Distribution of cholinergic neurotransmitter enzymes in the hippocampus and the dentate gyrus of the adult and the developing mouse

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 <‘. ‘Thereafter. the tissue hccemploying the method of BLK~ & SILV~K (197%). The tions were stamed in the complctc incubation medium for -22 h at 37 c‘. This medium contained 3 mM HEPFS I \‘ results arc correlated with the distribution pattern of hydroxycthylpiperarine-,V-Z-cthanesulphonic acid) butftl-. AC‘hE as well as with the quantitative data available pH 6.0. 4 mM choline chloride. I mM Ph (NO,)‘. 0 .I mu on the activity of ChAc within discrete layers of the acetylcoenzyme A and I WMcscrine or DFP at final conrat hippocampal region (FONNIIM. 1970). centrations. rinsed

FXPERIMENTAL from the Simonsen’s were maintained

ammonium

PROCEDURES

Pregnant CS7B1:6 mice used laboratory

(Gilroq.

and

California)

rooms for a short

period

of time prior to parturition. The day of birth offspring’s

was taken

age. Young

as postnatal

animals

day

were killed

5. 7. 10, IS. 20 and 35. The mother

I of

on da)s

the

I. 3.

was used as the adult

control. Animals

wcrc anesthetized and killed

paraformaldehyde 7.5. containing formation

in 0.1 M sodium hemisphere

containing

followed

sllcrose for 24 36 h. The on chucks.

Frozen

soaking

tissue blocks

cryostat

sections.

20pm

(American

and horizontal

30”,,

sulfide and rc-washed. with

wcrc then dehydrated. For establishing

Majer’s

controls

5”,,

Alternate

in the absence

uf choline

The

sections

In Permount.

staining. the follow-

(a) Sections were mcuhated

or acetqlcocnzgmc

(b) CuCIZ was added to the preincubatlon media at a final concentration

were

.~qurous

Fcctions wcrc

hcmatoxylin.

for ChAc

were adopted:

preincubated

sections

with

cleared and mounted

Ing procedures

.4 or both:

and incuhatlon

of 0. I mM: (c) sections wcrc

in the presence ot

I v IO ’\I

4.(I-naphthkl-

for AChE

of the incubation

the

Final

incubation

containing

mixture,

with

concentrations medium

were.

sodium maleate buffer, pH 6.0. 5 mM sodium citrate. isoOMPA

potassium

ferricyanidc,

(tetramonoisopropyl

and 3.5m~

scetylthiocholine

hncsterase)

activit).

Control

the

of the 65 mM 3 mM

X x 10 ’ M

pyrophosphortetramrde) iodide.

to inhibit cholinesterasc

and incubated

medium

of precipitation;

animals wcrc injected intraperironeallj vinyl) pqridinc

dissolved

weight _ 30 mg:ml alone

After

NaCl).

in O.V”,, NaCI C‘ontrols

(26Omg,kg

received

O.Y’,,

body Na(‘1

I h. the animals were killed dnd C‘hAc activity

in the hippocampus.

caudate

radiochemically

Hlstochcmical

staining

nucleus and the cerchellum bj the method of FoN\*~ .M for C’hiic was also carrlcd

out using hrains from the control tiny])

ld) a few

with 4-( I-naphthll-

pyrldine-treated

and the 3-(I-naphthyl-

animals

isoOMPA

was

(EC 3.1.1.X., pseudocho-

sections

in media containing

The

RESlJLTS

Sections were

for 30 min ar 37’ C in a medium

5.0 rnM

was not pass-

to the mcuhation

tempcraturc

was a moditica-

& ROOTS (1964).

of the substrate.

CUSO,.

thglvinyl)pyridine

ible owing to the development

(1975).

at room

WANG.

of 4-(l-naph-

at

on

of ChAc (HAI HKICH &

1976; KK~I L & GoL.mn.KC;. 1975). Addition

was determined

C. Prior to staining.

air-dried

to achicbe partial inhibition

were cut in a

planes. Sections were mounted

procedure

all the components

included

treated

to the slide.

The histochemical

of

frozen

C) and mounted in

tion of that of KAKNOVXY

components

(WV)

~~ IX‘C.

the sections were quickly

exception

pH

were then

Co.)

subbed glass shdes and stored at --I5 to ensure adherence

buffer.

in the same in

thick.

