Imaging of brain serotonergic neurotransmission involving phospholipase A2 activation and arachidonic acid release in unanesthetized rats

Brain Research Protocols 12 (2003) 16–25 www.elsevier.com / locate / brainresprot

Protocol

Imaging of brain serotonergic neurotransmission involving phospholipase A 2 activation and arachidonic acid release in unanesthetized rats Ying Qu*, Lisa Chang, Justin Klaff, Ruth Seeman, Andrea Balbo, Stanley I. Rapoport Brain Physiology and Metabolism Section, National Institute on Aging, National Institutes of Health, Building 10, Rm. 6 N202, Bethesda, MD 20892, USA Accepted 6 May 2003

Abstract In vitro studies have shown that 5-HT 2 receptors can be coupled via G-proteins to phospholipase A 2 (PLA 2 ) activation, releasing arachidonic acid from phospholipids. To examine this signaling pathway in brain, we developed an in vivo method to image regional brain PLA 2 activation in unanesthetized rats given different types of serotonergic drugs. Increased arachidonate incorporation from plasma, in response to drug-induced PLA 2 -activation, can be quantified with autoradiography, following the intravenous injection of radiolabeled arachidonate. For example, a 5-HT 2A / 2C receptor agonist, (6)-2,5-dimethoxy-4-iodophenyl-2-aminopropane, produced widespread increases in incorporation of labeled arachidonate by directly binding to 5-HT 2A / 2C receptors. Fluoxetine, a selective serotonin reuptake inhibitor, selectively increased incorporation of arachidonic acid by increasing 5-HT availability in the synaptic cleft and thus indirectly activating phospholipase A 2. The detailed method is described.  2003 Elsevier B.V. All rights reserved. Theme: Cellular and molecular biology Topic: Staining, tracing, and imaging techniques Keywords: Phospholipase A 2 ; Serotonin; Arachidonic; Imaging; Quantitative autoradiography; Signal transduction

1. Type of research The 5-HT 2 receptor is a G protein-coupled receptor and is recognized to be coupled to the phospholipase C (PLC) signaling pathway [1]. Less recognized, but equally important, is the ability of the 5-HT 2 receptor to couple to the phospholipase A 2 (PLA 2 ) pathway, stimulating release of the second messenger, arachidonic acid (AA) [2,3]. This signaling pathway is illustrated in Fig. 1. PLA 2 activation can be initiated by serotonergic 5-HT 2 receptors via a G-protein, as well as by cholinergic muscarinic M 1 and M 3 receptors and dopaminergic D 2 receptors [3,4].

*Corresponding author. Present address: Neuroscience, Johnson & Johnson Pharmaceutical Research and Development 3, 3210 Merryfield Row, San Diego, CA 92130, USA. Tel.: 11-858-320-3418; fax: 11-858450-2040. E-mail address: [email protected] (Y. Qu). 1385-299X / 03 / $ – see front matter  2003 Elsevier B.V. All rights reserved. doi:10.1016 / S1385-299X(03)00057-6

The in vivo fatty acid method described in this paper was developed to measure regional brain incorporation of a radiolabeled fatty acid, including [5,6,8,9,11,12,14,153 H]arachidonic acid ( 3 H-AA) in conscious rats. Tracer incorporation, represented as the incorporation coefficient k*, reflects PLA 2 -mediated AA release. Activation of PLA 2 in brain is revealed as increments in k* in different regions in response to serotonergic drugs acting at 5-HT 2 receptors or to changing serotonergic neurotransmission (Fig. 1). The fatty acid method can be used to evaluate serotonergic neurotransmission mediated by PLA 2 in awake rats. It can quantify and localize brain PLA 2 signaling in response to different drugs administered acutely or chronically. These studies involved rats chronically treated with the antidepressant mianserin (a 5-HT 2 receptor antagonist), acutely with (6)-2,5-dimethoxy-4-iodophenyl-2-aminopropane (DOI, a 5-HT 2A / 2C agonist), or acutely with fluoxetine (which inhibits 5-HT reuptake by binding to the

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Wilmington, MA, USA), weighing 290–320 g and 12 weeks old, were housed in standard laboratory conditions with a 12 h light–12 h dark cycle, with access to standard laboratory chow and water. The experimental protocol (No. 99-011) was approved by the National Institute of Child Health and Human Development, Animal Care and Use Committee, and conformed to the Guide for the Care and Use of Laboratory Animals (National Institute of Health Publication 86-23).

