A membrane-anchored cytoplasmic domain of the human insulin receptor mediates a constitutively elevated insulin-independent uptake of 2-deoxyglucose.

Insulin stimulates the autophosphorylation of the beta-subunit of the insulin receptor (IR) on tyrosine residues. Mutations which compromise IR autophosphorylation in vivo result in a decrease of the insulin-activated uptake of 2-deoxyglucose. These results are consistent with previous results which implicate IR autophosphorylation in the generation of the insulin response by cells. To further explore the specificity of the IR tyrosine phosphokinase (TPK) domain in IR function, we have altered the human IR (hIR) cDNA to encode truncated insulin-independent TPKs, which are expressed in chinese hamster ovary (CHO) cells as either membrane-anchored or cytosolic proteins. Both mutant hIRs exhibit TPK activity in vitro, although the cytosolic form is approximately 20 times more active. The carbohydrate moiety of the membrane-anchored form is of the high mannose type, consistent with an intracellular localization for this mutant hIR. The two mutant hIRs mediate very different physiological responses in transfected cells: the membrane-anchored, but not the cytosolic, hIR TPK mediates a constitutively elevated (135% the maximum insulin-stimulated response in CHO cells) insulin-independent uptake of 2-deoxyglucose. These results thus suggest that the hIR TPK is in fact specific for this aspect of IR function and, when membrane-associated, can mediate the insulin-independent uptake of 2-deoxyglucose. Neither of these mutant hIRs appears to transform CHO cells.

Kinetic model of 2-deoxyglucose metabolism using brain slices.

A six-compartment, nine-parameter kinetic model of 2-deoxyglucose (2DG) metabolism, which includes bidirectional tissue transport, phosphorylation, two-step dephosphorylation, phosphoisomerization, and conjugation to UDP and macromolecules, has been derived. Data for analysis were obtained from 540- and 1,000-microns-thick hippocampal and hypothalamic brain slices, which were incubated in buffer containing [14C]2DG, frozen, extracted with perchlorate, and separated on anion-exchange columns. Solutions of the equations of the model were fit to the data by means of nonlinear least-squares analysis. These studies suggest that dephosphorylation is adequately described by a single reaction so that the model reduces to eight parameters. The in vitro rate constants for transport, phosphorylation, and dephosphorylation are very similar to prior in vivo results. The phosphoisomerization rate constant is similar to dephosphorylation, so glycosylated macromolecules slowly accumulate and gradually assume larger relative importance as other compounds disappear more rapidly. Rate constants for 540-microns slices from hypothalamus and hippocampus are similar, while 1,000-microns slices have smaller tissue transport constants and larger phosphorylation constants. The rate equation for glucose utilization of this model is relatively insensitive to uncertainties regarding the rate constants. Including later metabolic components in kinetic models improves the calculations of glucose utilization with long isotope exposures.

The deoxyglucose method in the ferret brain. I. Methodological considerations.

In the brain of the anesthetized ferret, the 2-deoxyglucose (2-DG) transfer rate constants required to determine cerebral glucose utilization by the deoxyglucose method were calculated from regional gray matter time-radioactivity curves measured for 180 min after tracer injection. Results suggest that loss of metabolized tracer from brain occurs at a rate of about 1%/min for the first 180 min after injection if the rate constant of the rate-limiting step for loss of metabolized tracer (k4*) represents a first-order kinetic process. A simulation experiment shows that, whether k4* is assumed to be 0 or 0.01 min-1, has a negligible influence on glucose utilization rates obtained in conventional 45 min autoradiographic experiments provided that the entire analysis, including lumped constant determination, is carried out in a consistent way. The 2-DG lumped constant for k4* = 0 is 0.54, and 0.68 for k4* = 0.01 min-1.

The deoxyglucose method in the ferret brain. II. Glucose utilization images and normal values.