Optical

I”,,

perfusion, the

immersed

by

with

the hippocampal

chilled with dry ice (-65

preincubatcd

(sodium pen-

perfusion

cacodylate

was dissected out and overnight.

in isopentane

coronal

with Nemhutal

by intracardiac

IO?,, (w/v) sucrose. Following

block of cerebral

Cryo-cut

water,

vinyl) pqridine which has hcen used hq some invcstlgators

tobarbital)

fixative

At the end of the Incubation. distilled

counter-stamed

in this study were obtained

in the animal

in

were

preincubated

tinal concentrations

of

Histochemical activity of AChE appeared in the form of a reddish-brown precipitate, well localized to tissue components. The staining was totally abolished by incubation with eserine or with the combination of isoOMPA and BW284C51. One of the notable features of the enzyme staining was its presence as droplets in the neuronal and neuroglial nuclei throughout the brain. This nuclear reaction also was sensitive to eserine and to BW284C51. Comparison of stained sections prepared from perfused-fixed. sucrose-treated

Histochemistry

of cholinergic

enzymes m the mouse hlppocampus

brains and from fresh-frozen brains revealed slight inhibition of AChE staining intensity only as a result of the treatments employed in the routine procedure. On the other hand. perfusion-fixation resulted in excellent preservation of tissue structure and good localization of the reaction product.

In general. the regio inferior demonstrated darker AChE staining when compared to the regio superior (Fig. I). In the distribution of AChE also. these two subdivisions exhibited some differences which are described below in reference to the specific sublaminae. The u11~eu.sexhibited many scattered, stained axons, intermixed with almost an equal proportion of unstained ones. In the srrat~rm oricm, AChE activity was most evident in the infrapyramidal zone where an intensely staining band could be found close to the pyramidal cell layer (Fig. I, arrow). This band of AChE was one of the strongest AChE-reactive zones in the entire hippocampal region. At the transition from the regio superior to the regio inferior, the band lost its compactness and became diffuse, almost filling up the stratum oriens. Here, the infrapyramidal staining could be traced directly to the AChE-stained fibers of the fimbria. In the regio inferior also, the band appeared somewhat broad and continued into the hilus of the dentate gyrus. In the regio superior, deeper to the infrapyramidal band, the remaining stratum oriens displayed diffuse and moderate neuropi1 reaction in the form of stained fibrils. presumably preterminal axons. The pwmidal cells did not contain AChE activity in their cytoplasm. There was, however, intense intercellular staining of fibrils which appeared to ramify between the cells. This was very pronounced in the regio inferior where the pyramidal neurons are well separated from each other. The staining pattern here did not correspond to the axonal thickenings and terminal expansions characteristic of the mossy fiber endings. The str~ltum radiutum presented low AChE activity in its neuropil except at the junction with the pyramidal ceil layer. Here. a moderately dark-stained suprapyramidal band of AChE activity was visible (Fig. 1). In the regio superior. this band remained narrow and in close proximity to the beginning of the pyramidal cell apical dendrite. At the transition from the regio superior to the inferior, this zone deviated from the vicinity of the pyramidal cell layer and became separated from it by the mossy fiber zone which displayed lower AChE activity. The AChE staining associated with the mossy fiber zone appeared to be least at the septal levels, but increased progressively toward the temporal levels. The stratum radiatum narrowed at the entrance to the hilus of the dentate gyrus and became continuous with the neuropil of the hilus at a sharp boundary. The srruturn /acunosur,l-moleculare stained lightly

1’3

and diffusely throughout most of its extent. Toward the junction of the hippocampal and the subicular cortices. for a short distance, the entire layer displayed homogeneous staining for AChE. This staining was more evident in sections through the temporal hippocampal formation than at septal levels. Simslarly. starting at the Junction of the rcgio superior and the inferior and continuing into the regio mfcrior. the most distal part of this layer exhibited a continuous oval zone of AChE reaction (Fig. I). This zone abutted against the free end of the outer lcat of the dentate gyrus and was very clearly apparent at all levels of the hippocampal formation. The limit between the stratum radiatum and the stratum lacunosum-moleculare was marked by a discontinuous and moderately staining band of AChE activity which coursed through the regio superior (Fig. 1). At the distal limit of the regio superior. this band became confluent with the aforementioned oval area of homogeneous staining in the stratum lacunosum-moleculare. Scattered throughout the stratum oriens. radiatum and lacunosum-moleculare, there were many large. fusiform or triangular neurons which contained some intracellular AChE activity. No stained processes were seen extending from these cell bodies into the surrounding neuropil. Adult denture