3.2. Chemicals and materials

Fig. 1. Model explaining PLA 2 activation in response to serotonergic drugs. Under normal conditions, the 5-HT that is released from presynaptic vesicles into the synaptic cleft binds to postsynaptic 5-HT 2 receptors coupled via a G-protein to PLA 2 , thus hydrolyzing arachidonic acid (AA) from membrane phospholipids (PL). Administration serotonergic drugs activates PLA 2 and increases incorporation of AA by different routes. (1) 5-HT 2A / 2C agonist, DOI directly binds to 5-HT 2 receptors to activate this signal; (2) fluoxetine (SSRI) inhibits 5-HT uptake, thus increasing 5-HT in the synaptic cleft so as to increase PLA 2 activation and AA release.

serotonin selective reuptake transporter, SSRI). Each of these drugs can modulate serotonergic neurotransmission.

2. Time required • Rats chronically treated with mianserin for 2 weeks. • Surgery for arterial and venous catheterizations: 30 min. • Recovery from anesthesia: 4–5 h. • Intravenous [ 3 H]AA tracer infusion: 5 min. • Plasma sample collection: 20 min. • Measurement of radioactivity of plasma samples: 2 h. • Exposure of brain slices with internal standards: 15– 18 weeks for [ 3 H]hyperfilm (described in Section 3) or 2 weeks for the [ 3 H]phosphor imaging plate (described in Section 3). • Quantitative densitometry: 1 h per brain.

3. Materials

3.1. Animals Male Fischer-344 rats (Charles River Laboratories,

• 3 H-labeled [5,6,8,9,11,12,14,15- 3 H]AA ([ 3 H]AA) (catalog No. MT 901) was purchased from Moravek Biochemicals (Brea, CA, USA). Its purity was tested by thin-layer chromatography (TLC) before use and a purity of 96% or above is acceptable. • Mianserin, fluoxetine and DOI were purchased from Sigma-Research Biochemicals (Natick, MA, USA). • Pentobarbital sodium was purchased from Richmond Veterinary Supply (Richmond, VA, USA). • [ 3 H]Methylmethacrylate autoradiographic standards and [ 3 H]Hyperfilm (Amersham, Arlington Heights, IL, USA). • Bovine serum albumin (BSA) / 4-(2-hydroxyethyl)-1piperazine-ethanesulfonic acid (HEPES) buffer: 597 mg HEPES, 4.5 g NaCl, 25 g fatty acid-free BSA were added to 500 ml distilled water and adjusted pH to 7.4. • PE 50 polyethylene catheters (Clay Adams, Lincolnshire, IL, USA). • Kodak developer and replenisher (catalog No. 1900984, Kodak GBX, Eastman Kodak, Rochester, NY, USA). (Dilution of 1 / 5). • Kodak fixer and replenisher (catalog No. 1902485, Kodak GBX, Eastman Kodak). (Dilution of 1 / 5).

3.3. Surgical equipment • Operating microscope (Zeiss, OpMi-1, Carl Zeiss, Thornwood, NY, USA). • Fine surgical instruments: for example, surgical blade, bone rongeur forceps, micro-scissor and scissors (Fine Science Tools, Foster City, CA, USA). • Equipment for halothane anesthesia for animals (MDS Matrx, Orchard Park, NY, USA). • Electric coagulator with jeweler forceps (Codman & Shurtleff, Randolph, MA, USA) • Blood pressure and heart rat monitor (Gould, Recorder 2400, Gould, Cleveland, OH, USA). • Blood pH, CO 2 ,O 2 analyzer (Blood Gas Analyzer Model 238 pH, CIBA Corning, Medfield, MA, USA). • Infusion pump (Syringe infusion pump 22, Harvard Instruments, Holliston, MA, USA).

3.4. Imaging systems Imaging system 1: CCTV camera (Model WV-BL200,

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Pansonic, Japan) and light box (Northern Light, Model B90, Imaging Research, Ontario, Canada). Image analysis software: NIH Image (version 1.62) created by Wayne Rasband (National Institutes of Health, Bethesda, MD, USA). Imaging system 2: [ 3 H]phosphor imaging plates (Fuji Medical System, Stamford, CT, USA) were scanned by a BAS 5000 scanner system (West Lafayette, IN, USA). Image Gauge V3.45 software for phosphor imaging. It can be installed on a Macintosh computer (Apple Computer, Cupertino, CA, USA).

