0306-4522/91 $3.00 + 0.00 PergamonPress plc 0 1991IBRO
NeuroscienceVol. 45, No. 2, pp. 261-272, I991 Printedin Great Britain
REGIONAL HETEROGENEITY IN THE DISTRIBUTION NEUROTRANSMITTER MARKERS IN THE RAT HIPPOCAMPUS H.
H~RTNAGL,*~
M. L. BEXGER,* G. SPERK$
OF
and CH. PIPL*
*Institute of Biochemical Pharmacology, University of Vienna, Borschkegasse 8a, A-1090, Vienna, Austria SDepartment of Pharmacology, University of Innsbruck, Innsbruck, Austria Abstract--A detailed neurochemical analysis of the distribution of markers for the most relevant neurotransmitter systems within the rat hippocampal formation has been performed. The hippocampi, obtained from unfrozen brains of male Sprague+Dawley rats were subdissected into tissue parts containing mainly CAl, CA3 or the dentate gyms, respectively. Each part was further divided into ventral and dorsal halves. In these six hippocampal subregions the concentrations of noradrenaline, dopamine, serotonin, 3-methoxy-4-hydroxyphenylglycol, 5-hydroxyindoleacetic acid and the putative neurotransmitter amino acids glutamate, aspartate, GABA, glycine and taurine, and the levels of somatostatin and neuropeptide Y and the activities of choline acetyltransferase, acetylcholinesterase and glutamate decarboxylase were measured. A marked heterogeneity in the subregional distribution of markers for various neurotransmitter systems within the hippocampal formation was observed. Each neuronal marker was characterized by an individual pattern of distribution. Most of the markers showed a concentration-gradient, increasing from dorsal to ventral; only taurine was more abundant in the dorsal than in the ventral parts and no dorsoventral difference was seen for aspartate, glycine and neuropeptide Y. The highest molar ratios of total 3-methoxy4hydroxyphenylglycol to noradrenaline and 5-hydroxyindoleacetic acid to serotonin were found in the dorsal hippocampus. The levels of noradrenaline, GABA and glutamate decarboxylase
activity were highest in the dentate gyms and lowest in CAI. The concentrations of somatostatin were highest in CAI; those of serotonin were highest in CA3. Highest activities of choline acetyltransferase and acetylcholinesterase were found in the dentate gyrus; lowest activities were found in CA3. In CA3 the lowest values of glutamate, aspartate, taurine and somatostatin were also found. The heterogeneity in the distribution of individual neurochemical markers allows insights into possible functional differences of hippocampal subregions and provides a relevant basis for future neurochemical investigations in this brain-area. _
The rat hippocampal formation has been thoroughly studied with respect to its neuroanatomy, electrophysiology, neuropathology and neurochemistry. Its three-dimensional shape is relatively complex. It appears grossly as an elongated structure with its longitudinal axis bending in a C-shaped manner from the septal nuclei rostrodorsally to the incipient temporal lobe caudoventrally (e.g. Ref. 2). The hippocampal formation comprises four cortical regionsthe dentate gyrus, the hippocampus proper, the subiculum and the entorhinal cortex. This complexity implies a rather heterogeneous structural and functional organization. As a consequence of the elongated structure, the inputs of various neurotransmitter systems reach their destination via several main pathways; thus, subregional differences in the tTo whom correspondence should be addressed. Abbreoiarions: AChE, acetylcholinesterase; CbAT, choline acetyltransferase; DA, dopamine; EDTA, ethylenediaminetetra acetate; GAD, glutamate decarboxylase; 5HIAA, 5-hydroxyindoleacetic acid; 5-HT, serotonin; HPLC, high-performance liquid chromatography; MHPG, 3-methoxy4hydroxyphenylglycol; iEo-MHPG, 3-hydroxy4methoxyphenylglycol; NA, noradrenaline; NMDA, N-methyl-o-aspartate. 261
concentration of neuronal markers and in neuronal activity may be expected. Whereas most neuroanatomical work has focused on the cellular localization of neurotransmitter systems in the transverse axis so far, much less is known about the distribution of neurotransmitter markers along the dorsoventral (septotemporal) axis of the hippocampus. The importance of this longitudinal axis for the architecture of intrinsic hippocampal connections has been emphasised in a review by Amaral and Wittet.2 and arises from recent neuroanatomical work. Recently, gradients for various neuronal markers with concentrations increasing from dorsal to ventral have been described for cholinergic neurons,12~2L~62 for noradrenaline (NA),33,62 serotonin (5-HT)32*62and somatostatin.” The overall density of the hippocampal noradrenergic innervation was reported to be 20% higher ventrally than dorsally and marked subregional differences in the density of NA varicosities were obvious.45 Choline acetyltransferase (ChAT)positive cells of the medial septal nucleus project more heavily to the ventral than to the dorsal levels of the hippocampus and dentate gyrus,’ consistent with the lower concentrations of acetylcholine and ChAT activity’9*2’*62and lower density of
262
1-i. H~RTNACL cv ~1