yyrus

Both the external and the internal leaves of the dentate gyrus demonstrated a similar pattern and intensity of AChE staining. In the moleculuu IUJU. the most remarkable feature was the presence of a zone of intense AChE activity close to the layer of granule cell bodies. which assumed the form of a supragranular band (Figs 2 and 3). Along with the infrapyramidal band. this zone was one of the areas of maximum AChE content in the entire hippocampal region. Outer to this band. the entire molecular layer appeared to stain diff‘usely throughout its width. There was no AChE-negatibc zone immediately superior to the supragranular band. The yrururlr cells. as such, were devoid of cytoplasmic enzyme activity (Fig. 3). However. as in the case of the pyramidal cells, intercellular staining was present. In contrast with the former. the staining was lighter and more patchy. appearing randomly at intervals throughout the granular layer. The intercellular reaction assumed the form of stained strands or fibrils percolating between the granule cells in continuity with both the supragranular band and with the hilar staining (see below). The hi/us of the dentate gyrus demonstrated moderate AChE activity uniformly throughout its neuropii (Fig. 2). The staining was not localized to the infragranular zone and was sharply demarcated, at the limit of the hilus, from the lighter staining. terminal portions of the stratum oriens and radiatum of the regio inferior. When the supra- and infrapyramidal bands of AChF activity in the regio inferior were fol-

FIGS

I 10.Coronal

dentate

sections through

of KAKNOVX~

by the method

gyrus: mf. mossy fiber lone; of the hippocampus;

FIG

the septal hippocampal

& ROOTS (1964).

I. Hippocampal

in the stratum

region

oriens.

ori. stratum

arrows

FIG;. 2. Dentate FIG FIG. 4. Hilus

3. External

of the dentate

cell layer:

oriens of the hippocampus;

arrow

denotes

the suprapyramidal

band

gyrus of the adult mouse.

x 425.

mouse.

of the

radiatum

the infrapyramidal in the stratum

band

radiatum.

x 6X.

leaf of the dentate gyrus of the adult mouse.

gyrus of the adult

hi. hihi\

rad. stratum

Regio sup. regio superior.

mouse. x 40. The

indicate

stained for acetylcholinesterase

gran. granule

Regio inf. regio inferior:

of the adult

Doubic

formation

fi, fimbria:

The

arrow

x 170 indicates

a polymorphic

neuron. FIG. 5. Hippocampal intracellular

region

and pcricullular

of the developing enqme

mouse.

postnatal

surfaces of the cerebral FIG

6. Hippocampal

FIG. 7. Dentate

gqrus of the developing

x 40. The

arrow

mouse. postnatal

mouse. postnatal

day IO.

day IO. x 130.

region of the developing

mouse. postnatal

day 15. * 40.

FIG. 9. Hippocampal

region of the developing

mouse. postnatal

da!

Coronal

radiatum

Hippocampal

of 0.1 rnM Cu”.

formatIon

of BURT & SILVER (1973~).

formation

Regio inf, regio inferior:

of the adult mouse.

of both choline

x 42. Control

and acetylcoenzqme

20. x 40.

day 35. x 42.

of the adult fi, timbria:

gyrus: mf, mossy fiber zone: or,, stratum

of the hippocampus:

Omission

mouse. postnatal

the septal hippocampal

bq the method

layer; hil. hilus of the dentate rad, stratum

region of the developing

vzctlons through

indicates

x 57.

FIG. 8. Htppocampal

for choline acetyltransferasc

FIG. Il.

5.

fissure and the medial and dorsal

hemisphere.

region of the developing

FIG. IO. Hippocampal FIGS. llLl6.

day

activity along the hippocampal

mouse stained

gran. granule

cell

oriens of the hippocampus; Regio sup. regio superior.

section incubated

in the presence

A from the incubation

medium

gave

similar resulls. FIG. 12. Hippocampal

formation FIG. I3

FIG.

of the adult mouse.