4. Detailed procedure

4.1. Prepare radiolabeled chemicals for unanesthetized animal To use radiolabeled chemicals in unanesthetized animals, [ 3 H]AA’s specific activity needs to be 200 Ci / mmol and its purity by TLC should exceed 96%; follow the procedure to test the purity of stock radioisotopes. The stock radioisotopes are stored in ethanol, which needs to be evaporated under nitrogen flow; isotopes then are resuspended by 15 min of sonication in BSA / HEPES buffer to a final concentration of 1.75 mCi / kg. After an experiment, the radioactive animal’s carcass is subjected to proper precautions and disposed of as radioactive waste.

4.2. Procedure for testing the purity of stock radioisotopes • Dilute 10 ml stock radioisotope with 500 ml chloroform–methanol (2:1). • An AA standard can be used for all fatty acids and made at 20 mg / ml. • Prepare TLC plate (Silica Gel 60, EM Science, A Division of EM Industries, Gibbstown, NY, USA) by cutting one plate in half. With a pencil, mark a horizontal line from the bottom of plate. Then, mark off four 2-cm rows beginning at the left edge, then two smaller rows. Label large rows A, B, C and D. Skip the next row and label the last one S (standard). • Load TLC plate by adding 5 ml of lipid standard just above horizontal line at bottom of each row. Keep dot as small as possible by adding a little at a time and drying in between. • Change tips and then load 5 ml of diluted isotope on top of the standard in rows A through B only. • Spot 5 ml of diluted isotope onto a bottom corner of the plate and cut out before running in solvent. Place in a scintillation vial. • Place TLC plate into loaded solvent (30 ml light petroleum / 20 ml diethyl ether / 0.5 ml acetic acid) chamber with the standard and isotope at the bottom. • Remove from chamber when solvent is about 1 cm

from the top of the plate (about 6–7 min). Use iodine fumes to develop plate for about 20 s. • After developing, the lipid standards will mark where the fatty acid bands are. There will be one large band that marks the intact fatty acid. Staining below the intact band will mark oxidized fatty acid (fatty acid gets heavier and thus does not travel as far). Above the band will be staining of fatty acid that has broken down (fatty acid gets lighter as bonds break down (off) therefore it travels farther). With a pencil, mark four sections in row A. A15dotted section, A25area below intact fatty acid, A35area of intact fatty acid, and A45area above intact fatty acid. Repeat for rows B, C and D. • Counting radioactivity. Starting at the bottom of row A, cut out the four sections. A1, A2, A3 and A4. Repeat for rows B, C and D. Place the large band, 3, in one vial and all the other pieces (1, 2 and 4) into one vial (two vials per column). Label vials. To all vials, add 2 ml tissue solubilizer, Soluene-350 (Packard Instruments, Meriden, CT, USA) (under hood or above sink) to release fatty acid, then add 12 ml of scintillation cocktail. Shake to mix, let settle and count. • 100% radioactivity will be read from the isotope that was not run through solvent. Then, add up the DPM from each column and get the average from the four columns (A, B, C, D). This equals 100%. Then add all the 3 band DPM from each column and average them. Divide this average by the 100% average to equal % of pure isotope. A percent purity of 96% or above is acceptable for use in experiments.

4.3. Animal preparation and chronic drug administration • Control rats were treated for 14 days (or 21 days) with 10 ml / kg i.p. saline daily. • Experimental rats were treated for 14 days with 10 mg / kg i.p. mianserin daily, then 24 h to allow the drug to be washed from brain.

4.4. Surgery for arterial and venous catheterizations On the morning of the experiment, the rat is anesthetized in a chamber saturated with halothane vapor. To ensure light anesthesia, the rat is removed from the chamber upon becoming unconscious. Body weight is measured to determine appropriate amount of isotope for infusion. The rat is mounted to a halothane scavenger system (Fluovac, International Market Supply, Stoelting Physiology Research Instruments, Wood Dale, IL, USA) during surgery. The halothane / oxygen regulator is set at an oxygen flow rate of 0.5–0.6 ml / min and the anesthetic level is maintained at 1–3% (v / v) in O 2 . The animal is placed on its back. Lidocaine 1% is