To measure cerebral glucose utilization with the autoradiographic deoxyglucose method, the tracer transfer rate constants and lumped constants must be known. 2-Deoxyglucose (2-DG) and fluorodeoxyglucose (FDG) constants were determined in 18 gray and white matter brain structures of the anesthetized ferret. The ferret is a domestic carnivore particularly suitable for deoxyglucose studies because of its small brain size and low body weight. The average gray matter rate constants for tracer transfer across the blood-brain barrier are similar for 2-DG and FDG in the ferret brain (K*1 = 0.21 ml/g/min and k*2 = 0.39 min-1). The rate constant for the rate-limiting step of tracer phosphorylation, k*3, is 1.6 times higher for FDG than for 2-DG (0.21 vs. 0.13 min-1). Loss of metabolized tracer is about 1-1.5%/min throughout the ferret brain for both tracers as estimated for a 180 min experimental period. Taking into account this loss, the lumped constant is 0.92 for FDG and 0.68 for 2-DG. Glucose utilization values in the brain of the anesthesized ferret range from 33 mumol/100 g/min in the corpus callosum to 104 mumol/100 g/min in the caudate nucleus. Representative glucose utilization images of coronal sections of the ferret brain are shown. Brain structures are identified on the same slices counterstained with thionin.

Tracer 2-deoxyglucose kinetics in brain regions of rats given kainic acid.

The initial distribution of tracer amounts of 2-deoxyglucose between plasma and brain tissue, relative to native glucose, and the rate of accumulation of 2-deoxyglucose-6-phosphate were determined in brain regions of rats given kainic acid intravenously. Regional plasma flow was measured in a comparable group of animals. A previously described compartmental model was used to obtain estimates of rates of glucose transport and of glucose phosphorylation. Both rates were significantly increased in entorhinal cortex, hippocampus, amygdala, and septal nucleus. From measured brain tissue and plasma glucose concentrations, glucose fluxes were also calculated in terms of either irreversible or reversible Michaelis-Menten kinetics. In all brain regions of control rats and in six of the ten regions studied in rats given kainic acid, rates of glucose transport calculated in terms of the Michaelis-Menten models were consistent with those estimated by the tracer 2-deoxyglucose procedure. However, in the four regions in which glucose metabolism was stimulated, rates of glucose transport calculated from the behaviour of tracer 2-deoxyglucose were considerably higher than rates calculated from measured concentrations of glucose in plasma and brain tissue using Michaelis-Menten models. The possibility is considered that in those regions that are metabolically stimulated by kainate, there is an increasing asymmetry between the luminal and abluminal membranes of the capillary endothelium in the permeability to glucose and its analogs. An alternative proposal is that in the model used to analyse the tracer 2-deoxyglucose data, the assumption of a rapid mixing of tracer throughout the endogenous pool of tissue glucose prior to phosphorylation becomes invalid. The discrepancies between tracer and native glucose in these particular regions of rats given kainate are consistent with an apparent metabolic compartmentation. The influence of kainate on plasma flow was found to differ regionally, with flow in entorhinal cortex, hippocampus, and amygdala being unchanged. There is some evidence for increased rates of glycolysis relative to oxidative metabolism in these regions.

Cortical hypometabolism and its recovery following nucleus basalis lesions in baboons: a PET study.

The cerebral metabolic rate for glucose was measured serially with positron emission tomography and [18F]fluorodeoxyglucose in five baboons with stereotactic electrocoagulation of the left nucleus basalis of Meynert (NbM). Four days after lesion, a significant metabolic depression was present in the ipsilateral cerebral cortex, most marked in the frontotemporal region, and which recovered progressively within 6-13 weeks. These data demonstrate that adaptive mechanisms efficiently compensate for the cortical metabolic effects of NbM-lesion-induced cholinergic deafferentation. Moreover, unilateral NbM lesions also induced a transient reduction in contralateral cortical metabolic rate, the mechanisms of which are discussed. Explanation of these effects of cholinergic deafferentation in the primate could further our understanding of the metabolic deficits observed in dementia of the Alzheimer's type.

Deoxyglucose lumped constant estimated in a transplanted rat astrocytic glioma by the hexose utilization index.