Hippocampal

14. Regio superior of the hlppocampus reactive cell body in the stratum

FIG. 16. External

leaf of the dentate

x 42. Section incubated of the adult mouse.

of the adult mouse.

oriens. Double

FIG. IS. Regio inferior of the hippocampus cell body and apical dendrite.

formatlon

arrows

of the adult mouse.

both containing

reaction

gyl-us of the adult

in the absence of choline. x 42.

x 130. The arrow points to an cn~ymcindicate stained tufts of processes. x 640. The arrow product.

mouse.

indicates a pyramIdal

Note nuclear

x 325. The

tufts of processes. ,4Iso note stained cells in the hilus.

arrow

reaction. indicates

stained

125

.

i

??

,

‘.

?

. i

127

I28

129

Histochemistry

TAl3Ll

I.

Dt VI.LOPMENT

OF

AChE

of cholinergic

ACTIVITY

IN

THE

HIPPOCAMPUS

I

Subdivisions

enzymes

3

in the mouse

Of

THE

5

hippocampus

MOlJSt

DURING

Days. postnatal 7 + + +

Fimbria Dorsal fornix (fornirc wperlor) Al\eus Infrapyramidal hand Interpyramldal stainmg Suprapyramidal band Band at the Junction of stratum radiatum and lacunosummoleculare Stratum lactlnosum-moleculare. regio superior. near subiculum Stratum lacunc~ciltn-moleculare. regio inferiol Scattered cells in stratum oriens. radiatum and lacunosummolecularc

+

+

+

++

++

II

THE

I0 + + + + +++ +-

POSTNATAL

I5 ++

+++ ++ +++ ++A ++A

Pf~KIOI)*

20

tt + -/-i tC *+ c+ t Jmi -1+ + c

+

++

+++

+

++

-t’-

+

++

++ t

+++

+++

-t +

* The developmental sequence of events depicted in this table is based mostly on the findings in the major, AChE-positivc iones in the rcgio inferior. The regio superior presented a similar pattern but lagged behind the regio inferior in acquirmg these features. The staining intensity of the various layers and zones is expressed as: light.+ : moderate. + + : dark, + t f : and intense. + + + + as judged by scanning AChE-stained frozen sections under the microscope F-or delalls see the text.

lowed into the hilus, the staining of these bands merged with the dense neuropil AChE activity there (Fig. 1). The neurons of the polymorphic layer displayed only mmimal intracellular AChE activity (Fig. 4, arrow). The firr~hrici was mostly devoid of AChE activity. However. along its outer edge there were many stained fibers directed toward the stratum oriens of the hippocampus and the molecular layer of the inner leaf of the dentate gyrus (Fig. I). The dorsal fornix (fornix superior) was seen to contain a large number of stained fibers (Fig. 1).

The time-course of development of AChE reaction in the major. enzyme-positive zones of the regio inferior is shown in Table 1. The regio superior also acquired enzyme in a similar manner but at a slower rate. No differences were noticed in the pattern of appearance of the enzyme at different septotemporal levels of the hippocampus. On postnatal day 1. the hippocampus of the mouse presented fairly well-organized morphological features. Both the regio superior and the inferior could be recognized. The pyramidal cell layer was formed but the cells m it appeared to be small and fusiform in outline. The sublaminae of the neuropil were not clearly delineated and most of the neuropil was filled cells. with undifferentiated The

greater

part

of

to stain

for AChE

the

hippocampal

formation

on day 1. The most obvious feature at this stage was the presence of a diffuse, patchy rone of AChE staining along and on either side of the hippocampal fissure. This staining, at high magnification. could be seen to consist of intracellular and pericellular AChE reaction associated with failed

groups of immature neurons lying scattered throughout the presumptive stratum lacunosum-molecuiare of the hippocampus. The staining continued along the molecular layer of the subicular cortices and could be traced into similar AChE-positive areas along the medial and dorsal surfaces of the cerebral hemisphere. This diffuse AChE reaction was clearly visible on postnatal day 5 (Fig. 5, arrows). Thereafter, it became less pronounced and was no longer apparent on day 10. With the exception of the diffuse AChE reaction mentioned above, the earliest appearance of AChE activity in the hippocampus was in the regio inferior where fine droplets of the stain precipitate could be visualized between the pyramidal cell bodies and dendrites on days 3 and 5 (Fig. 5). This interpyramidal staining was quite marked on day 10 and increased further in intensity between days IO and 35 (Figs 6, 8-10). The next in the order of appearance of AChE reaction was the intracellular activity in neurons scattered throughout the stratum oriens, radiatum and lacunosum-moleculare. Very few neurons of a similar nature were detected within the pyramidal cell layer also. These AChE-positive cells were first evident on day 5 but rapidly increased in number by day 10 (Fig. 5). On days 10 and 15, they appeared very conspicuous (Figs 6 and 8). in striking contrast to the predominantly non-reactive neuropil. Starting on day 20, these cells became less conspicuous (Fig. 9) and in the 35-day-old animal they were no longer a remarkable feature even though few of them could be still identified amidst the strong neuropil staining (Fig. 10). The supra- and the infrapyramidal bands of AChE activity appeared somewhat later than the interpyra-

