Effect of the 5-HT1A receptor agonist ipsapirone on the local cerebral glucose utilization of the rat hippocampus

Brain Research. 436 (1987) 283-290 Elsevier

283

BRE 13133

Effect of the 5-HTIA receptor agonist ipsapirone on the local cerebral glucose utilization of the rat hippocampus A n d r e a s W r e e I , K a r l Z i U e s I , A x e l S c h l e i c h e r l, E r v i n H o r v f i t h 2 a n d J 6 r g T r a b e r 2 tInstitute of Anatomy, University of Cologne, Cologne (F.R. G.) and 2Neurobiology Department, Troponwerke, Cologne 80 (F.R. G. )

(Accepted 2 June 1987) Key words: Glucose utilization; Autoradiography; 2-Deoxyglucose; Serotonin; 5-HTIAreceptor; Ipsapirone; Hippocampus; Rat

Local cerebral glucose utilization (LCGU) was measured in the hippocampus of the rat brain followingi.p. injection ot the anxiolytic drug and 5-HTIAreceptor agonist ipsapirone (TVX Q 7821). Administration of ipsapirone (5 mg/kg) reduced glucose utilization in the various hippocampal areas to variable extent. The most subtle reduction took place, in the dorsal subiculum, while the most pronounced decrease was found in sector CA4 of Ammon's horn. The degree of LCGU reduction can be related to the 5-HTIA receptor density in the respective areas.

INTRODUCTION Various subtypes of 5-HT 1 rec~:ptors have been described in the rat brain. They differ qualitatively and quantitatively in their topographic distribution 7"11" 14.20.23.29-34.55.57. The 5-HTIA sites are enriched in structures of the limbic system, e.g. the hippocampal formation and the entorhinal area, whereas the 5HTIa sites predominate in the basal ganglia, the dorsal subiculum and the substantia nigra; the 5-HTIc sites are most concentrated in the choroid plexus. Due to the availability of very selective compounds such as 8-hydroxy-2-(di-n-prop)lamino)tetralin (8OH-DPAT) or ipsapirone (formerly TVQ Q 7821) the 5-HTIA recognition sites have been the object of intensive investigations. They play a major role in a variety of centrally mediated functions such as anxiety and aggression 52, sexual behavior iS, food intake t3 and other behaviors 53. Neuronal inhibition has been demonstrated with electrophysiological methods 3.46. In spite of the intensive investigation of this subject, very little is known about the functional consequences of 5-HTIA receptor activation by selective

drugs on the metabolic level. One possibility to gain insights into the actions of these compounds is to determine their influence on the local cerebral glucose utilization (LCGU) in rats. The LCGU has proven to be a valid measure of neuronal activity in the brain (for review see refs. 41, 42). In the present study the effect of th, 5-HTtA receptor agonist ipsapirone on the LCGU ~ter systemic administration in rats was investigated in different regions of the hippocampal formation. It was of interest to determine whether 5HTIA receptor stimulation caused a uniformly reduced LCGU in the hippocampus as has been proposed for other 5-HT agonists 17 or whether the anatomically different hippocampal areas were differently influenced. We found that ipsapirone treatment causes a distinct pattern of LCGU reduction in the rat hippocampal formation. Furthermore, we present detailed basic data on the glucose consumption of the hippocampal areas of conscious rats. MATERIALS AND METHODS Animal preparation Male Wistar rats (200-300 g) were anesthetized

Correspondence: K. Zilles, Institute of Anatomy, Universityof Cologne, Joseph-Stelzmann-Str.9, D-5000 Cologne 41, F.R.G.

0006-8993/87/$03.50 © 1987Elsevier Science PublishersB.V. (Biomedical Division)

284 with halothane (1.2 vol%) and polyethylene catheters were inserted into the femoral arteries and veins. According to standard procedures, the rats were immobilized from the abdomen to the hindlimbs by plaster casts; the forelimbs and thorax were left unrestrained. The rats were allowed to recover from the effect of anesthesia for 2 h before drug treatment. Drug administration Ipsapirone (2-[4-[4-(2-pyrimidinyl)- 1-piperazinyl]-

butyl]- 1,2- benzoisothiazole-3- (2H)one- 1,1 -dioxidehydrochloride) was injected i.p. in a pharmacologically effective dose of 5 mg/kg. Either this dose dissolved in 0.5 ml distilled water or, in the controls, the vehicle alone was administered 30 rain before the i.v. injection of the [14C]2-deoxyglucose. This dose has been shown to inhibit aggressive behavior and anxiety in animal models 39"52. With this dose the animals exhibited normal active behavior like exploration and social investigation and did not show sedation, ataxia or muscle relaxation 39,52.