acetylcholinesterase (AChE) staining in the dorsal hippocampus4’ In the transverse axis of the hippocampus, however, regional differences in the tissue levels of neurotransmitter markers are less defined. It is of particular interest that in this hippocampal axis, regional variations of the extracellular space exist among the subfields of the hippocampus, the CA1 region exhibiting the lowest extracellular volume.3’ Moreover, the hippocampus is also not homogeneous in its response to various stimuli and noxes neither in the transverse nor in the lon~tudinal axis. NA is known to exert differing effects on Ammon’s horn and dentate gyrus neurons.34,U It is also well established that ischemia causes neuronal necrosis only in selectively vulnerable sectors of the hippocampus, such as the CA1 field.26,27Colchicine, on the other hand, exerts a preferential ne~otoxicity towards dentate granule cells while sparing hippocampal pyramidal cells. I8 In the longitudinal axis it appears that the age-related loss in ~ptohippocampal afferents is mainly confined to the ventral hippocampus; an age-related increase in muscarinic binding sites has been observed only in the ventral pole.57 Furthermore, electrolytic lesions of the septum are associated with an increase in nerve growth factor levels only in the ventral but not in the dorsal hippocampus. Preliminary data indicate that the ventral portion is also more affected by the eholinotoxin ethylcholine aziridinium.20 Such a heterogeneity in neurochemical parameters, in function and in vulnerability to exogenous noxes, prompted us to assess a more detailed topographical pattern in the concentration of markers for various neurotransmitter systems as well in the transverse as in the longitudinal axis. For this purpose the rat hippocampus was dissected along its longitudinal axis into the subfields CAl, CA3 and dentate gyrus and further subdivided into dorsal and ventral parts. In the present study we focused on the choliner~c, monoaminergic and GABAergic systems as well on the distribution of the other putative neurotransmitter amino acids gIutamate, aspartate, glycine and taurine, and of somatostatin and neuropeptide Y immunoreactivities. EXPERIMENTAL
PROCEDURES
Male SpragueDawley rats (Forschungsinstitut fur Versuchstierzucht, Himberg, Austria) with a body weight of 310-44Og were used for the study. The animals were housed in groups of three to four per cage on a 12-h light-dark cycle, and had free access to laboratory chow and tap water. fUuteri& f’4C]Acetyl-coenzyme A and L-l-[14C]glutamic acid (specific activities: 55 mCi/mmol) were purchased from Amersham Laboratories (U.K.), acetyicoenzyme A from Boehringer Mannheim. Bovine serum albumin, bacitracin, acetylthiocholine, physostigmine, dithiobis-2-dinitrobenzoic
acid, NA, dopamine (DA), 5-HT, S-hydroxylndoiraccil~, acid (S-HIAA), sulphatase (from Helix pomatia. type H-5). 3-methoxy-4-hydroxyphenylgiycoi (MHPG) and 3.4-dihydroxybenzylamine hydrobromide were obtained from Sigma Chemie GmbH (Deisenhofen, F.R.G.), amino acids from Merck AG (Darmstadt, F.R.G.) and o-phthalaldehyde from Serva Feinbiochemica {~eidel~rg, F.R.G.). Aminex A-9 (sodium form) was provided by Bio-Rad Laboratorics (Richmond, CA, U.S.A.). 3-Hydroxy-4-methoxyphenylgiycol (iso-MHPG) was kindly donated by D. 1. Heal Dissection of the hippocampus Rats were killed by decapitation. The brains were rapidly removed and ~mm~iately chilled in iced-water kept at -0.8”C with NaCI. After 10 min of chilling, the hippocampi were dissected from the unfrozen brains and gently extended on a cold-plate (f 1°C) the inner surface facing upwards (Fig. I). The extended hippocampi were dissected into tissue parts containing mainly CAI, CA3 and the dentate gyrus, respectively. The extension of the hippocampus in its dorsoventral axis greatly facilitated the separation of the three subfields ~roughout the hip~rnp~ from rostrodorsal to caudoventral. The dissection procedure is described in detail in the legend to Fig. I. The anatomical markings of the small longitudinal fissures, which are additionally marked by blood vessels, allow a reproducible separation between the three subregions. Using this dissection procedure, a small part of CA3 and CA4 is contained within the dentate gyrus part. The CA3 part contains partly CA2. All three regions were additionallv cut into ventral and dorsal parts and the corresponding samples of the left and right hippocampus were combined. After dissection, the tissue samples were weighed and immediately frozen on dry ice and stored at -80°C until biochemical analyses. The average weights (mg rt S.E.M.) of the six subregions per hippocampus (n = 17) were: CA1 ventral: 13.0 + OS: CA I dorsal: 11.8+ 0.4; CA3 ventral: 8.6 kO.2: CA3 dorsal: 7.3 k 0.2; dentate gyrus ventral: 7.1 & 0.2; dentate gyrus dorsal: 7.8 + 0.3. B~o~herni~a[analwes Tissue samples were homogenized at 4°C by ultrasonication in 30-40 vols of N,-saturated deionized water. Immediately after sonication an aliquot of the homogenate (2~3~~1) was added to an equal volume of Cl.2M perchloric acid containing 0.8 mM NaHSO, and centrifuged at 25,000 g at 4°C for 15 min. The supernatant was used for the measurement of NA. DA. S-HT. S-HIAA and the putative neurotransmitte~ amino acids. The remaining aqueous homogenate was used for the measurement of activities of glutamate decarboxylase (GAD), ChAT and AChE. In a group of animals (n = 12). aliquots of the homogenates equivalent to 8mg of tissue were added to acetic acid to give a final volume of 1ml (final concentration of acetic acid: 2.5 M), resonicated and centrifuged at 25,OOBg for 15 min. The resulting supernatant was usd for quantification of somatostatin and neuropeptide Y. Aliquots for determination of each peptide were transferred into separate Eppendorf vials and stored at -80’ C. Before anaIysis samples were Iyop~liz~ in a speed vat concentrator (Savant, Farmingdale, NY, U.S.A.). For measurement of MHPG, tissue samples were sonicated in 12 vols of 0.12 M sodium acetate-citrate, pH 5.0 ~ntaining 3,4-dihydroxy~nzylamine hydrobromide and iso-MHPG as internal standards. Enzyme activities Choline ~cet~ftran~ferase. ChAT activity was measured according to the method of Fonnum,‘$ with minor modifications using substrate concentrations of 8 mM choline chloride and tritiated 0.2 mM acetykoenzyme A.” glutamate ~eearbo~~~a.~e.GAD was measured in the supernatant after centrifugation of the aqueous homogenate