*Both the external and internal leaves of the dentate gyrus exhibited similar pattern of AChE acquisition. The staining intenaltj of the various layers and Lanes is expressed as: light. + : moderate. + + dark. T i- + : and lntcnsc. t -t + +. JS judged by scanning AChE-stained frozen sections under the microscope. For details. SW the text

midal or the intracellular enzyme activity. The beginnings of these AChE-reactive zones could be visualiled on da> 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<‘hE-positive axons may reach the hippocampal region via this route also. Indeed. the findings of LFWIS & SHI TI (1967) suggested the possibility that septal arcrents may travel via both the dorsal fornix and the fimbria. They contended that the dorsal fornix carried a&rents mainly to the dorsal pole of the hippocampal formatlon while the fimbria carried septnl axons to

fhc ventral portions. Recent studies using anterograde transport of labeled leucine (MEIBACH& SIEGEL. 1977) have confirmed this observation and demonstrated a mediolatcral spatial organization in the dorsal fornix and in the outer edge of the fimbria of septal afferents destined for successive rostrocaudal levels of the hippocampal formation. The staining for AChE in the stratum lacunosummolecuiarc of the proximal regio superior and the adjacent molecular layer of the subiculum conjointly referred to as ‘zone 31’ (STORM-MATHISEN& BLACKSTALL 1964) has been shown to be resistant to septal lesions in the rat (MELLGREN & SKEBRO. 1973). On this basis, this enzyme activity has been attributed to non-septal cholinergic afferents. mostly travelling in the cingulum (STORM-MATHISEN. 1972). Even though earlier studies suggested that cingulum fibers may terminate in this zone (ALKESNE. BLACKSTAD, WALBERG & WHIX 1966; BLACKSTAD. 1967), recent investigations of the distribution of the cingulum support terminations in the presubiculum rather than in the subiculum or the hippocampus proper (DOMESICK. 1970; 1973). Moreover. lesions transecting all afferent pathways do not result in a loss of AChE activity in ‘lone 31’. which may owe its enzyme activity to the terminals of intrinsic cholinergic neurons (STORMMATHISEN. 1977). The mossy fiber area in the mouse and the rat (M~LLGREN, 1973; STORM-MATHISEN& BLACKSTAI). 1964) exhibits low AChE reactivity. However. in the mouse. at the sites of termination of these axons. AChE reaction did not assume the form of the coarse houton en pas.sugr, characteristic of the mossy fiber terminals. Since the granule cell mossy fiber system is most likely to be non-cholinergic (see ST-ORM-MATHISEN,1972; 1977). the low AChE reaction detected within the mossy fiber zone may be interpreted to represent AChE-positive septohippocampal axons travelling through the zone. toward the suprapyramidal band of enzyme activity. Such a course of septohippocampal fibers may also explain the intense interpyramidal straining particularly cvident at the transition between the regio superior and the inferior.

The progressive sequence of AChE acquisition in the various sublaminae of the hippocampus and the dentate gyrus of the mouse bears resemblance to similar events occurring in the rat (MATTHEWSet ul., 1974; M~FLLGRFN& SREBRO, 1973). The early appearance of intracellular enzyme activity in scattered neurons of the neuropil laminae of the hippocampus and in the hilar neurons of the dentate gyrus is very remarkable in the mouse. Although a few stained cells were detected here and there in the pyramidal cell layer, there was no indication of transient AChE activity in the cytoplasm of the pyramidal neurons in general, as reported by WENDER & KOZIK (1968). In the adult. only very little enzyme activity may be detected in the early-reactive neurons even though it is possible

