Long Withdrawal of Methylphenidate Induces a Differential Response of the Dopaminergic System and Increases Sensitivity to Cocaine in the Prefrontal Cortex of Spontaneously Hypertensive Rats

Methylphenidate (MPD) is one of the most prescribed drugs for alleviating the symptoms of Attention Deficit/Hyperactivity Disorder (ADHD). However, changes in the molecular mechanisms related to MPD withdrawal and susceptibility to consumption of other psychostimulants in normal individuals or individuals with ADHD phenotype are not completely understood. The aims of the present study were: (i) to characterize the molecular differences in the prefrontal dopaminergic system of SHR and Wistar strains, (ii) to establish the neurochemical consequences of short- (24 hours) and long-term (10 days) MPD withdrawal after a subchronic treatment (30 days) with Ritalin® (Methylphenidate Hydrochloride; 2.5 mg/kg orally), (iii) to investigate the dopaminergic synaptic functionality after a cocaine challenge in adult MPD-withdrawn SHR and Wistar rats. Our results indicate that SHR rats present reduced [³H]-Dopamine uptake and cAMP accumulation in the prefrontal cortex (PFC) and are not responsive to dopaminergic stimuli in when compared to Wistar rats. After a 24-hour withdrawal of MPD, SHR did not present any alterations in [³H]-Dopamine Uptake, [³H]-SCH 23390 binding and cAMP production; nonetheless, after a 10-day MPD withdrawal, the results showed a significant increase of [³H]-Dopamine uptake, of the quantity of [³H]-SCH 23390 binding sites and of cAMP levels in these animals. Finally, SHR that underwent a 10-day MPD withdrawal and were challenged with cocaine (10 mg/kg i.p.) presented reduced [³H]-Dopamine uptake and increased cAMP production. Wistar rats were affected by the 10-day withdrawal of MPD in [³H]-dopamine uptake but not in cAMP accumulation; in addition, cocaine was unable to induce significant modifications in [³H]-dopamine uptake and in cAMP levels after the 10-day withdrawal of MPD. These results indicate a mechanism that could explain the high comorbidity between ADHD adolescent patients under methylphenidate treatment and substance abuse in adult life.     

Autoradiographic Localization of [125I-Tyr0]Bradykinin Binding Sites in Brains of Wistar-Kyoto and Spontaneously Hypertensive Rats

1. The present study was undertaken to localize and characterize bradykinin (BK) binding sites in brains from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR).2. Serial sections of brains were cut from adult WKY and SHR and specific [¹²⁵I-Tyr⁰]bradykinin ([¹²⁵I-Tyr⁰]BK) binding was determined using in vitro quantitative receptor autoradiographic techniques.3. Specific binding of [¹²⁵I Tyr⁰]BK was localized in the medulla oblongata to the regions of the nucleus of the solitary tract (NTS), area postrema (AP), dorsal motor nucleus of the vagus (X), and caudal subnucleus of the spinal trigeminal nucleus in both strains of rat. The specific binding (85–90% of total binding) was of high affinity and saturable with K _D values in the range of 100 pM and a B _max of 0.75 fmol per mg tissue equivalent in the NTS–X–AP complex of both the WKY and SHR. In competition studies, the rank order of potencies was similar in both strains with BK = Lys-BK > icatibant >>> DesArg⁹-BK. The B₂ receptor antagonist icatibant inhibited [¹²⁵I-Tyr⁰]BK binding with a K _i value of 0.63 ± 0.19 nM in WKY and 0.91 ± 0.73 nM in SHR, while K _i values for the B> ₁ receptor agonist DesArg⁹-BK were 1475 ± 1055 and 806 ± 362 nM in WKY and SHR, respectively.4. Our finding of specific high-affinity [¹²⁵I-Tyr⁰]BK B₂ binding sites in the NTS, AP, and the X of WKY and SHR is important because these brain areas are associated with central cardiovascular regulation. However, alterations in BK B₂ receptors in the medulla that could contribute to the hypertensive state in the SHR were not detected.     

