Can a belly board reduce respiratory-induced prostate motion in the prone position?--assessed by cine-magnetic resonance imaging.

The purpose of this study is to evaluate the real-time respiratory motion of the prostate and surrounding tissues/organs in the supine and prone positions and to investigate, using cine-MRI, whether a belly board can reduce respiratory-induced motion in the prone position. Cine-MRI scans were made of 13 volunteers in the supine and prone positions on a flat board and in two different prone positions using a belly board. Images in cine mode were recorded for 20 seconds. For each session, the points of interest (POIs) were located at the apex, base, mid-anterior surface and mid-posterior surface of the prostate; the tip of the seminal vesicle; the pubic symphysis; and the sacrum. The maximum range and standard deviation (SD) of the displacement from the mean value were calculated. The SDs for each of the four different positions were compared using a paired t-test. Respiratory-induced prostate motion was significantly larger in the prone position than in the supine position. However, when a belly board was used in the prone position, motion in the prostate and surrounding tissues/organs was significantly reduced. There were no significant differences between the two different positions using a belly board in any of the POIs.

Ginkgo biloba leaf extract (EGb 761®) and its specific acylated flavonol constituents increase dopamine and acetylcholine levels in the rat medial prefrontal cortex: possible implications for the cognitive enhancing properties of EGb 761®.

Experimental and clinical data suggest that the Ginkgo biloba standardized extract EGb 761® exerts beneficial effects in conditions which are associated with impaired cognitive function. However, the neurochemical correlates of these memory enhancing effects are not yet fully clarified. The aim of this study was to examine the effect of repeated oral administration of EGb 761® and some of its characteristic constituents on extracellular levels of dopamine (DA), noradrenaline (NA), serotonin (5-HT), acetylcholine (ACh) and the metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in the medial prefrontal cortex (mPFC) of awake rats by use of in vivo microdialysis technique. Subacute (14 days, once daily), but not acute, oral treatment with EGb 761® (100 and 300 mg/kg) or the flavonoid fraction, which represents about 24% of the whole extract caused a significant and dose-dependent increase in extracellular DA levels in the mPFC. Repeated administration of EGb 761® also caused a modest but significant increase in the NA levels, whereas the concentrations of 5-HT and those of the metabolites DOPAC, HVA and 5-HIAA were not affected. The same treatment regimen was used in a subsequent study with the aim of investigating the effects of two Ginkgo-specific acylated flavonols, 3-O-(2''-O-(6'''-O-(p-hydroxy-trans-cinnamoyl)-ß-D-glucosyl)-α-L-rhamnosyl)quercetin (Q-ag) and 3-O-(2''-O-(6'''-O-(p-hydroxy-trans-cinnamoyl)-ß-D-glucosyl)-α-L-rhamnosyl)kaempferol (K-ag). Both compounds together represent about 4.5% of the whole extract. Repeated oral treatment with Q-ag (10 mg/kg) for 14 days caused a significant increase in extracellular DA levels of 159% and extracellular acetylcholine (ACh) levels of 151% compared to controls. Similarly, administration of K-ag (10 mg/kg) induced a significant rise of DA levels to 142% and ACh levels to 165% of controls, whereas treatment with isorhamnetin, an O-methylated aglycon component of EGb 761® flavonol glycosides had no effect. None of the tested flavonoids had a significant effect on extracellular DOPAC and HVA levels. The present findings provide evidence that the subacute treatment with EGb 761® and its flavonol constituents increases DA and ACh release in the rat mPFC, and suggest that the two Ginkgo-specific acylated flavonol glycosides Q-ag and K-ag are active constituents contributing to these effects. As seen for isorhamnetin, the effect on neurotransmitter levels seems not to be a general effect of flavonols but rather to be a specific action of acylated flavonol glycosides which are present in EGb 761®. The direct involvement of these two flavonol derivatives in the increase of dopaminergic and cholinergic neurotransmission in the prefrontal cortex may be one of the underlying mechanisms behind the reported effects of EGb 761® on the improvement of cognitive function.

Determination of histamine in microdialysis samples from Guinea pig skin by high-performance liquid chromatography with fluorescence detection.