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applied topically. A superficial skin incision of about 1.5 cm is made at the medial side of the upper hind limb to expose the femoral artery and vein. Muscles and fascia are gently teased aside with blunted instruments (scissors or forceps) to isolate the femoral artery and vein. The artery appears white and thinner; the vein is thicker and darker due to the deoxygenated blood. Collateral branches of both vessels are coagulated to prevent hemorrhage. A tight ligature (4.0 silk, Ethicon, Somerville, NJ, USA) is placed at the lower level of the vessel to stop circulation to the area. Two loose ligatures are placed around the upper and middle parts of the vessel. Finally a temporary occlusion clip is placed just below the loose ligature to occlude blood back-flow and prevent blood loss. From this point on, it is safe to make small incisions with a microscissor in the lower part of the vessels. A beveled PE 50 polyethylene catheter filled with heparinized saline (100 IU / ml) is inserted into the incisions of the artery or vein. The upper and middle ligatures are tightened to secure the catheter in the vessels. The two vessels are then tied together to secure them. The wound is flushed and cleaned with normal saline before closing with 9 mm autoclips (Clay Adams).

4.5. Recovery of animal from anesthesia • A wooden block, wrapped with absorbent paper, is used to immobilize the rat after the surgical procedure. Only the hindlimbs and the posterior body part of the rat are restrained. Prior to immobilization, half a roll of dry plaster cast is moisturized in lukewarm water to prevent thermal shock to the anesthetized animal. The lower body including the canulated leg of the animal is wrapped in a loose-fitting plaster caster. The cast is taped on the wooden block with complete immobilization of the hindlimbs, while allowing free movement of the animal’s head and forequarters. This stage should be fast enough to avoid waking the animal. • For the recovery phase, animals are kept inside a temperature-controlled and sound-dampened box for 4 to 5 h, for the effects of anesthesia to disappear. • During recovery, body temperature is maintained at 37 8C by using a rectal thermometer and a feedback heating device (Tekmar Control System, Vernon, Canada).

4.6. Acute drug administration and tracer infusion After 4–5 h, 125 ml arterial blood is withdrawn to measure pH, pO 2 and pCO 2 . Control rats and experimental rats received one of following treatments: • DOI (2.5 mg / kg i.p. 20 min before infusion). • Fluoxetine (10 mg / kg i.p. 30 min before infusion).

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• No treatment (saline (i.p. injection volume same as drug)). 1.75 mCi / kg [ 3 H]AA in 2 ml BAS / HEPES buffer is infused through the venous canula with an infusion pump, at a rate of 400 ml / min for 5 min. Timed 125-ml arterial blood samples are collected at the following intervals: 0.0, 0.25, 0.50, 0.75, 1.5, 3.0, 5.0, 6.5, 7.5, 19.0 min. At the end of 20 min, the rat is killed with 65 mg i.v. sodium pentobarbital. Brains are removed and frozen at 250 8C in 2-methylbutane, then stored at 280 8C until further analysis.

4.7. Curve of plasma radioactivity • Plasma is separated from arterial blood sample by 1 min of centrifugation. • Place 30 ml plasma into a test-tube containing 3 ml CHCl 3 –CH 3 OH (2:1). Cap test-tube and vortex. Add 1.5 ml 0.1 M KCl to extracted samples, cap and vortex. • Centrifuge samples for 10 min to separate lipid phase (bottom) from aqueous phase (top). • Remove 23100 ml from liquid phase and place in scintillation vial. Add 5 ml scintillation cocktail to vial, then vortex and count radioactivity using liquid scintillation spectroscopy. The method is modified from the method of Folch et al. [5].

4.8. Autoradiography and histology • Frozen brains are sectioned coronally on a cryostat at 220 8C. Sets of three adjacent 20-mm sections are collected and mounted on glass coverslips at 140-mm intervals and dried on a hot plate for 6 min. • Sections are exposed together with [ 3 H]methylmethacrylate autoradiographic standards (Amersham) to [ 3 H]Hyperfilm (Amersham) for 15–18 weeks, then developed following the manufacturer’s instructions. Alternatively, sections are exposed together with [ 3 H]methylmethacrylate autoradiographic standards to [ 3 H]phosphor imaging plates for 2 weeks and the plates are scanned by a BAS 5000 scanner system. • One of the adjacent sections is collected and stained with cresyl violet to identify brain regions by comparison with a rat-brain atlas [6]. The brain region in an autoradiogram can be identified from the section stained with cresyl violet. • Radioactivity in different brain regions is measured in sextuplicate by quantitative densitometry using the public domain image analysis program NIH Image (version 1.55) created by Wayne Rasband (National Institutes of Health) and installed on a Macintosh computer (Apple Computer) or by quantitative den-

