DISTRIBUTION OF CHOLINERGIC NEUROTRANSMITTER ENZYMES IN THE HIPPOCAMPUS AND THE DENTATE GYRUS OF THE ADULT AND THE DEVELOPING MOUSE VIJAYA K. VIJAYAN Department of Human Anatomy. School of Medicine. Universitk of California. Davis. CA 95616, U.S.A. Abstract--~The distribution of the cholinergic neutrotransmitter enzyme. acetylcholinesterase, in the hippocampus and the dentate gyrus of the adult and the developing mouse was examined using a histochemical procedure. The pattern of acetylcholinesterase staming in the hippocampal region of the adult mouse closely resembled that reported for the rat by other investigators. Enzyme activity was present predominantly in the neuropil, where it was concentrated in the supra- and the infrapyramidal zones of the hippocampus as well as in the supragranular region and the hilus of the dentate gyrus. In contrast to the adult pattern, during the first week of postnatal development acetylcholinesterase activity appeared to be largely intracellular. Strong staining was observed in the cytoplasm of scattered neurons throughout the neuropil laminae, particularly in the hilus of the dentate gyrus. During the succeeding weeks, the characteristic neuropil reaction developed in a slow and progressive manner, reaching the adult pattern by the end of the third postnatal week. Between the third and the fifth weeks, there was a substantial increase in the staining intensity of the enzyme. As a result of the increased neuropil reaction, acetylcholinesterase-positive cells became less conspicuous after the second postnatal week. The progressive acquisition of staining for acetylcholinesterase in the neuropil of the hippocampus and the dentate gyrus of the mouse during the early postnatal period compared well with the proposed model of development of septohippocampal connections in the rat. The histochemical distribution of choline acetyltransferase in the hippocampus and the dentate gyrus of the adult mouse was also examined. The reaction was largely intracellular, in the cytoplasm of the pyramidal and the granule cells. Neuropil staining was confined to the mossy fibers and their terminals. This distribution profile is in conflict with the localization of this enzyme in the hippocampal region established by other investigators on the basis of microdissection and assay. The significance of the results of choline acetyltransferase histochemistry in relation to methodological problems is discussed. THIZ MAMMALIANhippocampal

formation has become a useful target for the investigations of normal development as well as of the effects of experimental manipulations such as deafferentation (MATTHEWS, LYNCH & COTMAN, 1974; NADLER, NADLXR. MATTHEWS, COTMAN & LYNCH, 1974; MELLGREN & SRERRO, 1973; SREBRO & MELLGREN, 1974). The unique architectural features of the hippocampus and the dentate gyrus have facilitated these intensive efforts. Moreover, the hippocampal formation is an ideal example of cholinergically innervated cortex where the distribution of the cholinergic afferents is in good correspondence with the localization of the cholinergic neurotransmitter enzymes, acetylcholinesterase (EC 3.1.1.7. AChE) and choline acetyltransferase (EC 2.3.1.6. ChAc) (FONNUM, 1970; MELLGREN & SREBRO. 1973:

Moxo,

LYNCH &

COTMAN, 1973:

STORM-MATHISEN, 1970). Since the hippocampus

the dentate

gyrus undergo

considerable

postnatal

and de-

___-__. ilhbreviutions: AChe, acetylcholinesterase;

ChAc, choline acetyltransferase: DFP. di-isopropylphosphorofluoridate: HEPES. .V-2-hgdroxyethylpiperazine-N-2-ethanesulphonic acid; isoOMPA. tetramonoisopropyl pyrophosphortetramide.

velopment

(AL-WAN & DAS, 1965; 1966; GRAIN, COT-

MAN, TAYLOR & LYNCH, 1973; DAS & KREUTZBERG, 1967), they are appropriate models for establishing the progressive sequence of events associated with cholinergic innervation in the central nervous system. The histochemical distribution of AChE in the hippocampus and the dentate gyrus of the adult and the developing rat has been described in detail by various investigators (STORM-MATHISEN & BLACKSTAD, 1964; MATTHEWS et ul., 1974; MELLGREN, 1973). The localization of AChE in the hippocampal

formation of the adult guinea-pig has revealed interesting species difference which may reflect species-specificity in the architectural organization of the dentate gyrus (GENSER-JENSEN,1972). The presence and the distribution of AChE activity in the hippocampus and the dentate gyrus of the Macaw mulatta (MANOCHA & SHANTHA, 1970) and the human (FRIEDE, 1966; MWLGREN. HARKMARK & SKEBRO, 1977) have also been reported. To date, very little attention has been directed to the histochemistry of cholinergic neurotransmitter enzymes in the mouse hippocampal formation with the exception of a few reports (GEREBTZOFF. 1959, WENDEK & KOZIK. 1968). A recent

