Darrell A. Jackson et al- Choline Enhances Scopolamine-Induced Acetylcholine Release In Dorsal Hippocampus of Conscious, Freely-Moving Rats

8/3/2019 Darrell A. Jackson et al- Choline Enhances Scopolamine-Induced Acetylcholine Release In Dorsal Hippocampus of Co

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PergamonLife Sciences, Vol. 56, No. 1 , pp. 45-49, 1995Copyright 1994 Elsevier Science Ltd

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CHOLINE ENHANC ES SCOPOLAMINE-INDUCED ACETYLCHOLINE RELEASE INDORSAL HIPPOCAMPUS OF CONSCIOUS, FREELY-MOVING RATS

Darrell A. Jackson, Udo Kischka and Richard J. WurtmanDepartment of Brain and Cognitive Sciences, Massachusetts Institute of Technology

E25-604, Cambridge, Massachusetts 02139, USA(Received in final form October 13, 1994)

SummaryWe examined the effects of exogenous choline (30, 60, 120 mg/kg, i.p.) on basaland scopolam ine-evoked acetylcholine (AC h) release in awa ke animals, using inv i v o microdialysis. After collection of 3-4 baseline d ialysate samp les (1 5 mineach) , rat s received either saline or choline ch loride and 4 additional samp leswere collected. All animals then received scopolamine hydrochloride (0.5 mg/kg,i.p.) and 6 additional samples were collected. Basal ACh release in animalsreceiving choline did not differ from that in rats given saline, nor from A Chrelease prior to choline administration. Scopolam ine alone increased averag e AChlevels in dialysates from 1.22 + 0.54 to 11.18 f 3.07 pmol/l5 min (mean fSD; p = 0.001); administration of 60 mg/kg or 120 mg/kg of choline chloridesignificantly enhanced maxim al scopolamine respons es by about 55% . Thes eresults suggest that supplemental choline enhances evoked AC h release inhippocampus of freely-moving rats.

KAYword : choline, acetylcholine, microdialysis, scopolamine, hippocampus

Brain acetylcholine (AC h) levels are maintained within relatively narrow limits, indicatingthat the rate of AC h synthesis is adequ ate for sustaining the neurotransmitters release undernormal conditions. How ever, when ACh release is sustained, its levels can decre ase significantlyas shown in who le brain (1,2 ) and brain slices (3,4), indicating that synthesis is then unable tokeep up with demand. Some (3-8) but not all investigators (9-13) have observed that cholinesupplementation can increase brain AC h concentrations i n v i v o , and can prevent the depletionof AC h tissue levels otherw ise observe d in brain slices subjected to prolonged stimulation.Mor eover , preincubation of hippoca mpal slices with choline can significantly increase the releaseof ACh evoked by incubation with potassium (14) or with aminopyridines (15).

We now repor t that choline, given peripherally, causes a dose-depen dent increase inscopolamine-evoked ACh release in hippocampus of awake, freely-moving rats.

MethodsANIM ALS: Male Sprague-Dawley rats (250-300 g) obtained from Charles River Laboratories(Wilmington, MA ), were housed in groups of three and kept in a 12: 12 hour light/dark cycle.


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Water and food were provided ad libitum.SUR GER Y A ND DIALY SIS: Rats were anesthetized with equithesin (chlornembutolO.3 ml/100g, i.p.) and placed in a Kopf stereo taxic small animal frame . Concentric 3 mm microdialysisprobes with a molecular weight cutoff of 5000-6000 g were chronically implanted in theventrolateral aspect of the right hippocampus. Implantation coordinates used with reference tobregma and dura were: P = 5.8 mm, L = 5.5 mm and V = 6.0 mm (16). Probes werepermanently implanted using dental cement and jeweler screw s anchore d to the skull. Prior toprobe implantation, a polyethylene tube (0.51 mm internal diame ter and 1.56 mm outerdiame ter) was implanted chronically within the peritoneal cavity and allowed to exit the bodyat the top of the head, along with microdialysis tubing. Animals were allowed to recover fromanesthesia and surgery for 24-36 h. Approximately 3 h prior to collecting dialysate samples forbasal choline and acetylcholine determinations, probes were perfuse d with artificial cerebrosp inalfluid (containing, in mM: NaCl, 121; NaH C03, 25; KCl, 3.5 CaCl,, 1.2; MgC l,, 1.2;NaH,PO ,,l; Neostigmine, 10 PM: bubbled with 95% O2 and 5% CO2 for 15 min, pH 7.4) ata rate of 2 $/min. During the experiment, animals were allowed to have free access to waterand food and were expo sed to ambient air. All experiments were carried out between 11:OO amand 6:00 pm. Dialysates wer e collected in fractions representing 15 min intervals. Aftercollection of 4 basal extracellular samp les, rats with chronically implanted hippocampalmicrodialysis probes received choline chloride (60 mg/ kg, i.p.). Four additional dialysatesamples were collected with animals then receiving scopolamine hydrochloride (0.5 mg/kg, i.p.)and sample collection continuing for an additional 90 min.