AIM: To develop a sensitive and selective liquid-chromatographic method for the determination of histamine in microdialysis samples from guinea pig skin following allergenic provocation. METHODS: The novel fluorescence derivatization method is based on an intramolecular excimer-forming reaction between 2 amino moieties of histamine and 2 molecules of 4-(1-pyrene)butanoyl chloride (PBC) yielding the corresponding dipyrene-labeled derivative. RESULTS: The PBC derivative of histamine was separated within 20 min, and the detection limit (signal-to-noise ratio = 3) of histamine was 0.6 fmol/20 µl volume injected. The basal extracellular levels of histamine in guinea pig skin microdialysates were 20.6 ± 1.7 fmol/10 µl. Subcutaneous administration of histamine liberator compound 48/80 (3 mg/kg) increased the extracellular histamine levels in the skin dialysates by about 860%, whereas ovalbumin challenge (2 mg/kg i.v.) in the sensitized guinea pigs increased the extracellular histamine levels by about 3,030%. CONCLUSION: The novel technique for histamine determination in microdialysis samples from the guinea pig skin may be utilized in preclinical research of antihistaminergic drugs and evaluation of allergenic properties of various dermal preparations such as transdermal drug delivery systems.

SU-E-J-26: Automated Estimation Method of Patient Setup Errors Using Simulated Portal Images for Prostate Cancer Radiotherapy.

PURPOSE: We developed a novel automated estimation method for patient setup errors based on simulated and real portal images for prostate cancer radiotherapy. METHODS: The estimation of patient setup errors in this study was based on a template matching technique with a cross-correlation coefficient and Sobel filter between the real portal image and localized pelvic template of reference image, which were DRR (digitally reconstructed radiography) images and simulated portal images. The simulated portal image was derived by projecting a CT image according to an inverse exponential power law of x-ray attenuation for a water-equivalent path length of each voxel of the CT image on each ray from a source to each pixel on the EPID (electric portal imaging device). A localized pelvic template of each patient in AP (anterior-posterior) or lateral view was automatically extracted from the DRR or simulated portal images by cropping a rectangular region, which was determined by using the mean pelvic template and four anatomical feature points. We applied the proposed method to three prostate cancer cases, and evaluated it using the residual error between the patient setup error obtained by proposed method and the gold standard setup error determined by two radiation oncologists. RESULTS: The average residual errors of the patient setup error for the DRR and simulated portal images were 0.79 and 1.26 mm in the left-right (LR) direction, 3.17 and 2.05 mm in the superior-inferior (SI) direction, 1.69 and 5.82 mm in the anterior-posterior (AP) direction, 3.84 and 6.94 mm in Euclidean distance (ED), respectively. If we used the simulated portal image for LR and SI directions and the DRR image for AP direction, the Euclidean distance was 3.22 mm. CONCLUSIONS: The proposed method has a potential to correctly estimate patient setup errors for prostate cancer radiotherapy.

Galanin differentially regulates acetylcholine release in ventral and dorsal hippocampus: a microdialysis study in awake rat.

The purpose of the present study was to investigate, by use of in vivo microdialysis technique, the regulatory role of galanin on acetylcholine (ACh) release in the CA1, CA3, and dentate gyrus (DG) subregions of rat dorsal and ventral hippocampus. In the ventral hippocampus, local infusions of galanin (1.5 nmol) into CA1, and CA3, but not DG (3 nmol), decreased basal ACh release to 58.6% and 68.4%, respectively. In addition, local infusion of galanin (1.5 nmol) into the ventral DG, and CA3 areas decreased basal ACh levels in the CA1 to 51.2% and 84%, respectively. This observation implies that the effects of galanin are unlikely to be mediated via galanin autoreceptors on the cholinergic terminals, but rather via mechanisms involving galanin internalization and modulation of hippocampo-septo-hippocampal loops, attenuation of the excitability of the principal cells, or indirect modulation by galanin-containing vasopressin terminals to the ventral and/or dorsal hippocampus. In the dorsal hippocampus, galanin infusion (1.5 nmol) into the CA1 region increased ACh release to 128.2% of the control levels, but infusions of galanin had no effects in the CA3 and DG. In all cases, the ACh levels returned to basal values within 100 min after the galanin infusion. It is concluded that the attenuating effects of galanin on ACh release in the ventral hippocampus and increase in ACh release in the dorsal hippocampus are in line and support the current view on molecular and functional distinction between the ventral hippocampus being involved preferentially in motivational and emotional behavior, whereas the dorsal hippocampus is primarily implicated in cognitive processes of learning and memory.