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sitometry using Image Gauge V3.45 software for phosphor imaging. • Regional brain incorporation coefficients, k*, of [ 3 H]AA are calculated using the formula:

* (20 min) c brain k* 5 ]]]] 20

Ec*

plasma

dt

0

* (20 min) in where k* is in units of ml s 21 g 21 , c brain units of nCi / g and equals brain radioactivity at 20 min * determined by densitometry. c plasma in units of nCi / ml is the plasma fatty acid radioactivity determined by scintillation counting and t is time (s), after onset of [ 3 H]AA infusion.

5. Results

Fig. 2. Plasma organic radioactivity at different times after [ 3 H]AA injection.

In this study, all experiments were carried out in unanesthetized rats. After surgery, body temperature, arterial blood pressure, pulse, blood pH and the CO 2 and O 2 concentrations in blood were monitored throughout the experiment to be sure that the animal was maintained under normal physiological conditions. Table 1 shows the physiological measures of one group of control rats after surgery. Fig. 2 illustrates mean total plasma organic radioactivity at different times after the start of a 5-min intravenous infusion of [ 3 H]AA in rats. The mean value in each case peaked at approximately 5 min and declined precipitously, reaching less than 10% of the peak by 7.5 min. Brain regions were identified as shown in Fig. 3 and Table 2. Table 2 shows the mean basal [ 3 H]AA incorporation coefficients in one group of control rats. Highest values of k* are in the cerebral cortex. The signal in the cerebral cortex presents a laminar distribution in different bands, corresponding to layers I, II–III, IV, deep part IV of prefrontal cortex, frontal cortex, motor cortex, somatosensory cortex, auditory cortex and visual cortex. Highest values of k* are in layer VI of motor cortex, somatosensory cortex and layer I of auditory cortex and visual cortex. The

rest of neocortex also has high incorporation coefficients for [ 3 H]AA, as does the olfactory and pyriform cortex. Individual midbrain areas such as the habenular nucleus, median eminence, supraoptic nuclei and subfornical organ nuclei also show high values of k*. Nuclei in the brain stem and spinal cord such as colliculus nucleus, cochlear nucleus, vestibular nucleus, pretectal area flocculus and interpedunclear nucleus show high values. Intermediate values are seen in the following areas: caudate-putamen, amygdala, substantia nigra, diagonal band, septal nucleus, pyramidal cell layer of the CA1, CA2, CA3 in the hippocampus, most nuclei of thalamus, most parts of the hypothalamus, raphe nuclei, spinal tract V nucleus and locus coeruleus. Low values are seen in the globus pallidus, bed nucleus stria terminals, entopeduncular, internal capsule and pedunculopontine nucleus. Bar graphs, which compare effects of single dose DOI (2.5 mg / kg i.p.), chronic mianserin and chronic mianserin plus single dose DOI (2.5 mg / kg i.p.) in several representative brain regions, are shown in Fig. 4. The detailed results have been published elsewhere [7,8]. The effects of acute fluoxetine on k* have also been published [8,9].

6. Discussion

Table 1 Physiological parameters of control rats after surgery Body temperature** (8C) Arterial blood pressure (mm Hg) (systolic pressure / diastolic pressure) Heart rate (beats / min) Arterial pH Arterial blood gas (pCO 2 ) Arterial blood gas (pO 2 )

36.160.3 a 12562 / 7861 41664 7.4160.02 41.961.3 99.865.0

Animal number58. **Temperature was measured with a rectal thermoprobe. a Mean6SEM.

