w24,29x. Therefore, in order to better understand the relationship between local cerebral functional changes, cellular activation, stress and damage, in the present study, we mapped the expression of the c-Fos and 72-kDa heat shock protein ŽHSP72.. Indeed, the c-fos proto-oncogene undergoes a rapid and transient increase following a variety of external
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stimuli. The expression of its encoded protein product, Fos, appears in specific brain regions after seizures induced by various convulsants or by kindling w3,4,16,21,31,38x and allows the tracing of the specific pathways involved in brain activation. Likewise, the regional immunoreactivity of the inducible HSP72 is expressed in immature rat brain by stressed cells w8x in response to various types of insults, such as hyperthermia w17x, hypoxia-ischemia w7,8,40x and seizures w19,30,42x. The expression of HSP72 represents a sensitive and specific marker of excitation-induced stress and is known to influence the ability of cells to survive to various forms of toxicity, including seizures w18x. Thus, in the present study, we mapped the immunoreactive response of c-Fos and HSP72 to SE induced by PTZ in P10 or P21 rats.
min. Sections were then sequentially incubated twice in PBS, once in 0.6% hydrogen peroxide in PBS and twice in PBS containing 0.4% normal goat serum ŽVector Laboratories, Burlingame, CA, USA. for Fos or horse serum ŽVector. for HSP, 0.25% Triton-X100 and 1.5% bovine serum albumin ŽBSA.. Sections were then incubated overnight at 208C with the primary antibody, a rabbit
2. Materials and methods 2.1. Animals and pharmacological treatment Adult Sprague–Dawley rats ŽIffa–Credo Breeding Laboratories, L’Arbresle, France., one male and two females by cage were housed together in mating groups for 5 days and constantly maintained under standard laboratory conditions on a 12r12 h lightrdark cycle Žlights on at 06.00 h.. After delivery, litter sizes were reduced to 10 pups for homogeneity Žday of birth was considered as day 0.. Experiments were performed on P10 or P21 rats. To reach a SE of controlled intensity and quite long duration, the animals received repeated i.p. injections of PTZ Ž10 mgrml in saline., as previously described w10x. At P10 and P21, the rats were first injected with 40 mgrkg PTZ, followed by 20 mgrkg 10 min later and then, every 10 min, additional i.p. administrations of 10 mgrkg PTZ until SE was reached. This injection procedure induced EEG and behavioral changes characteristic of each age group, P10 or P21 w6,24x. Control animals received the same number of saline injections as their paired PTZ-exposed congeners. For the study of HSP72 expression, the animals were put back with their mother in their normal environment at about 6–8 h after the onset of SE. 2.2. Fos and HSP72 immunohistochemistry The immunohistochemical detection of c-Fos protein was performed in P10 and P21 rats at 2 Ž6–8 animals at each age. and 24 h Ž3 animals at each age. while the expression of HSP72 was measured at 24 Ž6–8 animals at each age. and 72 h Ž3 animals at each age. after the onset of SE. The expression of both proteins was revealed simultaneously in 2 matched control animals at each age. Brains were removed, immediately frozen in isopentane and stored at y808C. Serial coronal sections, 20 m m thick, were cut in a cryostat. Brain sections were fixed in 4% paraformaldehyde dissolved in phosphate buffer saline ŽPBS. at pH 7.4 for 7
Fig. 1. Expression of c-Fos in the hippocampus of P21 control rats ŽA., and of rats subjected to PTZ-induced SE at P10 ŽB. or P21 ŽC.. Note the total absence of labelling in the control hippocampus as well as marked labelling in the granular cell layer of the dentate gyrus in rats exposed to PTZ at both ages. Immunoreactivity of c-Fos is moderate in area CA3 and rather weak in areas CA2 and CA1 of the hippocampus and shows also an overall stronger expression at P21 than at P10. Scale bar s100 m m.