Regional heterogeneity of neuronal markers in hippocampus
d hlr#xKaKJL fissure
. longttuditKll axis by 180’ /
263
dissect CA3 bmrt
r tssure \
=-I”L& Y c-a-1, .f lssure
dissect out\
Fig. 1. Dissection of the hippocampal subregions. The position of the hippocampal formation in the rat brain after removal of the overlying cortex is indicated in the schematic drawing adapted according to Amaral and Witter.2 The whole hippocampus was dissected out @ and turned around its longitudinal axis by 180” and extended 0. The inner surface of the ~p~rnpus, facing the thalamus, is structured by two fissures: a ‘minor” fissure close to the timbria and the hippocampal fissure. Firstly, after removal of the fimbria, the CA3 part was dissected by cutting the extended hippocampus along the minor fissure 0. The remaining CAldentate gyrus (DG) part was turned by 80” clockwise. The separation between CA1 and DG @ was achieved by carefully opening the hippocampal fissure. The inserted light microscopic photo~ap~ (caption, x 10) were obtained from transverse slices cut from the ventral part of the extended hippocampus. For this purpose the hippocampi were dissected out of the brain after perfusion of the deeply anesthetized (150 mg/kg, i.p., thiopental) rat with 4% paraformaldehyde solution. The hippocampi were extended and separated into ventral and dorsal parts. The tissue parts were postlixed in the same hxative for 90 min, then immersed in 20% sucrose (24 h, 4°C) and subsequently frozen in isopentane (-70°C). Slices of 20 pm thickness were cut on a mi~otome and stained in Creayl Violet (0.5%). at 1S,OOO g for 10 min by trapping [“C]COz formed from as described by Roberts and Simonson” at substrate concentrations of 15 mM glutamic acid and 0.5 mM pyridoxalphosphate. Acetylcholinesterase. For calorimetric measurement the homogenates were treated with Triton X-108 (final concentration 0.4% + 10 mM EDTA) and centrifuged at 10,000 g for 10 min. The enxymatic reaction was performed according to Ellman ef ai.
L-1-[14CJgIutarnic acid
Determination of manoamines, 3-methoxy-bhydroxyphenylg&o1 and S-hy~oxy~o&acetic acid NA and DA were measured by high-performance liquid chromatoaranhv iHPLC1 with electrochemical detection after extr&ibn~to‘alumina according to Felice et aLI with minor modifications.” 5-HT and IHIAA were determined by HPLC with electrochemical detection as described previouslv.53 Total MHPG was determined after enxymatic hydrolysis by sulphatase and after prepurification on small Senhadex G 10 columns bv HPLC with coulometric detection, procedures modified &cordii to previously published methods.g6’*6J Homogenates were centrifuged for 15 min at 4°C at 18,OOOg.Suiphatase (14mg/ml) in 0.12M sodium
acetate-citrate @H 5.0) was added to the supernatants to give a final concentration of 1.5 mg/ml and samples were incubated for IS h at 37°C. Perchloric acid was added to a final concentration of 0.3 M, denaturated protein was centrifuged (15 min, 4”C, 18,OOOg)and supematants (volume made up to 0.6 ml with 0.01 M formic acid and foe4 M EDTA) were put on Sephadex G 10 columns (5 x 70 mm) prepared in long Pasteur pipettes p~~~~brat~ and washed as described previ~usly.~~ NA and MHPG were eluted with mobile-phase buffer of HPLC. This buffer contained 0.1 M sodium acetate, 0.05 M citric acid (PH 4.85 adjusted by sodium hydroxide), 50mg of EDTA and 13Omg of ~~~~phonic acid sodium salt/l and 5% methanol. The liquid chromatographic system consisted of a Model 5700 Solvent Delivery Module (1.2ml/min), a 30 x 4.6mm Spheri-5 RP-18 guard column (Brownlee Labs), a 250 x 4.6mm Hype&l ODS Sprn analytical column (FZ Seibersdort) and a Model 5100 A Coulochem ESA electrochemicaI detector with a Model 5021 conditioning cell and a Model 5011 analytical cell. The potential of the conditioning cell and the first detector was set at +0.03 V. The potential of the second detector, at which NA and MHPG were measured, was set at +0.46 V. The NA levels measured in the same chromatographic run as MHPG
264
H. HGRTWAGL rf al.
were used for the calculation of the molar ratio MHPG to NA. Determination of putative neurotransmirler amino acids The levels of glutamate, aspartate, GABA, glycine and tamine were measured after HPLC separation and post-column derivatization with the fluorogenic reagents o-phthalaldehyde and mercaptoethanol, according to the method described by S&mid et al.5’ with minor modifications. The column (length 250 x 4mm i.d.) was packed with Aminex A-9 (sodium form) and maintained at 76°C. The two-step buffer-latent consisted of 0.02 M sodium formateH,PO, (pH 3.3, IO min) and 02 M sodium acetate-H,PO.,, pH 5.6 (7min). The buffers and the o-phthalaldehyde reagent (post-column) were pumped at a rate of 1.0 ml/min. The amino acid derivatives were quantified in an Aminco fluoromonitor (360/450 nm).