Nitric Oxide Synthase in Spontaneously Hypertensive Rats

Since its discovery by Furchgott and Zawadzki in 1980 [18], endothelium-derived relaxing factor (EDRF) has been shown to play a central role in the cardiovascular system [10]. The endothelial product is chemically equivalent to nitric oxide (NO) [23, 40] or a biochemical congener thereof [48]. Fifteen years ago, this small, simple and highly toxic molecule was known as a lengthy list of environmental pollutants found in unsavory haunts such as smoke and smog, and even as destroyer of ozone, suspected carcinogen, and precursor of acid rain. In addition, NO seems an unlikely biological jack of all trades for most of the body's functions are regulated by extraordinarily large and complex proteins and compounds. But over the past decade, diverse lines of evidence have converged to show that this sometime poison is a fundamental player in the everyday business of the human body.     

Characterization of Excitatory Amino Acid Modulation of Dopamine Release in the Prefrontal Cortex of Conscious Rats

Abstract: The effect of various classes of excitatory amino acid agonists on the release of dopamine in the medial prefrontal cortex (PFC) of awake rats was examined using intracerebral microdialysis. Local infusion of 20 µ M α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), through the microdialysis probe, produced a significant increase of more than twofold in extracellular levels of dopamine. Application of 100 µ M AMPA increased these levels nearly 15 fold. The AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (50 µ M ) blocked the increase in dopamine release produced by 20 µ M AMPA. Local infusion of kainate at concentrations of 5 and 20 µ M increased dopamine release by nearly 150 and 500%, respectively. Local application of CNQX (50 µ M ) before 20 µ M kainate significantly attenuated the stimulatory effect of kainate on dopamine levels. In contrast to AMPA and kainate, infusion of N -methyl- d -aspartate (NMDA) at 20 or 100 µ M did not increase dopamine release. In fact, a trend toward a decrease in dopamine release was evident after 100 µ M NMDA. The present study indicates that the in vivo release of dopamine in the PFC is facilitated by AMPA and kainate receptors. This modulation is more profound than that previously reported in the basal ganglia. The lack of an excitatory effect of NMDA is in agreement with recent reports that the NMDA receptor may inhibit indirectly dopaminergic neurotransmission in the PFC.     

Dopamine Autoreceptors Modulate Dopamine Release from the Prefrontal Cortex

Electrical stimulation (at 0.3, 1, or 10 Hz, 120 pulses each) produced a calcium-dependent overflow of radioactivity from slices of the rabbit prefrontal cortex preloaded with [3H]3,4-dihydroxyphenylethylamine ([3H]DA, [3H]dopamine) in the presence of desipramine. Flat frequency-release curves were observed. Apomorphine and LY-171555 inhibited in a concentration-dependent fashion the evoked overflow of DA, an effect antagonized by haloperidol. Stimulation frequencies comparable to normal firing rates of mesocortical neurons (10 Hz) drastically reduced apomorphine-induced inhibition of DA overflow. Haloperidol produced greater facilitation of DA overflow at 10 than at 1 Hz. Nomifensine, a neuronal uptake inhibitor, enhanced DA overflow. These results indicate that mesocortical DA neurons projecting to the prefrontal cortex have release modulatory autoreceptors of the D2 subtype.     

Acute Stress Increases Depolarization-Evoked Glutamate Release in the Rat Prefrontal/Frontal Cortex: The Dampening Action of Antidepressants

Background

Behavioral stress is recognized as a main risk factor for neuropsychiatric diseases. Converging evidence suggested that acute stress is associated with increase of excitatory transmission in certain forebrain areas. Aim of this work was to investigate the mechanism whereby acute stress increases glutamate release, and if therapeutic drugs prevent the effect of stress on glutamate release.

