Effects of fluoxetine on ischemic cells and expressions in BDNF and some antioxidants in the gerbil hippocampal CA1 region induced by transient ischemia

Fluoxetine, a selective serotonin reuptake inhibitor, alters several physiological processes, for example, elevating intracellular cAMP level, in the hippocampus. We examined the effect of fluoxetine on ischemia-induced neuronal death, the expression of brain-derived neurotrophic factor (BDNF) and changes in some antioxidative enzymes in the hippocampal CA1 region induced by transient ischemia. In addition, we also studied the effect of fluoxetine on locomotor activity in gerbils after ischemia/reperfusion. Animals were administered with various doses of fluoxetine (10, 20, and 40 mg/kg, i.p.) once daily for 3 days before the ischemic surgery. The treatment of 10 mg/kg and 20 mg/kg fluoxetine did not show significant neuroprotective effects on CA1 pyramidal cells 4 days after ischemia/reperfusion, while the treatment with 40 mg/kg fluoxetine in ischemic animals showed about 77% neuronal survival rate compared to the control group. The treatment of 40 mg/kg fluoxetine in ischemic animals enhanced significantly BDNF, catalase (CAT), glutathione peroxidase (GPX), and superoxide dismutase-1 (SOD1) immunoreactivity in the CA1 region compared to those in the saline-treated group 4 days after ischemia/reperfusion. In addition, the treatment of fluoxetine (10, 20, 40 mg/kg) significantly inhibited post-ischemic hyperactivity. In brief, treatment with fluoxetine protects neuronal damage after transient ischemia, and the neuroprotective effect of fluoxetine in an ischemic animal model may be related with the up-regulation of BDNF, CAT, GPX, and SOD1 expression.     

Changes in immunoreactivity of HSP60 and its neuroprotective effects in the gerbil hippocampal CA1 region induced by transient ischemia

Heat shock proteins act as molecular chaperones and are involved in protein folding, refolding, transport, and translocation. In the present study, we observed changes in heat shock protein 60 (HSP60) immunoreactivity and protein level in the gerbil hippocampal CA1 region after 5 min of transient forebrain ischemia and its neuroprotective effect against ischemic damage. HSP60 immunoreactivity in the CA1 region began to increase in the stratum pyramidale at 30 min after ischemia/reperfusion, and peaked 24 h after ischemia/reperfusion. Thereafter, HSP60 immunoreactivity was decreased in the CA1 region with time. Seven days after ischemia/reperfusion, HSP60 immunoreactivity was increased again in the CA1 region: at this time point after ischemia/reperfusion, HSP60 immunoreactivity was expressed in glial cells in the ischemic CA1 region. HSP60 immunoreactive glial cells were astrocytes containing glial fibrillar acidic protein. In contrast, change in HSP60 immunoreactivity in the ischemic CA2/3 region was not significant compared with that in the ischemic CA1 region. In Western blot study, HSP60 protein level in the CA1 region was increased after ischemia/reperfusion and highest 24 h after ischemia/reperfusion. Animals treated with recombinant adenoviruses expressing Hsp60 (Ad-Hsp60) showed the neuroprotection of CA1 pyramidal neurons from ischemic damage. These results suggest that HSP60 may be associated with delayed neuronal death of CA1 pyramidal neurons after transient ischemia, and the induction of HSP60 protects the neurons from ischemic damage.     

The immunoreactivity and activity of adenylate cyclase type I are changed in the hippocampal CA1 region after transient forebrain ischemia in gerbils

Adenylate cyclase (AC) has a specific sensitivity to Ca²⁺/calmodulin. AC-I, one of the mediator of learning and memory, plays an important role in signal transduction underlying learning and memory function. In the present study, we found ischemia-related changes of AC-I in the hippocampal CA1 region, but not in the CA2/3 region, after 5 min of transient forebrain ischemia in gerbils. In the sham-operated group, AC-I immunoreactive neurons were detected in pyramidal and non-pyramidal cells in the hippocampus proper. AC-I immunoreactivity was significantly increased at 3 h in the CA1 region after ischemic insult. Thereafter, AC-I immunoreactivity was gradually decreased. Four days after ischemic insult, AC-I-immunoreactive CA1 pyramidal cells in the stratum pyramidale were very few due to delayed neuronal death. The results of Western blot analysis showed that changes of AC-I protein contents were similar to immunohistochemical data after ischemic insult. Gpp(NH)p-dependent AC-I activity in hippocampal CA1 region was not changed in all groups, while Ca²⁺/calmodulin-dependent AC-I activity in hippocampal CA1 region was significantly decreased 24 h after ischemia–reperfusion. These results suggest that the decrease of AC-I activity may be associated with impairment of neurodevelopment and neuroplasticity including learning and memory although the AC-I immunoreactivity was maintained 24 h postischemic group compared to that of the sham-operated group.     