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Fig. 2. Expression of c-Fos in the piriform cortex of control P10 rats ŽA. or of P10 ŽB. and P21 rats ŽC. subjected to PTZ-induced SE, and in the entorhinal cortex of P10 ŽD. and P21 rats ŽE. subjected to PTZ-induced SE. Note the lack of immunoreactivity in the control piriform cortex and the quite marked labelling in the piriform and entorhinal cortex of seizing animals which exhibits a similar distribution at both ages that is stronger in the most superficial layer of rats seizing at P21 compared to those seizing at P10. Scale bar s 100 m m.
affinity-purified polyclonal antibody ŽSanta Cruz Biotechnology, Santa Cruz, CA, USA, dilution 1:500 in PBS containing goat serum. for Fos or a monoclonal anti-72 Kd HSP ŽAmersham, Les Ulis, France, dilution 1:200 in PBS containing horse serum. for the detection of HSP72. The sections were rinsed twice in PBS containing the appropriate serum and incubated for 1 h at 208C with the secondary antibody, biotinylated goat anti-rabbit antibody, dilution 1:400 for Fos ŽVector. and biotinylated anti-mouse antibody, dilution 1:50 for HSP72 ŽVector. in the corresponding serumrTriton-X100rBSArPBS mixture. Sections were rinsed twice in the latter medium and covered with the ABC reagent ŽVectastain Kit, Vector. for 1 h at 208C. Sections were rinsed twice in PBS and incubated for 5–8 min in a mixture of 0.025% diaminobenzidine, 0.01% nickel chloride and 0.05% hydrogen peroxide in PBS. Then, sections were dehydrated in ethanol and coverslipped. In addition to control animals exposed only to saline, immunocytochemical control sections from 2 animals subjected to SE Ž2 for Fos and 2 for HSP72. underwent the procedures described above except for the exposure to the primary antibody which eliminated all staining.
The distribution of positive neurons was recorded from forebrain to cerebellum. Direct visual counting of Fos or HSP72 expressing neurons was performed using a 4-point grading scale of 0–3, with 0 corresponding to the absence of reactive cells and 1, 2 and 3 to low, moderate and high density labelled cells, respectively. 2.3. Rating of cell surÕiÕal Cell density was measured on adjacent sections from P10 and P21 rats taken at 24, 72 and 144 h after SE. Sections were stained by the hematoxylin-eosin method for cell counting that was performed under light microscopy. 3. Results 3.1. Expression of c-Fos In control P10 and P21 rats, all cerebral regions were devoid of c-Fos immunoreactivity ŽFigs. 1 and 2.. At both ages and at 2 h after the onset of SE, the expression of c-Fos induced by PTZ was prominent in the cerebral
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cortex and the dentate gyrus ŽFigs. 1 and 2.. Within the cerebral cortex, the piriform, entorhinal and parietal areas were strongly labelled at both ages and c-Fos expression was also high in the prefrontal cortex at P21. Conversely, c-Fos immunoreactivity was weak in the cingulate cortex, especially at P21. Within forebrain regions, labelling was weak in the septum and the caudate nucleus in which region it was absent at P21. c-Fos expression was moderate in the amygdala at both ages. Within the hippocampus, labelling was virtually absent in the CA1 area, weak in the CA3 region and strong in the dentate gyrus, already at P10 ŽFigs. 1 and 2.. The ventromedian hypothalamus and the medial thalamus were weakly marked at both ages while the paraventricular hypothalamus and the lateral thalamus expressed only c-Fos at P21 and P10, respectively. Within brainstem regions, weak c-Fos labelling appeared only at P21 in the medial geniculate body and substantia nigra, was moderate in the other regions and only strong in the cerebellar nuclei after PTZ-induced SE at P21. Conversely, at 24 h after the onset of SE, no c-Fos immunoreactivity could be detected either at P10 or at P21. 3.2. Expression of HSP72 PTZ-induced SE did not lead to any expression of HSP72 in any brain area and at any time, 24 or 72 h after SE, either at P10 or at P21. The lack of expression of HSP72 in the present model was not related to a technical problem since the same method allowed us to map HSP72 immunoreactivity in adult rats subjected to lithium-pilocarpine-induced SE w23x. 3.3. Cell density The analysis of hematoxylin-eosin stained sections did not reveal any difference in cell density at 24, 72 or 144 h after PTZ-induced SE in P10 or P21 rats. Very few scattered pycnotic nuclei could be seen at both ages at 72 h together with less well delineated cell borders but there was absolutely no visible difference in cell shape and density between control and PTZ-exposed P10 or P21 rats at 144 h. 4. Discussion The present data show that PTZ-induced SE lasting for 60 min leads to an early widespread expression of c-Fos that is quite strong in the cerebral cortex and the dentate gyrus of both P10 and P21 rats. Conversely, there is no late expression of c-Fos or induction of HSP72 by PTZ seizures in the immature rat brain. 4.1. Early expression of c-Fos during seizures in the immature rat brain Our data are in good accordance with previous studies concerning the induction of c-Fos following the adminis-
