Induction of DNA fragmentation and HSP72 immunoreactivity by adenovirus-mediated gene transfer in normal gerbil hippocampus and ventricle

Foreign genes have been successfully transferred and expressed in experimental animal brains using adenoviral vectors. However, it is not fully understood whether adenovirus-mediated gene transfer causes stressful or cytotoxic injury in brain. A replication-defective adenoviral vector containing the Escherichia coli lacZ gene (AdCMVnLacZ) was directly injected into right hippocampus and lateral ventricle of normal gerbil brains. Temporal and spatial profiles of the expression of lacZ gene products, DNA fragmentation detected by terminal deoxynucleotidyl d-UTP nick end labeling (TUNEL) staining, and heat shock protein 72 (HSP72) immunoreactivity were examined until 21 days after the injection. In the ventricle, lacZ gene was immediately and strongly expressed at 8 hr after the injection of AdCMVnLacZ, with a peak at 1–3 days, and disappeared by 21 days. Although a small number of choroid plexus cells were TUNEL positive at 3 and 7 days, no HSP72 immunostaining was observed in the ventricle. Small-to-moderate expression of lacZ gene was found in the needle route from 8 hr to 3 days after the injection, and a small number of TUNEL-positive cells were detected at the needle track at 1–3 days. In the hippocampus, lacZ gene was markedly expressed around the dentate gyrus (DG) at 8 hr to 3 days with a peak at 1 day. Large number of TUNEL or moderate-to-dense HSP70 staining cells were also detected in the same area. CA1 neuronal cells just adjacent to the needle route showed TUNEL positivity at 1 to 3 days. However, the TUNEL staining was not associated with lacZ gene expression. The majority of lacZ-expressing cells were discriminated from the TUNEL-positive cells, whereas some were double-positive with HSP72 staining in DG. Cellular loss was observed in the CA1 layer around the needle route. An apoptotic change was morphologically observed in the marginal region of the DG at 1–3 days and in the ventricle at 3–7 days. In the sham control group, TUNEL-positive or HSP72-staining cells were only detected around the needle track including CA1 cells adjacent to the needle route. These data suggest that adenoviral gene transfer may induce direct traumatic injury in the CA1 sector near the needle route, indirect apoptotic cell loss in the DG and ventricle, and stressful effect on the dentate granule cells in association with adenovirus infection in normal gerbil brain. J. Neurosci. Res. 54:38–45, 1998. © 1998 Wiley-Liss, Inc.     

Expression and changes of galanin in neurons and microglia in the hippocampus after transient forebrain ischemia in gerbils

In the present study, we investigated chronological changes of galanin (GAL), well known as the potassium channel opener, immunoreactivity and GAL protein level in the hippocampus of the gerbil at the various times after 5 min transient forebrain ischemia. In the sham-operated group, weak GAL immunoreactivity was found in non-pyramidal cells. At 12 h after ischemia-reperfusion, the number of GAL-immunoreactive neurons and GAL immunoreactivity were significantly increased in the hippocampus compared to 3 h after ischemic insult, especially in the hippocampal CA1 region. Thereafter the number of GAL-immunoreactive neurons and GAL immunoreactivity decrease time-dependently in the hippocampus. Four days after transient ischemia, GAL immunoreactivity was low as compared with the sham-operated group. At this time point after ischemic insult, GAL immunoreactivity was shown in microglia in the CA1 region because delayed neuronal death happened in the CA1 pyramidal cells. The result of Western blot showed the pattern of GAL expression similar to that of immunohistochemical data. These results suggest that the early increase of GAL in the CA1 pyramidal cells may be associated with the reduction of the excitotoxic damage, that long-lasting enhanced expression of endogenous GAL at 12 h-2 days after ischemia may be associated with efflux of potassium ion into the extracellular space, and that GAL expression in microglia 4 days after ischemia may be associated with reduction of ischemic damage.     

Differential expressions of glycine transporter 1 and three glutamate transporter mRNA in the hippocampus of gerbils with transient forebrain ischemia.