Elevation of Na+-K+ ATPase immunoreactivity in GABAergic neurons in gerbil CA1 region following transient forebrain ischemia

In a previous study, we suggested that GABAergic neurons might be resistant to ischemic insult, because of the maintenance of the GABA shunt, which is one of the ATP synthetic pathways in neurons. In the present study, we identified Na(+)-K(+) ATPase immunoreactivity in the gerbil hippocampus in order to determine whether changes in Na(+)-K(+) ATPase immunoreactivity correlate with GABA shunt following ischemic insult. At 12 h after ischemia-reperfusion, Na(+)-K(+) ATPase immunoreactivity accumulated in some neurons in the CA1 region. However, the protein content of Na(+)-K(+) ATPase was not altered. Interestingly, the density of Na(+)-K(+) ATPase immunoreactivity in neurons and the protein content in the CA1 region was intensified in the 24 h post-ischemic group. As a result of double immunofluorescence study, Na(+)-K(+) ATPase immunoreactive neurons were identified with GABAergic neurons. Therefore, our findings suggest that the increase of Na(+)-K(+) ATPase in GABAergic neurons may be able to explain the resistance of these cells to ischemic insult, and support our previous hypothesis that GABA may play an important role as a metabolite in the survival of GABAergic neurons after ischemic insult.     

Characterization of Na+/H+ exchange activity in cultured rat hippocampal astrocytes

Astrocytes actively maintain their intracellular pH (pH_i) more alkaline than expected by passive distribution of H⁺. Acid extruding transporters such as the amiloride-sensitive Na⁺/H⁺ exchanger (NHE) are necessary for pH regulation. Currently, four mammalian NHEs (NHE1-NHE4) have been cloned, with a fifth (NHE5) partially cloned. We attempted to determine which isoform(s) of NHE was present in cultured hippocampal astrocytes using amiloride sensitivity and immunospecificity as criteria. In the absence of HCO₃⁻, amiloride blocked pH_i recovery after an acid load with an IC₅₀ of ∼3.18 μM, similar to values reported for the amiloride-sensitive isoforms NHE1 and NHE2. Immunoblotting with a highly specific antibody for NHE1 identified a 100 kDa protein, indicating the presence of NHE1 in whole brain, hippocampus, and cultured hippocampal astrocytes. Further probing for an additional amiloride-sensitive NHE failed to detect evidence of the presence of NHE4. Surprisingly, application of the potent analog of amiloride, ethylisopropylamiloride (EIPA), caused a reversible alkalinization of pH_i, suggesting the presence of an additional acid/base transport mechanism that is EIPA-sensitive. © 1996 Wiley-Liss, Inc.     

Characterization of astrocytes and microglial cells in the hippocampal CA1 region after transient focal cerebral ischemia in rats treated with Ilexonin A

Ilexonin A is a compound isolated from the root of Ilex pubescens,a traditional Chinese medicine.Ilexonin A has been shown to play a neuroprotective role by regulating the activation of astrocytes and microglia in the peri-infarct area after ischemia.However,the effects of ilexonin A on astrocytes and microglia in the infarct-free region of the hippocampal CA1 region remain unclear.Focal cerebral ischemia models were established by 2-hour occlusion of the middle cerebral artery in rats.Ilexonin A(20,40 or 80 mg/kg)was administered immediately after ischemia/reperfusion.The astrocyte marker glial fibrillary acidic protein,microglia marker Iba-1,neural stem cell marker nestin and inflammation markers were detected by immunohistochemistry and western blot assay.Expression levels of tumor necrosis factor-αand interleukin 1βwere determined by enzyme linked immunosorbent assay in the hippocampal CA1 tissue.Astrocytes were activated immediately in progressively increasing numbers from 1,3,to 7 days post-ischemia/reperfusion.The number of activated astrocytes further increased in the hippocampal CA1 region after treatment with ilexonin A.Microglial cells remained quiescent after ischemia/reperfusion,but became activated after treatment with ilexonin A.Ilexonin A enhanced nestin expression and reduced the expression of tumor necrosis factor-αand interleukin 1βin the hippocampus post-ischemia/reperfusion.The results of the present study suggest that ilexonin A has a neuroprotective effect in the hippocampus after ischemia/reperfusion,probably through regulating astrocytes and microglia activation,promoting neuronal stem cell proliferation and reducing the levels of pro-inflammatory factors.This study was approved by the Animal Ethics Committee of the Fujian Medical University Union Hospital,China.     