tration of PTZ. Indeed, a major expression of the protein is present in the cerebral cortex, the dentate gyrus of the hippocampus, the amygdala and some thalamic and hypothalamic nuclei, and virtually absent from most midbrain and brainstem areas, as previously shown with PTZ in adult w3,5,14,21,34,37x or immature rodents w14,34x. However, in previous studies with immature animals, the induction of c-Fos either was not apparent before P20 in the mouse w34x or occurred only at 4 h after a prolonged seizure episode in the P10 rat w14x. However, in the other studies on immature animals, seizures were induced by the injection of a unique dose of the convulsant w14,34x while, in the present study, we injected several subconvulsive doses of PTZ in order to reach a 60–80 min SE. With this injection schedule, there is a time lag of 40–70 min before the onset of SE during which alterations of the EEG and isolated seizures occur w6,12,28x. After the onset of SE, there was no return to basal EEG activity for 30–50 min and numerous spontaneous seizures occurred thereafter w12,28x. Therefore, the expression of c-Fos in P10 rats at 2 h after the onset of SE in the present study could be related to the episodic seizure activity preceding SE in addition to the severe and sustained convulsive activity induced by our injection procedure. The basal expression of the c-fos gene during postnatal development is low until P10 in the cerebral cortex and P13 in the cerebellum and then doubles in both structures at P20 w10x. Likewise, the expression of c-fos mRNA induced by kainate administration is close to basal levels until P7 and doubles in hippocampus between P7 and P13 w35x. The marked expression of the c-Fos protein after PTZ prolonged seizures at P10 ww14x, the present studyx is probably not related to the immaturity of the transcriptional apparatus that facilitates stimulus-transcription coupling since the c-fos gene is inducible in the brain of the P7–P8 rat after hypoxia ischemia w9x and in the brain of the P3 mouse after brain tissue disruption w33x. In the present study, the early c-Fos immunoreactivity at P21 presents many similarities with that recorded in the adult rat after PTZ seizures w37x. Indeed, c-Fos expression is moderate to high in the cerebral cortex, the amygdala, the dentate gyrus, the hypothalamic paraventricular nucleus and the medial thalamus. There is also a slight labelling in the substantia nigra in P21 and in adult rats w37x. 4.2. Relationship between early c-Fos immunoreactiÕity and local cerebral glucose utilization during seizures There is some mismatch between the spatial distribution of early c-Fos immunoreactivity and the changes in LCMRglcs and LCBF induced by acute PTZ seizures in P10 or P21 rats w26,27x. Indeed, in P10 rats, LCMRgls and rates of LCBF largely increased over control values in all brain areas but hippocampus and inferior colliculus where they were similar to control values. In P21 rats, LCMRglcs and
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LCBF rates remained higher than control values in most brainstem areas while they decreased in hippocampus, cerebral cortex, white matter and some thalamic and motor areas w26,27x. However, c-Fos protein is expressed in almost the same regions at both ages, mainly in cerebral cortex, hippocampus and amygdala. Thus, there is no direct correlation between the areas of metabolic activation and those expressing c-Fos but it is interesting to notice that c-Fos is mainly expressed in the areas where LCMRglcs are decreased during acute PTZ seizures at P21 w26,27x or in the long term w13x, i.e., cerebral cortex, amygdala and hippocampus. However, these functional changes occur without any change in cell density, as reported in the present study and could rather reflect a functional reorganization induced by seizures and related cellular activation. Moreover, the lack of expression of c-Fos at 24 h after PTZ-induced seizures recorded at both P10 and P21 is in accordance with the absence of lesions recorded in this model of seizures at both ages. Indeed, it has been shown that the second wave of c-Fos labelling usually occurs 24–48 h after acute seizures in cells destined to die w22x. The anatomical discrepancy between changes in LCMRglcs and regional c-Fos immunoreactivity can be explained by the fact that c-Fos immunohistochemistry reveals gene activation within specific cell nuclei at the site of activation while