Methodology/Findings

Rats were chronically treated with vehicle or drugs employed for therapy of mood/anxiety disorders (fluoxetine, desipramine, venlafaxine, agomelatine) and then subjected to unpredictable footshock stress. Acute stress induced marked increase in depolarization-evoked release of glutamate from synaptosomes of prefrontal/frontal cortex in superfusion, and the chronic drug treatments prevented the increase of glutamate release. Stress induced rapid increase in the circulating levels of corticosterone in all rats (both vehicle- and drug-treated), and glutamate release increase was blocked by previous administration of selective antagonist of glucocorticoid receptor (RU 486). On the molecular level, stress induced accumulation of presynaptic SNARE complexes in synaptic membranes (both in vehicle- and drug-treated rats). Patch-clamp recordings of pyramidal neurons in the prefrontal cortex revealed that stress increased glutamatergic transmission through both pre- and postsynaptic mechanisms, and that antidepressants may normalize it by reducing release probability.

Conclusions/Significance

Acute footshock stress up-regulated depolarization-evoked release of glutamate from synaptosomes of prefrontal/frontal cortex. Stress-induced increase of glutamate release was dependent on stimulation of glucocorticoid receptor by corticosterone. Because all drugs employed did not block either elevation of corticosterone or accumulation of SNARE complexes, the dampening action of the drugs on glutamate release must be downstream of these processes. This novel effect of antidepressants on the response to stress, shown here for the first time, could be related to the therapeutic action of these drugs.     

Excess Salt Increases Infarct Size Produced by Photothrombotic Distal Middle Cerebral Artery Occlusion in Spontaneously Hypertensive Rats

Cerebral circulation is known to be vulnerable to high salt loading. However, no study has investigated the effects of excess salt on focal ischemic brain injury. After 14 days of salt loading (0.9% saline) or water, spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) were subjected to photothrombotic middle cerebral artery occlusion (MCAO), and infarct volume was determined at 48 h after MCAO: albumin and hemoglobin contents in discrete brain regions were also determined in SHR. Salt loading did not affect blood pressure levels in SHR and WKY. After MCAO, regional cerebral blood flow (CBF), determined with two ways of laser-Doppler flowmetry (one-point measurement or manual scanning), was more steeply decreased in the salt-loaded group than in the control group. In SHR/Izm, infarct volume in the salt-loaded group was 112±27 mm³, which was significantly larger than 77±12 mm³ in the control group (p = 0.002), while the extents of blood-brain barrier disruption (brain albumin and hemoglobin levels) were not affected by excess salt. In WKY, salt loading did not significantly increase infarct size. These results show the detrimental effects of salt loading on intra-ischemic CBF and subsequent brain infarction produced by phototrhombotic MCAO in hypertensive rats.     

Glutamatergic Regulation of Basal and Stimulus-Activated Dopamine Release in the Prefrontal Cortex

Abstract: The present study was undertaken to determine whether basal and stimulus-activated dopamine release in the prefrontal cortex (PFC) is regulated by glutamatergic afferents to the PFC or the ventral tegmental area (VTA), the primary source of dopamine neurons that innervate the rodent PFC. In awake rats, blockade of NMDA or α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors in the VTA, or blockade of AMPA receptors in the PFC, profoundly reduced dopamine release in the PFC, suggesting that the basal output of dopamine neurons projecting to the PFC is under a tonic excitatory control of NMDA and AMPA receptors in the VTA, and AMPA receptors in the PFC. Consistent with previous reports, blockade of cortical NMDA receptors increased dopamine release, suggesting that NMDA receptors in the PFC exert a tonic inhibitory control on dopamine release. Blockade of NMDA or AMPA receptors in the VTA as well as blockade of AMPA receptors in the PFC reduced the dopaminergic response to mild handling, suggesting that activation of glutamate neurotransmission also regulates stimulus-induced increase of dopamine release in the PFC. In the context of brain disorders that may involve cortical dopamine dysfunction, the present findings suggest that abnormal basal or stimulus-activated dopamine neurotransmission in the PFC may be secondary to glutamatergic dysregulation.     