The extracellular concentrations of glutamate and its co-agonist for the N-methyl-d-aspartate (NMDA) receptor, glycine, may be under the control of amino acid transporters in the ischemic brain. However, there is little information on changes in glycine and glutamate transporters in the hippocampal CA1 field of gerbils with transient forebrain ischemia. This study investigated the spatial and temporal expressions of glycine transporter 1 (GLYT1) and three glutamate transporter (excitatory amino acid carrier 1, EAAC1; glutamate/aspartate transporter, GLAST; glutamate transporter 1, GLT1) mRNA in the gerbil hippocampus after 3 minutes of ischemia. The GLYT1 mRNA was transiently upregulated by the second day after ischemia in astrocytelike cells in close vicinity to hippocampal CA1 pyramidal neurons, possibly to reduce glycine concentration in the local extracellular spaces. The EAAC1 mRNA was abundantly expressed in almost all pyramidal neurons and dentate granule cells in the control gerbil hippocampus, whereas the expression level in CA1 pyramidal neurons started to decrease by the fourth day after ischemia in synchrony with degeneration of the CA1 neurons. The GLAST and GLT1 mRNA were rather intensely expressed in the dentate gyrus and CA3 field of the control hippocampus, respectively, but they were weakly expressed in the CA1 field before and after ischemia. As GLAST and GLT1 play a major role in the control of extracellular glutamate concentration, the paucity of these transporters in the CA1 field may account for the vulnerability of CA1 neurons to ischemia, provided that the functional GLAST and GLT1 proteins are also less in the CA1 field than in the CA3 field. This study suggests that the amino acid transporters play pivotal roles in the process of delayed neuronal death in the hippocampal CA1 field.     

Expression of Bax and Bcl-2 protein in the gerbil hippocampus following transient forebrain ischemia and its modification by phencyclidine

Abstract

To determine the effect of phencyclidine (a noncompetitive NMDA receptor antagonist) on expression of Bax and Bcl-2 (apoptosis-regulating proteins) in gerbil hippocampus after transient forebrain ischemia, brain sections were immunohistochemically evaluated 48, 72, 96 hand 7 days following ischemia. In ischemic control animals, the expression of Bax in CA 7 neurons was increased with time and peaked at 72 h, then disappeared at 96 h. In the phencyclidine (5 mg kg^-1 , intraperitoneally)-treated animals, the intensity of Bax expression at 72 h was weaker than that of ischemic control animals. Furthermore, at 96 h, Bax expression was still observed in CA1 neurons. No expression of Bcl-2 in the CA1 neurons was detected in either control or phencyclidine-treated animals. From these results, it is possible that NMDA receptor antagonists exert their preventive effect against delayed neuronal death through inhibition of Bax protein expression, although the precise relationship between the function of Bax protein and delayed neuronal death is still unclear. [Neural Res 1997; 19: 629-633]     

Expression of Heat Shock Protein-70 and Limbic Seizure-induced Neuronal Death in the Rat Brain

The effect of MK-801, a non-competitive N-methyl-D-aspartate (NMDA) antagonist, on the kainic acid-induced expression of the inducible heat shock protein 70 kDa (HSP70) and on neuronal death in the rat hippocampus was investigated. HSP70 is expressed in ?80% of the pyramidal neurons in the CA1 field 1 day after kainic acid injection. The majority of these HSP70-immunopositive neurons exhibited swelling and a hollow appearance in the perikaryon, indicating that they had been injured following kainic acid-elicited limbic seizures. Four days after administration of kainic acid, 87% of the pyramidal neurons in the CA1 field were dead. When a single dose of MK-801 was administered 1 h before kainic acid injection, the number of rats suffering with seizures was reduced, the severity of limbic seizures was attenuated and seizure onset was delayed. Neither HSP70 expression on day 1 nor neuronal loss on day 4 in the CA1 pyramidal cell layer was observed in these animals. A considerable number of HSP70-immunopositive neurons was detected in the dentate hilus, however, and somewhat fewer in the CA3a and CA3c subfields on day 1. Severe neuronal damage in these regions followed on day 4. Interestingly, little HSP70 expression or neuronal loss was observed in the CA3b subfield in these same animals. When a single dose of MK-801 was given 4 h after kainic acid treatment, HSP70 expression was partially blocked; 18% of neurons expressed HSP70 on day 1 and 37% on day 4 in CA1 pyramidal neurons in comparison to the kainic acid controls. About 50% neuronal death was detected in the CA1 pyramidal cell layer 4 days after kainic acid treatment followed by MK-801. When the animals were treated with MK-801 4 h after kainic acid treatment followed by additional daily administration for 3 days, a negligible number of pyramidal neurons expressed HSP70, and the survival of pyramidal cells was significantly increased in the CA1 field. Limbic seizure-induced HSP70 expression not only indicates neuronal injury in the pyramidal cell layer of the hippocampus but also predicts delayed neuronal death, at least in the case of the CA1 field of animals that suffered stage IV—V seizures.     