Development of a web-based CT dose calculator: WAZA-ARI.

A web-based computed tomography (CT) dose calculation system (WAZA-ARI) is being developed based on the modern techniques for the radiation transport simulation and for software implementation. Dose coefficients were calculated in a voxel-type Japanese adult male phantom (JM phantom), using the Particle and Heavy Ion Transport code System. In the Monte Carlo simulation, the phantom was irradiated with a 5-mm-thick, fan-shaped photon beam rotating in a plane normal to the body axis. The dose coefficients were integrated into the system, which runs as Java servlets within Apache Tomcat. Output of WAZA-ARI for GE LightSpeed 16 was compared with the dose values calculated similarly using MIRD and ICRP Adult Male phantoms. There are some differences due to the phantom configuration, demonstrating the significance of the dose calculation with appropriate phantoms. While the dose coefficients are currently available only for limited CT scanner models and scanning options, WAZA-ARI will be a useful tool in clinical practice when development is finalised.

Mephedrone, compared with MDMA (ecstasy) and amphetamine, rapidly increases both dopamine and 5-HT levels in nucleus accumbens of awake rats.

BACKGROUND AND PURPOSE: The designer drug 1-(4-methylphenyl)-2-methylaminopropan-1-one (4-methylmethcathinone, mephedrone) is reported to possess psychostimulant, entactogenic and hallucinogenic effects. The purpose of this study was to examine the effects of acute administration of mephedrone on extracellular levels of dopamine (DA) and 5-HT in the nucleus accumbens of awake rats and compare these effects with those induced by 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) and amphetamine. EXPERIMENTAL APPROACH: Microdialysis sampling was performed while simultaneously recording locomotor activity in rats and the monoamines were determined by HPLC with electrochemical detection. KEY RESULTS: Mephedrone (3 mg·kg(-1) s.c.) and (+)-amphetamine (1 mg·kg(-1) s.c.) caused rapid increases in extracellular DA levels of 496% and 412%, respectively, whereas MDMA (3 mg·kg(-1) s.c.) showed only a moderate effect (235%). The corresponding 5-HT levels increased to 941% (mephedrone) and 911% (MDMA), but only to 165% following amphetamine. The calculated t(1/2) values for elimination rate of mephedrone, MDMA and amphetamine-induced increases in extracellular DA levels were 25, 303 and 51 min, the corresponding t(1/2) values for 5-HT were 26, 48 and 84 min, respectively. Locomotor activity was increased most by amphetamine, whereas both mephedrone and MDMA showed about three times lower and shorter-lasting effects. CONCLUSIONS AND IMPLICATIONS: The neurochemical and functional properties of mephedrone resemble those of MDMA, but it also shows an amphetamine-like effect in that it evokes a rapid release and elimination of DA in the brain reward system, a feature that may contribute to its potent reinforcing properties.

Estimation of the number of CT procedures based on a nationwide survey in Japan.

In 2007, a nationwide survey was conducted to determine the frequency of CT procedures in Japan in order to compare the current use of CT among developed countries. The frequency of adult and pediatric CT scans was estimated using a model based on the results of the survey. Survey questionnaires were sent to 2,266 CT facilities: 1,068 government hospitals and 1,198 other hospitals and non-hospital medical centers. The questionnaire requested information including the number of beds, outpatients per day, type of CT scanner, various body regions scanned, and the number of scans performed. The results of the study indicate that the number of CT procedures was closely correlated with the number of hospital beds. The authors estimate that approximately 20.5 million procedures were performed in 2005 and 21.2 million in 2006. The number of pediatric CT procedures was calculated by multiplying the total number of CT procedures by the estimated fraction of pediatric (0-15 y) CT procedures. Annual pediatric CT procedures were estimated to have been approximately 580,000 in 2005 and 600,000 in 2006. The present study indicates that the number of procedures per thousand of population, 166 for total CT and 32-34 for pediatric CT, is lower in Japan than in the U.S.

Diffusion and clearance of superparamagnetic iron oxide nanoparticles infused into the rat striatum studied by MRI and histochemical techniques.