rTMS of the Left Dorsolateral Prefrontal Cortex Modulates Dopamine Release in the Ipsilateral Anterior Cingulate Cortex and Orbitofrontal Cortex

Background

Brain dopamine is implicated in the regulation of movement, attention, reward and learning and plays an important role in Parkinson''s disease, schizophrenia and drug addiction. Animal experiments have demonstrated that brain stimulation is able to induce significant dopaminergic changes in extrastriatal areas. Given the up-growing interest of non-invasive brain stimulation as potential tool for treatment of neurological and psychiatric disorders, it would be critical to investigate dopaminergic functional interactions in the prefrontal cortex and more in particular the effect of dorsolateral prefrontal cortex (DLPFC) (areas 9/46) stimulation on prefrontal dopamine (DA).

Methodology/Principal Findings

Healthy volunteers were studied with a high-affinity DA D2-receptor radioligand, [¹¹C]FLB 457-PET following 10 Hz repetitive transcranial magnetic stimulation (rTMS) of the left and right DLPFC. rTMS on the left DLPFC induced a significant reduction in [¹¹C]FLB 457 binding potential (BP) in the ipsilateral subgenual anterior cingulate cortex (ACC) (BA 25/12), pregenual ACC (BA 32) and medial orbitofrontal cortex (BA 11). There were no significant changes in [¹¹C]FLB 457 BP following right DLPFC rTMS.

Conclusions/Significance

To our knowledge, this is the first study to provide evidence of extrastriatal DA modulation following acute rTMS of DLPFC with its effect limited to the specific areas of medial prefrontal cortex. [¹¹C]FLB 457-PET combined with rTMS may allow to explore the neurochemical functions of specific cortical neural networks and help to identify the neurobiological effects of TMS for the treatment of different neurological and psychiatric diseases.     

Corticotropin-Releasing Factor Stimulates Catecholamine Release in Hypothalamus and Prefrontal Cortex in Freely Moving Rats as Assessed by Microdialysis

Abstract: In vivo microdialysis was used to measure changes in extracellular concentrations of catecholamines and indoleamines in freely moving rats in response to administration of corticotropin-releasing factor (CRF). Dialysis probes were placed stereotaxically in either the medial hypothalamus or the medial prefrontal cortex. We used a repeated-measures design in which each rat received artificial CSF or one dose of CRF 3–4 h apart, and each subject was retested with the same treatments in the reverse order 5–7 days later. With the dialysis probe in the hypothalamus, intracerebroventricular administration of CRF (17 or 330 pmol) dose-dependently increased dialysate concentrations of norepinephrine (NE), dopamine (DA), and all their measurable catabolites except normetanephrine. The effects on NE were substantially greater than those on DA. Dialysate concentrations of serotonin could not be measured reliably, but those of its catabolite, 5-hydroxyindoleacetic acid, were also elevated. Concentrations of NE and DA were elevated within the first one or two (20 min) collection periods, with a peak response at ∼ 1–2 h. Dialysate concentrations of catecholamines and metabolites normally returned to baseline within 3 h. Similar data were obtained with dialysis probes in the medial prefrontal cortex after intracerebroventricular administration of 17 or 167 pmol of CRF, except that the increases in DA exceeded those of NE in this region. Intraperitoneal administration of CRF (1 nmol) similarly elevated dialysate concentrations of NE, DA, 5-hydroxyindoleacetic acid, and all catecholamine catabolites except normetanephrine in both medial hypothalamus and medial prefrontal cortex. These results support earlier neurochemical data suggesting that CRF administered both centrally and peripherally stimulates the release of both DA and NE in the brain.     

