U-83836E prevents kainic acid-induced neuronal damage

Tsukuba College of Technology is the first national university established as an institute of higher education for the visually and hearing impaired. We have been systematically conducting a University Personality Inventory (UPI) survey on our students since 1989 to understand their mental health. In this study, we compared the UPI scores of the new students of Tsukuba College of Technology in 1993 and 1994 with unimpaired students from the University of Tsukuba (control group), but found no significant difference in the UPI scores of the visually impaired and the control group. However, we noticed a significant difference in the average UPI scores between the hearing impaired and the control group. The visually impaired group were divided into four subgroups, UPI scores descended in order from degree 1 (total blindness), to degrees 2 and 3 (amblyopia), to degree 4 (visual acuity > or = 0.3). The UPI scores of the degree 4 subgroup were significantly lower than those of the control group. An investigation of the items for which the check rate was at least 50% showed that the visually impaired students had a variety of psychological problems, most of which seemed to concern depression or anxiety as did the normal control group. The number of affirmative responses increased with low visual acuity. The only one belonging to the 'lie' scale item was observed in the group of hearing impaired students. Thus, comparing these three groups from the viewpoint of mental health, we noticed the hearing impaired group was slightly different from the other two groups, but the visually impaired group was similar to the normal control group.     

Dendritic atrophy in the dentate gyrus of the senescent rat

Quantitative electron microscopic analysis of the supragranular zone of the dentate gyrus molecular layer has shown that the number, volume fraction and surface area of dendritic shaft profiles are significantly decreased in senescent rats, relative to young adults. These modifications of dendritic morphology, which are not associated with age-related changes in dimensions of the molecular layer or in numbers of granule cells, may result from a decrease in the number and/or length of dendrites. In either case, the decreases in the number, volume fraction and surface area of dendritic shaft profiles found in the dentate gyrus of senescent rats signify an age-related atrophy of dendrites. Comparison of changes in the number and volume fraction of dendritic shaft profiles has demonstrated that age-related dendritic atrophy involves predominantly dendritic branches.     

Extracellular recordings in the colchicine-lesioned rat dentate gyrus following transplants of fetal dentate gyrus and CA1 hippocampal subfield tissue

Grafts of fetal dentate gyrus (DG) and CA1 hippocampal subfield tissue were extruded into the dentate gyri of adult male Sprague-Dawley rats, 7-10 days after lesioning the granule cells with colchicine (0.06 microliter of 7 mg/ml solution at each of 5 sites/hippocampus). Graft area-host and host-graft area connectivities were investigated 4-6 months post-transplantation by recoding extracellular evoked response in hippocampal slice preparations. Following stimulation of the host mid-molecular layer, evoked field potential responses, showing considerable variation, were recorded in both types of graft. Evoked responses in the lesioned DG without grafts were recorded in very few slices. Stimulation of the area of DG tissue grafts occasionally evoked responses in the host CA3/CA4 and there was no evidence for CA1 graft area-CA3/CA4 connectivity; stimulation of DG and CA1 graft areas occasionally evoked responses in the host CA1. Responses in the area of both DG and CA1 grafts supported short-term potentiation following stimulation of the host mid-molecular layer but only DG graft areas supported long-term potentiation of the population spike amplitude. In the area of both types of transplant a tonic bicuculline-sensitive inhibition was present and paired-pulse stimulation paradigms provided some evidence for inhibition. It is possible that responses recorded within the area of grafted tissue to stimulation of the host are attributable to host-graft connectivity and similarly, responses recorded in the host to stimulation of the area of the graft may be attributable to graft-host connectivity. Only DG graft areas received host inputs which were capable of sustaining a long-term potentiation and establishing efferent contacts with the host CA3/CA4 subfield, suggesting that these would be more likely than CA1 grafts to reinstate normal functional circuitry.     

Hypertrophy of astroglial processes in the dentate gyrus of the senescent rat

Quantitative electron microscopic analysis of the supragranular zone of the dentate gyrus molecular layer has shown that the number and volume fraction of profiles of astroglial processes are significantly increased in senescent rat relative to young adults. These ultrastructural modifications, which are not associated with significant age-related changes in the number of astrocytes or in the width of the molecular layer, may result from a formation of new astroglial processes and/or elongation of existing ones. In either case, the increase in the number and volume fraction of astroglial process profiles is an indicator of age-related astroglial hypertrophy. Hypertrophy of astroglial procecesses, which seems to develop with advanced age as a response to partial deafferentation of neurons, may compensate for a decrease in the dendritic volume fraction, thereby preventing changes in the dimensions of the dentate gyrus molecular layer in senescence.     

