Immunoreactivities and Levels of Mineralocorticoid and Glucocorticoid Receptors in the Hippocampal CA1 Region and Dentate Gyrus of Adult and Aged Dogs

Corticosteroids are important factors in the maintenance of homeostasis in the brain. They are regulated via the interaction with two corticosteroid receptor systems—the mineralocorticoid (MR) and glucocorticoid receptor (GR). In the present study, we observed age-related changes in serum cortisol levels, and immunoreactivities and protein levels of MR and GR in the hippocampal CA1 region and dentate gyrus. The serum cortisol levels were significantly high (about twofold) in the aged group compared to that in the adult group. In the adult dog (2–3 years old), MR and GR immunoreactivity was detected in neurons in the pyramidal layer of the CA1 region, and in the granular and multiform layers of the dentate gyrus. In the aged dog (10–12 years old), MR immunoreactivity in the CA1 region was significantly decreased, especially, in the dentate multiform layer. In contrast, GR immunoreactivity in the aged dog was slightly decreased in the CA1 region and dentate gyrus. In the Western blot analysis, MR protein level in the aged dog was significantly lower compared to that of the adult dog; GR protein level in the aged dog was not significantly decreased. This result indicates that the reduction of MR immunoreactivity and protein level in the hippocampus of the aged dog may be associated with neural dysfunction in the aged hippocampus.     

Differences in Doublecortin Immunoreactivity and Protein Levels in the Hippocampal Dentate Gyrus Between Adult and Aged Dogs

Doublecortin (DCX), a microtubule-associated protein, specifically expresses in neuronal precursors. This protein has been used as a marker for neuronal precursors and neurogenesis. In the present study, we observed differences in DCX immunoreactivity and its protein levels in the hippocampal dentate gyrus between adult and aged dogs. In the adult dog, DCX immunoreactive cells with well-stained processes were detected in the subgranular zone of the dentate gyrus. Numbers of DCX immunoreactive cells in the dentate gyrus of the aged dog were significantly decreased compared to those in the adult dog. DCX immunoreactive cells in both adult and aged dog did not show NeuN (a marker for mature neurons) immunoreactivity. NeuN immunoreactivity in the aged dog was poor compared to that in the adult dog. DCX protein level in the aged dentate gyrus was decreased by 80% compared to that in the adult dog. These results suggest that the reduction of DCX in the aged hippocampal dentate gyrus may be involved in some neural deficits related to the hippocampus.     

Ionized Calcium-binding Adapter Molecule 1 Immunoreactive Cells Change in the Gerbil Hippocampal CA1 Region after Ischemia/Reperfusion

Ionized calcium-binding adapter molecule 1 (iba-1) is specifically expressed in microglia and plays an important role in the regulation of the function of microglia. We observed chronological changes of iba-1-immunoreactive cells and iba-1 level in the gerbil hippocampal CA1 region after transient ischemia. Transient forebrain ischemia in gerbils was induced by the occlusion of bilateral common carotid arteries for 5 min. Immunohistochemical and Western blot analysis of iba-1 were performed in the gerbil ischemic hippocampus. In the sham-operated group, iba-1-immunoreactive cells were detected in the CA1 region. Thirty minutes after ischemia/reperfusion, iba-1 immunoreactivity significantly increased, and its immunoreactive cells were well ramified. Three hours after ischemia/reperfusion, iba-1 immunoreactivity and level decreased, and thereafter they increased again with time after ischemia/reperfusion. Three days after ischemia/reperfusion, iba-1-immunoreactive cells had well-ramified processes, which projected to the stratum pyramidale of the CA1 region. Seven days after ischemia/reperfusion, iba-1 immunoreactivity and level were highest in the CA1 region, whereas they significantly decreased in the CA1 region 10 days after ischemia/reperfusion. Iba-1-immunoreactive cells in the ischemic CA1 region were co-localized with OX-42, a microglia marker. In brief, iba-1-immunoreactive cells change morphologically and iba-1 immunoreactivity alters in the CA1 region with time after ischemia/reperfusion. These may be associated with the delayed neuronal death of CA1 pyramidal cells in the gerbil ischemic hippocampus.     