6.1. Alternative and support protocols The in vivo fatty acid method involves the intravenous administration of a radiolabeled long-chain fatty acid, then measuring its incorporation into brain lipids some 20 min later by quantitative autoradiography. Regional incorporation coefficients, k*, are calculated as regional brain radioactivity divided by integrated plasma radioactivity (the plasma ‘input’ function). Labeled arachidonate is

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Fig. 3. [ 3 H]Arachidonate incorporation coefficients k* in different brain regions in coronal sections from brain of control rat. k* is color-coded.

incorporated mainly into the sn-2 position of phosphatidylinositol and phosphatidylcholine within brain synaptic membranes [10,11]; its incorporation can be affected by changes in brain functional activity [12–15]. Because k* is independent of cerebral blood flow [13], tracer incorporation reflects brain phospholipid metabolism but not delivery by blood. Direct activation of PLA 2 in brain can be revealed as increments in k* in different regions in response to agonists acting at specific receptors coupled to PLA 2 [11,12,14,15]. We have applied this method to imaging PLA 2 activation in response to drugs acting at cholinergic muscarinic M 1 and M 3 receptors [15], dopaminergic D 2 receptors [14], or serotonergic 5-HT 2 receptors [8], all of which are coupled to PLA 2 activation via a G protein. PLA 2 activation also can be initiated by Ca 21 influx into brain, due to glutamate acting at NMDA receptors or acetylcholine at nicotinic receptors. Thus, the fatty acid

method is a very useful tool for investigating animal models of brain disease involving neurotransmitter loss or imbalance. Indirect activation of PLA 2 can be produced in response to a drug that can change the quantity of a neurotransmitter in the synaptic cleft. An increased neurotransmitter concentration in the synaptic cleft will act as an endogenous agonist for PLA 2 -coupled receptors. In this regard, we have applied the fatty acid method to study the effect of acute fluoxetine on PLA 2 activation. Fluoxetine is a selective 5-HT reuptake inhibitor [8] that mainly binds to serotonin reuptake sites so as to increase the 5-HT concentration in the synaptic cleft. Therefore, the method can identify pharmacological processes and image neurotransmitter-initiated signaling beyond the receptor by using appropriate drugs that change the neurotransmission of acetylcholine, dopamine or serotonin. Additionally, the method has been modified for studying the awake mouse.

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Fig. 3. (continued)

For example, we are able to quantify and localize PLA 2 signaling changes in response to DOI in the serotonin transporter knockout mouse. We also are extending the method to the in vivo brain imaging of PLA 2 -mediated 11 signaling in monkeys and humans, by using [ C]AA and positron emission tomography (PET) [16,17]. The theoretical descriptions have been published [18].

6.2. Trouble shooting • Successful tracer infusion depends on the surgery for arterial and venous catheterizations. To test whether a catheter is in the right position, check whether the animal received enough radioactivity by looking at the curve for plasma radioactivity to see if it is within 2–3 standard deviations of normal curves. • Avoiding bleeding during surgery. After surgery,

keeping the animal in a stable physiological condition is important. • Instead of using [ 3 H]AA, [ 14 C]AA (Moravek, catalog No. NC 364) can be used as a radiotracer with Kodak Diagnostic Film (Ektascan姠 MC EMC-1, Eastman Kodak). [ 3 H]AA autoradiographs that are exposed to [ 3 H]Hyperfilm (Amersham) have a better resolution, although they demand a much longer exposure. • [ 3 H]Phosphor imaging plates can give the quickest results. However, one imaging plate only can be used for 4–5 exposures, even if tissue is fixed in formaldehyde vapor for 24 h to prevent fragmentation.

7. Quick procedure • Chronic drug or saline treatment for control and experimental animals.

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Table 2 Regional [ 3 H] arachidonic acid incorporation coefficients k* (ml / s / g brain310 4 ) in control rat brain Brain region

Abbrev.a

Control

Brain region

Abbrev.

Cerebral cortex prefrontal cortex IV frontal cortex layer II–III frontal cortex layer IV motor cortex layer II–III motor cortex layer IV motor cortex layer V–VI somatosensory cortex layer II–III somatosensory cortex layer IV somatosensory cortex layer V–VI anterior cingulate cortex auditory cortex layer II–III auditory cortex layer IV auditory cortex layer V–VI visual cortex layer II–III visual cortex layer IV visual cortex layer V–VI

PFr Fr II–III Fr IV FrPaM II–III FrPaM IV FrPaM V–VI Soms II–III Soms IV Soms V–VI Acg Aud II–III Aud IV Aud V–VI Str (Ast) II–III Str (Ast) IV Str (Ast) V–VI

9.160.3 9.560.5 10.660.5 9.160.4 10.560.5 8.960.4 9.560.5 10.260.5 8.960.4 9.860.4 10.361.2 11.961.5 9.661.1 10.061.0 10.761.0 10.060.9

pars compacta

SNC

8.260.8

Septum lateral septal nucleus medial septal nucleus dorsal diagonal band ventral diagonal band