Time- and cell-type speciifc changes in iron,ferritin, and transferrin in the gerbil hippocampal CA1 region after transient forebrain ischemia

In the present study, we used immunohistochemistry and western blot analysis to examine changes in the levels and cellular localization of iron, heavy chain ferritin (ferritin-H), and transferrin in the gerbil hippocampal CA1 region from 30 minutes to 7 days following transient forebrain ischemia. Relative to sham controls, iron reactivity increased signiifcantly in the stratum pyramidale and stratum oriens at 12 hours following ischemic insult, transiently decreased at 1–2 days and then increased once again within the CA1 region at 4–7 days after ischemia. One day after ischemia, ferritin-H immunoreactivity increased significantly in the stratum pyramidale and decreased at 2 days. At 4–7 days after ischemia, ferritin-H immunoreactivity in the glial components in the CA1 region was signiifcantly increased. Transferrin im-munoreactivity was increased signiifcantly in the stratum pyramidale at 12 hours, peaked at 1 day, and then decreased signiifcantly at 2 days after ischemia. Seven days after ischemia, Transferrin immunoreactivity in the glial cells of the stratum oriens and radiatum was signiifcantly increased. Western blot analyses support-ed these results, demonstrating that compared to sham controls, ferritin H and transferrin protein levels in hippocampal homogenates significantly increased at 1 day after ischemia, peaked at 4 days and then decreased. These results suggest that iron overload-induced oxidative stress is most prominent at 12 hours after ischemia in the stratum pyramidale, suggesting that this time window may be the optimal period for therapeutic intervention to protect neurons from ischemia-induced death.     

Late expression of Na+/H+ exchanger 1 (NHE1) and neuroprotective effects of NHE inhibitor in the gerbil hippocampal CA1 region induced by transient ischemia

Although acidosis may be involved in neuronal death, the participation of Na⁺/H⁺ exchanger (NHE) in delayed neuronal death in the hippocampal CA1 region induced by transient forebrain ischemia has not been well established. In the present study, we investigated the chronological alterations of NHE1 in the hippocampal CA1 region using a gerbil model after ischemia/reperfusion. In the sham-operated group, NHE1 immunoreactivity was weakly detected in the CA1 region. Two and 3 days after ischemia/reperfusion, NHE1 immunoreactivity was observed in glial components, not in neurons, in the CA1 region. Four days after ischemia/reperfusion, NHE1 immunoreactivity was markedly increased in CA1 pyramidal neurons as well as glial cells. These glial cells were identified as astrocytes based on double immunofluorescence staining. Western blot analysis also showed that NHE protein level in the CA1 region began to increase 2 days after ischemia/reperfusion. The treatment of 10 mg/kg 5-(N-ethyl-N-isopropyl) amiloride, a NHE inhibitor, significantly reduced the ischemia-induced hyperactivity 1day after ischemia/reperfusion. In addition, NHE inhibitor potently protected CA1 pyramidal neurons from ischemic damage, and NHE inhibitor attenuated the activation of astrocytes and microglia in the ischemic CA1 region. In addition, NHE inhibitor treatment blocked Na⁺/Ca²⁺ exchanger 1 immunoreactivity in the CA1 region after transient forebrain ischemia. These results suggest that NHE1 may play a role in the delayed death, and the treatment with NHE inhibitor protects neurons from ischemic damage.     

Effect of ischemic preconditioning on antioxidant status in the gerbil hippocampal CA1 region after transient forebrain ischemia