glucose utilization reflects changes in the activity of the whole cell body and can reflect either the activity at the site of action, along the whole neuronal pathway affected, or only at the target area w39x. Moreover, as pointed out by others, c-Fos immunoreactivity is unlikely to represent all the structures involved in the seizure events. Indeed, some structures like the substantia nigra, the cerebellar cortex and the reticular formation do not seem to be able to express the c-Fos protein. The lack of immunoreactivity in those regions has been shown in seizures induced by PTZ w2,3,21x, kainic acid w16,31x or lithium-pilocarpine w23x and could be related to the fact that the changes in neuronal activity associated with these drug-induced seizures do not provide the critical stimulus for the expression of c-Fos immunoreactivity w36,37x. 4.3. Lack of expression of HSP72 after PTZ-induced SE in immature rats The lack of expression of HSP72 at 24 and 72 h after PTZ-induced SE in the brain of immature rats is not related to the immaturity of the transcriptional apparatus that facilitates stimulus-transcription coupling since HSP70 and HSP72 genes and proteins are inducible in the brain of the P7 rat following hypoxic-ischemic injury w1,7,15x. However, to our knowledge there is no evidence of the expression of HSP72 after seizures in the immature brain, except in P21 rats after lithium-pilocarpine-induced SE wMotte et al., unpublished datax. But, compared to PTZ seizures that do not induce any damage in the immature rat
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brain w24,29x, lithium-pilocarpine SE leads to extensive neuronal damage in P21 and adult rats w11,32,41x. Thus, the lack of HSP72 expression after PTZ-induced SE in immature rats recorded in the present study could be related to the fact that the changes in neuronal activity induced by these seizures do not provide the critical stimulus for the expression of HSP72, as previously shown for c-Fos immunoreactivity w36,37x. Indeed, in the lithium-pilocarpine model of seizures in the adult rat, HSP72 immunoreactivity never appears before 50 min of severe seizure activity at which time only a very limited number of neurons are labelled while 2–3 h of status epilepticus are necessary to induce a more widespread and strong response of HSP72 w23x. Likewise, in the adult rat brain, PTZ-induced seizures do not lead to the expression of HSP70 mRNA w30x. The lack of expression of HSP after PTZ-induced seizures could relate to the lack of neuronal damage in this model of seizures w24,29x, as recorded in the present study at 24, 72 and 144 h after the induction of SE. In conclusion, the data of the present study show that PTZ-induced SE lasting for 60–80 min induce the early expression of c-Fos immunoreactivity mainly in the dentate gyrus, amygdala and cerebral cortex of P10 and P21 rats while there is no late expression of c-Fos or induction of HSP72 at any age. Thus, c-Fos immunoreactivity can be considered as a good marker of neuronal activation while the lack of late c-Fos and HSP72 expression could reflect the fact that cellular activation does not reach the critical level for induction of the stress protein and of neuronal damage in the present model of PTZ-induced SE in the immature rat. References w1x K.S. Blumenfeld, F.A. Welsh, V.A. Harris, M.A. Pesenson, Regional expression of c-fos and heat shock protein-70 mRNA following hypoxia-ischemia in the immature rat brain, J. Cereb. Blood Flow Metab. 12 Ž1992. 987–995. w2x M. Dragunow, R. Faull, The use of c-fos as a metabolic marker in neuronal pathway tracing, J. Neurosci. Methods 29 Ž1989. 261–265. w3x M. Dragunow, H.A. Robertson, Generalized seizures induce c-fos proteinŽs. in mammalian neurons, Neurosci. Lett. 82 Ž1987. 157–161. w4x M. Dragunow, H.A. Robertson, Kindling stimulation induces c-fos proteinŽs. in granule cells of the rat dentate gyrus, Nature 329 Ž1987. 441–442. w5x M. Dragunow, H.A. Robertson, Localization and induction of c-fos protein-like immunoreactive material in the nuclei of adult mammalian neurons, Brain Res. 440 Ž1988. 252–260. w6x G. El Hamdi, A. Pereira de Vasconcelos, P. Vert, A. Nehlig, An experimental model of generalized seizures for the measurement of local cerebral glucose utilization in the immature rat I. Behavioral characterization and determination of lumped constant, Dev. Brain Res. 69 Ž1992. 231–241. w7x D.M. Ferriero, H.Q. Soberano, R.P. Simon, F.R. Sharp, Hypoxiaischemia induces heat shock protein-like ŽHSP72. immunoreactivity in neonatal rat brain, Dev. Brain Res. 53 Ž1990. 145–150. w8x M.F. Gonzalez, K. Shiraishi, K. Hisanaga, S.M. Sagar, M. Mandabach, F.R. Sharp, Heat shock proteins as markers of neuronal injury, Mol. Brain Res. 93 Ž1989. 93–100.
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