The lumped constant (LC) for calculating the regional glucose (glc) metabolic rate by the deoxyglucose (DG) method was estimated in a transplanted rat glioma and normal rat brain. First, the hexose utilization index (HUI) was measured at 1.5, 3.0, and 4.5 min in right hemisphere glioma implants and uninvolved contralateral hemisphere following bolus intravenous injections of [3H]DG and [14C]glucose. At these times, the glioma HUI values were 0.639, 0.732, and 0.712, respectively, and the coordinate left hemisphere values were 0.432, 0.449, and 0.418. Second, the volumes of distribution of DG and glucose were determined to be 0.436 and 0.235 in glioma implants and 0.402 and 0.237 in left hemisphere, respectively. Third, following simultaneous intracarotid injections of [3H]DG and [14C]glucose, the ratio K1/K1 was 1.1 in glioma grafts and 1.3 in left hemisphere. With these values for HUI, volume of distribution, and K1 ratio, the LC in this rat glioma was estimated to be 2.1 times higher than the left hemisphere LC (p less than 0.02). These results suggest that measurement of brain tumor CMRglc using a normal brain LC may significantly overestimate the true tumor CMRglc.

c-fos expression and (14C) 2-deoxyglucose uptake in the caudal cerebellum of the rat during motor/sensory cortex stimulation.

Fos, the protein product of the c-fos gene, is induced in neurons in response to a variety of stimuli. In order to see if Fos could be used to map activity in the brain, the pattern of Fos staining was compared to the pattern of (14C) 2-deoxyglucose (2DG) uptake in the seventh and eighth lobules of the cerebellum during electrical stimulation of the cerebral cortex. Electrical stimulation of hindlimb motor/sensory cortex of awake rats increased 2DG uptake in the contralateral and ipsilateral cerebellum. The largest increases occurred in granule cell patches in the contralateral copula pyramidis (Cop P) and pyramis (P), the hemispheric and vermal portions of the eighth cerebellar lobule, respectively. The granule cell patches formed parasagittal bands that extended short distances mediolaterally, and extended long distances anteroposteriorly over much of the Cop P. Forelimb motor/sensory cortex stimulation increased 2DG uptake bilaterally in the seventh, paramedian (PM) cerebellar lobule. The greatest increases occurred in the granule cell layer contralateral to the stimulation. These and the above results generally agree with classical studies that localize forelimb on the seventh lobule anterior to the hindlimb on the eighth lobule. However, hindlimb cortical stimulation activated parts of the PM, and forelimb cortical stimulation activated portions of the rostral Cop P. In general, nonoverlapping portions of Cop P and PM were activated during the two types of cortical stimulation. These results are consistent with a fractured somatotopy (Welker and Shambes, '85) in which nonadjacent body parts are consistently represented in adjacent granule cell patches on each lobule, with the fractured somatotopy being different for every lobule. No region of cerebellum expressed Fos in unstimulated, electrode implanted, control subjects. However, following 15 minutes of electrical stimulation of hindlimb cortex, Fos was expressed 4 hours later in patches of granule cell nuclei in Cop P and P. These patches of Fos immunostained granule cells occurred in similar locations in Cop P to the patches of highest glucose metabolism observed with the 2DG method. Zones of Purkinje cell nuclei also expressed Fos. These Purkinje cell zones were often directly over similar sized granule cell patches in P. In the hemisphere however, the zones of Purkinje cells in ventrolateral Cop P expressing Fos only partially overlapped underlying granule cell patches that expressed Fos. Moreover, Fos was not unduced in any Purkinje cells adjacent to the Fos- stained granule cell patch in dorsolateral Cop P.(ABSTRACT TRUNCATED AT 400 WORDS)

In vivo measurement of [18F]fluorodeoxyglucose rate constants in rat brain by external coincidence counting.

The operational equation for the double-label deoxyglucose method described in the following paper requires the knowledge of the rate constants for transfer of fluorodeoxyglucose across the blood-brain barrier (K1* and K2*), and those for phosphorylation of fluorodeoxyglucose (K3*) and dephosphorylation of fluorodeoxyglucose-6-phosphate (k4*). These rate constants were determined in anesthetized rats by external coincidence counting. Radioactivity in parietal brain was measured for a 110 min experimental period after a bolus injection of 18F-labeled fluorodeoxyglucose. Apparent rate constants were obtained by fitting the resulting tissue radioactivity curves to the tissue radioactivity function of the deoxyglucose model modified to take into account the dephosphorylation of fluorodeoxyglucose-6-phosphate. The apparent fluorodeoxyglucose rate constants in rat brain are K1* = 0.195 ml g-1 min-1, k2* = 0.379 min-1, k3* = 0.088 min-1, and k4* = 0.009 min-1.