121

investigation

of age-associated

alterations

in the hippocampal formation of the mouse demon0.8 x IO ’ \I cscrmc (physoatigmmc) sulfate in place 111 iso0MP.A or of 5 x IO ’M BW?84(‘5 I ( I .5-h (+;111~Idstrated a decline in C’hAc activity. unaccompanied by meth! I an~moniumphen~l)-prntan-i-one dlhromlde) iti a change in AC‘hE level. during scncscence (VlJ.4Yh\. addition to isoOMPA. The mcubatlon was carried out ‘II 1977). This finding initiated a series of s;ludies to 37 (‘ for 2 h Following thih. rhc sectlons were rlnscd m dctcrmine whether discrete modifications in the acdistilled hater. Alternate scctlonh wcrc counterstaincd \vlth tivities of AChE and ChAc in specific sublaminae of Mayer’s hematox]lin. .A11scctlons were dchqdrated. cle.lrcd the hippocampus and the dentate gyrus contributed and mounted in Permount. to this net cfrcct. As a preliminary part of this work. In order to dcterminc the ctfccts of fixation and ,ucrosc it was considered essential to establish the localizatreatment on the histochemical reaction. :I limited number tion of AChE and ChAc in the hippocampal region of hrains were frozen immediatclq after removal. sectioned of the mouse. The present report describes the results and sections wcrc stamod for AChE. Some of these \cctlons. prior- to staining. were briefly tixed hq immcrslon of AChE histochemistry in the dcvcloping and the in the same lixatibe which wab used for the perfusion adult animal. The data are compared with the distriThe histochemlcal pl-occdurc for C‘hAc was based on bution of the enzyme in the rat (STOKM-MATHISFIV the method of BCRI. & StLvrK (19731). Air-drtcd sections 8~ BLACKSTAD.1964; MF.LI.GKIN. 1973; MAITHF~WS(‘1 were transferred to a prcincubation medium contaming al.. 1974) and are correlated with the available inforI rnM eberinc sulfate or DFP (di-isopropyl phosphot-ofluorimation on the afferent connections of the hippodate Sigma C‘hemical Co.1 in 25 rnM bodlum cacodqlate campus and the dentate gyrus. In addition. the histobuffer. pH 6.0, containing 100 mht sucrose. Preincubation chemical localization of ChAc was also examined. was carried out for I h ;~t 4 IO but they continued to react lightly until day 15 when the AChE staining intensity increased considerably (Fig. 8). On day 20 (Fig. 9) they were the darkest-staining elements in the hippocampus and continued to remain likewise on day 35 (Fig. IO). With the exception of these bands. the remainder of the stratum oriens and radiatum seemed to acquire AChE activity relatively slowly and the adult pattern was reached by postnatal day 20 (Fig. 9). The stratum lacunosum-moleculare did not become conspicuousIF delimited from the stratum radiatum until day 10. At this time. the discontinuous band of AChE activity bordering these zones started to stain for AChE (Fig. 6). At the same time. AChE activity became evident in the stratum lacunosum-moleculare of the regio inferior and in the part of the stratum lacunosum-moleculare of the regio superior which adjoins the subiculum. By day 20, these three zones of AChE activity assumed their adult pattern of enzyme distribution (Fig. 9).