Comparison of Ionized Calcium-binding Adapter Molecule 1-Immunoreactive Microglia in the Spinal Cord Between Young Adult and Aged Dogs

Microglia are main form of active immune defense, and they are constantly moving and analyzing the CNS for damaged neurons and infectious agents. In this study, we compared microglia in the spinal cord of the young adult (1–2 years old) and aged (10–12 years old) German Shepherd dogs via immunohistochemistry and western blot analysis for ionized calcium-binding adapter molecule 1 (Iba-1), a microglial marker. In addition, we also observed the interferon-γ (IFN-γ), a pro-inflammatory cytokine, and interleukin-1β (IL-1β), produced by activated microglia/macrophage, protein levels in these groups. At first, we found that neuronal nuclei (NeuN, a neuronal marker)-immunoreactive neurons were distributed throughout the grey mate of the spinal cord, and there were no significant differences between the adult and aged groups. Most of Iba-1-immunoreactive microglia were morphologically ramified microglia (resting form) in the adult group, while some Iba-1-immunoreactive microglia were morphologically activated microglia in the aged group. In western blot analysis, Iba-1, IFN-γ and IL-1β expression were increased in the aged group. This result may be associated with age-dependent changes in the spinal cord.     

Comparison of Glucocorticoid Receptor and Ionized Calcium-Binding Adapter Molecule 1 Immunoreactivity in the Adult and Aged Gerbil Hippocampus Following Repeated Restraint Stress

Stress leads to changes in homeostasis and internal balance of the body and is known to be one of important factors in the development of several diseases. In the present study, we investigated changes in glucocorticoid receptor (GR) and ionized calcium-binding adapter molecule 1 (Iba-1) immunoreactivity in the adult and aged gerbil hippocampus after chronic restraint stress. Serum corticosterone level was much higher in both the stress-groups than the control groups. No neuronal death was found in all hippocampal subregions of the adult and aged gerbil after chronic restraint stress. GR immunoreactivity was decreased in both the adult and aged groups after repeated restraint stress; however, GR immunoreactivity in the adult-stress-group was decreased much more than that in the aged-stress-group. Iba-1 immunoreactive microglia were hypertrophied and activated in the adult group after repeated restraint stress; in the aged-stress-group, there was no any significant change in Iba-1 immunoreactive microglia. In brief, level of GR, not Iba-1, in the hippocampus was much decreased in the adult gerbil compared to the aged gerbil following chronic restraint stress.     

Oroxylin A,a Flavonoid,Stimulates Adult Neurogenesis in the Hippocampal Dentate Gyrus Region of Mice

Previously, we reported the cognitive enhancing effects of oroxylin A in unimpaired mice and its memory ameliorating activity in various memory impaired mice. To elucidate the mechanism mediating the cognitive effects of oroxylin A, this study examined the consequences of oroxylin A administration on neurogenesis in the hippocampal dentate gyrus using immunostaining for 5-bromo-2-deoxyuridine (BrdU) incorporation. In addition, we determined whether the new cells adopted a neuronal or glial fate by examining the co-localization of BrdU staining with neuronal or glial markers. Administration of oroxylin A in a dose-dependent and time-dependent manner increased the number of BrdU-incorporating cells. Moreover, the percentage of BrdU-incorporating cells co-localized with neuronal markers, neuronal nuclei, was significantly increased by the oroxylin A administration. These results suggest that the increased neurogenesis induced by the administration of oroxylin A could be, at least in part, associated with its positive effects on cognitive processing.     

Comparison of Alpha-Synuclein Immunoreactivity in the Hippocampus Between the Adult and Aged Beagle Dogs

Alpha-synuclein (α-syn), as a neuroprotein, is expressed in neural tissue, and it is related to a synaptic transmission and neuronal plasticity. In this study, we compared the distribution and immunoreactivity of α-syn and related gliosis in hippocampus between young adult (2–3 years) and aged (10–12 years) beagle dogs. In both groups, α-syn immunoreactivity was detected in neuropil of all the hippocampal sub-regions, but not in neuronal somata. In the aged hippocampus, α-syn immunoreactivity was apparently increased in mossy fibers compared to that in the adult dog. In addition, α-syn protein level was markedly increased in the aged hippocampus. On the other hand, GFAP and Iba-1 immunoreactivity in astrocytes and microglia, respectively, were increased in all the hippocampal sub-regions of the aged group compared to that in the adult group: especially, their immunoreactivity was apparently increased around mossy fibers. In addition, in this study, we could not find any expression of α-syn in astrocytes and microglia. These results indicate that α-syn immunoreactivity apparently increases in the aged hippocampus and that GFAP and Iba-1 immunoreactivity are also apparently increased at the regions with increased α-syn immunoreactivity. This increase in α-syn expression might be a feature of normal aging.     