Topographical assessment of accumulated radioactivity from [14C]2-deoxyglucose and [6(-14C)]glucose in rat forebrain at different survival periods.

The uptake and retention of radioactivity was measured in discrete areas of rat brain at different times after i.v. injection of [14C]2-deoxyglucose or [6(-14)C]glucose, in unrestrained rats. In most brain regions, the accumulation of radioactivity from the two compounds was similar when a 30-min survival period for [6(-14)C]glucose was compared to a 45-min survival period for [14C]2-deoxyglucose. However, at those times, autoradiographic images of the hippocampus and piriform cortex appeared distinctly different for [14C]2-deoxyglucose and [6(-14)C]glucose. Relatively more radioactivity accumulated from [14C]2-deoxyglucose, compared to [14C]glucose, in the stratum lacunosum-moleculare of the hippocampus and in layer 4 of the isocortex. In contrast, relatively more radioactivity accumulated from [6(-14)C]glucose, compared to [14C]2-deoxyglucose, in the molecular and granule cell layers of the dentate gyrus, the CA1 pyramidal cell layer of the hippocampus, and in layer 2 of the piriform cortex. When rats were killed 5 min after injection of [6(-14)C]glucose, the relative neuroanatomical distribution of radioactivity was similar to the 30-min survival period, except in layer 4 of the isocortex, where relatively more radioactivity was present at the early time. When rats were killed 5 min after injection of [14C]2-deoxyglucose, in 20 of 24 brain regions examined, the absolute and relative amounts of accumulated radioactivity were similar when compared to that of the 45-min survival period. In contrast, the absolute and relative amounts of radioactivity were significantly greater for the 5-min compared to the 45-min survival period, in the CA1 pyramidal cell field, dentate gyrus, and layer 2 of the piriform cortex. For those regions, the appearance of autoradiograms prepared from rats killed 5 min after administration of [14C]2-deoxyglucose is remarkably similar to the appearance of autoradiograms prepared from rats killed 5 or 30 min after injection of [6(-14)C]glucose. Possible mechanisms are discussed to explain the observed differences in the accumulation of radioactivity in discrete brain regions after injection of [6(-14)C]glucose and [14C]2-deoxyglucose at the different survival times examined.

The patterns of [14C]2-deoxyglucose uptake in female rat brain produced by electrical stimulation of hypothalamic and limbic brain areas.

The aim of this study was to investigate the pattern of [14C]2-deoxyglucose uptake in anaesthetized rat brain produced by electrical stimulation of brain areas implicated, by previous electrical stimulation studies, in the neural control of pituitary hormone and especially gonadotrophin secretion. Stimulation of the median eminence led to a significant increase in the relative metabolic activities of the arcuate, ventromedial hypothalamic, supraoptic and paraventricular nuclei and the preoptic area. Stimulation of the suprachiasmatic or paraventricular nuclei or the medial preoptic area, anterior hypothalamic area, the dorsal or ventral hippocampus or amygdala led to an increase in the relative metabolic activity of many brain regions known to have direct connections with these areas, but in addition produced increases in the relative metabolic activity of areas which have secondary connections. Hippocampal stimulation confirmed previous neuroanatomical findings of major intrinsic functional connections between different fields of the ipsilateral and contralateral hippocampus. Stimulation of the amygdala, unexpectedly, did not change the relative metabolic activity of the arcuate nucleus and medial preoptic area which have neuroanatomical connections with the amygdala. Similarly, stimulation of the medial preoptic area did not change significantly the relative metabolic activity of the mamillary body and dorsomedial thalamic area. The effect of preoptic area stimulation on the relative metabolic activity of several brain regions was changed by ovariectomy and by injection of oestradiol benzoate. Stimulation of the preoptic area and suprachiasmatic nuclei, but not the anterior hypothalamic area or other brain regions, increased significantly the plasma concentrations of luteinizing hormone. These results show that (i) electrical stimulation of brain areas concerned with the control of gonadotrophin and other pituitary hormone secretion changes the metabolic activity of nuclei and neural pathways extrinsic as well as intrinsic to the hypothalamic-pituitary system, (ii) the [14C]2-deoxyglucose method can detect changes in antidromic as well as orthodromic activity and in multi-synaptic pathways, (iii) neuroanatomical pathways are not always activated metabolically by electrical stimulation, and (iv) the preoptic-suprachiasmatic nucleus gonadotrophin control system is discrete and is little affected by increased metabolic activity of the hypothalamus produced by stimulation of the anterior hypothalamic area or other brain areas.