The purpose of the present study was to investigate, by MRI and histochemical techniques, the diffusion and clearance abilities of superparamagnetic iron oxide nanoparticles (SPION) coated with dextran (Dextran-SPION) and gold (Au-SPION) following their local infusions into the rat brain. In separate groups of anesthetized rats, the Dextran-SPION and Au-SPION were infused at concentrations of 0.01, 0.1, 1 and 5 µg Fe/0.5 µl and at the flow rate of 0.5 µl min(-1) into the left and right striata, respectively. Repetitive T2-weighted spin-echo MRI scans were performed at time intervals of 1, 6, 12, 24, 48, 72 h, and one, two and eight weeks after inoculation. Following infusion of Dextran-SPION (0.1 µg and 1 µg Fe), the maximal distribution volume was observed at about 12-24 h after inoculation and two weeks later the Fe signals were undetectable for the lower dose. On the other hand, Au-SPION remained tightly localized in the closest vicinity of the infusion site as revealed by unchanged MRI signal intensities and strong histochemical staining of Fe(2+) and Fe(3+) ions in the corresponding brain slices. Immunohistochemical staining of astrocytic and microglial reactions revealed that there were no marked differences in GFAP, VIM or OX-42 labeling observed between the nanoparticle types, however the astrocytic reaction was more pronounced in rats receiving nanoparticles compared to the control (aCSF-infused) rats. In conclusion, the present data demonstrate that the viral-sized Dextran-SPION were able to diffuse freely through the interstitial space of the brain being progressively cleared out from the infusion site within two weeks. Thus, Dextran-SPION could be beneficially used in MRI-guided diagnostic applications such as in experimental oncology or as labels and carriers for targeted drug delivery, whereas Au-SPION could be used for labeling and tracking the transplanted stem cells in experimental MRI.

The Ginkgo biloba extract EGb 761(R) and its main constituent flavonoids and ginkgolides increase extracellular dopamine levels in the rat prefrontal cortex.

BACKGROUND AND PURPOSE: Experimental and clinical data suggest that extracts of Ginkgo biloba improve cognitive function. However, the neurochemical correlates of these effects are not yet fully clarified. The purpose of this study was to examine the effects of acute and repeated oral administration of the standardized extract EGb 761((R)) on extracellular levels of dopamine, noradrenaline and serotonin (5-HT), and the dopamine metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the prefrontal cortex (PFC) and striatum of conscious rats. EXPERIMENTAL APPROACH: Monoamines and their metabolites were monitored by the use of microdialysis sampling and HPLC with electrochemical or fluorescence detection. KEY RESULTS: A single oral dose of EGb 761 (100 mg.kg(-1)) had no effect on monoamine levels. However, following chronic (100 mg.kg(-1)/14 days/once daily) treatment, the same dose significantly increased extracellular dopamine and noradrenaline levels, while 5-HT levels were unaffected. Chronic treatment with EGb 761 showed dose-dependent increases in frontocortical dopamine levels and, to a lesser extent, in the striatum. The extracellular levels of HVA and DOPAC were not affected by either acute or repeated doses. Treatment with the main constituents of EGb 761 revealed that the increase in dopamine levels was mostly caused by the flavonol glycosides and ginkgolide fractions, whereas bilobalide treatment was without effect. CONCLUSIONS AND IMPLICATIONS: The present results demonstrate that chronic but not acute treatment with EGb 761 increased dopaminergic transmission in the PFC. This finding may be one of the mechanisms underlying the reported effects of G. biloba in improving cognitive function.

MitoPark mice mirror the slow progression of key symptoms and L-DOPA response in Parkinson's disease.

The MitoPark mouse, in which the mitochondrial transcription factor Tfam is selectively removed in midbrain dopamine (DA) neurons, is a genetic model for Parkinson's disease (PD) that replicates the slow and progressive development of key symptoms. To further validate this model, we have extended both behavioral and biochemical analyses in these animals. We found that vertical movements decline earlier and faster than horizontal movements, possibly modeling the early occurrence of axial, postural instability in PD. L-DOPA induces different locomotor responses depending on the age: in young MitoPark mice the L-DOPA-induced motor activation is small; middle-aged MitoPark mice respond in a dose-dependent manner to L-DOPA, whereas aged MitoPark mice display a double-peaked locomotor response to a high dose of L-DOPA that includes an intermittent period of very low motor activity, similar to the 'on-off' phenomenon in PD. To correlate behavior with biochemical data, we analyzed monoamine levels in three different brain areas that are highly innervated by the DA system: striatum, anterior cortex and olfactory bulb. DA levels declined earlier and faster in striatum than in cortex; only at the latest time-point analyzed, DA levels were found to be significantly lower than control levels in the olfactory bulb. Interestingly, the ratio between homovanillic acid (HVA) and DA differed between regions over time. In striatum and olfactory bulb, the ratio increased steeply indicating increased DA turnover. In contrast, the ratio decreased over time in cortex, revealing important differences between DA cells in substantia nigra and the ventral tegmental area.