Cholinergic systems
The distribution of markers for cholinergic neurons was uneven in the various subregions of the hippocampal formation and is summarized in Table 1. The ventral parts of CAI, CA3 and dentate gyrus exhibited higher activities of ChAT as well as of AChE, compared to the dorsal portion (P < 0.01). Among the three subregions, highest activities of ChAT were measured in dentate gyrus (P < 0.01 vs CA1 and CA3), lowest in CA3 fP < 0.01 vs CA1 and dentate gyrus). For both enzymes the ventrodorsal difference was least prominent in the dentate gyrus. This subregional difference was less pronounced for AChE than for ChAT.
Radioimmunoassay of somatostatin and neuropeptiak Y The ~yophiliz~ samples were r~onstitut~ in 500~1 of ~eur~chern~~~l markers for rn5~5~~nerg~~ inner assay buffer, consisting of 0.15 M sodium phosphate bu&Ter, vaticms of the hip~acumpaI formation pH 7.4, 0.15 M sodium chloride, 0.1% bovine serum albumin, 0.1% gelatin, 0.02% sodium azide, 0.002% phenyl The regional distribution of NA, DA, 5-HT and red and 0.02% bacitracin. Standard curves were determined 5-HIAA is shown in Tables 2 and 3. by including the respective synthetic peptide in amounts Cutecholamines. Levels of NA exhibited marked between 5 and 5OOfmol. Somatostatin and neuropeptide subregional differences. The highest concentration Y immunoreactivit~es were determined in duplicate at was measured in dentate gyrus (P < 0.01 vs CA 1 and two different tissue ~ncentra~ons as described in detail elsewhere.3s*” In various brain areas, somatostatin imCA3) and the lowest one in CA1 (P < 0.01 vs CA3 munoreactivity comprises somatostatin-14 (about SO%), and dentate gyrus). A considerable increase in NA somatostatin-28 (about 10%) and pro-somatostatin (about concentrations from dorsal to ventral was only ob10%) as revealed by HPLC analysis previously.56 Neuroserved in CA 1 and CA3 areas, whereas this difference peptide Y immunoreactivity co-migrates in a single peak with synthetic neuropeptide Y in HPLC.” was not significant in dentate gyrus. In the dorsal Data ana@& All data are presented as means + S.E.M. For statistical analyses the two-factor analysis of variance (ANOVA; factors: subregion and dorsoventral location) was applied. The two-factor ANOVA indicated the significance of subregional and dorsoventral differences aa well as of the interaction between subregions and dorsoventral location. In the case of significant sub~gion~ diilerences Nap-Keuls test was applied as aposteriori test for more detailed analysis of eventual differences among the subregions, using the mean value of the corresponding dorsal and ventral parts of each subregion. Accordingly, the probability values for subregional differences are related to the subregion in its whole extension. Significant differences in the dorsoventral location were followed by paired Student’s t-test for each indi~dual subregion, wrrected with a-adjustment accord-
ing to Bonferroni (see Ref. 38). RESULTS
For the individual neuronal systems, the following distribution of their representative markers was observed.
hippocampus the NA content was about two-fold higher in dentate gyrus than in CAI. For the main metabolite of NA, MHPG, a much smaller but still significant difference between dorsal and ventral parts of CA1 and CA3 was observed. The highest MWPG levels were measured in dentate gyrus (P ~0.01 vs CA1 and CA3). In CA1 and CA3 but not in the dentate gyrus, the moIar ratio of MHPG to NA was significantly higher in the dorsal parts than in the ventral parts. The highest MHPG to NA ratio was present in CA1 (P < 0.01 vs CA3 and dentate gyrus). In general, the MHPG to NA ratio followed a dist~bution which was opposite to that of the NA levels, i.e. the higher the MHPG to NA ratio, the lower the NA levels. The levels of DA were remarkably low in the entire hippocampal formation. An increase from dorsal to ventral was seen in all three subfields. In contrast to NA, lowest levels of DA were found in the dentate gyrus (P < 0.05vsCA3). The NA to DA ratio was, therefore, considerably higher
Table I. Regional differences in the distribution of markers for the cholinergic systems in the hippoeampal formation of the rat CA1 ChAT (nmol/h per mg tissue) AChE (nmol/h per mg tissue)
Dorsal Ventral Ventrodorsal ratio Dorsal Ventral Ventrodorsal ratio
5.86 f 7.80 f I .43 + 365 f 502 + 1.40 *
0.13 0.14** 0.03 12 28*+ 0.10
CA3
Dentate gyrus
5.27 + 0.12 6.81 tO.lS** 1.30 + 0.04 364* 1s 479 + 16** 1.33 & 0.04
7.20+0.15 8.54fO.t7** 1.19+0.02 422+ 11 497 + 18** 1.18&0.03
Data are mean + S.E.M. values (n = 17 for ChAT and n = 11 for AChE). **P
NeuroscienceVol. 45, No. 2, pp. 261-272, I991 Printedin Great Britain
REGIONAL HETEROGENEITY IN THE DISTRIBUTION NEUROTRANSMITTER MARKERS IN THE RAT HIPPOCAMPUS H.