Comparison of Neurogenesis in the Dentate Gyrus Between the Adult and Aged Gerbil Following Transient Global Cerebral Ischemia

In the present study, we compared differences in cell proliferation, neuroblast differentiation and neuronal maturation in the hippocampal dentate gyrus (DG) between the adult and aged gerbil induced by 5 min of transient global cerebral ischemia using Ki-67 and BrdU (markers for cell proliferation), doublecortin (DCX, a marker for neuroblast differentiation) and neuronal nuclei (NeuN, a marker for mature neuron). The number of Ki-67-immunoreactive (⁺) cells in the DG of both the groups peaked 7 days after ischemia/reperfusion (I/R). However, the number in the aged DG was 40.6 ± 1.8% of that in the adult DG. Thereafter, the number decreased with time. After ischemic damage, DCX immunoreactivity and its protein level in the adult and aged DG peaked at 10 and 15 days post-ischemia, respectively. However, DCX immunoreactivity and its protein levels in the aged DG were much lower than those in the adult. DCX immunoreactivity and its protein level in the aged DG were 11.1 ± 0.6% and 34.4 ± 2.1% of the adult DG, respectively. In addition, the number of Ki-67⁺ cells and DCX immunoreactivity in both groups were similar to those in the sham at 60 days postischemia. At 30 days post-ischemia, the number of BrdU⁺ cells and BrdU⁺/NeuN⁺ cells in the adult-group were much higher (281.2 ± 23.4% and 126.4 ± 7.4%, respectively) than the aged-group (35.6 ± 6.8% and 79.5 ± 6.1%, respectively). These results suggest that the ability of neurogenesis in the ischemic aged DG is much lower than that in the ischemic adult DG.     

Comparison of Phosphorylated Extracellular Signal-Regulated Kinase 1/2 Immunoreactivity in the Hippocampal Ca1 Region Induced by Transient Cerebral Ischemia Between Adult and Aged Gerbils

In this study, the authors examined the difference of phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) in the hippocampal CA1 region (CA1) between adult and aged gerbils after 5 min of transient cerebral ischemia. Delayed neuronal death in the CA1 of the aged group was much slower than that in the adult group after ischemia/reperfusion (I/R). pERK1/2 immunoreaction was observed in the CA1 region of the sham-operated adult gerbil. pERK1/2 immunoreactivity and protein levels in the ischemic CA1 region of the adult group were markedly increased 4 days after I/R, and then reduced up to 10 days after I/R. In contrast, pERK1/2 immunoreaction was hardly detected in the CA1 region of sham-operated aged gerbils, and the immunoreactivity increased from 1 day after the ischemic insult, and still observed until 10 days post-ischemia. In addition, pERK1/2-immunoreaction was expressed in astrocytes in the ischemic CA1 region: The expression in the ischemia-operated aged gerbils was later than that in the ischemia-operated adult gerbils. These results indicate that different patterns of ERK1/2 immunoreactivity may be associated with different processes of delayed neuronal death in adult and aged animals.     

Inositol 1,4,5-Trisphosphate 3-Kinase mRNA: High Levels in the Rat Hippocampal CA1 Pyramidal and Dentate Gyrus Granule Cells and in Cerebellar Purkinje Cells

The distribution of inositol 1,4,5-trisphosphate (InsP3) 3-kinase mRNA in the rat brain is reported using oligonucleotides based on a cDNA clone sequence that encodes rat brain InsP3 3-kinase and the in situ hybridization technique. Moderate levels were found in CA2-4 pyramidal neurons, in the cortex, and in the striatum. The cerebellar granule cells, thalamus, hypothalamus, brainstem, spinal cord, and white matter tracts were almost negative. The levels of InsP3 3-kinase mRNA were highest in the hippocampal CA1 pyramidal neurons, granule cells of the dentate gyrus, and cerebellar Purkinje cells. These results contrast with the lower concentration of the InsP3 receptor already reported in the hippocampus versus the Purkinje cells and suggest a special role for inositol 1,3,4,5-tetrakisphosphate in Ammon's horn.     