AnalysisDialysate ACh and choline concentrations were determined by HPL C with an enzymereacto r containing acetylcholineste rase and choline oxidase, c oupled to an electrochem ical

detector for hydrogen peroxide (Bioanalytical Systems Inc., West Lafayette, IN), as describedby Potter et al. (17). At the end of the experiment, the animals were sacrificed and their brainsremo ved to verify pro be placement and to asses s the condition of tissue surrounding theimplantation site. The percentage recovery of each microdialysis probe measured for Ach andcholine w ere determined to be 24% and 26%, respectively. Choline chloride was obtained fromthe Sigma Chemical Company (St. Louis, M O).STA TISTIC S: Significance of difference between the saline control and choline group s wa sdetermined by two-way analysis of variance (ANO VA) and Tukey test. Statistical tests wereperformed with the aid of SYSTA T version 5 software (Systat Inc., Evanston, IL) on aMacintosh IIci personal computer.

ResultsStability in baseline acetylcholine and choline levels was achieved prior to either saline

or choline supplementation. Eac h sample represented 15 min collection time (n =4, saline; n = 8,choline); basal choline levels were determined to be 13.4 7 + 2.2 for saline and 12.01 + 1.28for choline. System ic administration of choline chloride (60 mg/ kg, i.p.) elevated choline levelsin hippoca mpal dialysates maximally (almost 3-fold) 15 min post-injection (Fig. 1). Two -wayANO VA followed by a Tukey test was used to determine the statistical significance of differencebetween the two groups . Ex tracellular choline levels wer e significantly elevated 15 minfollowing choline administration compared with those of saline-treated control rats (P = 0.004).No other statistically significant differences we re found. These elevated levels returned tobaseline values (i.e., within 36 % of pre-injection choline lev els) by 60 min. Systemic injection


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60Saline, n=4

50

60mgIkg

1 I I I

0 30 A 60 90 Al20 150 180 210Time, min

FIG. 1Effect of choline or administration (60 m g/kg i.p.) on extracellular hippocampalcholine levels. The results are expressed as means f SEM . *P = 0.004 TukeyHSD multiple comparisons matrix of pairedwise comparison probabilities; Systatstatistical program.

of the muscarinic antagonist scopolam ine decre ased extracellular choline concentrations incholine-pretre ated rats, and in rats that had received saline (Fig. 1 ). B asal acetylcholine levelsranged from 0.41 to 1.87 pmol/l5 min for choline groups and 0.71 to 1.96 pmol/l5 min forsaline control. Pretreatment of rats with 30, 60 or 120 mg/kg, i.p., choline chloride resulted inno significant differences in basal ACh levels compare d with those in rats receiving equalvolumes of saline (Fig. 2).

Administration of 30 mg/k g, i.p., of choline chloride 1 h prior to scopolam ine, i.p., didnot potentiate scopolam ine-evoked AC h release (i.e., comp ared with that in rats receiving salineprior to the scopolamine; Fig. 2). How ever, administration of 60 o r 120 mg/kg choline chloridedid significantly (P


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aIm;

6 l a nt:; S W

_ cs _ z c c oe n . 4 Choline, 30 mgk g n=S5 Choline, 120 mgk g

0 30 60 90 1 2 0 150 0 30 M) 90 1 2 0 15 0

Time, min.

FIG. 2Dose-dependent effect of choline pretreatment on scopolamine-evoked AChrelease within rat hippocampus. The solid arrows indicate when saline or cholinewas administered and the clear arrows indicate when rats received scopolamine(0.5 mg/kg, i.p.). Percent values were derived from the last basal ACh valuepreceding choline or saline administration. Values are expressed as means +SEM.In accord ance with previous findings (2 2), peripher al administration of the non-selective

muscarinic antagonist, scopolamine, caused a pronounced enhancement of hippocampal AChrelease. E xtracellular ACh levels were maximally elevated 30 min following scopolamineadministration, gradually returning to near-basal levels by 120 min. In the present study,pretreatment with choline produced a dose-dependent enhancement of scopolamine-evoked AChrelease. It was previously observed that atropine-evoked release of ACh in the striatum isenhanced by pretreatm ent with choline (13) and it is well established that presynaptic muscarinicautoreceptors modulate AC h release (23,24).

In conclusion, ACh release in the awake rats hippocampus, when m uscarinicautore ceptors are blocked, is affecte d by the availability of choline.

AcknowledamentsThese studies were supported in part by a grant (MH -28783) from the National Institute ofMental Health. Dr. Jackson is also the recipient of support from Training Grant (T32M H1576 1).


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Darrell A. Jackson et al- Choline Enhances Scopolamine-Induced Acetylcholine Release In Dorsal Hippocampus of Conscious, Freely-Moving Rats