that the cytoplasmic AChF. IS morel> 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<‘hE-positive interneurons in the rat neostriatum and pars rcticulata of the substantia nigra also acquire A<‘hI- reaction during the early postnatal period (BI ‘IUI~K & HOIIGE, 1976). These interneurons. like the neurons in the hippocampal region. arc not casill detected in the adult unless the surrounding strong ncuropil AChE activity is inhibited h> DFP (Brr(,mI< & HODG~. 1976). The development of AChE activity in the supraand infrapyramidal bands and in the supragranular zone occurs late, on postnatal day IO, in tht: mouse. This is tenable with the view that medial septal afferents enter the supragranular zone of the dentate gyrus beginning on day IO (Lov. LYNCH & COTMAN. 1977). The timing of the appearance of staining in these zones is comparable to the results of Mt LLGKPU (1973) for the rat. but MAIT~J~~S VI trl. ( 1974) were able to demonstrate the infrapyramidal and the supragranular bands much earlier. on day 6. Both MATTHEWSet ul. (1974) and the present study utilized comparable histochemical procedures for AChE localization. However. differences in the method of fixation. tissue sectioning and staining may have conthe discrepancy tributed to ohscrbcd. Thus. MA~THFWS et N/. (1974) employed a strong fixative for perfusion (4”. paraformaldehydr and OS”,, glutaraldehyde) followed by a brief period (2 hl of post-lixation. In the present study. a neaker fixative was used but the post-fixfor perfusion ( 1‘I,, paraformaldehyde) ation was long (24 h). MA-I-IN~WSer uI. (1974) utihred a prolonged incubation period (24 h) and greatel thickness of tissue sections both of which would facilitate the detection of low levels of enryme activltc. Such differences in methodology need to be considered in establishing absolutely the earliest possible time of AChE positivity in the septohippocampal axons, which may or may not coincide with the earliest time of entry of these afferents into the hippocampus. Histochrmistry

of choline ucqvltrunsf;trtrsc

Histochemical methods for demonstrating C’hAc. based on the splitting of acetyl coenzyme A and the subsequent precipitation of coenzyme A as a metal mercaptide, have been developed by B~JRT (1970) and by K.&A, MANN & HEBB (1970). However. these procedures are replete with problems and have not yet found extensive applications in the study of choliner-

Histochemistry

of cholinergic

enqmcs

gic systems. LEBRIN & WASER (1975) compared the many modifications of the histochemical method for ChAc and obtained the most impressive results with the method of BURT & SILVER (19736). The present study. employing the same method, resulted in positive histochemical reaction which was stable in the presence of 1.OmM DFP and was sensitive to 0.1 mM C‘u’ ‘. The reaction thus fulfilled the criteria for ChAc applied by BURN & SILVER (1973h). The persistence of the histochemical staining at lower rates. even in the absence of choline. has also been pointed out by BURN (1971) and by L~WRIN& WAS~R (1975). In the present study. the use of DFP in place of eserine markedly reduced the intensity of the histochemical coloration. in agreement with the conclusion by BURT & SILVF.R (lY73a) that DFP was most effective in eliminating non-specific hydrolysis of acetyl coenzyme A. Thus. the general characteristics of the ChAc histochemical reaction reported here agree with the results of other investigators. However. the histochemical distribution of the enzyme obtained in the present study is at variance with the accepted topographical localization of ChAc in the hippocampus and the dentate gyrus. Biochemical assay of microdissected samples has revealed high ChAc levels in the infrapyramidal zone in the stratum oriens. in the suprapyramidal area of the stratum radiatum. as well as in the supragranular portion of the molecular layer and in the hilus of the dentate gyrus (FONNIIM. 1970). The histochemicai staining demonstrated in the present study was visible in the hilus, both in the neuropil and in the polymorphic neurons located there. However. no infrapyramidai or supragranular accumulations of the reaction product could be detected in the stained sections. On the contrary, maximum ChAc staining was located intraneuronally, in the cytoplasm of the pyramidal and the granule cells, where minimal or no AChE activity is believed to exist. L~BHIN& WASER (1975) also commented on the high ChAc content of the pyramidal neurons. Biochemically, both the pyramidal and the granule cell layers demonstrate high ChAc activity; however, owing to the AChE-negative nature of these neurons, the reaction has been attributed to contamination from adjacent layers or to the presence of the enzyme in the neuropil (FONNUM. 1970). There is very little evidence for the cholinergic nature of the pyramidal and granule cells. On the contrary, a large body of evidence based on lesions of afferent pathways. supports a presynaptic location of ChAc in the hippocampal region (LEWIS, SHUTE & SILVER, 1967; STORMMATHIS~N, 1973; see review by STORM-MATHISEN. 1977). The demonstration, in the present study, of histochemical staining associated with the mossy fibers and their terminals also conflicts with the current concepts of nemotransmission in the hippocampus (STORM-MATHISEN.1977). The lack of correlation between the localization of ChAc obtained by biochemical methods and by the histochemical method may indicate non-specificity of