Global cerebral ischemia due to cardiocirculatory arrest in mice causes neuronal degeneration and early induction of transcription factor genes in the hippocampus

To analyze the role of specific genes and proteins in neuronal signaling cascades following global cerebral ischemia, it would be useful to have a reproducible model of global cerebral ischemia in mice that potentially allows the investigation of mice with specific genomic mutations. We first report on the development of a model of reversible cardiocirculatory arrest in mice and the consequences of such an insult to neuronal degeneration and expression of immediate early genes (IEG) in the hippocampus. Cardiocirculatory arrest of 5 min duration was induced via ventricular fibrillation in mechanically ventilated NMRI mice. After successful cardiopulmonary resuscitation (CPR), animals were allowed to reperfuse spontaneously for 3 h (n=7) and 7 days (n=7). TUNEL staining revealed a selective degeneration of a subset of neurons in the hippocampal CA1 sector at 7 days. About 30% of all TUNEL-positive nuclei showed condensed chromatin and apoptotic bodies. Immunohistochemical studies of IEG expression performed at 3 h exhibited a marked induction of c-Fos, c-Jun, and Krox-24 protein in all sectors of the hippocampus, peaking in vulnerable CA1 pyramidal neurons and in dentate gyrus. In contrast, sham-operated animals (n=3) did not reveal neuronal degeneration or increased IEG expression in the hippocampus when compared with untreated control animals (n=3). In conclusion, we present a new model of global cerebral ischemia and reperfusion in mice with the use of complete cardiocirculatory arrest and subsequent CPR. Following 5 min of ischemia, a subset of CA1 pyramidal neurons was TUNEL-positive at 7 days. The expression of IEG was observed in all sectors of the hippocampus, including selectively vulnerable CA1 pyramidal neurons. This appears to be a good model which should be useful in evaluating the role of various genes in transgenic and knockout mice following global ischemia.     

Regional and time‐dependent neuroprotective effect of hypothermia following oxygen‐glucose deprivation

The neuroprotective effect of hypothermia has been demonstrated in in vivo and in vitro models of cerebral ischemia. In regard to the hippocampus, previous studies have mainly focused on CA1 pyramidal neurons, which are very vulnerable to ischemia. But the dentate gyrus (DG), in which neuronal proliferation occurs, can also be damaged by ischemia. In this study, we explored the neuroprotective effect of postischemic hypothermia in different areas of the hippocampus after mild or severe ischemia. Organotypic hippocampal slice cultures were prepared from 6‐ to 8‐day‐old rats and maintained for 12 days. Cultures were exposed to 25 or 35 min of oxygen and glucose deprivation (OGD). Neuronal damage was quantified after 6, 24, 48, and 72 h by propidium iodide fluorescence. Mild hypothermia (33°C) was induced 1 h after the end of OGD and was maintained for a period of 24 h. Short OGD produced delayed neuronal damage in the CA1 area and in the DG and to a lesser extend in the CA3 area. Damage in CA1 pyramidal cells was totally prevented by hypothermia whereas neuroprotection was limited in the DG. Thirty‐five‐minute OGD induced more rapid and more severe cell death in the three regions. In this case, hypothermia induced 1 h after OGD was unable to protect CA1 pyramidal cells whereas hypothermia induced during OGD was able to prevent cell loss. This study provides evidence that neuroprotection by hypothermia is limited to specific areas and depends on the severity of the ischemia. © 2014 Wiley Periodicals, Inc.     

Cellular localization of epidermal‐type and brain‐type fatty acid‐binding proteins in adult hippocampus and their response to cerebral ischemia

This study aimed at an analysis of expression of epidermal‐type and brain‐type fatty acid‐binding proteins (E‐FABP and B‐FABP, also called FABP5 and FABP7, respectively) in adult hippocampus and their potential value as neuroprotective factors after ischemic brain damage in monkey model. The immunostaining and Western blotting results show that FABP5 was mainly expressed in neurons, whereas FABP7 was primarily expressed in astrocytes and progenitors of the subgranular zone (SGZ). Interestingly, FABP5 expression in neurons increased in cornu Ammonis 1 (CA1) and remains stable within dentate gyrus (DG) after ischemia; FABP7 expression increased within both CA1 and SGZ. This indicates a potential role for FABP5 and FABP7 in intracellular fatty acid transport within different neural cells. The change in FABP5–7 expression within CA1 and DG of the adult postischemic hippocampus was compatible with previous findings of downregulation in CA1 neurons and upregulation in SGZ progenitor cells after ischemia. Altogether, the present data suggest that polyunsaturated fatty acids, such as docosahexaenoic acid, may act via FABP5 or 7 to regulate adult postischemic hippocampal neuronal antiapoptosis or neurogenesis in primates. © 2009 Wiley‐Liss, Inc.     