H~RTNAGL,*~
M. L. BEXGER,* G. SPERK$
OF
and CH. PIPL*
*Institute of Biochemical Pharmacology, University of Vienna, Borschkegasse 8a, A-1090, Vienna, Austria SDepartment of Pharmacology, University of Innsbruck, Innsbruck, Austria Abstract--A detailed neurochemical analysis of the distribution of markers for the most relevant neurotransmitter systems within the rat hippocampal formation has been performed. The hippocampi, obtained from unfrozen brains of male Sprague+Dawley rats were subdissected into tissue parts containing mainly CAl, CA3 or the dentate gyms, respectively. Each part was further divided into ventral and dorsal halves. In these six hippocampal subregions the concentrations of noradrenaline, dopamine, serotonin, 3-methoxy-4-hydroxyphenylglycol, 5-hydroxyindoleacetic acid and the putative neurotransmitter amino acids glutamate, aspartate, GABA, glycine and taurine, and the levels of somatostatin and neuropeptide Y and the activities of choline acetyltransferase, acetylcholinesterase and glutamate decarboxylase were measured. A marked heterogeneity in the subregional distribution of markers for various neurotransmitter systems within the hippocampal formation was observed. Each neuronal marker was characterized by an individual pattern of distribution. Most of the markers showed a concentration-gradient, increasing from dorsal to ventral; only taurine was more abundant in the dorsal than in the ventral parts and no dorsoventral difference was seen for aspartate, glycine and neuropeptide Y. The highest molar ratios of total 3-methoxy4hydroxyphenylglycol to noradrenaline and 5-hydroxyindoleacetic acid to serotonin were found in the dorsal hippocampus. The levels of noradrenaline, GABA and glutamate decarboxylase
activity were highest in the dentate gyms and lowest in CAI. The concentrations of somatostatin were highest in CAI; those of serotonin were highest in CA3. Highest activities of choline acetyltransferase and acetylcholinesterase were found in the dentate gyrus; lowest activities were found in CA3. In CA3 the lowest values of glutamate, aspartate, taurine and somatostatin were also found. The heterogeneity in the distribution of individual neurochemical markers allows insights into possible functional differences of hippocampal subregions and provides a relevant basis for future neurochemical investigations in this brain-area. _
The rat hippocampal formation has been thoroughly studied with respect to its neuroanatomy, electrophysiology, neuropathology and neurochemistry. Its three-dimensional shape is relatively complex. It appears grossly as an elongated structure with its longitudinal axis bending in a C-shaped manner from the septal nuclei rostrodorsally to the incipient temporal lobe caudoventrally (e.g. Ref. 2). The hippocampal formation comprises four cortical regionsthe dentate gyrus, the hippocampus proper, the subiculum and the entorhinal cortex. This complexity implies a rather heterogeneous structural and functional organization. As a consequence of the elongated structure, the inputs of various neurotransmitter systems reach their destination via several main pathways; thus, subregional differences in the tTo whom correspondence should be addressed. Abbreoiarions: AChE, acetylcholinesterase; CbAT, choline acetyltransferase; DA, dopamine; EDTA, ethylenediaminetetra acetate; GAD, glutamate decarboxylase; 5HIAA, 5-hydroxyindoleacetic acid; 5-HT, serotonin; HPLC, high-performance liquid chromatography; MHPG, 3-methoxy4hydroxyphenylglycol; iEo-MHPG, 3-hydroxy4methoxyphenylglycol; NA, noradrenaline; NMDA, N-methyl-o-aspartate. 261
concentration of neuronal markers and in neuronal activity may be expected. Whereas most neuroanatomical work has focused on the cellular localization of neurotransmitter systems in the transverse axis so far, much less is known about the distribution of neurotransmitter markers along the dorsoventral (septotemporal) axis of the hippocampus. The importance of this longitudinal axis for the architecture of intrinsic hippocampal connections has been emphasised in a review by Amaral and Wittet.2 and arises from recent neuroanatomical work. Recently, gradients for various neuronal markers with concentrations increasing from dorsal to ventral have been described for cholinergic neurons,12~2L~62 for noradrenaline (NA),33,62 serotonin (5-HT)32*62and somatostatin.” The overall density of the hippocampal noradrenergic innervation was reported to be 20% higher ventrally than dorsally and marked subregional differences in the density of NA varicosities were obvious.45 Choline acetyltransferase (ChAT)positive cells of the medial septal nucleus project more heavily to the ventral than to the dorsal levels of the hippocampus and dentate gyrus,’ consistent with the lower concentrations of acetylcholine and ChAT activity’9*2’*62and lower density of
262
1-i. H~RTNACL cv ~1