Ischemic preconditioning (IPC) is a condition of sublethal transient global ischemia and exhibits neuro-protective effects against subsequent lethal ischemic insult. We, in this study, examined the neuroprotective effects of IPC and its effects on immunoreactive changes of antioxidant enzymes including superoxide dismutase (SOD) 1 and SOD2, catalase (CAT) and glutathione peroxidase (GPX) in the gerbil hippocampal CA1 region after transient forebrain ischemia. Pyramidal neurons of the stratum pyramidale (SP) in the hippocampal CA1 region of animals died 5 days after lethal transient ischemia without IPC (8.6%(ratio of remanent neurons) of the sham-operated group);however, IPC prevented the pyramidal neurons from subsequent lethal ischemic injury (92.3%(ratio of remanent neurons) of the sham-operated group). SOD1, SOD2, CAT and GPX immunoreactivities in the sham-operated animals were easily detected in pyramidal neurons in the stratum pyramidale (SP) of the hippocampal CA1 region, while all of these immunoreac-tivities were rarely detected in the stratum pyramidale at 5 days after lethal transient ischemia without IPC. Meanwhile, their immunoreactivities in the sham-operated animals with IPC were similar to (SOD1, SOD2 and CAT) or higher (GPX) than those in the sham-operated animals without IPC. Furthermore, their immunoreactivities in the stratum pyramidale of the ischemia-operated animals with IPC were steadily maintained after lethal ischemia/reperfusion. Results of western blot analysis for SOD1, SOD2, CAT and GPX were similar to immunohistochemical data. In conclusion, IPC maintained or increased the expression of antioxidant enzymes in the stratum pyramidale of the hippocampal CA1 region after subsequent lethal transient forebrain ischemia and IPC exhibited neuroprotective effects in the hippocampal CA1 region against transient forebrain ischemia.     

Effect of nilvadipine on the voltage-dependent Ca channels in rat hippocampal CA1 pyramidal neurons

Effects of nilvadipine on the low- and high-voltage activated Ca²⁺ currents (LVA and HVA I_Ca, respectively) were compared with other organic Ca²⁺ antagonists in acutely dissociated rat hippocampal CA1 pyramidal neurons. The inhibitory effects of nilvadipine, amlodipine and flunarizine on LVA I_Ca were concentration- and use-dependent. The apparent half-maximum inhibitory concentrations (IC₅₀s) at every 1- and 30-s stimulation were 6.3×10⁻⁷ M and 1.8×10⁻⁶ M for flunarizine, 1.9×10⁻⁶ M and 7.6×10⁻⁶ M for nilvadipine, and 4.0×10⁻⁶ M and 8.0×10⁻⁶ M for amlodipine, respectively. Thus, the strength of the use-dependence was in the sequence of nilvadipine>flunarizine>amlodipine. Nilvadipine also inhibited the HVA I_Ca in a concentration-dependent manner with an IC₅₀ of 1.5×10⁻⁷ M. The hippocampal CA1 neurons were observed to have five pharmacologically distinct HVA Ca²⁺ channel subtypes consisting of L-, N-, P-, Q- and R-types. Nilvadipine selectively inhibited the L-type Ca²⁺ channel current which comprised 34% of the total HVA I_Ca. On the other hand, amlodipine non-selectively inhibited the HVA Ca²⁺ channel subtypes. These results suggest that the inhibitory effect of nilvadipine on the neuronal Ca²⁺ influx through both LVA and HVA L-type Ca²⁺ channels, in combination with the cerebral vasodilatory action, may prevent neuronal damage during ischemia.     

The NCX3 isoform of the Na+/Ca2+ exchanger contributes to neuroprotection elicited by ischemic postconditioning

It has been recently shown that a short sublethal brain ischemia subsequent to a prolonged harmful ischemic episode may confer ischemic neuroprotection, a phenomenon termed ischemic postconditioning. Na⁺/Ca²⁺ exchanger (NCX) isoforms, NCX1, NCX2, and NCX3, are plasma membrane ionic transporters widely distributed in the brain and involved in the control of Na⁺ and Ca²⁺ homeostasis and in the progression of stroke damage. The objective of this study was to evaluate the role of these three proteins in the postconditioning-induced neuroprotection. The NCX protein and mRNA expression was evaluated at different time points in the ischemic temporoparietal cortex of rats subjected to tMCAO alone or to tMCAO plus ischemic postconditioning. The results of this study showed that NCX3 protein and ncx3 mRNA were upregulated in those brain regions protected by postconditioning treatment. These changes in NCX3 expression were mediated by the phosphorylated form of the ubiquitously expressed serine/threonine protein kinase p-AKT, as the p-AKT inhibition prevented NCX3 upregulation. The relevant role of NCX3 during postconditioning was further confirmed by results showing that NCX3 silencing, induced by intracerebroventricular infusion of small interfering RNA (siRNA), partially reverted the postconditioning-induced neuroprotection. The results of this study support the idea that the enhancement of NCX3 expression and activity might represent a reasonable strategy to reduce the infarct extension after stroke.     

