Neuroseienee Letters, 90 (1988) |07-112 107 Elsevier Scientific Publishers Ireland Ltd.

NSL 05445

Acetylcholinesterase in the human frontal associative cortex during the period of cognitive

development: early laminar shifts and late innervation of pyramidal neurons

Ivica Kos tov i6 l, Jos ip Skavi62 and D a v o r Strinovib 2

I Section o1' Neuroanatomy, Department o['A natomy and 2Department of Forensic Medicine, School o[ Medicine, University qf Zagreb, Zagreb (Yugoslavia)

(Received 21 March 1988; Revised version received 5 April 1988; Accepted 5 April 1988)

Key words: Acetylcholinesterase; Frontal cortex: Development: Pyramidal neuron: Associative layer; Cholinergic system; Man

Laminar preferences in fibrillar acetylcholinesterase (ACHE) staining change dramatically in the human frontal cortex during the first postnatal year and perikaryal reactivity is found only in non-pyramidal neu- rons. The AChE reactivity of layer III pyramidal cell bodies and surrounding fibrillar network begins to develop after the first postnatal year, increases gradually and reaches its peak intensity in young adults, displaying a cluster-like arrangement. These data suggest that AChE-rich elements participate in the inner- vation of cortical associative neurons and layers during the cognitive development in man.

The pattern of the neocortical distribution of AChE-reactive fibers and cells differs significantly in various adult mammals [3, 9, 14, 16, 21, 22, 23]. The AChE staining of cortical fibers probably reflects differences in the cholinergic innervation [10, 22] originating from the basal forebrain nuclei [3, 12], but the relationship between AChE-reactive cells and the cholinergic system is less clear. AChE-reactive, choliner- gic (ChAT-positive) non-pyramidal neurons were found in rodent cortex [7]; feline cortex contains both non-pyramidal and pyramidal AChE-reactive [2, 14] non-choli- nergic perikarya [5, 15]; few such AChE-reactive, non-cholinergic cells were found in monkey cortex [4, 9, 22]. Only in human frontal cortex are there numerous ACHE- reactive layer III pyramidal cells [16, 23]. Considering the associative-commissural nature of layer III pyramidal neurons [8], this unique finding seems to be important in the light of the cholinergic hypothesis of cognitive functions [1]. Therefore, we began to study the postnatal AChE-histochemical maturation of the human frontal cortex, expecting to find a temporal coincidence between the appearance and subse-

Correspondence." I. Kostovi6, Section of Neuroanatomy, Department of Anatomy, School of Medicine, University of Zagreb, 41001 Zagreb, Salata I 1, Yugoslavia.

0304-3940/88/$ 03.50 O 1988 Elsevier Scientific Publishers Ireland Ltd.

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quent shifts of AChE reactivity of layer Ill pyramidal neurons and the developmel~t of human cognitive functions [19]. Transient fetal, non-cholinergic AChE-reactivity disappears from the human frontal cortex during early postnatal life [I 7, 18] and it is likely that AChE staining will reflect postnatal developmental shifts of cholinergic and cholinoceptive elements (although AChE is not a selective marker for cholinergic neuronal elements). We have studied the entire perinatal and postnatal period oJ ~ frontal cortex development (from 22 weeks of gestation to 26 years), analyzing corti- cal tissue specimens of 9 premature infants~ 4 newborns, 22 infants, 11 children, 4 adolescents and 6 young adults. Autopsy material was taken from patients showing no signs of neurological disorders prior to death. Blocks of prefrontal cortex (area 9), removed 4 12 hours after death (postmortem intervals longer than 8 h yield no consistent results) were immersed in 0.15 M phosphate buffer (pH 7.4), containing 4% paraformaldehyde and 1.25% glutaraldehyde, for 18-48h and serially cut on a freezing microtome. We applied the AChE histochemical method of Koelle (Lewi's modification) as described by Krnjevi6 and Silver [14]. Specificity was tested by the inhibition of pseudocholinesterase with tetraisopropylpyrophosphoramide and the inhibition of specific cholinesterase by 1,5-bis(4-allyl-dimethylammonium-phenyl)- pentan-3-on dibromide (Sigma). Adjacent Nissl-stained sections were used to delin- eate cortical areas and layers.

Three structural aspects of AChE-reactive staining were observed: AChE-reactive granula in neuronal cell bodies, AChE-reactive fibers and diffuse AChE-reactive deposits in the neuropil. The proportion and laminar distribution of these types of AChE staining change permanently during perinatal development. The overall corti- cal AChE staining is maximal in late gestation (28-34 weeks) when its distribution shows strict laminar preferences: the most heavily stained deposits within the cortical plate in prospective layers IV and III (Fig. IA), narrow band in prospective layer I, and moderate staining in prospective layer VI and in so-called subplate zone (i.e. the deepest part of the cortical anlage). Individual, axon-like AChE-reactive fibers can be recognized only above or below the heavily stained zone within the cortical plate, where the strong neuropil deposits impede the identification of structural corre- lates of AChE reactivity. AChE-reactive exclusively non-pyramidal cell bodies have a bilaminar distribution: superficial Cajal-Retzius cells in the prospective layer I and deep AChE-reactive cell bodies in the subplate zone. In the first postnatal week, AChE staining intensity of the neuropil decreases remarkably, especially in layers IV and I (Fig. 1B). On the other hand, in layers VI and V the first non-pyramidal ACHE- reactive cell bodies appear and the number of well-defined axon-like AChE-reactive fibers gradually increases. During the first 3 postnatal months there is a gradual, deep-to-superficial increase in the density of AChE-reactive fibers which are by the end of the third postnatal month also present in a substantial number in layers III and II. AChE-reactive, non-pyramidal cell bodies in layers III and ti were also seen in the third postnatal month (Fig. 2A). During this whole period, AChE-reactivity disappears from the neuropil of the layer IV. By 9.5 months of age, the density of AChE-reactive fibers in layer VI appears to exceed the adult density. The first ACHE- reactive layer III pyramidal cells, concentrated in subtayer l llc, have been visualized

