D2-like receptor activation does not initiate a brain docosahexaenoic acid signal in unanesthetized rats

Background The polyunsaturated fatty acid, docosahexaenoic acid (DHA), participates in neurotransmission involving activation of calcium-independent phospholipase A2 (iPLA2), which is coupled to muscarinic, cholinergic and serotonergic neuroreceptors. Drug induced activation of iPLA2 can be measured in vivo with quantitative autoradiography using 14C-DHA as a probe. The present study used this approach to address whether a DHA signal is produced following dompaminergic (D)2-like receptor activation with quinpirole in rat brain. Unanesthetized rats were infused intravenously with 14C-DHA one minute after saline or quinpirole infusion, and serial blood samples were collected over a 20-minute period to obtain plasma. The animals were euthanized with sodium pentobarbital and their brains excised, coronally dissected and subjected to quantitative autoradiography to derive the regional incorporation coefficient, k*, a marker of DHA signaling. Plasma labeled and unlabeled unesterified DHA concentrations were measured. Results The incorporation coefficient (k*) for DHA did not differ significantly between quinpirole-treated and control rats in any of 81 identified brain regions. Plasma labeled DHA concentration over the 20-minute collection period (input function) and unlabeled unesterified DHA concentration did not differ significantly between the two groups. Conclusion These findings demonstrate that D2-like receptor initiated signaling does not involve DHA as a second messenger, and likely does not involve iPLA2 activation. Electronic supplementary material The online version of this article (doi:10.1186/1471-2202-15-113) contains supplementary material, which is available to authorized users.

The regulation of G-protein mediated activation of cPLA 2 or iPLA 2 depends on extracellular and intracellular calcium concentrations. cPLA 2 is activated by extracellular Ca 2+ entry into the cell and iPLA 2 by intracellular calcium release from the endoplasmic reticulum via phospholipase C (PLC) (reviewed in [18]). PLC, when activated, converts membrane phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP 3 ). IP 3 binding to IP 3 receptors stimulates the release of intracellular Ca 2+ [19,20] and of calcium influx factor [21] from the endoplasmic/sarcoplasmic reticulum (ER/SR). The calcium influx factor contributes to the activation of iPLA 2 by dissociating calmodulin from the active site of iPLA 2 [22,23]. This PLC-mediated activation of iPLA 2 results in the hydrolysis of DHA from membrane phospholipid. DAG can also be further hydrolyzed to release DHA, although this pathway likely contributes minimally to DHA release.
Intracellular calcium release associated with activation of the PLC pathway regulates vesicle transport and neurotransmitter release at synaptic terminals [19,24]. Muscarinic M 1,3,5 and serotonergic 5-HT 2A/2C neurotransmission is coupled to iPLA 2 via the PLC pathway, as is dopaminergic neurotransmission, although the directionality of this coupling is not agreed on. Activation of D2-like receptors in isolated neurons or dissected brain structures was reported to increase [19,20,25,26] or decrease [27,28] intracellular calcium release or IP 3 concentration [29]. A reduction in intracellular calcium release would not activate iPLA 2 , whereas an increase would activate iPLA 2 and increase DHA release.
In the present study, we wished to see if activation of brain D2-like receptors in unanesthetized rats would also lead to a DHA signal, as it does an AA signal [11,12]. Stimulation of dopaminergic receptors by the agonists quinpirole or apomorphine produced an AA signal that could be blocked by the D2 receptor antagonists raclopride or butaclamol in unanesthetized rats [11,12,14].
To test this, we used an established in vivo kinetic method in awake rats [16], to quantify the DHA signal in response to the D2-like receptor agonist quinpirole, compared with vehicle. With this method, radiolabeled DHA is infused to steady state levels in plasma, and brain radioactivity is measured with quantitative autoradiography to derive the regional incorporation coefficient, k*. We found that D2-like receptor activation with quinpirole did not change the DHA incorporation coefficient (k*) into brain compared to vehicle-treated controls, suggesting that D2-like receptor activation does not involve DHA release as a second messenger.