A comparison of the cutaneous microvascular properties of the Spontaneously Hypertensive rat and the Wistar-Kyoto rat

The Spontaneously Hypertensive rat (SHR) and its non-hypertensive companion strain, the Wistar-Kyoto (WKY) rat, provide an excellent comparative model to permit study of the differential properties of cutaneous microvascular beds. We explored the possibility that chronically elevated vascular pressures in the SHR rat might affect the microvascular constitution of the skin. We measured skin blood flow at the back and at the paw of a group of 20-week-old WKY rats and a contrast group of SHR rats. We then performed skin biopsies at these two locations and used the NIH Image program to count and measure the size of capillaries, arterioles, and venules. We also determined microvascular density as percentage of total tissue area. At basal temperature, skin blood flow was similar in the two rat strains at both the back and paw. Heat induced vasodilatation resulted in a 50% increase in blood flow at the back, reaching the same level in the two rat groups. However, at the paw site, thermal stimulation resulted in significantly greater flow (39.3 +/- 3.1 ml/100 gm tissue per min) in the SHR rats than the WKY rats (28.6 +/- 1.9 ml/100 gm tissue per min, P < 0.05). The ratio of systemic arterial pressure to skin blood flow was computed as an index of vascular resistance to flow. At basal temperature, this index was 50% greater for the SHR rats at both skin sites. At 44 degrees C, the resistance index decreased at both sites in both rat groups but was still approximately 50% higher at the back of the SHR than the WKY rats. In contrast, the resistance index at 44 degrees C at the paw site fell to the same level in both the SHR and WKY rats. There were twice as many capillaries at the back of the WKY rats than at the back of the SHR rats (9.2 +/- 2.0 per mm2 vs. 4.7 +/- 1.2 per mm2, P < 0.05). Expressed as a percentage of total tissue area, the capillary density at the back in the WKY rats was 0.064 +/- 0.010% as compared to 0.034 +/- 0.008% in the SHR rats (P < 0.05). There were five times more arterioles at the paw compared to the back in both rat groups with no significant difference between the groups. We measured the diameter of the lumen and the thickness of the wall of each arteriole and computed their ratio as an index of possible media hypertrophy. There were minimal differences seen in these parameters between the two rat groups at the back and paw sites. The venular density was significantly higher at the paw than at the back in both rat groups with no significant difference between them. Reduced capillary density at the back of the SHR rats may be a developmental adaptation to high blood pressure. Such a reduction in the pathways of blood flow may help account for increased flow resistance at that site, independent of arteriolar vasoconstriction.     

Spatial Memory in Spontaneously Hypertensive Rats (SHR)

The spontaneously hypertensive rat (SHR) is an established animal model of ADHD. It has been suggested that ADHD symptoms arise from deficits in executive functions such as working memory, attentional control and decision making. Both ADHD patients and SHRs show deficits in spatial working memory. However, the data on spatial working memory deficits in SHRs are not consistent. It has been suggested that the reported cognitive deficits of SHRs may be related to the SHRs’ locomotor activity. We have used a holeboard (COGITAT) to study both cognition and activity in order to evaluate the influence of the activity on the cognitive performance of SHRs. In comparison to Wistar-Kyoto (WKY) rats, SHRs did not have any impairment in spatial working memory and reference memory. When the rats’ locomotor activity was taken into account, the SHRs’ working memory and reference memory were significantly better than in WKY rats. The locomotor activity appears to be a confounding factor in spatial memory tasks and should therefore be controlled for in future studies. In the SHR model of ADHD, we were unable to demonstrate an impairment of working memory which has been reported in patients with ADHD.     

Alteration of Cerebral Taurine Biosynthesis in Spontaneously Hypertensive Rats

Abstract: Cerebral taurine biosynthesis in a spontaneously hypertensive rat (SHR) has been studied. Cysteine sulfinic acid (CSA) and cysteic acid (CA), possible key intermediates in taurine biosynthesis, were found in the rat brain, whereas no cysteamine-cystamine was detected. In the brain of SHR, a statistically significant decrease in the contents of CSA, CA, and taurine was noted in the cerebellum, hypothalamus, and striatum as compared with normotensive Wistar Kyoto rats. Similarly, it was demonstrated that the activity of cysteine dioxygenase, the enzyme catalyzing cysteine to CSA, was attenuated significantly in the same brain areas of SHR. In contrast, no alteration in the activity of CSA decarboxylase, the enzyme converting CSA to hypotaurine or CA to taurine, was observed. A decline in the percent conversion of [¹⁴C]cysteine to [¹⁴C]taurine was found also in tissue homogenates from the cerebellum, hypothalamus, and striatum of SHR, indicating that the declines in taurine content may be due to an attenuation of taurine biosynthesis, possibly at the step involving cysteine dioxygenase.     