Electrophysiological properties of neurones in cultures from postnatal rat dentate gyrus

Electrophysiological properties of neurofilament-positive neurones in dissociated cell cultures were prepared at postnatal days 4-5 from rat dentate gyrus and studied using the whole-cell patch-clamp technique. These cells expressed a fast-inactivating, 0.5 microM tetrodotoxin-sensitive Na+ current; a high-voltage-activated (HVA) Ca2+ current, which was 30 microM Cd(2+)- and partially 2 microM nicardipine-sensitive; and an inward rectifier current, which was sensitive to extracellularly applied 1 mM Cs+. The outward current pattern was composed of a delayed rectifier-like outward current sensitive to 20 mM tetraethylammonium (TEA) and a fast-inactivating, Ca(2+)-dependent outward current. This transient Ca(2+)-dependent K+ outward current was identified by a subtraction procedure. K+ currents recorded under conditions of blocked Ca2+ currents (after rundown of the HVA Ca2+ current or blocked by extracellularly applied Cd2+) were subtracted from control currents. By comparison with the current pattern of identified dentate granule cells, it is concluded that the investigated cell type originated from interneurones or projection neurones of the dentate hilus.     

Experience-induced neurogenesis in the senescent dentate gyrus

We demonstrate here that under physiological conditions neurogenesis continues to occur in the dentate gyrus of senescent mice and can be stimulated by living in an enriched environment. Neurogenesis was investigated by confocal microscopy of three-channel immunofluorescent staining for the proliferation marker bromodeoxyuridine (BrdU) and neuronal and glial markers. Quantification was performed with unbiased stereological counting techniques. Neurogenesis decreased with increasing age. Stimulation of adult and aged mice by switching from standard housing to an enriched environment with opportunities for social interaction, exploration, and physical activity for 68 d resulted in an increased survival of labeled cells. Phenotypic analysis revealed that, in enriched living animals, relatively more cells differentiated into neurons, resulting in a threefold net increase of BrdU-labeled neurons in 20-month-old mice (105 vs 32 cells) and a more than twofold increase in 8-month-old mice (684 vs 285 cells) compared with littermates living under standard laboratory conditions. Corresponding absolute numbers of BrdU-positive astrocytes and BrdU-positive cells that did not show colabeling for neuronal or glial markers were not influenced. The effect on the relative distribution of phenotypes can be interpreted as a survival-promoting effect that is selective for neurons. Proliferation of progenitor cells appeared unaffected by environmental stimulation.     

Endogenous glutamate levels regulate nerve growth factor mRNA expression in the rat dentate gyrus

The levels of nerve growth factor (NGF) mRNA can be regulated in vitro and in vivo in the hippocampal formation by events associated with pharmacological activation of glutamate receptors. In the present study, the level of NGF mRNA in the hippocampal formation was examined following an intrahippocampal injection of 1 nmole fluorocitrate, which temporarily inhibits the astrocyte metabolic activity in vivo. Consistent with previous findings, fluorocitrate treatment significantly increased glutamate levels and decreased glutamine levels in the dentate gyrus as determined by in vivo microdialysis. The increased ratio of glutamate to glutamine was followed by a significant increase in NGF mRNA expression selectively in dentate gyrus granule cells. The effects of increasing glutamate levels were blocked by pretreatment with 50 nmole 2-amino-5-phosphonovalerate (AP5), a competitive antagonist that acts at the N-methyl-D-aspartate (NMDA) glutamate receptor subtype. These findings suggest that NGF mRNA expression is regulated, in part, by changes in endogenous glutamate levels, partially through enhanced excitatory neurotransmission through NMDA receptors.     

Ultrastructural evidence for bouton proliferation in the partially deafferented dentate gyrus of the adult rat

A quantitative morphological study of the changes in the dentate gyrus molecular layer in response to the removal of perforant path afferents was made utilizing electron microscopic techniques. Alterations in 1. the population of remaining afferents, 2. glial cells, and 3. granule cell dendrites are reported. The major observation was an increase in intact bouton density in the region of denervation which began at 5 days post-lesion and continued through 11 days post-lesion, the longest post-lesion survival time studied.     