Synergistic induction of HSP40 and HSC70 in the mouse hippocampal neurons after cerebral ischemia and ischemic tolerance in gerbil hippocampus.

An ischemia-induced gene was screened using a differential display technique in mouse transient forebrain ischemia. One of the ischemia-responsive clones was found to encode mouse hsp40. HSP40 has a critical regulatory function in the HSC70 ATPase activity. Expression of hsp40 mRNA was low in the nonischemic mouse hippocampus, but it was significantly upregulated 4 hr after ischemia by Northern blot analysis. In situ hybridization analysis revealed hsp40 mRNA induction in the neuron. HSP40 protein expression was also enhanced in the pyramidal and dentate granular neurons from 2 to 4 days after ischemia. The temporal expression and distribution profile of HSC70 protein was similar to that of HSP40, and both proteins were colocalized in ischemic hippocampal neurons. In the gerbil transient forebrain ischemia model, both HSP40 and HSC70 proteins were expressed strongly in ischemia-resistant CA3 neurons and dentate granule cells 1 day after 5 min ischemia, but were not expressed in vulnerable CA1 neurons. However, both proteins were in parallel expressed in the tolerance-acquired CA1 neurons. Based on the current observation that both HSP40 and HSC70 proteins were synergistically expressed in the ischemia-resistant and tolerance-acquired neurons, cochaperone HSP40 may play a significant role against postischemic neuronal response and lead to cell survival through interaction with simultaneously induced HSC70.     

Multiple neurotrophic signals converge in surviving CA1 neurons of the gerbil hippocampus following transient forebrain ischemia

Delayed cell death involving the CA1 area of the hippocampus was produced following 5 minutes of transient forebrain ischemia in gerbils. Cell death mainly affected CA1 pyramidal neurons, whereas parvalbumin-immunoreactive (parv-ir) cells were spared. Synaptophysin immunoreactivity was observed in the strata oriens and radiatum of CA1 for months, although immunoreactivity decreased in gerbils surviving 1 year post-ischemia. Golgi studies disclosed a few pyramidal neurons with dendrites, variably covered with dendritic spines, in the CA1 area of 1-year surviving gerbils. In the normal gerbil, the majority of CA1 neurons expressed brain-derived neurotrophic factor (BDNF), tyrosine protein kinase C (TrkC), fibroblast growth factor receptor 1 (Flg), transforming growth factor-alpha (TGF-alpha), and epidermal growth factor-receptor (EGF-R), but only a minority of cells were tyrosine protein kinase B (TrkB)-immunoreactive. Marked reduction in the number of BDNF-, TrkC-, Flg-, TGF-alpha-, and EGF-R-ir cells was observed in CA1 from 24 hours to 1 year after ischemia. In contrast, TrkB-ir cells survived the ischemic insult. Double-labeling immunohistochemistry disclosed that about 90% of surviving BDNF-ir and 85% of TrkB-ir neurons co-localized parvalbumin in the CA1 area. In control gerbils, only about 5% of BDNF-ir cells in CA1 co-expressed TrkB. However, TrkB co-localized in about 95% of surviving BDNF-ir neurons in CA1 in ischemic gerbils. In addition, parvalbumin was co-expressed in about 90% of TrkC-, 95% Flg-, and 85% EGF-R-ir surviving neurons in the stratum pyramidale of CA1. Finally, basic fibroblast growth factor (bFGF) was expressed by reactive astrocytes from day 4 onwards. These data show that the subpopulation of TrkB-/parv-ir neurons in CA1 survive the ischemic episode and that multiple neurotrophic signals converge in surviving neurons of the gerbil hippocampus following transient forebrain ischemia. J. Comp. Neurol. 394:416–430, 1998. © 1998 Wiley-Liss, Inc.     