Derivatization chemistries for determination of serotonin, norepinephrine and dopamine in brain microdialysis samples by liquid chromatography with fluorescence detection.

The present paper provides an overview on currently developed derivatization chemistries and techniques for determination of monoamine neurotransmitters serotonin (5-HT), norepinephrine (NE) and dopamine (DA) in microdialysis samples by microbore liquid chromatography with fluorescence detection. In mild alkaline conditions, 5-hydroxyindoles and catecholamines react with benzylamine (BA), forming highly fluorescent 2-phenyl-4,5-pyrrolobenzoxazoles and 2-phenyl(4,5-dihydropyrrolo) [2,3-f]benzoxazoles, respectively. However, for derivatization of DA a higher fluorescence intensity was achieved for reaction with 1,2-diphenylethylenediamine (DPE) rather than with BA, therefore for simultaneous determination of 5-HT, NE and DA in brain microdialysates, a two-step derivatization with BA followed by DPE was developed. The detection limits for 5-HT, NE and DA were 0.2, 0.08 and 0.13 fmol, respectively, in an injection volume of 20 microL, which corresponds to concentrations of 30, 12 and 19.5 pm, respectively in standard solution prior to derivatization. The experimental data presented demonstrate the ability of the technique to simultaneously monitor neuronally releasable pools of monoamine neurotransmitters in the rat and mouse brains at basal conditions and following pharmacological treatments or physiological stimuli. These techniques play an important role in drug discovery and clinical investigation of psychiatric and neurological diseases such as depression, schizophrenia and Parkinson's disease.

Comparison of a digital flat-panel versus screen-film, photofluorography and storage-phosphor systems by detection of simulated lung adenocarcinoma lesions using hard copy images.

The purpose of this study was to compare hard copy images from a flat-panel detector digital radiography system with conventional radiography, photofluorographic radiography and storage phosphor radiography for the detection of simulated lung adenocarcinoma lesions and also for radiation dose. To test the diagnostic performance of these four systems, the authors used 15 types of lung adenocarcinoma phantom according to Noguchi's classification and an anthropomorphic chest phantom. The visual evaluation of tumour detectability by four radiologists and two general thoracic surgeons was examined with a five-level confidence scale. Lung doses were measured with glass dosemeters for the chest radiology systems under the conditions used by each hospital and centre. Our results indicated that flat-panel detector digital radiography and storage phosphor radiography are not necessarily superior to conventional radiography and photofluorographic radiography for detecting lung adenocarcinomas when only hard copy images are used, and this suggests a need to carefully optimize chest radiography.

Loss of heterozygosity alterations associated with progesterone therapy in endometrial hyperplasia and adenocarcinoma.

Loss of heterozygosity (LOH) was analyzed in four patients with endometrial hyperplasia (EH) with atypia (two patients) and without atypia (two patients) and in five patients with endometrial adenocarcinoma (EAC) to clarify the clinicopathologic relationship between genetic alterations and hormone therapy. Each patient was initially administered high-dose medroxyprogesterone acetate (MPA) as a uterine-sparing treatment. The five microsatellite markers used to analyze LOH were at chromosomal loci 8p22.1, 8p21, 8p21.3, 8p22, and 8p22. DNA was extracted from paraffin-embedded sections before, during, and after MPA therapy using laser capture microdissection. As a result, LOH was more frequently detected after MPA therapy (overall ratios were 16, 17, and 29% before, during, and after MPA therapy, respectively). LOH is more easily detected in EH loci than in EAC loci before MPA. For EAC, initial LOH detection on chromosome 8 may be related to an incomplete response to MPA, but negative LOH does not guarantee a favorable treatment outcome. For EH or atypical endometrial hyperplasia, it is unknown whether LOH alteration associated with MPA therapy is related to atypia of the disease.

Bilateral ovarian leiomyomas: CT and MRI features.