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Fig. 1. AChE-stained frozen sections of the human frontal granular cortex at different ages. Roman nu- merals indicate cortical layers. Bar (A C) = 1 mm. Strong AChE reactivity in the cortical plate of 28- week-old premature infant (A) displays marked laminar preferences; prospective layer IV is densely stained. SP, subplate zone. Layer IV staining decreases in the newborn (B). In 23-year-old young adult (C) layer l llc is darkly stained owing to the development of AChE reactivity of pyramidal neurons.

in a 4-year-old child; they have a stronger AChE reactivity in the frontal cortex of 11- and 14-year-old children (well-preserved specimens from the period between 4 and 11 years were not obtained) and their cell bodies are arranged in clusters showing a certain degree of spatial periodicity. Layer III pyramidal cell bodies have the stron- gest AChE reactivity in the young adult (23-26 years) cortex (Fig. 2B, C and D), where layer III is clearly visible even at low-power magnification (Fig. I C), due to the presence of both AChE-reactive cell bodies and related AChE-reactive fibers. Two other remarkable features of young adult cortex are: low AChE reactivity of the granular layer IV (Fig. I C) and well-pronounced clustering of layer III pyramidal AChE-reactive cell bodies. A high-power magnification (Fig. 2C, D) clearly shows the nature of the cellular granular and pericellular fibrillar staining in layer III: fine, densely arranged AChE-reactive granula cover pyramidal cell bodies and proximal parts of apical dendrites (Fig. 2D, arrow); this granular AChE reactivity appears more related to the cell surface and membrane than to the perinuclear cytoplasma. Some AChE-reactive fibers from the surrounding neuropil 'contact' pyramidal cell bodies or proximal parts of their dendrites (Fig. 2C).

Our results demonstrate an early laminar reorganization and prolonged postnatal maturation of the 'cholinesterasic' cortical system; AChE reactivity appears in layer Ill pyramidal neurons only after the first year. AChE reactivity appears much earlier in extrinsic fibers and intrinsic non-pyramidal neurons than in pyramidal cells. Thus, both systems may induce synthesis of AChE in pyramidal cells and, accordingly, both may give rise to the dense AChE-reactive network arround their bodies. GABAergic [6] or peptidergic [10] non-cholinergic local circuitry neurons may be an intrinsic source of the AChE-reactive network arround pyramidal dendrites. The cholinergic basal forebrain is the most likely extrinsic source of the AChE-reactive network in the mammalian cortex [3, 12, 22]. Thus, cortical AChE reactivity can be

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Fig. 2. AChE-stained frozen sections of the frontal granular cortex: a 3-month-old infant (A) and a 23- year-old man (B, C and D). Bar = 200 pm (A, B) and 25/lm (C, D). AChE-reactive non-pyramidal neu- rons (arrows) are present at 3 months (A). AChE-reactive layer III neurons arranged in clusters are the major feature of the young adult cortex (B). Note the granular appearance of AChE-reactive product in cell bodies and proximal dendrites (arrows) of layer lllc pyramidal neurons and in approaching ACHE- reactive fibres (double arrow) (C, D).

used as an ind ica tor o f the chol inocept ive na tu re o f commissu ra l - a s soc i a t i ve pyrami -

da l neurons o f layer III . The chol inocept ive na ture o f bo th p y r a m i d a l and non-py ra -

mida l cor t ica l neurons is well d o c u m e n t e d in some exper imenta l m a m m a l s [5]. W h a t -

ever neural e lements are the real source o f the cor t ical chol inesteras ic act ivi ty, the

findings o f the present s tudy remain very i m p o r t a n t because they lead to the fol low-

ing conclusions: the chemical m a t u r a t i o n o f layer I l i p y r a m i d a l neurons is a late

pos tna t a l event and their A C h E react iv i ty shows a spat ia l per iod ic i ty in the tangen-

tial plane; the late invo lvement o f A C h E react ive e lements in the innerva t ion o f effer-

ent co lumns o f commissu ra l - a s soc i a t i ve p y r a m i d a l neurons seems to be a unique

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feature of the human and possibly the primate brain. It is remarkable that these structural-histochemical changes coincide with the late stages of cognitive develop- ment in man [24]. In contrast, the early phase of cognitive development [8] coincides with the disappearance of transient AChE patterns [17, 18] and overproduction of AChE-reactive fibers and changes in muscarinic receptor densities [13] which occur during the first year of postnatal life. These early postnatal changes in AChE distri- bution share major features with the developmental pattern described for non- primate mammals [2, 11, 20, 25].

Supported by Yugoslav-U.S. Joint Board Grant No. 698 and SIZ za znanost SRH. It is a pleasure to thank Z. Cmuk and D. Budin~6ak for excellent technical assistance, and Drs. M.E. Molliver, M. Judag and L. Mrzljak for stimulating discus- sions.

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