acetylcholinesterase (AChE) staining in the dorsal hippocampus4’ In the transverse axis of the hippocampus, however, regional differences in the tissue levels of neurotransmitter markers are less defined. It is of particular interest that in this hippocampal axis, regional variations of the extracellular space exist among the subfields of the hippocampus, the CA1 region exhibiting the lowest extracellular volume.3’ Moreover, the hippocampus is also not homogeneous in its response to various stimuli and noxes neither in the transverse nor in the lon~tudinal axis. NA is known to exert differing effects on Ammon’s horn and dentate gyrus neurons.34,U It is also well established that ischemia causes neuronal necrosis only in selectively vulnerable sectors of the hippocampus, such as the CA1 field.26,27Colchicine, on the other hand, exerts a preferential ne~otoxicity towards dentate granule cells while sparing hippocampal pyramidal cells. I8 In the longitudinal axis it appears that the age-related loss in ~ptohippocampal afferents is mainly confined to the ventral hippocampus; an age-related increase in muscarinic binding sites has been observed only in the ventral pole.57 Furthermore, electrolytic lesions of the septum are associated with an increase in nerve growth factor levels only in the ventral but not in the dorsal hippocampus. Preliminary data indicate that the ventral portion is also more affected by the eholinotoxin ethylcholine aziridinium.20 Such a heterogeneity in neurochemical parameters, in function and in vulnerability to exogenous noxes, prompted us to assess a more detailed topographical pattern in the concentration of markers for various neurotransmitter systems as well in the transverse as in the longitudinal axis. For this purpose the rat hippocampus was dissected along its longitudinal axis into the subfields CAl, CA3 and dentate gyrus and further subdivided into dorsal and ventral parts. In the present study we focused on the choliner~c, monoaminergic and GABAergic systems as well on the distribution of the other putative neurotransmitter amino acids gIutamate, aspartate, glycine and taurine, and of somatostatin and neuropeptide Y immunoreactivities. EXPERIMENTAL
PROCEDURES
Male SpragueDawley rats (Forschungsinstitut fur Versuchstierzucht, Himberg, Austria) with a body weight of 310-44Og were used for the study. The animals were housed in groups of three to four per cage on a 12-h light-dark cycle, and had free access to laboratory chow and tap water. fUuteri& f’4C]Acetyl-coenzyme A and L-l-[14C]glutamic acid (specific activities: 55 mCi/mmol) were purchased from Amersham Laboratories (U.K.), acetyicoenzyme A from Boehringer Mannheim. Bovine serum albumin, bacitracin, acetylthiocholine, physostigmine, dithiobis-2-dinitrobenzoic
acid, NA, dopamine (DA), 5-HT, S-hydroxylndoiraccil~, acid (S-HIAA), sulphatase (from Helix pomatia. type H-5). 3-methoxy-4-hydroxyphenylgiycoi (MHPG) and 3.4-dihydroxybenzylamine hydrobromide were obtained from Sigma Chemie GmbH (Deisenhofen, F.R.G.), amino acids from Merck AG (Darmstadt, F.R.G.) and o-phthalaldehyde from Serva Feinbiochemica {~eidel~rg, F.R.G.). Aminex A-9 (sodium form) was provided by Bio-Rad Laboratorics (Richmond, CA, U.S.A.). 3-Hydroxy-4-methoxyphenylgiycol (iso-MHPG) was kindly donated by D. 1. Heal Dissection of the hippocampus Rats were killed by decapitation. The brains were rapidly removed and ~mm~iately chilled in iced-water kept at -0.8”C with NaCI. After 10 min of chilling, the hippocampi were dissected from the unfrozen brains and gently extended on a cold-plate (f 1°C) the inner surface facing upwards (Fig. I). The extended hippocampi were dissected into tissue parts containing mainly CAI, CA3 and the dentate gyrus, respectively. The extension of the hippocampus in its dorsoventral axis greatly facilitated the separation of the three subfields ~roughout the hip~rnp~ from rostrodorsal to caudoventral. The dissection procedure is described in detail in the legend to Fig. I. The anatomical markings of the small longitudinal fissures, which are additionally marked by blood vessels, allow a reproducible separation between the three subregions. Using this dissection procedure, a small part of CA3 and CA4 is contained within the dentate gyrus part. The CA3 part contains partly CA2. All three regions were additionallv cut into ventral and dorsal parts and the corresponding samples of the left and right hippocampus were combined. After dissection, the tissue samples were weighed and immediately frozen on dry ice and stored at -80°C until biochemical analyses. The average weights (mg rt S.E.M.) of the six subregions per hippocampus (n = 17) were: CA1 ventral: 13.0 + OS: CA I dorsal: 11.8+ 0.4; CA3 ventral: 8.6 kO.2: CA3 dorsal: 7.3 k 0.2; dentate gyrus ventral: 7.1 & 0.2; dentate gyrus dorsal: 7.8 + 0.3. B~o~herni~a[analwes Tissue samples were homogenized at 4°C by ultrasonication in 30-40 vols of N,-saturated deionized water. Immediately after sonication an aliquot of the homogenate (2~3~~1) was added to an equal volume of Cl.2M perchloric acid containing 0.8 mM NaHSO, and centrifuged at 25,000 g at 4°C for 15 min. The supernatant was used for the measurement of NA. DA. S-HT. S-HIAA and the putative neurotransmitte~ amino acids. The remaining aqueous homogenate was used for the measurement of activities of glutamate decarboxylase (GAD), ChAT and AChE. In a group of animals (n = 12). aliquots of the homogenates equivalent to 8mg of tissue were added to acetic acid to give a final volume of 1ml (final concentration of acetic acid: 2.5 M), resonicated and centrifuged at 25,OOBg for 15 min. The resulting supernatant was usd for quantification of somatostatin and neuropeptide Y. Aliquots for determination of each peptide were transferred into separate Eppendorf vials and stored at -80’ C. Before anaIysis samples were Iyop~liz~ in a speed vat concentrator (Savant, Farmingdale, NY, U.S.A.). For measurement of MHPG, tissue samples were sonicated in 12 vols of 0.12 M sodium acetate-citrate, pH 5.0 ~ntaining 3,4-dihydroxy~nzylamine hydrobromide and iso-MHPG as internal standards. Enzyme activities Choline ~cet~ftran~ferase. ChAT activity was measured according to the method of Fonnum,‘$ with minor modifications using substrate concentrations of 8 mM choline chloride and tritiated 0.2 mM acetykoenzyme A.” glutamate ~eearbo~~~a.~e.GAD was measured in the supernatant after centrifugation of the aqueous homogenate