Chronological alterations of calbindin D-28k immunoreactivity in the gerbil main olfactory bulb after ischemic insult

We investigated spatial and temporal alterations of calbindin D-28k (CB) immunoreactivity in the gerbil main olfactory bulb after transient ischemia-reperfusion. In sham-operated animals, CB-immunoreactive (IR) neurons were found in the periglomerular layer, external plexiform layer and granule cell layer. At 1-4 days after ischemic insult, the number of CB-IR neurons significantly increased. This result suggests that the increased CB may buffer the intracellular calcium at an early time point after the ischemic insult. In contrast, 10-30 days after the ischemic insult, the number of CB-IR neurons significantly decreased as compared to sham-operated animals. This result suggests that a malfunction in olfactory process may have occurred in the olfactory bulb at a later time point after the ischemic insult.     

Stimulation-induced RSA-like field activity in region CA1 of the hippocampal slice: Amplitude maxima and topography

In the present experiment RSA-patterned stimulation was applied to afferents in the CA1 region of the hippocampal slice preparation and depth profiles of the resultant field activity were performed. RSA-patterned stimulation applied to the stratum lacunosum-moleculare region resulted in field activity through the CA1 region with similar morphology, phase, and amplitude profiles to the type 1 RSA profile found in vivo. In contrast, RSA-patterned stimulation applied to the stratum oriens and midstratum radiatum regions did not result in field profiles characteristic of the RSA recorded in vivo. The results of the present study confirm predictions made by previous modelling experiments and CSD analyses performed in the freely moving animal, which suggest that the type 1 RSA profile is primarily the result of distal excitation onto CA1 pyramidal cells.     

Somatosensory stimulation suppresses the excitability of pyramidal cells in the hippocampal CA1 region in rats

The hippocampal region of the brain is important for encoding environment inputs and memory formation. However, the underlying mechanisms are unclear. To investigate the behavior of indi-vidual neurons in response to somatosensory inputs in the hippocampal CA1 region, we recorded and analyzed changes in local ifeld potentials and the ifring rates of individual pyramidal cells and interneurons during tail clamping in urethane-anesthetized rats. We also explored the mechanisms underlying the neuronal responses. Somatosensory stimulation, in the form of tail clamping, chan-ged local ifeld potentials into theta rhythm-dominated waveforms, decreased the spike ifring of py-ramidal cells, and increased interneuron ifring. In addition, somatosensory stimulation attenuated orthodromic-evoked population spikes. These results suggest that somatosensory stimulation sup-presses the excitability of pyramidal cells in the hippocampal CA1 region. Increased inhibition by local interneurons might underlie this effect. These ifndings provide insight into the mechanisms of signal processing in the hippocampus and suggest that sensory stimulation might have thera-peutic potential for brain disorders associated with neuronal hyperexcitability.     

Antioxidant-like protein 1 is altered in non-pyramidal cells and expressed in astrocytes in the gerbil hippocampal CA1 region after transient forebrain ischemia

In the present study, we observed chronological changes of antioxidant-like protein 1 (AOP-1) in the gerbil hippocampal CA1 region after 5 min of transient forebrain ischemia using immunohistochemistry and western blot. AOP-1 was significantly altered in the CA1 region after transient ischemia. In the sham-operated group, AOP-1 immunoreactivity was detected in pyramidal and non-pyramidal cells of the CA1 region. At 30 min after ischemic insult, AOP-1 immunoreactivity and protein level was decreased in the CA1 region. At 12 h after ischemic insult, AOP-1 immunoreactivity and protein level was highest in this region. At this time, after ischemia, AOP-1 immunoreactivity in non-pyramidal cells was high compared to the sham-operated group. Based on double immunofluorescence study, AOP-1-immunoreactive neurons were identified as GABAergic, which were stained with GAD or parvalbumin. Thereafter, AOP-1 immunoreactivity and protein levels were decreased time-dependently. From 4 days after ischemic insult, AOP 1 immunoreactivity was generally expressed in astrocytes. Five days after ischemic insult, AOP-1 immunoreactivity and protein level was increased again to 1.4 folds compared to that of the sham-operated group. In brief, AOP-1 immunoreactivity was increased in GABAergic non-pyramidal cells in the hippocampal CA1 region at early time after ischemic insult and was expressed in astrocytes at late time after ischemia. This result suggests that AOP-1 may be important role in homeostasis of GABAergic neurons because these neurons are resistant to ischemic damage.     