Experimental p r o c e d u r e

The determination of the L C G U with the [14C]2deoxyglucose technique has been previously described 19'37'41-45. In brief, 80-100/~Ci/kg of 2-deoxyo[1-14C]glucose (spec. act. 51.3-59.4 mCi/mmol, Amersham Buchler, Braunschweig, F . R . G . , or New England Nuclear, Dreieich, F . R . G . ) were injected as a bolus via the femoral venous catheter over a 20-s period. Within the 45 min after the beginning of the injection, 16 timed arterial blood samples of approximately 50 ld each were collected through the femoral arterial catheter. The samples were immediately centrifuged and the plasma levels of [l~C]deoxyglucose and glucose were determined by scintillation counting and by glucose oxidase assay. Following decapitation, the brains were rapidly removed and frozen in isopentane at - 4 0 °C and stored at - 7 0 °C until sectioning. Coronal sections (20/~m) were cut in a cryostat, thaw-mounted on coverslips, dried on a hot plate and exposed to x-ray film (Osray M3, Agfa Gevaert, Leverkusen, F . R . G . ) for two

TABLE I Effect o[ ipsapirone (5 mg/kg i.p.) on the local cerebral glucose utilization in the hippocarnpal region of the rat brain

The LCGU data are expressed as Flmol glucose/(100 g x min) of glucose utilization (means + S.D.). In all hippocampal structures the drug-treated rats exhibited significant lower LCGU values compared with the control animals (a = 0.05, Mann-Whitney U-test). The relative reduction of LCGU in ipsapirone-treated rats compared to control animals is calculated by: rel. red. = (LCGU controlsLCGU ipsapirone created) x 100/LCGU controls (%). Ammon's horn layers: Lmol, lacunosum molecular layer; Luc, lucidum layer; Or. oriens layer; Py, pyramidal layer; Rad, radiatum layer; n, number of animals used in each group. Structure

Sector CA 1 of Ammon's horn Rad and Lmol layers Py and Or layers Sector CA2/3 Gf Ammon's horn Luc Rad and Lmol layers Py and Or layers Sector CA4 of Ammon's horn Dentate gyrus External limb Internal limb Dorsa] subiculum Molecular layer Pyramidal layer Ventral subiculum Molecular layer Pyramidal layer Presubiculum Para~abiculum Entorhinai area

Controls LCGU (n = 8)

Ipsapirone LCGU (n = 13)

Relative reduction of LCGU

59.0 + 5.1 51.1 + 0,.8

41.6 + 8.6 35.6 + 7.1

29.5 30.3

63,1 +_6.7 56.0 + 6.0 57.9 + 3.1

41.3 + 9.4 37.2 + 7.9 38.0 + 7.8

34.5 33.6 34.4

64.3 + 5.1 55.4 + 4.0

45.2 + 10.1 38.7 +_8.1

29.7 30.1

68.7 + 6.4 63.4 + 5.5

61.8 + 5.7 52.6 + 10.9

10.0 17.0

65.0 + 6.2 61. | + 6.1 63.2 ,'- 7.4 57.5 + 5.3 59.6 + 5.5

45.6 + 8.8 43.1 + 7.7 51.0 + 9.6 43.3 + 4.8 43.7 + 7.8

29.8 29.5 19.3 24.7 26.7

285 weeks at room temperature in light-tight x-ray cassettes along with precalibrated [~aC]methylmethacrylate standards (Amersham Buchler, Braunschweig, F.R.G.). Serial sections adjacent to those which were used for autoradiography were mounted on chrome alum-gelatine-coated slides, fixed in buffered formaldehyde and stained either for perikarya (Cresyl fast violet s) or acetylcholinesterase *s to facilitate anatomical identification of the respective areas and laminae of the hippocampus.

brain hippocampai region were delineated according to the criteria given by Buyer t, Lorente de No >, Stephan 47 and Zilles s4. RESULTS In the control animals the L C G U measurements revealed areal and laminar differences in the glucose consumption of the hippocampal region (Table I). By