Comparison of GAD65 and 67 Immunoreactivity in the Lumbar Spinal Cord Between Young Adult and Aged Dogs

We investigated distribution and age-related changes in two isoforms of GABA synthesizing enzymes, glutamic acid decarboxylase (GAD) 65 and 67, in the lumbar levels (L(5)-L(6)) of the dog spinal cord. Male German shepherds were used at 1-2 years (young adult dogs) and 10-12 years (aged dogs) of age. GAD65 immunoreaction was observed in neuropil, not in cell bodies, in all laminae of the adult lumbar spinal cord: Many punctate GAD65-immunoreactive structures were shown in all laminae. The density of GAD65 immunoreactive structures was highest in laminae I-III, and lowest in lamina VII. In the aged dog, the distribution pattern of GAD65 immunoreactivity was similar to that in the adult dog; however the density of GAD65-immunoreactive structures and its protein levels were significantly increased in the aged lumbar spinal cord. GAD67 immunoreaction in the adult dog was also distributed in all laminae of the lumbar spinal cord like GAD65; however, we found that small GAD67-immunoreactive cell bodies were observed in laminae II, III and VIII. In the aged dogs, GAD67 immunoreactivity and its protein levels were also increased compared to those in the adult group. In conclusion, our results indicate that the distribution of GAD65-immunoreactive structures is different from GAD67-immunoreactive structures and that their immunoreactivity in the aged dogs is much higher than the adult dogs.     

Comparison of the Immunoreactivity of Trx2/Prx3 Redox System in the Hippocampal CA1 Region Between the Young and Adult Gerbil Induced by Transient Cerebral Ischemia

In the present study, we compared the immunoreactivities and levels of Trx/prx redox system, thioredoxin 2 (Trx2), thioredoxin reductase 2 (TrxR2) and peroxiredoxin 3 (Prx3), as well as neuronal death in the hippocampal CA1 region between the adult and young gerbil after 5 min of transient cerebral ischemia. At 4 days post-ischemia, pyramidal neurons (about 90%) in the adult stratum pyramidale of the CA1 region showed "delayed neuronal death (DND)"; however, at this time point, few pyramidal neurons showed DND in the young stratum pyramidale. At 7 days post-ischemia, about 56% of pyramidal neurons showed DND in the young stratum pyramidale. The immunoreactivities of all the antioxidants in the young sham-group were similar to those in the adult sham-group. At 4 days post-ischemia, the immunoreactivity of TrxR2, not Trx2 and Prx3 in the adult ischemia-group was dramatically decreased in CA1 pyramidal neurons. At this time point, the immunoreactivities of all the antioxidants in the young ischemia-group were apparently increased compared to the adult ischemia-group. From 7 days pots-ischemia, non-pyramidal cells showed the immunoreactivities of all the antioxidants in the ischemic CA1 region; however, in the young ischemia-groups, the immunoreactivities were much lower than those in the adult ischemia-groups. In brief, our results showed that the immunoreactivities of Trx2, TrxR2 and Prx3 were dramatically increased in CA1 pyramidal neurons of the young ischemia-groups at 4 days post-ischemia compared to those in the adult ischemia-groups induced by transient cerebral ischemia.     

Comparison of Glial Activation in the Hippocampal CA1 Region Between The Young and Adult Gerbils After Transient Cerebral Ischemia

It has been reported that young animals are less vulnerable to brain ischemia. In the present study, we compared gliosis in the hippocampal CA1 region of the young gerbil with those in the adult gerbil induced by 5?min of transient cerebral ischemia by immunohistochemistry and western blot for glial cells. We used male gerbils of postnatal month 1 (PM 1) as the young and PM 6 as the adult. Neuronal death in CA1 pyramidal neurons in the adult gerbil occurred at 4?days posti-schemia; the neuronal death in the young gerbil occurred at 7?days post-ischemia. The findings of glial changes in the young gerbil after ischemic damage were distinctively different from those in the adult gerbil. Glial fibrillary acidic protein-immunoreactive astrocytes, ionized calcium-binding adapter molecule (Iba-1), and isolectin B4-immunoreactive microglia in the ischemic CA1 region were activated much later in the young gerbil than in the adult gerbil. In brief, very less gliosis occurred in the hippocampal CA1 region of the young gerbil than in the adult gerbil after transient cerebral ischemia.     