Metabotropic glutamate receptors mediate activation of NPY-Y2 receptor expression in the rat dentate gyrus

Neuropeptide Y-Y2 receptor mRNA and binding were investigated after local injection of excitatory amino acid receptor agonists into the rat hippocampus. The general metabotropic glutamate receptor (mGluR) agonist (1S,3R)ACPD (200 and 400 nmol) and the group I mGluR agonist DHPG (50 nmol) enhanced Y2 receptor mRNA levels in granule cells (by up to 470%) and [125I]PYY(3-36) binding in mossy fibers. The group I mGluR antagonist 4-CPG (200 nmol) inhibited the action of (1S,3R)ACPD. On the other hand, AMPA and NMDA enhanced Y2 receptor expression only at neurodegenerative doses (> 0.3 and 3 nmol, respectively). It is suggested that seizure-induced Y2 receptor expression in granule cells may be mediated by group I mGluRs.     

In vivo chronoamperometric measurements of the clearance of exogenously applied serotonin in the rat dentate gyrus

The present study evaluated high-speed chronoamperometry as a method for measuring the clearance of serotonin (5-HT) from extracellular space in vivo. Male Sprague-Dawley rats were anaesthetized and a Nafion-coated, carbon fiber electrode, attached to a multibarrel pipette, was lowered into the subgranular layer of the dentate gyrus, a region which receives dense serotonergic innervation, or the corpus callosum, a fiber tract relatively devoid of the 5-HT transporter (SERT). Serotonin, pressure ejected into these regions, produced replicable electrochemical signals. The amplitude and time course of the signals were significantly prolonged in the corpus callosum compared to the dentate gyrus. Similarly, signals produced by locally applied 5-HT in the dentate gyrus of rats following destruction of hippocampal serotonergic innervation with 5,7-dihydroxytryptamine (5,7-DHT), were significantly enhanced compared to those observed in control animals. The time course of the 5-HT signal was significantly prolonged by local application of the selective 5-HT reuptake inhibitor, fluvoxamine, into the dentate gyrus. By contrast, fluvoxamine did not modify the clearance of 5-HT when locally applied into the dentate gyrus of 5,7-DHT lesioned rats or into the corpus callosum of intact rats. Taken together, these data demonstrate that in intact rats, the SERT contributes to the clearance of exogenously applied 5-HT from the extracellular space. Under the experimental conditions used in this study, high-speed chronoamperometry proved to be a reliable method for directly measuring extracellular 5-HT and appears to be a valuable tool for the study of 5-HT clearance by the SERT in vivo.     

Organization of the entorhinal projection to the rat dentate gyrus revealed by Dil anterograde labeling

It has been suggested that the entorhino-hippocampal circuit is involved in memory formation. To investigate the way that associative memory is elaborated in the circuit, the entorhino-dentate projection was studied with the fluorescent lipophilic tracer Dil. We investigated the projection originating in the dorsal part of the entorhinal cortex by injecting Dil along the rhinal sulcus. Anterograde fluorescent labeling allowed us to examine sections of the sample with a confocal microscope or in wholemount preparations with a fluorescence microscope. Quantitative analysis of the distribution of the Dil-labeled perforant path by confocal microscopy was performed in the septal one third level of the hippocampus. The analysis confirmed that the topographical map along the mediolateral dimension of the entorhinal cortex was transferred to the proximodistal level (from the inner one third to the edge of the molecular layer) of the granule cell dendrites in a gradually shifting manner. The fiber profile observed after lateral entorhinal injection was thick in the suprapyramidal blade and thin in the infrapyramidal blade. The fiber profile observed after medial entorhinal injection was thin in the suprapyramidal blade and thick in the infrapyramidal blade. Fluorescence microscopic observation of wholemount preparations showed that projections from the Dil injection site were distributed wider than half the dentate gyrus in the longitudinal direction. In transverse sections, the range of the labeled fiber distribution was confirmed to be more than two thirds of the dentate gyrus in the same direction regardless of the mediolateral level of the injection site. It has been suggested that the dorsoventral axis of the entorhinal cortex is represented in the septotemporal levels of the dentate gyrus, but that the topographical correspondence might be weak and vague. Although our investigation was limited to the projection from the dorsal entorhinal cortex to the dorsal part of the dentate gyrus, we conclude that the widely distributed projection covers the dentate gyrus in a nontopographic manner.     