The connections between the suprachiasmatic, ventrolateral geniculate and raphe nuclei studied by uptake of [14C]2-deoxyglucose.

The [14C]2-deoxyglucose method was used to investigate the role of the ventrolateral geniculate and raphe nuclei in the control of the metabolism of the suprachiasmatic nuclei in adult female Wistar rats anaesthetized with alphaxalone. Three to seven days before the [14C]2-deoxyglucose studies a stimulating electrode was implanted or a lesion was made in the ventrolateral geniculate nucleus, or the ascending projection from the raphe nuclei was severed. Stimulation of the ventrolateral geniculate nucleus (biphasic rectangular pulses, 30 s on and 30 s off, 50 Hz, 500 microA pulse amplitude and 1 ms pulse duration) led to a significant increase in the relative metabolic activity of the ipsilateral suprachiasmatic nucleus and a smaller increase in the relative metabolic activity of the contralateral suprachiasmatic nucleus. The stimulus also increased significantly the relative metabolic activities of mainly the ipsilateral hypothalamus, midbrain central gray and reticular formation, all of which are too remote from the ventrolateral geniculate nucleus to be affected by current spread. In animals in which the ventrolateral geniculate nucleus had been lesioned, the relative metabolic activity of the suprachiasmatic nuclei was not significantly different from normal. In animals in which the ascending projection from the raphe nuclei had been severed, there was a slight, though significant increase in the relative metabolic activity of the suprachiasmatic nucleus of one side. These results, together with the effects of stimulating the suprachiasmatic nuclei [R. C. Maxwell and G. Fink, Neuroscience 23, 241-263 (1987)], show that the connections between the ventrolateral geniculate, raphe nuclei and suprachiasmatic nuclei are "metabolically functional", but that the integrity of the ventrolateral geniculate nucleus is not essential for maintaining the relative metabolic activity of the suprachiasmatic nuclei. The raphe nuclei may reduce the relative metabolic activity of the suprachiasmatic nucleus.

Actions of novel antidiabetic thiazolidinedione, T-174, in animal models of non-insulin-dependent diabetes mellitus (NIDDM) and in cultured muscle cells.

1. The antihyperglycaemic effect and the possible mechanism of action of T-174, a novel thiazolidinedione derivative, was determined in vivo and in vitro. 2. Oral administration of T-174 markedly improved hyperglycaemia, hyperinsulinaemia, hyperlipidaemia, and glucose intolerance in genetically obese and diabetic yellow KK (KK-Ay) mice (0.2-15.5 mg kg(-1) day(-1), for 7 days) and Zucker fatty rats (1.4-11.4 mg kg(-1) day(-1), for 6 days). The ED50 values for the glucose lowering action of T-174 and pioglitazone, another thiazolidinedione antidiabetic, were 1.8 and 29 mg kg(-1) day(-1), respectively in KK-Ay mice; T-174 was about 16 times more potent than pioglitazone. 3. The hypoglycaemic effect of exogenous insulin in KK-Ay mice was enhanced after the administration of T-174. A hyperinsulinaemic euglycaemic clamp study in Zucker fatty rats showed an amelioration of whole-body insulin resistance by the T-174 treatment. 4. Insulin-stimulated glucose metabolism was enhanced in adipocytes from KK-Ay mice treated with T-174. The insulin receptor number of the adipocytes was increased without a change in the affinity of the receptor. 5. The hypomagnesaemia in KK-Ay mice was completely restored by T-174. 6. In cultured L6 myotubes, glucose consumption and [3H]-2-deoxy-glucose transport were enhanced by T-174 (EC50; 6 and 4 microM, respectively). Combination of insulin with T-174 was additive to stimulate glucose disposal. 7. These results suggest that the antihyperglycaemic effect of T-174 was mediated by enhanced insulin action. This was associated with amelioration of the hypomagnesaemia and T-174 directly increased basal and insulin-stimulated glucose utilization by cultured muscle cells.