Animals and diets
Experiments were conducted following the "Guide for the Care and Use of Laboratory Animals" (National Institutes of Health Publication No. 86-23) and were approved by the Animal Care and Use Committee of Eunice Kennedy Shriver National Institute of Child Health and Human Development. Two-month-old male Fischer CDF 344 rats (Charles River Laboratories, Wilmington, MA) were acclimated for one month in an animal facility with regulated temperature, humidity and 12-h dark/light cycle. Rats were maintained on the Rodent NIH-31 auto 18-4 diet (Zeigler Bros, Gardens, PA), which contained (as% of total fatty acid) 20.1% saturated, 22.5% monounsaturated, 47.9% linoleic, 5.1% α-linolenic, 0.02% arachidonic, 2.0% eicosapentaenoic, and 2.3% docosahexaenoic acid [30]. Water and food were provided ad libitum.

Tracer purification and drug preparation
Radiolabeled [1-14 C]DHA dissolved in ethanol (53 mCi/ mmol, Moravek Biochemicals, Brea, CA) was purified on 60 A°thin-layer chromatography (TLC) silica plates (~5 mg per 3 cm lane on each plate) alongside phospholipid, cholesterol, cholesteryl ester, triglyceride and unesterified fatty acid standards using diethyl ether: heptane: acetic acid (60:40:3 v/v) as a solvent. The [1-14 C]DHA was purified because the stock tracer bottles used for this study had been opened in the past, a factor which was previously found to reduce tracer purity over time despite storing it in a −80°C freezer, due to loss of the preservative argon gas blanket in the stock bottle once opened. The plate was sprayed with 0.03% 6-p-toluidine-2-naphthalene-sulfonic acid in 50 mM Tris-HCl buffer (pH7.4) (w/v), and the unesterified fatty acid band containing [1-14 C]DHA was identified under UV light, scraped and purified from the silica particles by the Folch method (in 30 ml 2:1 v/v chloroform/methanol and 7.5 ml 0.5 M KCl). The chloroform extract was dried under nitrogen, reconstituted twice with 10 ml ethanol, centrifuged to remove additional silica particles, and pipetted to a new 50 ml Pyrex tube. The ethanol extract was reconstituted in 5 ml of ethanol. Radioactive purity measured in a portion of the ethanol extract with HPLC using acetonitrile/water (90/10%) as a solvent (constant flow rate of 2 ml/min), confirmed that 93% of the radioactivity eluted at the same time as the unesterified DHA (unlabeled) standard. On the day of the experiment, a portion of the ethanol extract was dried under nitrogen and resuspended in HEPES buffer, pH 7.4, containing 50 mg/ml fatty acidfree bovine serum albumin (Sigma-Aldrich, St Louis, MO).
An acute 1 mg/kg i.v. dose of (−)-quinpirole hydrochloride dissolved in 0.9% saline (Sigma-Aldrich) was chosen because it produces widespread significant increments in the incorporation coefficient, k*, for AA in the brain of unanesthetized rats, which can be blocked by the D2-like receptor antagonists, butaclamol or raclopride, without causing convulsions [12,14,31].

Surgical procedures and tracer infusion
A total of 16 rats were randomized to saline or quinpirole treatment (n = 8 per treatment). Rats were anesthetized with halothane (2-3% v/v in O 2 ) and polyethylene (PE 50) catheters were surgically inserted into the right femoral artery and vein (4 rats per day, 2 saline and 2 quinpirole) [31]. The wound was closed with surgical clips and the rat was wrapped loosely, with its upper body remaining free, in a fast-setting plaster cast taped to a wooden block. Each surgery lasted 20-25 min. Rats were allowed to recover from anesthesia for 3-4 h in an environment maintained at 25°C. Rectal temperature was maintained at 36.5-37.5°C using a feedback-heating device and rectal thermometer. Arterial blood pressure and heart rate were measured with a blood pressure recorder (CyQ 103/302; Cybersense, Nicholasville, KY). One minute after an i.v. injection of quinpirole or saline,