Protective role of melatonin in domoic acid-induced neuronal damage in the hippocampus of adult rats

Domoic acid (DA), a kainite-receptor agonist and potent inducer of neurotoxicity, has been administered intravenously in adult rats in the present study (0.75 mg/kg body weight) to demonstrate neuronal degeneration followed by glial activation and their involvement with inducible nitric oxide synthase (iNOS) in the hippocampus. An equal volume of normal saline was administered in control rats. The pineal hormone melatonin, which protects the neurons efficiently against excitotoxicity mediated by sensitive glutamate receptor, was administered intraperitoneally (10 mg/kg body weight), 20 min before, immediately after, and 1 h and 2 h after the DA administration, to demonstrate its role in therapeutic strategy. Histopathological analysis (Nissl staining) demonstrated extensive neuronal damage in the pyramidal neurons of CA1, CA3 subfields and hilus of the dentate gyrus (DG) in the hippocampus at 5 days after DA administration. Sparsely distributed glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes were observed in the hippocampus at 4-24 h after DA administration and in the control rats. Astrogliosis was evidenced by increased GFAP immunoreactivity in the areas of severe neuronal degeneration at 5 days after DA administration. Along with this, microglial cells exhibited an intense immunoreaction with OX-42, indicating upregulation of complement type 3 receptors (CR3). Ultrastructural study revealed swollen or shrunken degenerating neurons in the CA1, CA3 subfields and hilus of the DG and hypertrophied astrocytes showing accumulation of intermediate filament bundles in the cytoplasm were observed after administration of DA. Although no significant change could be observed in the mRNA level of iNOS expression between the DA-treated rats and controls at 4-24 h and at 5-day time intervals, double immunofluorescense revealed co-expression of induced iNOS with GFAP immunoreactive astrocytes, but not in the microglial cells, and iNOS expression in the neurons of the hippocampal subfields at 5 days after DA administration. Expression of iNOS was not observed in the hippocampus of control rats. DA-induced neuronal death, glial activation, and iNOS protein expression were attenuated significantly by melatonin treatment and were comparable to the control groups. The results of the present study suggest that melatonin holds potential for the treatment of pathologies associated with DA-induced brain damage. It is speculated that astrogliosis and induction of iNOS protein expression in the neurons and astrocytes of the hippocampus may be in response to DA-induced neuronal degeneration.     

The chronic administration of cerebrolysin induces plastic changes in the prefrontal cortex and dentate gyrus in aged mice

Cerebrolysin (Cbl) is a mixture of neuropeptides with effects similar to the endogenous neurotrophic factors and is considered one of the best drugs used in the treatment of dementias such as Alzheimer's disease (AD). In brains with AD, morphological changes in the dendrites of pyramidal neurons of the prefrontal cortex (PFC) and hippocampus have been reported. These changes are reflected particularly in the decrement of both the dendritic tree and spine density. Here we evaluated the effect of this drug on the dendrites of pyramidal neurons of the PFC and CA1 dorsal hippocampus and granule cells from the dentate gyrus (DG) and medium spiny neurons of the nucleus accumbens (NAcc) of aged mice. Cbl (5 ml kg(-1) , i.p.) was administered daily for 60 days to 6-month-old mice. Dendritic morphology was studied by the Golgi-Cox stain procedure followed by Sholl analysis at 8 months ages. In all Cbl-treated mice a significant increase in dendritic spine density and dendritic length in pyramidal neurons of the PFC and granule cells of the DG was observed. Interestingly, the enhancement in dendritic length was close to the soma in pyramidal neurons of the PFC whereas in granule neurons of the DG the increase in dendritic length was further from the soma. Our results suggest that Cbl induces plastic modifications of dendritic morphology in the PFC and DG. These changes may explain the therapeutic effect seen in AD patients treated with Cbl.     

蛋白偶联受体GPR40在缺血性成年猴海马的表达

目的 利用猴短暂性全脑缺血模型,研究蛋白偶联受体40(GPR40)在海马的表达,从而探讨GPR40受体在缺血性卒中的作用.方法 参照Yamashima建立成年猴短暂性全脑缺血模型,24只猴随机数字表法分成4组,即对照组、缺血后4 d组(d4)、缺血后9 d组(d9)和缺血后15 d(d15),采用Western印迹和免疫荧光在蛋白质水平观察GPR40的表达.结果 Western blotting结果显示CA1区域GPR40在短暂性全脑缺血后表达逐渐下降(15 d内);而在DG区域表达升高,与对照组比较有统计学意义(P<0.05).免疫荧光分析显示CA1区域与GPR40共表达的神经元细胞数逐渐下降.到d15约下降50%;而DG区域则无明显变化,邻近SGZ区域与GPR40共表达的星形细胞数量迅速升高,d4升高约2.5倍.结论 脑缺血后海马CA1和DG两区域GPR40表达差异.特别SGZ星形细胞GPR40表达升高,提示可通过增加GPR40配体中长链不饱和脂肪酸如DHA来改善脑缺血后海马神经元的损伤.     