Lesions of excitatory pathways reduce hippocampal cell death after transient forebrain ischemia in the gerbil

Summary Transient forebrain ischemia produces a spatially and temporally selective pattern of neuronal degeneration in the hippocampal formation of the Mongolian gerbil. Ischemic neuronal death has been suggested to depend on the activation of excitatory hippocampal pathways that project to the vulnerable neurons. This idea was tested by examining the effect of a unilateral entorhinal cortical lesion or a unilateral knife cut lesion of intrahippocampal pathways on the neuropathology produced by 5 min of complete fore-brain ischemia. A prior lesion of either the ipsilateral entorhinal cortex or the mossy fiber and Schaffer collateral-commissural pathways partially prevented the destruction of CA1b pyramidal cells in most animals. It did not, however, reduce the extent of ischemic neuronal death in any other hippocampal subfield. Within area CA1b, an entorhinal lesion protected an average of 23% of the pyramidal cells and a transection of both mossy and Schaffer collateral-commissural fibers protected an average of 36.5%. CA1b pyramidal cells saved from ischemia-induced degeneration appeared clearly abnormal when stained with cresyl violet or by silver impregnation. It is suggested that lesions of excitatory pathways attenuate ischemic damage to area CA1b by directly or indirectly reducing the level of synaptic excitation onto the vulnerable neurons. However, only a relatively small percentage of hippocampal neurons can be protected by these lesions in the gerbil ischemia model and there is reason to believe that the neurons protected in this manner may not be electrophysiologically competent. Synaptic excitation therefore appears to play an important, but not an essential, role in this model of ischemic brain damage.Supported by NIH Stroke Center grant NS 06233     

Differential contributions of Ca2+‐activated K+ channels and Na+/K+‐ATPases to the generation of the slow afterhyperpolarization in CA1 pyramidal cells

In many types of CNS neurons, repetitive spiking produces a slow afterhyperpolarization (sAHP), providing sustained, intrinsically generated negative feedback to neuronal excitation. Changes in the sAHP have been implicated in learning behaviors, in cognitive decline in aging, and in epileptogenesis. Despite its importance in brain function, the mechanisms generating the sAHP are still controversial. Here we have addressed the roles of M‐type K⁺ current (I_M), Ca²⁺‐gated K⁺ currents (I_Ca(K)'s) and Na⁺/K⁺‐ATPases (NKAs) current to sAHP generation in adult rat CA1 pyramidal cells maintained at near‐physiological temperature (35 °C). No evidence for I_M contribution to the sAHP was found in these neurons. Both I_Ca(K)'s and NKA current contributed to sAHP generation, the latter being the predominant generator of the sAHP, particularly when evoked with short trains of spikes. Of the different NKA isoenzymes, α₁‐NKA played the key role, endowing the sAHP a steep voltage‐dependence. Thus normal and pathological changes in α₁‐NKA expression or function may affect cognitive processes by modulating the inhibitory efficacy of the sAHP.     

NADH in the pyramidal cell layer of hippocampal regions CA1 and CA3 upon selective inhibition and uncoupling of oxidative phosphorylation

N₂-laser-induced fluorescence in combination with the time and spectral resolution of fluorescent NADH molecules allows on-line measurement of relative NADH concentration with high spatial resolution (diameter of optical fibre 200 μm, λ_exc = 337 nm, λ_det = 460 nm). Energy metabolism was impaired in submerged rat hippocampal slices using the inhibitors amytal, 3-nitropropionate (3-np), sodium cyanide (1 mM each) and the uncoupling agent 2,4-DNP (200 μM). A microprocessor-controlled repeated positioning of the optical fibre in CA1 and CA3 pyramidal cell layers, and CA1 stratum radiatum (CA1_SR). Time-dependently, NADH fluorescence increased reversibly upon perfusion with amytal and cyanide. It was unchanged by perfusion with 3-np for 40 min and rapidly decreased upon perfusion with 2,4-DNP. The CAl/CA3 ratio of NADH fluorescence mildly decreased to 0.92 ± 0.04 (mean ± S.D.) at 10 min (P < 0.05) and 0.89 ± 0.05 at 211 min (P < 0.01) upon perfusion with amytal. The CA1/CA3 ratio increased to 1.56 ± 0.28 at 10 min (P < 0.01) and 1.29 ± 0.35 at 20 min (P < 0.05) upon application of 2,4-DNP. Fluorescence in CA1_SR, was similar to fluorescence in CA1 upon perfusion with 2,4-DNP and similar to CA3 upon perfusion with amytal. We conclude that NADH fluorescence can be measured with high regional selectivity and specificity in hippocampal slices. Selective inhibition of mitochondrial complex I and uncoupling of energy metabolism differentially impair NADH concentration in different hippocampal areas.