The postnatal development of AChE reaction in various parts of the dentate gyrus of the mouse is indicated in Table 2. On da) 1. the dentate gyrus presented an immature appearance with only its external leaf somewhat defined. A collection of undifferentiated granule cells could be detected in the place of the internal leaf. The diffuse cellular and neuropil reaction of AChE observed in the hippocampus at this time was seen to extend into the molecular layer of the external leaf of the dentate gyrus. This staining was clearly apparent on day 5 (Fig. 5. arrows) and subsided by day 7. On days 3 and 5. AChE activity could be detected in the cytoplasm of many neurons scattered in the hilus and in the molecular layer. These stained cells increased in number on day IO and were the most conspicuous features on postnatal days 10 and 15 (Figs 68). The majority of these AChE-positive cells were found in the hilus where they corresponded in si/e and shape to the polymorphic neurons located

there. On day 20, these neurons could still be identrfied. even though they were less obvious due to the greater neuropil reaction (Fig. 9). On day 35. the strong neuropil staining of the hilus made it difficult to locate these neurons (Fig. IO). The neuropil of the hilus also began to stain on postnatal day 3. However, it continued to stain light11 on day 5 (Fig. 5); only on day 10. the AChE reaction became moderate (Fig. 6). Between days 15 and 35, the hilar neuropil became progressively darker (Figs X&10). The supragranular band of AChE reaction in the molecular layer became first visible on day 10. On day 15, it increased in staining intensity (Fig. 8) and was clearly evident and dark on day 20 (Fig. 9). The molecular layer superficial to the supragranular band first demonstrated staining for AChE on day 7 and slowly increased in staining intensity to reach the adult pattern by day 20. The fimbria and the dorsal fornix exhibited identlcal patterns of AChE development. In both of these structures. light AChE reaction appeared on day 7. By day 15, they stained darker. more or Icss as in the adult. Even though the adult pattern of AChF distribution was reached by postnatal day 20. a substantial increase in the enzyme activity occurred between days 20 and 35 in all AChE-positive /ones (Figs 9 and 10).

ChAc activity which resulted from incubating tissue sections according to the procedure of BURT & SILVER (19736) appeared as light to dark brown coloration with a distinct granularity at high magnification. The reaction was resistant to I mM DFP. When eserinc was substituted for DFP, the staining intensity increased considerably. In the presence of 0.1 mM Cu’ + or in the absence of both substrates, moderate, agranular nuclear reaction alone remained (Fig. 1I ). Omission of choline from the incubation medium resulted in a decrease in the intensity of the reaction which retained its characteristic localization (Fig. 12). Inclusion of 4-(l-naphthylvinyl) pyridine in the preincubation medium alone did not cause any change in the staining pattern or intensity. Biochemical determinations of ChAc activity in the cerebellum. the hip-

Histochemistry

of cholinergic

pocampus and the caudate nucleus of control and 4-( 1-naphthylvinyl ) pyridine-treated animals demonstrated inhibition of enzyme activity in the order of 30, 49 and 60”/,, respectively. The most striking and common feature of ChAc staining in the mouse brain was the presence of the reaction product predominantly in neuronal cytoplasm and dendrites. Considerable nuclear staining was also present. most of which was resistant to Cu2-. This was true not only for the hippocampus but also for the cerebral cortex, caudate nucleus and the brainstem. Neuropil staining, in general. was low. The choroid plexus exhibited conspicuous staining reaction. In the hippocampus, the most intense histochemical reaction was present in the regio inferior (Fig. 13). The reaction was largely located in the cytoplasm of the pyramidal cells and scattered neurons in the neuropil (Figs 14 and 15). The various neuropil laminae stained weakly (Fig. 14) with the exception of the mossy fiber zone which exhibited considerable staining reaction (Fig. 15). In the dentate gyrus, the granule cells contained intracytoplasmic reaction product (Fig. 16). The molecular layer stained lightly only. In the hilus. both the neuropil and the scattered neurons demonstrated moderate histochemical staining (Fig. 16). DISCUSSION Distribution