Network properties of the dentate gyrus in epileptic rats with hilar neuron loss and granule cell axon reorganization

Neuron loss in the hilus of the dentate gyrus and granule cell axon reorganization have been proposed as etiologic factors in human temporal lobe epilepsy. To explore these possible epileptogenic mechanisms, electrophysiological and anatomic methods were used to examine the dentate gyrus network in adult rats that had been treated systemically with kainic acid. All kainate-treated rats, but no age-matched vehicle-treated controls, were observed to have spontaneous recurrent motor seizures beginning weeks to months after exposure to kainate. Epileptic kainate-treated rats and control animals were anesthetized for field potential recording from the dentate gyrus in vivo. Epileptic kainate-treated rats displayed spontaneous positivities ("dentate electroencephalographic spikes") with larger amplitude and higher frequency than those in control animals. After electrophysiological recording, rats were perfused and their hippocampi were processed for Nissl and Timm staining. Epileptic kainate-treated rats displayed significant hilar neuron loss and granule cell axon reorganization. It has been hypothesized that hilar neuron loss reduces lateral inhibition in the dentate gyrus, thereby decreasing seizure threshold. To assess lateral inhibition, simultaneous recordings were obtained from the dentate gyrus in different hippocampal lamellae, separated by 1 mm. The perforant path was stimulated with paired-pulse paradigms, and population spike amplitudes were measured. Responses were obtained from one lamella while a recording electrode in a distant lamella leaked saline or the gamma-aminobutyric acid-A receptor antagonist bicuculline. Epileptic kainate-treated and control rats both showed significantly more paired-pulse inhibition when a lateral lamella was hyperexcitable. To assess seizure threshold in the dentate gyrus, two techniques were used. Measurement of stimulus threshold for evoking maximal dentate activation revealed significantly higher thresholds in epileptic kainate-treated rats compared with controls. In contrast, epileptic kainate-treated rats were more likely than controls to discharge spontaneous bursts of population spikes and to display stimulus-triggered afterdischarges when a focal region of the dentate gyrus was disinhibited with bicuculline. These spontaneous bursts and afterdischarges were confined to the disinhibited region and did not spread to other septotemporal levels of the dentate gyrus. Epileptic kainate-treated rats that displayed spontaneous bursts and/or afterdischarges had significantly larger percentages of Timm staining in the granule cell and molecular layers than epileptic kainate-treated rats that failed to show spontaneous bursts or afterdischarges. In summary, this study reveals functional abnormalities in the dentate gyri of epileptic kainate-treated rats; however, lateral inhibition persists, suggesting that vulnerable hilar neurons are not necessary for generating lateral inhibition in the dentate gyrus.     

Pentylenetetrazol causes polysynaptic responses to appear in the dentate gyrus

In a particular brain region specific changes in inhibition or excitation may be the basis of seizure initiation. Alternatively, changes in the balance of excitation and inhibition in the circuit, which may be detectable as polysynaptic responses may be more important indicators of epileptogenesis. That the appearance of polysynaptic responses precedes the initiation and, therefore, may be necessary for the onset of epileptiform activity in the hippocampal-parahippocampal circuit was tested using the chemical convulsant pentylenetetrazol. Excitation and paired-pulse inhibition were measured in CA1 and the dentate gyrus of the urethane-anaesthetized rat before and after administration of pentylenetetrazol. In addition, three polysynaptic responses were monitored. In both CA1 and the dentate gyrus, pentylenetetrazol, 100 mg/kg, caused a trend towards increased excitability and caused a relatively mild loss of inhibition. Two polysynaptic responses appeared in the dentate gyrus after the administration of pentylenetratrazol, both apparently mediated through the entorhinal cortex. A polysynaptic response of the CA1 pyramidal neurons to contralateral angular bundle stimulation was not observed. These experiments demonstrate that pentylenetetrazol will facilitate only the appearance of polysynaptic responses mediated through the entorhinal cortex. These results support the hypothesis that pentylenetetrazol has a specific action within the entorhinal cortex that may facilitate the synchronization and spread of epileptiform activity. These results are also consistent with the hypothesis that the appearance of polysynaptic responses may be necessary for the onset of epileptogenesis in the hippocampal-parahippocampal circuit.     