Proteomic Response to Acupuncture Treatment in Spontaneously Hypertensive Rats

Previous animal and clinical studies have shown that acupuncture is an effective alternative treatment in the management of hypertension, but the mechanism is unclear. This study investigated the proteomic response in the nervous system to treatment at the Taichong (LR3) acupoint in spontaneously hypertensive rats (SHRs). Unanesthetized rats were subject to 5-min daily acupuncture treatment for 7 days. Blood pressure was monitored over 7 days. After euthanasia on the 7^th day, rat medullas were dissected, homogenized, and subject to 2D gel electrophoresis and MALDI-TOF analysis. The results indicate that blood pressure stabilized after the 5th day of acupuncture, and compared with non-acupoint treatment, Taichong-acupunctured rat’s systolic pressure was reduced significantly (P<0.01), though not enough to bring blood pressure down to normal levels. The different treatment groups also showed differential protein expression: the 2D images revealed 571±15 proteins in normal SD rats’ medulla, 576±31 proteins in SHR’s medulla, 597±44 proteins in medulla of SHR after acupuncturing Taichong, and 616±18 proteins in medulla of SHR after acupuncturing non-acupoint. In the medulla of Taichong group, compared with non-acupoint group, seven proteins were down-regulated: heat shock protein-90, synapsin-1, pyruvate kinase isozyme, NAD-dependent deacetylase sirtuin-2, protein kinase C inhibitor protein 1, ubiquitin hydrolase isozyme L1, and myelin basic protein. Six proteins were up-regulated: glutamate dehydrogenase 1, aldehyde dehydrogenase 2, glutathione S-transferase M5, Rho GDP dissociation inhibitor 1, DJ-1 protein and superoxide dismutase. The altered expression of several proteins by acupuncture has been confirmed by ELISA, Western blot and qRT-PCR assays. The results indicate an increase in antioxidant enzymes in the medulla of the SHRs subject to acupuncture, which may provide partial explanation for the antihypertensive effect of acupuncture. Further studies are warranted to investigate the role of oxidative stress modulation by acupuncture in the treatment of hypertension.     

Glutamatergic Control of Dopamine Release During Stress in the Rat Prefrontal Cortex

Abstract: In vivo microdialysis was used to assess the hypothesis that the stress-induced increase in dopamine release in the prefrontal cortex is mediated by stress-activated glutamate neurotransmission in this region. Local perfusion of an α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, blocked the stress-induced increase in dopamine levels, whereas an NMDA receptor antagonist, 2-amino-5-phosphonopentanoic acid, at the dose tested, was not able to alter this response significantly. These data indicate that the effect of stress on dopamine release in the prefrontal cortex is mediated locally by activation of AMPA/kainate receptors, which modulate the release of dopamine in this region.     

Characterization of Spontaneous,Transient Adenosine Release in the Caudate-Putamen and Prefrontal Cortex