11: the mouse

hlppocumpus

1.15

the latter procedure, for which, to date. an effective histochemical inhibitor has not been unequivocally identified. Chloroacetylcholine perchlorate. which was found to achieve complete inhibition of the enzyme by KASA & MORRIS (1972) was reported to be ineffective by Bt RT & SILVER (19736) and by LERRIN & WAS~K f 1975). 4-( I -naphthylvinyl) pyridine has been shown 10 produce partial inhibition of mouse hrain ChAc activity in r~‘o (HAUHRI(.H & WAN;. 1Y76; KKUI & Gc)I_I)H~R(;. 1975). In the present study. following treatment with 4-( I-naphthylvinyl) pyridine i,r riro, 49”,, inhibition of enzyme activity in the hrppocampus was obtained biochemically but no inhibition could be demonstrated histochemically. It remains to be clarified whether this observation implies non-specificity of the histochemical procedure or may he attributed to the possible reversible nature of the inhibition (KRITLL.& GOIJX~~RG, 1975) allowing substantial recovery of the enzyme during incubation. Alternatively. the lack of histochemical enzyme staining in the infrapyramidal, suprapyramudal. and the supragranular zones of the neurnpil may have resulted from solubilitation of the enzyme from tissue sections during the staining procedure. ChAc exists in both soluble and hound forms (FONN~IM & MN.THE-S•RNSSES. 107.3). The solubility of the enzyme is retained even in formalin-fixed tissues (BURT & DETTHARN. 1972). In sections of the hippocampus, only 43”,, of the total ChAc activity is retained under histochcmical conditions (BI:RT. 1972). Since the soluble form is histochemically identified with the enzyme associated with axons of cholinergic neurons (BURT. 1972), a prcfcrential loss of significant amounts of the soluble enzyme from the septohippocampal axons may be considered to result in an absence of the staining reaction at the sites of these axons. However. this reasoning neither explains the apparently spurious reaction in the cytoplasm of the pyramidal and the granule cells nor is tenable with the demonstration of staining in the mossy fibers and their terminals. In view of these facts. it would he of rnterest to compare the results of the present investigation with those of a recent study which employed a somrwhat different histochemical approach and demonstrated parallel distribution of AChE and ChAc m the rat hippocampus (see SIORM-MATHISI-IV.1977). At present, details of the latter study are not available. The immunofluorescence method for demonstrating ChAc (McGtk~, MC‘GEER,SINC;H& CHASM.1973) has not yet been applied to the mammalian hippocampus.

The results of the present study indicate that the distribution and the development of AChE, activity in the hippocampal region of the mouse may be attributed to the course, termination and postnatal maturation of the septohippocampal afferents. This conclusion, which requires substantiation. is based on similarities demonstrated between the mouse and the rat in the histochemical pattern and development of

,A(‘ht<

staining.

present study

The

Con to the elucidation c;mcc of the carI! contained

AC‘hF.-rcactivc.

in the hippocampus

of the rodent.

also directs

The

atten-

specificity

of the natur-e and the signitiintrinsic

neurons

and the dentate

results

of C‘hAc

presented here serve to emphasize

of the histochemxai

for detailed

application

methodology.

gyrus

to

histochcmistrq

ir‘ histochcnnc;il

bc consldcred

microdisscction

the need for greater

procedure

cholincrgic

;I

wei

;I~

of the immtlnohlstochenil~c~~l mapping

;15 a supplernent~~l

:ind micrwsxt!

of C’h:\c

:k

approach

10

in ths c\alu;ttw~t

111

s~stcms.

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Neurochrm,

22,

VIJAYAU V. K. C1977) Cholinergic 12.7 II.

793- 803.

enzymes

in the cerebellum

WI-NIXR M. & KUZIK M. (1968) Zur Chemoarchitektonik MPusegehtrns. Acru Histochcm Cyrochrm. 31. 166-181. (Acceptd

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