IMAGE ANALYSIS Autoradiographs were analyzed with a computer assisted imaging device, IBAS (Kontron, Munich, F.R.G.). Technical details concerning the densitometry of autoradiographs ~.2'~6"35.38, especially with the IBAS system equipped with a w-camera have previously been published 56. In brief, images of one hemisphere were digitized by the system with a spatial resolution of 20/~m x 20 p m per pixel. The original images of grey value data were then transformed into L C G U values, taking into account the readings of the calibrated standards, the integrated concentration of ~4C and glucose in the arterial plasma during the experimental period, and the appropriate constants for the rat 37'42"45, and using the operational equation given by Savaki et al. 37, which accounted for variations in the arterial olasma glucose concentration (values of constants used: k~* = 0.18'a rain, k2* = 0.245/min, k3* = 0.053/rain, (k2+k3) = 0.577/min, lumped constant = 0.464). For each brain region in each animal, 8-12 autorad;,ograpl~s were evaluated for measurement. The pixel values of each area, which was outlined (Fig. lb) and completely measured as previously described ~6, were finally gathered by computer programs. The mean rate of glucose consumption and the S.D. were calculated.

Data analysis The data are presented as means + S.D. Statistical differences in the calculated values of controls and drug-treated animals were tested by the M a n n Whitney U-test 36, ct = 0.05.

Delineation of cortical areas The different cortical areas and laminae of the rat

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Fig..l. Nissl-stained section (a), and autoradiograpi~ (b) of the directly adjacent section. The autoradiograph is transfo~in:~ into local cerebral glucose utilization ~umol glucose/(100 g :< min)) an.q different LCGU ranges are plotted in different print modes ac,'ording to the given scaling. The sections are taken fxom a control rat at the level c ¢ the dorsal hippocampus 3.3 mm posterior to bregma54. In the autoiadiograpi-i (b) areal ~ a laminar boundaries are traced by superimposing the print on the Nissi-stained section in (a). By this procedure, accurate boundaries of areas and laminae, which do not always give a corresponding contrast in the autoradiographs, can be established. CA. sectors of Ammon's horn; Lmol, lacunosum molecular laver: Luc, lucidum layer: Or, oriens layer: Py. pyramidal layer: Rad, radiatum laver: DGex. external limb of dentate gyrus: DGin, internal limb of dentate gyrus.

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Fig. 2. Autoradiographs at the level of the dorsal hippocampus (a, b, about 3.3 mm posterior to bregma) and temporal hiopocampus (c, d, about 5.8 mm posterior to bregma according to Zilles~'~)from a control rat (a, c) and a rat injected with 5 mg/kg ip~apirone i.p. (b, d). The autoradiographs are transformed into local cerebral glucose utilization Lumol glucose/(l(l('~ g x rain)) and different ranges are plotted in different print modes according to the scaling given in (d), which applies for a-d. Ipsapirone leads to a decreased glucose utilization. Note thc minor LCGU reduction in the dorsal subiculum (Sd) following ipsapirone administration (d). The architectonic bor-. der between the dorsal subiculum and the laterally adjoining sector CA1 of Ammon's horn is mart-~'~ (arrowhead) and clearly visible after ipsapirone treatment (d) due to the strong LCGU reduction in CA1 following drug admit,; , ation.