~1’acetylcholinesterase

in the hippocampus

The distribution of AChE in the hippocampus and the dentate gyrus of the adult mouse compares well with the pattern described for the rat (MELLGREN, 1973; STORM-MATHISEN & BLACKSTAD. 1964). The enzyme activity is located predominantly in association with axons. in the neuropil, and only to a small extent intracellularly in scattered neurons in the various neuropil laminae including the hilus of the dentate gyrus. Maximum AChE activity is concentrated in the infra- and the suprapyramidal bands in the hippocampus and in the supragranular region of the dentate gyrus. Moderate enzyme reaction is present in the outer molecular layer of the dentate gyrus. as well as in interstices between the pyramidal cells particularly at the transition between the regio superior and the inferior. The stratum radiatum and oriens stain lighter and the stratum lacunosum-moleculare is the least reactive. with certain exceptions. In both the rat and the mouse there is a band of moderate AChE activity at the boundary between the stratum radiaturn and the lacunosum-moleculare of the regio superior. Another common feature is the homogeneous enzyme staining of the stratum lacunosummoleculare of the regio inferior and of the superior, near the subiculum as well as near the transition to the regio inferior. Features of AChE staining which may be peculiar to the mouse include an increasing staining intensity of the mossy fiber zone along the

enzymes tn the

mouqe

hIppocampus

1::

septotemporal axis and the absence of a clear commissural zone in the molecular layer of the dentarc gyrus. Origin

of‘hippocampul

acc~tylcholillesterasc

Most of the AChE activity in the hippocampus and the dentate gyrus of the rat has been attributed to cholinergic septal afferents originating in the medial septal nucleus and in the nucleus of the diagonal band of Broca. This conclusion is based on the fact that following lesions of the septal afferents or of the septal nuclei of origin. there is substantial loss of .4ChE and/or ChAc in the hippocampal region (Fohur ~1. 1970; Ltrwls & SHCW. 1967; MCGEER. W.4114. TI KA\O & TWG. 1969: MLLLGREN & SREBRO. 197.1:Mosho 1973: OIXRFELD-NOWAK. NAKliIt,wI( /, rt al.. BIALO~AS. DABROU’SKA,WIERAS~KO Br GaADo\vsK .j. 1974: SREBRO & MI.LLC;REK.. 1974: SRFBRO. OnceFELL>-NOWAK.KI.o~os, DABROWSKA& NAKKIt ~I(‘L. 1973; STORM-MATHISEN. 1972: S’rORM-MAl’H1Sl.h & GIJLDHt:RG. 1974). Following irreversible inhibition q.)f AChE the recovery of the enlyme activity in the septurn precedes that in the hippocampal region ICIIII)PENDALI:, COTMAN, KOZAR & L> NCH. 1971). These and other studies supportive of a septohippocampal cholinergic projection have been reviewed in &tail recentI\, (STORM-MATHISEN, 1977). The sites 01 tcrmination of the septal afferents have been rcportzd somewhat differently b> different investigators. While earlier research indicates that terminal fields are !‘Ystricted to the stratum radiatum and orit’ns 01’ the regio inferior and to the hilus of the dentate g~rils (RAISMAN,COWAN & POWELL. 1965). additional septal input to the supragranular zone of the molecular layer of the dentate gyrus has been a recent discoLcr> (MOSKO et cd..1973). The results of these dcgeneratlon studies. however. do not correlate either with !he extensive AChE reactivity present in the intact hippocampal region (M~LLGREN & SR~BRO. 1973: MOSKO rt al.. 1973) or with the near total loss of A(‘hF in this region following septal lesions (MELI.GaI N bi SRI.BRO, 1973). In view of these discrepancies. it is of interest that more recent techniques of retrograde transport of horseradish peroxidase and antcrogrude :r;msport of labelled ieucinc have been applied to m;lp out the septohippocampal pathways (Sr-GII s( LANDIS. 1974: MIZBACH & SIFGI:L. 1977). The rcsuits of these studies support a distribution of septal a&rents extensively in all fields of the hippocampal region. in good correspondence with the iocaliration of AChE (see STORM-MATHISEN,1977). The presence of considerable AChE activity rn the dorsal fornix of the mouse indicates that A masked by the surrounding strong neuropil reaction. This VIM IS strengthened by the obscrvatlon that in the atiulr ~a{. following septal lesions. A(‘hE-positive neurons can be demonstrated with cast (MI I.L(;KI x & %+t~sKo. 1973). The significance of thcsr neurons. which appear early during postnatal maturation. remains to hr: established. The early AC’hF-reactive neuron\ of rhe hippocampus and the denate gyru\ correspond in their size. shape and locatlon to the cells with short axis cylinder originally described by LOHI.NII II Nti (1934). It is of interest that A