Regional heterogeneity of neuronal markers in hippocampus
d hlr#xKaKJL fissure
. longttuditKll axis by 180’ /
263
dissect CA3 bmrt
r tssure \
=-I”L& Y c-a-1, .f lssure
dissect out\
Fig. 1. Dissection of the hippocampal subregions. The position of the hippocampal formation in the rat brain after removal of the overlying cortex is indicated in the schematic drawing adapted according to Amaral and Witter.2 The whole hippocampus was dissected out @ and turned around its longitudinal axis by 180” and extended 0. The inner surface of the ~p~rnpus, facing the thalamus, is structured by two fissures: a ‘minor” fissure close to the timbria and the hippocampal fissure. Firstly, after removal of the fimbria, the CA3 part was dissected by cutting the extended hippocampus along the minor fissure 0. The remaining CAldentate gyrus (DG) part was turned by 80” clockwise. The separation between CA1 and DG @ was achieved by carefully opening the hippocampal fissure. The inserted light microscopic photo~ap~ (caption, x 10) were obtained from transverse slices cut from the ventral part of the extended hippocampus. For this purpose the hippocampi were dissected out of the brain after perfusion of the deeply anesthetized (150 mg/kg, i.p., thiopental) rat with 4% paraformaldehyde solution. The hippocampi were extended and separated into ventral and dorsal parts. The tissue parts were postlixed in the same hxative for 90 min, then immersed in 20% sucrose (24 h, 4°C) and subsequently frozen in isopentane (-70°C). Slices of 20 pm thickness were cut on a mi~otome and stained in Creayl Violet (0.5%). at 1S,OOO g for 10 min by trapping [“C]COz formed from as described by Roberts and Simonson” at substrate concentrations of 15 mM glutamic acid and 0.5 mM pyridoxalphosphate. Acetylcholinesterase. For calorimetric measurement the homogenates were treated with Triton X-108 (final concentration 0.4% + 10 mM EDTA) and centrifuged at 10,000 g for 10 min. The enxymatic reaction was performed according to Ellman ef ai.
L-1-[14CJgIutarnic acid
Determination of manoamines, 3-methoxy-bhydroxyphenylg&o1 and S-hy~oxy~o&acetic acid NA and DA were measured by high-performance liquid chromatoaranhv iHPLC1 with electrochemical detection after extr&ibn~to‘alumina according to Felice et aLI with minor modifications.” 5-HT and IHIAA were determined by HPLC with electrochemical detection as described previouslv.53 Total MHPG was determined after enxymatic hydrolysis by sulphatase and after prepurification on small Senhadex G 10 columns bv HPLC with coulometric detection, procedures modified &cordii to previously published methods.g6’*6J Homogenates were centrifuged for 15 min at 4°C at 18,OOOg.Suiphatase (14mg/ml) in 0.12M sodium
acetate-citrate @H 5.0) was added to the supernatants to give a final concentration of 1.5 mg/ml and samples were incubated for IS h at 37°C. Perchloric acid was added to a final concentration of 0.3 M, denaturated protein was centrifuged (15 min, 4”C, 18,OOOg)and supematants (volume made up to 0.6 ml with 0.01 M formic acid and foe4 M EDTA) were put on Sephadex G 10 columns (5 x 70 mm) prepared in long Pasteur pipettes p~~~~brat~ and washed as described previ~usly.~~ NA and MHPG were eluted with mobile-phase buffer of HPLC. This buffer contained 0.1 M sodium acetate, 0.05 M citric acid (PH 4.85 adjusted by sodium hydroxide), 50mg of EDTA and 13Omg of ~~~~phonic acid sodium salt/l and 5% methanol. The liquid chromatographic system consisted of a Model 5700 Solvent Delivery Module (1.2ml/min), a 30 x 4.6mm Spheri-5 RP-18 guard column (Brownlee Labs), a 250 x 4.6mm Hype&l ODS Sprn analytical column (FZ Seibersdort) and a Model 5100 A Coulochem ESA electrochemicaI detector with a Model 5021 conditioning cell and a Model 5011 analytical cell. The potential of the conditioning cell and the first detector was set at +0.03 V. The potential of the second detector, at which NA and MHPG were measured, was set at +0.46 V. The NA levels measured in the same chromatographic run as MHPG
264
H. HGRTWAGL rf al.
were used for the calculation of the molar ratio MHPG to NA. Determination of putative neurotransmirler amino acids The levels of glutamate, aspartate, GABA, glycine and tamine were measured after HPLC separation and post-column derivatization with the fluorogenic reagents o-phthalaldehyde and mercaptoethanol, according to the method described by S&mid et al.5’ with minor modifications. The column (length 250 x 4mm i.d.) was packed with Aminex A-9 (sodium form) and maintained at 76°C. The two-step buffer-latent consisted of 0.02 M sodium formateH,PO, (pH 3.3, IO min) and 02 M sodium acetate-H,PO.,, pH 5.6 (7min). The buffers and the o-phthalaldehyde reagent (post-column) were pumped at a rate of 1.0 ml/min. The amino acid derivatives were quantified in an Aminco fluoromonitor (360/450 nm).