Adenosine is a neuroprotective agent that inhibits neuronal activity and modulates neurotransmission. Previous research has shown adenosine gradually accumulates during pathologies such as stroke and regulates neurotransmission on the minute-to-hour time scale. Our lab developed a method using carbon-fiber microelectrodes to directly measure adenosine changes on a sub-second time scale with fast-scan cyclic voltammetry (FSCV). Recently, adenosine release lasting a couple of seconds has been found in murine spinal cord slices. In this study, we characterized spontaneous, transient adenosine release in vivo, in the caudate-putamen and prefrontal cortex of anesthetized rats. The average concentration of adenosine release was 0.17±0.01 µM in the caudate and 0.19±0.01 µM in the prefrontal cortex, although the range was large, from 0.04 to 3.2 µM. The average duration of spontaneous adenosine release was 2.9±0.1 seconds and 2.8±0.1 seconds in the caudate and prefrontal cortex, respectively. The concentration and number of transients detected do not change over a four hour period, suggesting spontaneous events are not caused by electrode implantation. The frequency of adenosine transients was higher in the prefrontal cortex than the caudate-putamen and was modulated by A₁ receptors. The A₁ antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine, 6 mg/kg i.p.) increased the frequency of spontaneous adenosine release, while the A₁ agonist CPA (N⁶-cyclopentyladenosine, 1 mg/kg i.p.) decreased the frequency. These findings are a paradigm shift for understanding the time course of adenosine signaling, demonstrating that there is a rapid mode of adenosine signaling that could cause transient, local neuromodulation.     

The Anxiogenic β-Carboline FG 7142 Selectively Increases Dopamine Release in Rat Prefrontal Cortex as Measured by Microdialysis

The effect of the anxiogenic beta-carboline methyl-beta-carboline-3-carboxyamide (FG 7142) on dopamine release in prefrontal cortex and striatum in the awake freely moving rat was determined using the technique of microdialysis. FG 7142 (25 mg/kg, i.p.) caused a time-dependent increase in dopamine release in prefrontal cortex which was statistically significantly greater than the response to vehicle administration. Dopamine release in striatum was unaltered by FG 7142. Pretreatment of animals with the benzodiazepine antagonist Ro 15-1788 (30 mg/kg, i.p., 15 min prior to FG 7142 administration) completely abolished the increase in dopamine release caused by FG 7142 in prefrontal cortex. These data indicate that the anxiogenic benzodiazepine inverse agonist FG 7142 can selectively increase dopamine release in prefrontal cortex, and that this effect appears to be mediated via the gamma-aminobutyric acid/benzodiazepine receptor complex.     

Circadian Rhythm of Glucose Uptake in Cultures of Skeletal Muscle Cells and Adipocytes in Wistar-Kyoto,Wistar, Goto-Kakizaki,and Spontaneously Hypertensive Rats

Hypertension and noninsulin-dependent diabetes mellitus are usually associated with marked glucose intolerance. Hypertensive and even nonhypertensive diabetic individuals display disturbances of the normal circadian blood pressure rhythm. However, little is known about circadian changes of the glucose uptake in muscle and fat cells, the major glucose utilizing tissues. Therefore, we investigated circadian rhythms of glucose uptake in primary muscle and fat cell cultures of hypertensive and type II diabetic rats and their respective control strains. 2-Deoxy-d-(1-³H)glucose uptake was measured over 48 h after synchronization of cells by means of medium change with and without addition of insulin, phloretine, and/or staurosporine. The circadian changes of glucose uptake were assessed by fitting cosine curves to the uptake values. Insulin stimulation of deoxyglucose uptake was only present in control animals, not in hypertensive and diabetic rats. Deoxyglucose uptake displayed a circadian rhythm in control animals, and was markedly disturbed in hypertensive and diabetic animals. Blocking of glucose transporters by phloretine abolished the circadian pattern of deoxyglucose uptake indicating a role of glucose transporters in its generation. Inhibition of kinases by staurosporine inhibited the insulin-stimulated deoxyglucose uptake, but did not dampen the circadian rhythmicity of basal deoxyglucose uptake. The generation of the circadian rhythm of glucose uptake in muscle and fat cell cultures is therefore probably insulin independent and independent of protein kinases. In summary, our results show for the first time: (a) a circadian rhythm of deoxyglucose uptake in glucose utilizing muscle and fat cells in vitro, (b) a disruption of this rhythm in cells of hypertensive and diabetic rats.