We recently treated a 21-year-old woman with leiomyomas arising from the bilateral ovaries, a very rare condition. On magnetic resonance imaging, more than half of the left adnexal mass showed low signal intensity on T2-weighted images and good enhancement by gadolinium-DTPA, and the remaining part showed high signal intensity on T2-weighted images, so the lesions initially were diagnosed as ovarian fibromas or as thecomas with a certain degree of degeneration. Pathologic examination of the excised tumors proved that they were bilateral ovarian leiomyomas; in addition, the tumor from the left side showed hemorrhagic and myxoid changes with torsion of 180 degrees.

Effects of isoflurane on prefrontal acetylcholine release and hypothalamic Fos response in young adult and aged rats.

This experiment investigated the influence of age on prefrontal acetylcholine (ACh) release and Fos response in the hypothalamic paraventricular nucleus and the nucleus tractus solitarius (NTS) of rats following isoflurane anesthesia. It is known that isoflurane decreases acetylcholine release in most brain regions. In the present study, we found that the level of prefrontal acetylcholine was significantly lower in 28-month-old rats (14% of baseline) than in 3-month-old rats (38% of baseline) during 2 h of isoflurane anesthesia (P < 0.05). The old rat group showed significantly greater Fos induction in the paraventricular nucleus compared to the young adult rat group (P < 0.05), indicating that the old rats were subjected to stress. No difference in Fos response was noted in the nucleus tractus solitarius. The old rats displayed a significant increase in feeding behavior during the 3-h recovery period (P < 0.05), but there was no difference in overall acetylcholine levels. Taken together, these findings suggest that isoflurane anesthesia influences old rats more profoundly than young adult rats with regard to reductions in acetylcholine release and stress responses. This may have implications for understanding the development of postoperative delirium in aged patients.

Intraseptal muscarinic ligands and galanin: influence on hippocampal acetylcholine and cognition.

The cholinergic neurons in the septohippocampal projection are implicated in hippocampal functions such as spatial learning and memory. The aim of this study was to examine how septohippocampal cholinergic transmission is modulated by muscarinic inputs and by the neuropeptide galanin, co-localized with acetylcholine (ACh) in septohippocampal cholinergic neurons, and how spatial learning assessed by the Morris water maze test is affected. Muscarinic inputs to the septal area are assumed to be excitatory, whereas galanin is hypothesized to inhibit septohippocampal cholinergic function. To test these hypotheses, compounds were microinjected into the medial septum and hippocampal ACh release was assessed by microdialysis probes in the ventral hippocampus of the rat. Blockade of septal muscarinic transmission by intraseptal scopolamine increased hippocampal ACh release suggesting that septal cholinergic neurons are under tonic inhibition. Stimulation of septal muscarinic receptors by carbachol also increased hippocampal ACh release. Despite this increase, both scopolamine and carbachol tended to impair hippocampus-dependent spatial learning. This finding also suggests a revision of the simplistic notion that an increase in hippocampal ACh may be facilitatory for learning and memory. Galanin infused into the medial septum enhanced hippocampal ACh release and facilitated spatial learning, suggesting that septal galanin, contrary to earlier claims, does not inhibit but excites septohippocampal cholinergic neurons. Galanin receptor stimulation combined with muscarinic blockade in the septal area resulted in an excessive increase of hippocampal ACh release combined with an impairment of spatial learning. This finding suggests that the level of muscarinic activity within the septal area may determine the effects of galanin on hippocampal cognitive functions. In summary, a limited range of cholinergic muscarinic transmission may contribute to optimal hippocampal function, a finding that has important implications for therapeutic approaches in the treatment of disorders of memory function.

Activation of 5-HT(1A) autoreceptors enhances the inhibitory effect of galanin on hippocampal 5-HT release in vivo.