Chemical analysis
Blood samples, collected before, during or after [1-14 C] DHA infusion, were centrifuged immediately at 18,000 g for 30 s at room temperature. Total lipids were extracted from plasma (30 μl) using a modified Folch procedure [32]. One hundred μl of the lower organic phase was used to determine the radiolabeled unesterified plasma [1-14 C]DHA concentration by liquid scintillation counting.
Concentrations of unlabeled, unesterified fatty acids were determined from 100 μl of ice-thawed arterial plasma collected at 20 minutes. Unesterified heptadecanoic acid (17:0) was added as an internal standard to the plasma and total lipids were extracted with the Folch method. The total lipid extract was separated by thin layer chromatography on 60 A°silica gel plates (Whatman, Clifton, NJ) alongside phospholipid, unesterified fatty acids, triglyceride and cholesteryl ester standards using the solvent system heptane: diethylether: glacial acetic acid (60:40:3, v/v/v). The plates were sprayed with 0.03% 6-p-toluidine-2-naphthalene-sulfonic acid in 50 mM Tris-HCl buffer (pH7.4) (w/v), and the unesterified fatty acids band was identified under UV light, scraped and methylated with 1% H 2 SO 4 (by vol) in anhydrous methanol (3 h at 70°C) after adding 0.2 ml toluene. The fatty acid methyl esters were extracted with 3 ml heptane after terminating the reaction with 1.5 ml water [33], reconstituted in 25 μl of isooctane and quantified by gas chromatography as described [30]. The injection volume was 2 μl.

Quantitative autoradiography
Quantitative autoradiography was performed on a total of 81 brain regions from autoradiographs of coronal brain sections. The regions were identified from a stereotaxic rat brain atlas [34], and were sampled in both hemispheres. The average of bilateral measurements for each region from three consecutive brain sections was used to calculate regional radioactivity (nCi/g wet brain) by digital quantitative densitometry, using the public domain 1.62 Analysis NIH Image program. Regional brain incorporation coefficient k* (ml plasma/s/g wet brain) of [1-14 C] DHA were calculated as follows [35]: where c Ã brain (nCi/g wet brain wt) is brain radioactivity 20 min after infusion, c Ã plasma (nCi/ml plasma) is arterial unesterified [1-14 C]DHA concentration, and t (min) is time after beginning [1-14 C]DHA infusion. Integrated plasma radioactivity (integral of c* plasma .dt), which corresponds to the input function, was determined by trapezoidal integration and used to calculate k*.
The regional rate of incorporation of unesterified DHA from plasma into brain phospholipids, J in (nmol/s/g), was calculated as follows [30,36,37]: where c plasma is the plasma concentration (nmol/ml) of unlabeled unesterified DHA. Since negligible amounts of DHA (<1%) can be synthesized de novo by the brain from its precursor, alpha-linolenic acid [30,36], J in represents the metabolic loss of DHA by the brain [35].

Statistical analysis
All data are presented as mean ± SD. An unpaired t-test was used to assess significant changes in body weight, baseline body temperature, arterial blood pressure and heart rate, input function, the incorporation coefficient (k*) and the incorporation rate, J in . A paired t-test was applied to compare mean body temperature, blood pressure and heart rate in the same animal before and after saline or quinpirole injection. Statistical significance was accepted at p < 0.05.

Physiological parameters
One control rat died after surgery and prior to infusion with saline due to unknown causes. During the brain slicing, one control and one quinpirole brains were not sectioned uniformly, resulting in a saturated, unquantifiable signal, and a sample size of 6 saline and 7 quinpirole. Physiological parameters, plasma input function and fatty acid concentrations were therefore obtained from 6 saline and 7 quinpirole treated rats. Table 1 shows bodyweight, body temperature, arterial blood pressure, heart rate and orofacial activity, before and after saline or quinpirole treatment. Body weight did not differ significantly between the groups. Baseline body temperature was within physiological range (36.5-37.5°C) for both groups. Baseline body temperature, heart rate and systolic and diastolic blood pressure did not differ significantly (P > 0.05 by unpaired t-test).
Heart rate and systolic and diastolic blood pressures did not significantly change after saline or quinpirole injection, compared to baseline (P > 0.05 by paired t-test). Body temperature did not change also after saline injection, but was significantly reduced by 0.6°C after quinpirole injection (P < 0.01 by paired t-test; Table 1).
All quinpirole-treated rats but not controls exhibited orofacial head-and sniffing activity between 2 to 10 minutes after quinpirole injection, as reported [16]. No convulsions were observed in either group.