Cholinergic systems
The distribution of markers for cholinergic neurons was uneven in the various subregions of the hippocampal formation and is summarized in Table 1. The ventral parts of CAI, CA3 and dentate gyrus exhibited higher activities of ChAT as well as of AChE, compared to the dorsal portion (P < 0.01). Among the three subregions, highest activities of ChAT were measured in dentate gyrus (P < 0.01 vs CA1 and CA3), lowest in CA3 fP < 0.01 vs CA1 and dentate gyrus). For both enzymes the ventrodorsal difference was least prominent in the dentate gyrus. This subregional difference was less pronounced for AChE than for ChAT.
Radioimmunoassay of somatostatin and neuropeptiak Y The ~yophiliz~ samples were r~onstitut~ in 500~1 of ~eur~chern~~~l markers for rn5~5~~nerg~~ inner assay buffer, consisting of 0.15 M sodium phosphate bu&Ter, vaticms of the hip~acumpaI formation pH 7.4, 0.15 M sodium chloride, 0.1% bovine serum albumin, 0.1% gelatin, 0.02% sodium azide, 0.002% phenyl The regional distribution of NA, DA, 5-HT and red and 0.02% bacitracin. Standard curves were determined 5-HIAA is shown in Tables 2 and 3. by including the respective synthetic peptide in amounts Cutecholamines. Levels of NA exhibited marked between 5 and 5OOfmol. Somatostatin and neuropeptide subregional differences. The highest concentration Y immunoreactivit~es were determined in duplicate at was measured in dentate gyrus (P < 0.01 vs CA 1 and two different tissue ~ncentra~ons as described in detail elsewhere.3s*” In various brain areas, somatostatin imCA3) and the lowest one in CA1 (P < 0.01 vs CA3 munoreactivity comprises somatostatin-14 (about SO%), and dentate gyrus). A considerable increase in NA somatostatin-28 (about 10%) and pro-somatostatin (about concentrations from dorsal to ventral was only ob10%) as revealed by HPLC analysis previously.56 Neuroserved in CA 1 and CA3 areas, whereas this difference peptide Y immunoreactivity co-migrates in a single peak with synthetic neuropeptide Y in HPLC.” was not significant in dentate gyrus. In the dorsal Data ana@& All data are presented as means + S.E.M. For statistical analyses the two-factor analysis of variance (ANOVA; factors: subregion and dorsoventral location) was applied. The two-factor ANOVA indicated the significance of subregional and dorsoventral differences aa well as of the interaction between subregions and dorsoventral location. In the case of significant sub~gion~ diilerences Nap-Keuls test was applied as aposteriori test for more detailed analysis of eventual differences among the subregions, using the mean value of the corresponding dorsal and ventral parts of each subregion. Accordingly, the probability values for subregional differences are related to the subregion in its whole extension. Significant differences in the dorsoventral location were followed by paired Student’s t-test for each indi~dual subregion, wrrected with a-adjustment accord-
ing to Bonferroni (see Ref. 38). RESULTS
For the individual neuronal systems, the following distribution of their representative markers was observed.
hippocampus the NA content was about two-fold higher in dentate gyrus than in CAI. For the main metabolite of NA, MHPG, a much smaller but still significant difference between dorsal and ventral parts of CA1 and CA3 was observed. The highest MWPG levels were measured in dentate gyrus (P ~0.01 vs CA1 and CA3). In CA1 and CA3 but not in the dentate gyrus, the moIar ratio of MHPG to NA was significantly higher in the dorsal parts than in the ventral parts. The highest MHPG to NA ratio was present in CA1 (P < 0.01 vs CA3 and dentate gyrus). In general, the MHPG to NA ratio followed a dist~bution which was opposite to that of the NA levels, i.e. the higher the MHPG to NA ratio, the lower the NA levels. The levels of DA were remarkably low in the entire hippocampal formation. An increase from dorsal to ventral was seen in all three subfields. In contrast to NA, lowest levels of DA were found in the dentate gyrus (P < 0.05vsCA3). The NA to DA ratio was, therefore, considerably higher
Table I. Regional differences in the distribution of markers for the cholinergic systems in the hippoeampal formation of the rat CA1 ChAT (nmol/h per mg tissue) AChE (nmol/h per mg tissue)
Dorsal Ventral Ventrodorsal ratio Dorsal Ventral Ventrodorsal ratio
5.86 f 7.80 f I .43 + 365 f 502 + 1.40 *
0.13 0.14** 0.03 12 28*+ 0.10
CA3
Dentate gyrus
5.27 + 0.12 6.81 tO.lS** 1.30 + 0.04 364* 1s 479 + 16** 1.33 & 0.04
7.20+0.15 8.54fO.t7** 1.19+0.02 422+ 11 497 + 18** 1.18&0.03
Data are mean + S.E.M. values (n = 17 for ChAT and n = 11 for AChE). **P