Transport of radioactive deoxyglucose by the isolated perfused human placental cotyledon as measured by the paired-tracer dilution method. Inconsistency of the transfer rates across the two trophoblast borders with the simultaneously measured rate of transplacental transfer.

The uptake of [3H]2-deoxy-D-glucose ([3H]2DG) by the maternal and fetal borders of the trophoblast was measured in the dually perfused human placental cotyledon by use of the paired-tracer dilution method. The uptake was estimated from the maximum extraction of the tracer. The transcellular component of transplacental transport of [3H]2DG was measured simultaneously. From the rates of uptake by the two trophoblast borders the rate of transcellular transport was predicted. The rate predicted was significantly lower than the rate observed. This was taken to indicate that the paired-tracer dilution method based on the maximum extraction underestimated the rates of uptake.

Transport of 1,5-anhydro-D-glucitol across plasma membranes in rat hepatoma cells.

The transport of 1,5-anhydro-D-glucitol (AG) across plasma membranes was investigated in rat hepatoma cells, Reuber H-35. The AG uptake by the cells showed a concentration gradient dependency: the uptake was saturated within 40 s, which was less than one-third of the saturation time for 2-deoxy-D-glucose (DG) uptake. Furthermore, the Km value of the transport system for AG was higher than 100 mM. Though AG has a pyranoid structure resembling that of glucose, AG did not compete for cellular uptake with DG, D-glucose or 3-O-methyl-D-glucose, which are taken into cells through the glucose transporters. Conversely, the DG transport was not inhibited by AG at concentrations up to 50 mM. AG transport was hardly inhibited by 10 microM cytochalasin B, which strongly inhibits glucose transporters. In contrast, the AG transport was inhibited by 100 microM phloretin much more strongly than the DG transport when cells were preincubated with the inhibitor; the inhibition constant was 28.0 microM. The AG transport was not inhibited by 100 microM phloridzin, while the DG uptake was slightly inhibited by phloridzin. On the basis of these observations we propose that the AG uptake into rat hepatoma cells is mediated by a carrier distinct from glucose transporters.

Cerebral glucose utilization during sleep-wake cycle in man determined by positron emission tomography and [18F]2-fluoro-2-deoxy-D-glucose method.

Using the [18F]fluorodeoxyglucose method and positron emission tomography, we studied cerebral glucose utilization during sleep and wakefulness in 11 young normal subjects. Each of them was studied at least thrice: during wakefulness, slow wave sleep (SWS) and rapid eye movement sleep (REMS), at 1 week intervals. Four stage 3-4 SWS and 4 REMS fulfilled the steady state conditions of the model. The control population consisted of 9 normal age-matched subjects studied twice during wakefulness at, at least, 1 week intervals. Under these conditions, the average difference between the first and the second cerebral glucose metabolic rates (CMRGlu was: -7.91 +/- 15.46%, which does not differ significantly from zero (P = 0.13). During SWS, a significant decrease in CMRGlu was observed as compared to wakefulness (mean difference: -43.80 +/- 14.10%, P less than 0.01). All brain regions were equally affected but thalamic nuclei had significantly lower glucose utilization than the average cortex. During REMS, the CMRGlu were as high as during wakefulness (mean difference: 4.30 +/- 7.40%, P = 0.35). The metabolic pattern during REMS appeared more heterogeneous than at wake. An activation of left temporal and occipital areas is suggested. It is hypothetized that energy requirements for maintaining membrane polarity are reduced during SWS because of a decreased rate of synaptic events. During REMS, cerebral glucose utilization is similar to that of wakefulness, presumably because of reactivated neurotransmission and increased need for ion gradients maintenance.