Incorporation coefficient (k*)
Mean values of DHA incorporation coefficient k* in acute saline-and quinpirole-treated rats are presented in Table 2. Regional k* values ranged between 3.4 to 15.9 ml/s/g × 10 −4 in saline-treated control rats, consistent with our previous report [16]. There was no significant difference in k* between quinpirole-treated and control rats in any of the 81 brain regions examined. A representative autoradiogram comparing the DHA signal following saline or quinpirole is shown in Figure 2.
Incorporation rate (J in ) Regional rates of unesterified DHA incorporation into the brain, calculated from the product of k* and unesterified plasma DHA concentration (2), did not differ significantly between the groups in the 81 regions examined (data not shown). Mean J in values in vehicle and quinpirole treated rats were 58.8 ± 20.8 and 73.6 ± 22.5 nmol/s/g×10 −4 , respectively (P >0.05 by unpaired t-test).

Discussion and conclusions
D2-like receptor activation by acute quinpirole did not significantly change brain DHA incorporation coefficient (k*) or rate (J in ) in any of the 81 regions studied. The Orofacial activity, consisting of head tremors and sniffing was observed for 10 minutes after saline or quinpirole injection. All quinpirole-treated rats exhibited characteristic head tremors and sniffing activity within 2 minutes of drug injection. Values are mean ± SD of n = 6 saline and 7 quinpirole treated rats per group. **P < 0.01 compared to before quinpirole injection by paired t-test.   incorporation coefficient k* reflects the metabolic loss of DHA by the brain following its release from membrane phospholipid and J in reflects net loss [35]. The lack of a quinpirole effect on each parameter suggests that D2like receptor activation does not involve DHA release from membrane phospholipid. DHA release is controlled by iPLA 2 [3], which is activated following displacement of bound calmodulin protein by calcium influx factor [23]. Intracellular calcium increases concurrently because it is released with the calcium influx factor from the ER/SR in response to IP 3 formation following PLC stimulation [19][20][21]. Unlike iPLA 2 , cPLA 2 requires extracellular calcium for activation [3], consistent with in vivo evidence of increased AA but not DHA incorporation into the brain following glutamatergic NMDA receptor activation, which allows extracellular calcium into the cell [16]. D2-like receptor stimulation also allows extracellular calcium into the cell, similar to NMDA [28]. Thus, the increase in AA [12,38] but not DHA incorporation ( Table 2) following acute quinpirole confirms the independence of iPLA 2 of extracellular calcium and likely reflects selective coupling of D2-like receptors to cPLA 2 but not iPLA 2 . Several in vitro studies reported a reduction in intracellular calcium levels following dopaminergic activation [27,28]. The lack of effect of quinpirole on k* agrees with these observations. This study does not rule out the possibility that intracellular calcium levels increased following D2-receptor stimulation with quinpirole, as reported in vitro [25][26][27][28]. Such elevations if present, however, were not significant enough to produce a measurable DHA signal with our method.
This study demonstrates that there is no DHA signal following activation of D2-like receptors, although there is a signal following activation of muscarinic and serotonergic receptors [9,17]. It is likely, therefore, that iPLA 2 is activated by the latter two but not D2 receptors. The difference in G-protein receptor coupling remains to be clarified, but it may be related to limited intracellular calcium release and dissociation of calmodulin from iPLA 2 following   dopaminergic receptor stimulation, compared to muscarinic stimulation [39][40][41][42].
In summary, this study showed that D2-like receptor activation does not involve DHA release as a second messenger. This observation, along with our previous finding that D2-like receptor activation stimulates AA release, suggest that D2 receptors are selectively coupled to AA but not DHA as a second messenger.

Additional file
Additional file 1: Table S1. Plasma unesterified fatty acid concentrations (nmol/ml) in rats treated with saline or quinpirole.