Selective vulnerability in the gerbil hippocampus following transient ischemia

Summary Following brief ischemia, the Mongolian gerbil is reported to develop unusual hippocampal cell injury (Brain Res 239:57–69, 1982). To further clarify this hippocampal vulnerability, gerbils were subjected to ischemia for 3, 5, 10, 20, and 30 min by bilateral occlusion of the common carotid arteries. They were perfusion-fixed after varying intervals of survival time ranging from 3 h up to 7 days. Following brief ischemia (5–10min), about 90% of the animals developed typical hippocampal damage. The lesion was present throughout the extent of the dorsal hippocampus, whereas damage outside the hippocampus was not observed. Each sector of the hippocampus showed different types of cell reaction to ischemia. Ischemic cell change was seen in scattered CA4 neurons, and reactive change was found in CA2, whereas CA1 pyramidal cells developed a strikingly slow cell death process. Ischemia for 3 min did not produce hippocampal lesion in most cases. Following prolonged ischemia (20–30min), brain injury had a wide variety in its extent and distribution. These results revealed that the gerbil brief ischemia model can serve as an excellent, reliable model to study the long-known hippocampal selective vulnerability to ischemia. Delayed neuronal death in CA1 pyramidal cells was confirmed after varying degrees of ischemic insult. These findings demonstrated that the pathology of neuronal injury following brief ischemia was by no means uniform nor simple.     

Microglial reaction and neuronal death in the hippocampus of rat models of epilepsy

Reaction of microglial cells as well as DNA fragmentation in pyramidal cells was investigated using immunohisto-chemistry and in situ end-labeling method (TUNEL) in the hippocampus of rats after rapid kindling or kainic acid treatment. In intact rats, no or very little DNA fragmentation was detected in the hippocampus. Resting microglia distributed evenly throughout the hippocampus. Neither major histocompatibility complex antigens class I (MHC I) nor class II (MHC II) immunoreactivity was seen in the hippocampus. In the rapid-kindling model, no DNA fragmentation, reactive microglia or MHC antigen-positive cells were present in the hippocampus. In rats given an intraperitoneal injection of kainic acid (12 mg/kg), reactive microglial cells were seen around pyramidal neurons in the CA1 and CA3 field of the hippocampus as well as in the hilus of the dentate gyrus at 3 h. At that point in time, DNA fragmentation was not detected. DNA fragmentation was clearly observed, mainly in the CA1 region of the hippocampus, from 24 h to 4 weeks after the kainic acid injection. The number of reactive microglia was quickly increased and reached a maximum at 7 days after the injection, and continued until 8 weeks thereafter. During this period, many reactive microglia expressed MHC I and MHC II. The present study indicates that epileptic seizures do not depend on microglial activation and that microglial activation is closely related to the neuronal death process induced by kainic acid.     

Non-phosphorylated neurofilament protein immunoreactivity in adult and developing rat hippocampus: specificity and application in grafting studies

Neurofilament proteins are critical to the development and maintenance of neuronal shape in the nervous system. These proteins are developmentally regulated and several transition forms are expressed, prior to full neuronal stabilization. We have studied the spatial distribution and time course of expression of non-phosphorylated neurofilament protein (NPNFP) immunoreactivity in several preparations of rat hippocampus, using a mixture (SMI 311) of several monoclonal antibodies directed against NPNFP epitopes. Differential staining was observed in young and adult hippocampus. Large pyramidal neurons in CA3 and CA4 subfields were strongly immunoreactive in adult hippocampus whereas the smaller CA1 pyramidal neurons, most interneurons and dentate granule cells were immunonegative. SMI 311 staining initially appeared at postnatal day (P) 5 with positive staining in apical dendrites and soma in a few pyramidal neurons in CA3, but almost reached the adult pattern by P10. Compared to adult hippocampus, the number of immunoreactive interneurons in all subfields appeared increased at P10 and P15. In cultures of embryonic hippocampus, all neurons, regardless of their morphology, were SMI 311 positive, suggesting loss of differential expression in tissue culture conditions. However, SMI 311 expression in fetal hippocampal neurons grafted to adult hippocampus was similar to hippocampal neurons which had developed in situ. These results suggest that SMI 311 antibody identifies a distinct group of primarily CA3 and CA4 pyramidal cells in adult hippocampus. The application of SMI 311 immunostaining appears suitable for identification of large CA3 and CA4 pyramidal neurons within hippocampal transplants grafted to adult CNS but not in tissue culture.