Local cerebral glucose utilization following unilateral and bilateral lesions of the nucleus basalis magnocellularis in the rat.

To investigate to what extent the loss of cholinergic projections to the neocortex results in functional impairment in the target areas, local rates of cerebral glucose utilization were measured following excitotoxin lesions of the nucleus basalis magnocellularis (NBM) in the rat. Both unilateral and bilateral lesions of NBM resulted in reversible depression of cerebral metabolism. The effects of unilateral lesions were limited to the cortical areas which receive most of the cholinergic projections from NBM. The metabolic defect produced by bilateral lesions was spread to the whole brain. Within 4 months, however, normal metabolic values coexisted with marked changes of the presynaptic cholinergic markers and impairment of conditioned behavior.

Local cerebral glucose utilization following subacute and chronic diazepam pretreatment: differential tolerance.

Local cerebral glucose utilization (LCGU) was determined in parallel groups of conscious rats receiving diazepam (0.3 mg/kg i.v.) either acutely or following subacute (5 mg/kg i.p. daily for 3 days) or chronic (5 mg/kg i.p. daily for 28 days) diazepam pretreatment, using 2-deoxyglucose quantitative autoradiography. Acute administration of diazepam reduced LCGU in 44 of the 66 structures examined compared to vehicle-treated controls. These included limbic, cortical and extrapyramidal structures, and areas associated with sensory processing. These data are consistent with many brain regions being functionally involved in the diverse acute pharmacological effects of diazepam and with the widespread distribution of benzodiazepine receptors throughout the neuroaxis. Following subacute treatment, when animals were tolerant to the sedative effects of diazepam, glucose use remained depressed in the majority of areas studied. However, in the locus coeruleus, dorsal tegmental nucleus and most structures associated with auditory processing, tolerance to the depressant effect of diazepam upon glucose use had occurred suggesting the importance of these structures in the sedative effect of diazepam. The most striking feature of the patterns of LCGU after chronic diazepam treatment was that tolerance had occurred in the mammillary body and subiculum. However, glucose use remained depressed in hippocampal layers and in structures that provide input to the hippocampus (e.g. raphe nuclei). These data suggest that the outflow of neuronal activity from the hippocampus to the mammillary body via the subiculum is restored after chronic treatment, and may implicate these pathways in the anxiolytic action of diazepam. Overall, it would appear that different neuroanatomical substrates underlie the various pharmacological effects of diazepam and that there may be regional differences in tolerance mechanisms.

Postischemic glucose utilization in rat hippocampal layers.

The influence on hippocampal glucose utilization was determined in male Wistar rats 7 days after a 10-min forebrain ischemia. Ischemia was induced by clamping of the carotid arteries and lowering blood pressure to 40 mm Hg. Despite severe neuronal damage as assessed by histological techniques, local cerebral glucose utilization (LCGU) was significantly increased in the pyramidal and radiatum layer of the CA1 sector, while in layers of the CA2, CA3 and CA4 sector and dentate gyrus. LCGU was reduced compared to non-ischemic controls. The increases in LCGU are suggested to reflect long-lasting hyperexcitation in the selectively vulnerable CA1 sector, implicating a correlation between cellular hypermetabolism and neuronal damage.

Cerebral glucose utilization during diazepam withdrawal in rats.

The diazepam withdrawal syndrome in rats was characterized behaviorally by an increase in spontaneous motor activity, slight body tremor and a lack of convulsions. The 2-deoxyglucose (2-DG) technique was used to measure quantitatively cerebral glucose utilization during diazepam withdrawal and revealed changes in glucose utilization in 30% of the 54 structures evaluated. Areas of increased glucose utilization included medial geniculate, inferior colliculus, visual cortex, mammillary body, dorsal hippocampus, cerebellar flocculus, and zona reticulata and globus pallidus, olfactory cortex, nucleus accumbens and internal capsule. There was no single or consistent relationship between reported benzodiazepine receptor densities and glucose utilization.