Long-term potentiation and dual-component quantal signaling in the dentate gyrus

Long-term potentiation (LTP) of excitatory transmission is an important candidate cellular mechanism for the storage of memories in the mammalian brain. The subcellular phenomena that underlie the persistent increase in synaptic strength, however, are incompletely understood. A potentially powerful method to detect a presynaptic increase in glutamate release is to examine the effect of LTP induction on the rate at which the use-dependent blocker MK-801 attenuates successive N-methyl-D-aspartic acid (NMDA) receptor-mediated synaptic signals. This method, however, has given apparently contradictory results when applied in hippocampal CA1. The inconsistency could be explained if NMDA receptors were opened by glutamate not only released from local presynaptic terminals, but also diffusing from synapses on neighboring cells where LTP was not induced. Here we examine the effect of pairing-induced LTP on the MK-801 blocking rate in two afferent inputs to dentate granule cells. LTP in the medial perforant path is associated with a significant increase in the MK-801 blocking rate, implying a presynaptic increase in glutamate release probability. An enhanced MK-801 blocking rate is not seen, however, in the lateral perforant path. This result still could be compatible with a presynaptic contribution to LTP in the lateral perforant path if intersynaptic cross-talk occurred. In support of this hypothesis, we show that NMDA receptors consistently sense more quanta of glutamate than do alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors. In the medial perforant path, in contrast, there is no significant difference in the number of quanta mediated by the two receptors. These results support a presynaptic contribution to LTP and imply that differences in intersynaptic cross-talk can complicate the interpretation of experiments designed to detect changes in transmitter release.     

Expression of GABA(A) receptor subunits in the hippocampus of the rat after kainic acid-induced seizures

The GABA(A) receptor is a ligand gated chloride channel consisting of five membrane spanning proteins for which 13 different genes have been identified in the mammalian brain. The present review summarizes recent work from our laboratory on the characterization of the immunocytochemical distribution of these GABA(A) receptor subunits in the rat brain and changes in immunoreactivity and mRNA expression after kainic acid-induced status epilepticus. A heterogeneous distribution of immunoreactive GABA(A) receptor subunits was observed. The most abundant ones were: alpha1, alpha2, alpha4, alpha5, beta2, beta3, gamma2, and delta. Alpha1, beta2, and gamma2 were about equally distributed in all subfields of the hippocampus; alpha4- and delta-subunits were preferentially found in the dentate molecular layer and in CA1; alpha2 was localized to the dentate molecular layer and CA3; alpha5 was found in the dendritic areas of CA1 to CA3; and beta1 was preferentially seen in CA2. Alpha1, beta2, gamma2 and delta were highly concentrated in interneurons. Kainic acid-induced seizures caused acute and chronic changes in the expression of mRNAs and immunoreactive proteins. Acute changes included decreases in alpha2, alpha5, beta1, beta3, gamma2 and delta mRNA levels (by about 25-50%), accompanied by increases (by about 50%) in alpha1, alpha4, and beta2 messages in granule cells (after 6-12 h). Chronic changes, characterized by losses in mRNA and immunoreactive proteins in CA1 and CA3, are undoubtedly due to seizure-related cell damage. However, compensatory expression of alpha2 and beta3 subunits, especially in CA3b/c, was observed. Furthermore, increases in mRNAs and immunoreactive proteins were seen for alpha1, alpha2 alpha4, beta1, beta2, beta3 and gamma2 in granule cells and in the molecular layer of the dentate gyrus at 7-30 days after kainic acid injection. The changes in the expression of GABA(A) receptor subunits, observed in practically all hippocampal subfields, may reflect altered GABA-ergic transmission during development of the epileptic syndrome. Increased expression of GABA(A) receptor subunits in the dendritic field of granule cells and CA3 suggest that GABA-ergic inhibition may be augmented at these levels. However, the lasting preservation of alpha1-, beta2-, and gamma2-subunits in interneurons could provide a basis for augmented inhibition of GABA-ergic interneurons, leading to net disinhibition.     

Metabotropic glutamate receptor-induced homosynaptic long-term depression and depotentiation in the dentate gyrus of the rat hippocampus in vitro

We have investigated the role of metabotropic glutamate receptors (mGluR) in the induction of homosynaptic long-term depression (LTD) and depotentiation (DP) in the dentate gyrus of the adult rat. Perfusion of the mGluR agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) for a prolonged period (20 min) induced long-term depression (LTD) of field excitatory postsynaptic field potentials (epsps) from the baseline level and also depotentiation (DP) from the long-term potentiated level. Both the ACPD-and the low frequency stimulation (LFS)-induced LTD and DP were inhibited in the presence of the mGluR antagonist (+)-alpha-methyl-4-carboxyphenylglycine (MCPG), demonstrating the necessity for the activation of metabotropic glutamate receptors in the induction of LTD/DP. The LFS and ACPD-induced LTD were independent of the activation of N-methyl-D-aspartate (NMDA) receptors, as they were not blocked by the NMDA receptor antagonist D-2-amino-5-phophonopentanoate (AP5).     