The microdialysis technique was used to examine interactions between 5-HT(1A) and galanin receptors in the dorsal raphe nucleus (DRN), by measuring the extracellular levels of 5-HT in the ventral hippocampus of awake rats. The rats were pretreated with the 5-HT(1A) receptor agonist (R,S)-8-OH-DPAT (0.3 mg/kg, s.c.) or saline. 8-OH-DPAT caused a time-dependent reduction of basal 5-HT levels down to 43-48% at 40 min while at 140 min, the hippocampal 5-HT had returned to control values. At that time point, the rats received a second injection of 8-OH-DPAT or galanin (0.15, 0.5 and 1.5 nmol/0.5 microl) infused into the lateral ventricle. The second injection of 8-OH-DPAT caused a significantly smaller reduction of hippocampal 5-HT levels. In contrast, galanin at all three doses in the 8-OH-DPAT-pretreated groups, was significantly more potent in reducing 5-HT levels (maximal reduction to 74%, 52% and 49%, respectively) than it was in saline-pretreated rats (maximal reduction to 96%, 85% and 69%, respectively). The inhibitory effect of galanin (1.5 nmol) on extracellular 5-HT levels in the rat hippocampus was significantly attenuated by co-administration of the 5-HT(1A) receptor antagonists WAY-100635 (0.3 and 0.6 mg/kg s.c.) and, to a lesser extent, with pindolol (20 mg/kg s.c.). These data provide direct in vivo evidence of agonistic 5-HT(1A)-galanin receptor interaction at the presynaptic level. Furthermore, the findings indicate that a down-regulation of the somato-dendritic 5-HT(1A) autoreceptors, following their stimulation with 8-OH-DPAT and possibly also indirectly with 5-HT reuptake inhibitors, may be compensated by a subsequent 'sensitization' of the inhibitory galanin receptors in the DRN. Thus, the enhanced galanin receptor-mediated inhibition of 5-HT neurotransmission may contribute to the pathophysiology of depression or to the reduced and delayed efficacy of antidepressant therapies.

Determination of serotonin in microdialysis samples from rat brain by microbore column liquid chromatography with post-column derivatization and fluorescence detection.

The present paper describes a new method for on-line determination of 5-HT in brain microdialysates from awake rats by microbore column liquid chromatography with post-column derivatization and fluorescence detection. The derivatization reagent contained 1 mM benzylamine and 0.5 mM potassium hexacyanoferrate (III), both dissolved in a mixture of acetonitrile and 25 mM borate buffer (pH 11.0) (1:1, v/v). The limit of detection (S/N=3) for 5-HT was 0.5 fmol/20 microl. The samples were injected every 20 min onto a microbore column packed with C18 silica gel. The method exhibits an excellent stability over the periods of at least 12-24 h. The basal levels of 5-HT from 25 awake rats were 7.10+/-1.06 fmol/20 microl in the dorsal hippocampus and 4.64+/-0.91 fmol/20 microl (mean+/-SD) in the striatum. The 5-HT release increased to about 1500% during the perfusion with 100 mM K(+) containing Ringer solution or it was reduced to 60 or 40% during the perfusion with 1 microM tetrodotoxin or calcium free Ringer, respectively. The new method can be used to monitor extracellular 5-HT following acute systemic drug administration.

Microdialysis in freely moving mice: determination of acetylcholine, serotonin and noradrenaline release in galanin transgenic mice.

In the present study, we describe micro-surgical methods for simultaneous implantation of a microdialysis probe and an intraventricular injection cannula via their respective guide cannulas into the mouse brain. Basal and stimulated release of acetylcholine (ACh), serotonin (5-HT) and noradrenaline (NA) was determined in the ventral hippocampus of freely moving mice. NA and 5-HT were determined in one run by a newly developed HPLC method based on precolumn derivatization with benzylamine and fluorescence detection. The mice with a loss-of-function mutation of the galanin gene (KO) and the mice that over-expressed galanin (OE) were studied. No significant differences in basal, potassium-stimulated or scopolamine-induced extracellular ACh levels were observed in 4-month-old wild-type (WT) and KO mice. In the aged, 10-month-old animals, the basal extracellular ACh levels were significantly reduced in both WT and KO groups. Galanin (1 nmol i.c.v.) caused a significant reduction of basal extracellular NA by about 40% in both WT and galanin OE mice, however, in the latter group the effect was delayed by almost 2 h. A 10-min forced swimming stress caused a higher increase in release of NA and 5-HT in the OE group than in the corresponding WT mice. Finally, venlafaxin (10 mg/kg i.p.) increased extracellular NA to 400% of the control values in the CBA mice, but only to 250% in the C57BL mice. It is concluded that galanin may play an important role in the cholinergic mechanisms underlying cognitive disorders. Furthermore, modulation by galanin and by behavioral activation, of NA and 5-HT neurotransmission in galanin over-expressing mice indicates its possible role in the aetiology of mood disorders.