围产期食物限制对子代成年大鼠海马CA1区突触可塑性的影响

围产期食物限制导致子代大鼠学习和记忆能力等的神经生物学变化,但其机制并不清楚。将成年Wistar雌性大鼠与雄性大鼠同笼,受孕后随机分为对照组 (n=9) 和食物限制组 (n=8) 。对照组母鼠在妊娠期和哺乳期自由进食和饮水,食物限制组母鼠从妊娠的第7天到子代大鼠出生后21天进行食物限制,食物限制量为对照组大鼠的50%。子代雄性大鼠成年后,通过Morris 水迷宫测试空间学习和记忆能力。之后,在海马CA1区在体记录场兴奋性突触后电位 (field excitatory postsynaptic potential,fEPSP),并采用免疫组织化学方法观察海马CA1区神经元型一氧化氮合酶 (nNOS) 阳性细胞密度的变化。结果表明,围产期食物限制降低了子代大鼠出生后第1、7、10、14和21天的体重,并减弱了成年子代大鼠的学习和记忆能力,海马CA1区fEPSP的斜率和nNOS阳性细胞的密度也明显降低。结果提示,围产期食物限制可能通过抑制NO的产生降低了海马突触可塑性,从而影响了子代大鼠的学习和记忆能力。     

Comparison of Trophic Factors Changes in the Hippocampal CA1 Region Between the Young and Adult Gerbil Induced by Transient Cerebral Ischemia

In the present study, we investigated neuronal death/damage in the gerbil hippocampal CA1 region (CA1) and compared changes in some trophic factors, such as brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor (VEGF), in the CA1 between the adult and young gerbils after 5?min of transient cerebral ischemia. Most of pyramidal neurons (89?%) were damaged 4?days after ischemia?Creperfusion (I?CR) in the adult; however, in the young, about 59?% of pyramidal neurons were damaged 7?days after I?CR. The immunoreactivity and levels of BDNF and VEGF, not GDNF, in the CA1 of the normal young were lower than those in the normal adult. Four days after I?CR in the adult group, the immunoreactivity and levels of BDNF and VEGF were distinctively decreased, and the immunoreactivity and level of GDNF were increased. However, in the young group, all of their immunoreactivities and levels were much higher than those in the normal young group. From 7?days after I?CR, all the immunoreactivities and levels were apparently decreased compared to those of the normal adult and young. In brief, we confirmed our recent finding: more delayed and less neuronal death occurred in the young following I?CR, and we newly found that the immunoreactivities of trophic factors, such as BDNF, GDNF, and VEGF, in the stratum pyramidale of the CA1 in the young gerbil were much higher than those in the adult gerbil 4?days after transient cerebral ischemia.     

Deficits in the Activation and Phosphorylation of Hippocampal Tyrosine Hydroxylase in the Aged Fischer 344 Rat Following Intraventricular Administration of 6-Hydroxydopamine

Abstract: Tyrosine hydroxylase activity was measured under optimal and suboptimal assay conditions in hippocampal extracts from young (2 month), mature (12 month), and old (24 month) Fischer 344 male rats 72 h after the infusion of 200 µg of the neurotoxin 6-hydroxydopamine or vehicle into the lateral ventricle. The lesion resulted in a 45–55% decrease of tyrosine hydroxylase activity measured under optimal conditions (pH 6.1, 3.0 m M 6-methyl-5,6,7,8-tetrahydropterin) and an ∼35% decrease in the relative concentration of immunoreactive tyrosine hydroxylase. When measured under suboptimal conditions (pH 6.6, 0.7 m M 6-methyl-5,6,7,8-tetrahydropterin), tyrosine hydroxylase activity in 2- and 12-month-old lesioned animals was twice that measured in vehicle-treated animals. However, in the old lesioned animals, tyrosine hydroxylase activity measured under suboptimal conditions was not different from that measured in age-matched vehicle-treated animals. Isoforms of tyrosine hydroxylase were identified on immunoblots after two-dimensional gel electrophoresis using enhanced chemiluminescence. The relative proportion of lower pl isoforms of tyrosine hydroxylase in the 2-month-old lesioned animals was greater than that observed in vehicle-treated controls. In contrast, no difference was seen in the relative proportion of tyrosine hydroxylase isoforms in the 24-month-old lesioned versus control animals. These data indicate that the ability of locus ceruleus neurons to rapidly respond to and compensate for insult is attenuated in 24-month-old Fischer 344 rats due to a deficit in stimulus-evoked enzyme phosphorylation.     