Multiple subpial transection in kainic acid-induced focal cortical seizure

Hypoxia is a potent activator of the sympathetic nervous system by stimulating arterial chemoreceptors. However, out of 15 laboratory studies on the effects of acute and prolonged hypoxia on catecholamines, 14 failed to show any changes in plasma or urinary noradrenaline and only four studies showed significant increases in plasma or urinary adrenaline. By contrast, six out of eight studies on MSNA showed increased sympathetic nerve activity to the leg. An increased clearance of plasma catecholamines during hypoxia may be a possible explanation. Furthermore, many of the studies had limitations in a number of subjects and catecholamine assays used. Emotional aspects of the study protocols, which could contribute to the increase in adrenaline, was only assessed by sham runs in one chamber study. However, 13 out of 14 reviewed field studies on subjects staying for more than 1 week at high altitude, reported increased plasma or urinary excretion of noradrenaline which may be compatible with increased sympathetic activity. Adrenaline changed to a lesser degree. Out of seven studies on more short-term (4 h to 3 days) exposure to high altitude, only one demonstrated significantly increased plasma noradrenaline. In this study, however, several subjects had been exposed to high altitude less than 1 week before the experiment. In a new study on 12 climbers reported in this paper, a temporary reduction in plasma catecholamines was found 2 days after arrival at 4200 m. There was a steady increase towards normal levels after 1 week. Plasma vasopressin (AVP) increased suggesting a compensatory mechanism. Both plasma noradrenaline and adrenaline were positively correlated with oxygen saturation in these subjects. Thus, in previously unacclimatized subjects, short-term exposure to high altitude does not increase plasma catecholamines, rather plasma levels decreased. In addition to increased clearance, there is some evidence of reduced synthesis of catecholamines during short-term hypoxia. The oxygen sensitivity of tyrosine hydroxylase (TH) activity, may be one possible mechanism.     

Expression of the 27,000 mol. wt heat shock protein following kainic acid-induced status epilepticus in the rat

Western analysis and immunohistochemistry were used to determine the time-course and the distribution of the 27,000 mol. wt heat shock protein, Hsp27, in rat brain following systemic administration of kainic acid. No Hsp27 immunoreactivity was detected in naive control animals or in rats that failed to develop status epilepticus. Hsp27 immunoreactivity was detected as early as 12 h in the parietal cortex, piriform cortex and the hippocampus of rats that developed status epilepticus. The number of cells expressing Hsp27 and the intensity of Hsp27 immunoreactivity were increased 24 h after kainic acid administration. Hsp27 immunoreactivity was still observed seven days post-kainic acid injection. The morphology of the Hsp27-positive cells and double immunofluorescence against Hsp27 and glial fibrillary acidic protein revealed that Hsp27-positive cells were astrocytes. In addition, the distribution of Hsp27 suggested that astrocytic Hsp27 was dependent on excitation-induced metabolic stress rather than the direct effect of kainic acid on astrocytes.     

Depletion of NOS activity in the rat dentate gyrus neurons by DSP-4 and protection by deprenyl

Insulin resistance and hyperinsulinemia have been linked with essential hypertension. Age-associated increases in glucose intolerance and hypertension are also well established. To clarify the influence of aging on the insulin sensitivity, euglycemic hyperinsulinemic glucose clamp technique was carried out in 41 normotensive subjects and 42 patients with essential hypertension. The subjects of these groups were divided into two subgroups: young (< 40 years old) and middle-elderly (> or = 40 years old). Insulin sensitivity was assessed as M-value, the rate at which glucose must be infused to maintain a basal blood glucose level. In normotensive subjects, the young subgroup had a significantly higher M-value than did the middle-elderly subgroup. There was a significant negative correlation between age and M-value in normotensive subjects. On the other hand, there was no significant difference in M-value between the young and middle-elderly subgroups in the patients with essential hypertension. The age did not correlate with M-value in the hypertensive group. The normotensive subjects showed a significantly lower M-value than the hypertensive patients in the young group, but not in the middle-elderly group. These results indicate that 1) insulin sensitivity declines with age in normotensive subjects and that 2) insulin sensitivity is already diminished in the early stage of hypertension, and no further decrease in insulin sensitivity occurs with aging in essential hypertensive patients.