287 comparing the autoradiographs (Fig. lb) with the directly adjacent Nissi-stained sections (Fig. la) it can be seen that the dark band seen in the autoradiograph, which indicates high glucose utilization, defines the border region between the sector CA! of Ammon's horn and the external limb of the dentate gyrus. The hippocampal fissure is located in the middle of the dark band (Fig. lb). Glucose consumption v, as found to be significantly higher in the laminae of sectors CA2/3 of Ammon's horn than in sector CA1. The print giving the LCGU (Fig. lb) clearly identifies the cytoarchitectonic border between these sectors. Furthermore, in the dentate gyrus, the external and internal subfields showed different glucose consumptions (Table I). The external limb exhibited a significantly higher glucose utilization than the internal limb (Table I, Fig. lb) .... The effect of ipsapirone on the LCGU is seen from autoradiographs (Fig. 2), in which the grey values have been transformed into glucose utilization values. Obviously, in several brain areas of the ipsapirone-treated animals (Fig. 2b,d), the LCGU was reduced as compared with the control rats (Fig. 2a,c). This was, beside other regions, especially pronounced in most areas of the hippocampal region. This paper focusses ell the drug-induced LCGU alterations in the hippocampus. In the control rat (Fig. 2c) the c:,,toarchitectonic border (marked by arrowhead) between the dorsal subiculum and the laterally adjoining sector CAI of Ammon's horn is barely visible. In contrast, in the ipsapirone-treated rat (Fig. 2d), the architectonic border can be clearly seen, because of the lov~ LCGU reduction in tae dorsal subiculum and the more pronounced effect in CA1. A quantitative analysis is given in Table I. Ipsapirone oroduced a significant decrease in glucose utilization in all the hippocampal areas (Table I). Interestingly, the degree of this reduction in LCGU (calculated as relative reduction in the treated animals compared to the controls) differed in the various areas and laminae under study (Table I) and ranged between 10% and 34%. The smallest reduction was found in the molecular and pyramidal layers of the dorsal subiculum (10% and 17%, respectively). The inhibitory effect of ipsapirone on the LCGU was most pronounced in the CA2/3 and CA4 fields, which showed a reduction in glucose consumption of about 34%. An LCGU re-

duction of about 30% was found in the CA 1 field, the dentate gyrus and the ventral subiculum. Glucose utilization was reduced in the entorhinal area by 27%, in the parasubiculum by 25%. and i~1 the presubiculum by 19%. DISCUSSION

In the control rats the hippocampal region e×hibited a heterogeneous LCGU distribution. The comparison of the autoradiographs with the adjacent Nissl-stained sections is necessary for determining area-specific glucose utilization. First, it must be mentioned, that the dark band seen in the autoradiographs of the hippocampal region, which was assumably interpreted in the literatureS.6.21.22.26-28. 40.45.48-51 as the molecular layer of the hippocampus, is situated in the border region between the CA sectors of Ammon's horn and the external limb of the dentate gyrus. This band, therefore, spans two different areas° On the other hand, changes in LCGU are often indicative of areal boundaries. The border between sectors CA 1 and CA2/3 is clearly visible in the autoradiogram, and corresponds with the cytoarchitecture. Based on our results, there are differences between the glucose consumption in sectors CA 1 and CA2/3 of Ammon's horn. In the literature, sector CA3 was reported by two groups "'2~ to exhibit a higher glucose utilization than CA 1. whereas Celik et al. 9 found similar LCGU values in these two sectors. According to our results, there are differences as well in the LCGU values for the external and internal limbs of the dentate gyrus: but. to our knowledge, these have not yet been reported in the conscious rat. The results presented in this paper provide evidence, that, in the hippocampai region of the rat, the 5-HT~A ligand ipsapirone reduces glucose utilization. The amount of LCGU reduction, however, differs considerably among the different hippocampal structures. The glucose consumption is only slightly reduced in the dorsal subiculum, but it is heavily decreased in the sectors CA2/3 and CA4 of Ammon's horn. The general decrease in hippocampai glucose consumption following ipsapirone administration is roughly in line with recent results of the effect of other 5-HT agonists 17. These authors reported a 25-50% reduction of the LCGU in the molecular

288 layer of the hippocampus and the dentate gyrus after treatn'~ent of rats with 5-HT agonists such as LSD, 5MeODMT (5-methoxy-N,N-dimethyltryptamine), quipazine and 6-CPP (6-chloro-2-(1-piperazinyl)pyrazine). These substances, however, simultaneously bind to various subtypes of the 5-HT binding sites and, in part, to non-5-HT binding sites also. Therefore, Grome and Harper's 17results cannot be directly related to this study, as ipsapirone binds selectively to the 5-HT)A sitesl2'14. Electrophysiological studies 3'46 have shown that systemic or local administration of ipsapirone potently inhibited single-unit activity of neurons in the dorsal raphe nucleus, the dentate gyrus and the sector CA1 of A m m o n ' s horn. It can be argued, that the reduction in L C G U in the hippocampal region, presented in this study, reflects the depression of neuronal activity seen in the electrophysiological studies. The differences in the relative reduction of glucose consumption in the hippocampal areas following ipsapirone treatment might be related to the local distribution of 5-HTIA binding sites. Ipsapirone had the least effect on the L C G U in the dorsal subiculum, where the density of 5-HTIA binding sites is very low 14'30. Correspondingly, areas rich in 5-I-ITIA rec-

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