Activity of Ca2+/Calmodulin-Dependent Protein Kinase II Following Ischemia: A Comparison Between CA1 and Dentate Gyrus in a Hippocampal Slice Model

Abstract: Both CA1 and dentate gyrus regions of the hippocampal slice exhibit an irreversible loss of synaptic transmission after exposure to in vitro ischemic conditions (buffer without oxygen and glucose). However, after shorter durations of ischemia (8–10 min) the CA1 region shows an irreversible loss of synaptic responses, whereas the dentate gyrus region completely recovers synaptic responses upon reoxygenation. To determine biochemical mechanisms underlying this differential susceptibility, we have examined changes in Ca²⁺/calmodulin-dependent protein kinase II (CaM-KII) and cyclic AMP-dependent protein kinase activities in homogenates from CA1 and dentate gyrus regions of the hippocampal slice after increasing durations of in vitro ischemia. Time-dependent changes in CaM-KII activities were correlated with changes in electrophysiological responses. CA1 homogenates from slices exposed to 1 min of ischemia showed significant increases in CaM-KII activity, whereas there was no significant change in kinase activity in dentate homogenates after 1 min of ischemia. However, after longer durations of ischemia (5, 10, and 20 min) we found a time-dependent reduction in CaM-KII activity in both CA1 and dentate gyrus regions, whereas no change was detected in cyclic AMP-dependent protein kinase activity. Irreversible depression of CaM-KII activity was seen at shorter durations of ischemia (10 min) in the CA1 region than in dentate region (20 min), which correlated with irreversible effects on synaptic responses. Immunoblot analysis showed that the decrease in CaM-KII activity was not due to degradation of CaM-KII protein. However, the microtubule-associated protein MAP2, known to be a substrate for the Ca²⁺-dependent proteases (calpains), showed extensive proteolysis evident after 90 min of reoxygenation after ischemia.     

Isoform-Specific Redistribution of Calcineurin Aα and Aβ in the Hippocampal CA1 Region of Gerbils After Transient Ischemia

Abstract: To investigate isoform-specific roles of Ca²⁺/calmodulin-dependent phosphatase [calcineurin (CaN)] in ischemia-induced cell death, we raised antibodies specific to CaN Aα and CaN Aβ and localized the CaN isoforms in the hippocampal CA1 region of Mongolian gerbils subjected to a 5-min occlusion of carotid arteries. In the nonischemic gerbil, immunoreactions of both isoforms were highly enriched in CA1 regions, especially in the cytoplasm and apical dendrites of CA1 pyramidal neurons. At 4–7 days after the induced ischemia, immunoreactivities of the CaN Aα isoform in CA1 pyramidal cells were markedly reduced, whereas they were enhanced in the CA1 radiatum and oriens layers. In contrast, CaN Aβ immunoreactivities were reduced in all layers of the ischemic CA1 region, whereas they were enhanced in activated astrocytes, colocalizing with glial fibrillary acidic protein. These findings suggest that up-regulation of CaN Aα in afferent fibers in CA1 and up-regulation of CaN Aβ in reactive astrocytes may be involved in neuronal reorganization after ischemic injury.     

Glutamate Activates Phospholipase D in Hippocampal Slices of Newborn and Adult Rats

Abstract: Phospholipase D (PLD) is activated by many neuro-transmitters in a novel signal transduction pathway. In the present work, PLD activity was studied comparatively in hippocampal slices of newborn and adult rats. Basal PLD activity in adult rats was almost three times higher than in newborn rats. In newborn rats, L-glutamate and 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid (1 S ,3 R -ACPD) time- and concentrationdependently enhanced the formation of [³H]phosphatidylpropanol ([³H]PP) and of [³H]phosphatidic acid in the presence of 2% propanol. N -MethylD-aspartate and kainate (both 1 m M ) caused small, but significant increases (∼50%). whereas α-amino-3-hydroxy-5-methylisoxazole-4-propionate (100 μ M ) was ineffective. Maximally effective concentrations of glutamate (1 m M ) and of 1 S ,3 R -ACPD (300 μ M ) increased the PLD activity to almost 300% of basal activity; the EC₅₀ values were 199 and 47 μ M , respectively. Glutamate receptor antagonists, such as DL-2-amino-3-phosphonopropionic acid (AP3). DL-2-aminc-5-phosphonovalenic acid, and kynurenate (all 1 m M ) did not inhibit the glutamate-evoked increase of PP formation. In slices of adult rats, the response to 1 S ,3 R -ACPD was significant, but small, whereas glutamate was effective only in the presence of the glutamate uptake inhibitor L-aspartate-β-hydroxarnate. It is concluded that glutamate activates PLD in rat hippocampus through an AP3-resistant metabotropic receptor. This effect is subject to ontogenetic development, with one important factor being glutamate uptake.