LSI MS VDBD VDBV

6.560.3 7.360.4 7.660.5 7.660.4

Hippocampal formation Ammon’s horn CA1 Ammon’s horn CA2 Ammon’s horn CA3 dentate gyrus

Hip CA1 Hip CA2 Hip CA3 DG

9.260.3 8.560.4 8.760.5 9.961.2

White matter corpus callosum internal capsule anterior commissure

cc ic aca

4.460.4 4.260.4 5.360.5

Olfactory system olfactory cortex pyriform cortex

PO Pir

11.160.5 9.760.4

Thalamus and related areas paratenial nuclei anteroventral nuclei anteromedial nuclei reticular nuclei paraventricular nuclei ventroposterior medial nucleus ventroposterior lateral lateral habenular nucleus medial habenular nucleus medial geniculate nucleus dorsolateral geniculate nucleus parafascicular nucleus inferior colliculus superior colliculus

PT AV AM Rt PVA VPM VPL LHb MHb MG DLG PF CICVL SuG

8.260.3 10.260.4 8.560.3 8.460.5 7.360.6 7.960.3 7.760.3 8.660.3 9.360.4 10.461.0 8.460.4 7.8460.30 11.960.97 11.761.9

Hypothalamus supraoptic nucleus subfornical organ lateral nuclei anterior nuclei periventricular nucleus arcuate nucleus ventromedial nucleus posterior nucleus medial forebrain bundle mammillary body deep layers of superior colliculus interpeduncular nucleus spinal Tract V nucleus

SO SFO LH Ahy Pe Arc VMH PH mfb MM SC IPC Sp51

13.062.5 9.160.6 6.760.5 6.860.8 8.060.5 6.860.4 6.560.4 7.660.3 6.860.4 7.460.4 13.061.8 10.360.8 8.360.8

Cerebellum cerebellar gray matter molecular layer, gray matter granular layer, gray matter flocculus cerebellar white matter

CbG MolCbG GrCbG FI CbW

10.460.9 10.760.9 11.960.7 10.360.7 5.360.6

Choroid plexus

ChP

46.963.1

Basal ganglia and related areas nucleus accumbens caudate putamen dorsal caudate putamen ventral caudate putamen lateral caudate putamen medial bed nucleus stria preoptic nucl. suprachiasmatic nucleus bed nucleus stria terminalis entopeduncular nucleus globus pallidus subthalamic nucleus amygdala basolateral / basomedial nucl. substantia nigra pars reticulata median eminence Brainstem and spinal cord raphe magnus nuclei raphe pallidus nuclei raphe median nuclei raphe dorsal nuclei locus coeruleus cochlear nucleus vestibular nucleus (medial) pretectal area pedunculopontine nucleus

Acb CPU CPU CPU CPU LPO / MPO PSCH BSTPO EN GP Sth BL / BM

8.160.4 8.160.3 8.360.3 8.060.4 7.960.3 7.460.3 7.760.3 6.660.4 6.260.5 6.260.5 7.560.4 6.660.3

SNR ME

8.560.8 14.062.5

RMg Rpa MnR DR LC VCO, GrCo MVe PPT PPTGg

7.560.6 8.560.7 8.560.8 9.160.7 9.961.0 14.062.5 11.760.8 12.661.5 7.260.7

Control

k* values are means6SEM (n58). a From Paxinos (Paxinos, 1987)

• • • •

Rat surgery for arterial and venous catheterizations. Acute administration of drug on saline. Intravenous infusion of [ 3 H]AA. Measure plasma radioactivity

• Measure brain radioactivity with quantitative autoradiography • Calculate regional incorporation coefficients k* (brain radioactivity / integrated plasma radioactivity) of AA

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Fig. 4. Bar graphs of comparing regional incorporation coefficients k*. Effects of control, single dose DOI treatment (2.5 mg / kg i.p.), chronic mianserin treatment and chronic mianserin treatment plus single dose DOI (2.5 mg / kg i.p.) on k* [(ml s 21 g 21 )310 4 ] of [ 3 H]arachidonic acid in rats: (A) comparison between different layers of somatosensory cortex; (B) comparison in different brain areas. Abbreviations as in Table 2. One-way ANOVA, Dunnett’s multiple comparison test were used. The control rat was used as the control group to compare with each group. *** P,0.001.

8. Essential literature references [7], [8], [14], [15] and [18].

[9]

[10]

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