Distribution of mRNA for the α4 subunit of the nicotinic acetylcholine receptor in the human fetal brain

Molecular Brain Research 58 Ž1998. 123–131

Research report

Distribution of mRNA for the a 4 subunit of the nicotinic acetylcholine receptor in the human fetal brain Cendra Agulhon a , Yves Charnay a , Philippe Vallet a , Daniel Bertrand b, Alain Malafosse a

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DiÕision de Neuropsychiatrie, Belle-Idee, de Psychiatrie, Hopitaux UniÕersitaires de GeneÕe ´ Departement ´ ˆ ` (HUG), Ch. du Petit-Bel-Air 2, CH-1225 Chene-Bourg, GeneÕa, Switzerland ˆ b Departement de Physiologie, Centre Medical UniÕersitaire, UniÕersite´ de GeneÕe, ´ ´ ` 1211 GeneÕa, Switzerland Accepted 21 April 1998

Abstract Neuronal nicotinic acetylcholine receptors ŽnAChRs. present in the central nervous system ŽCNS., are multimeric proteins constituted of two different subunits, a and b , with different subtype arrangements and different pharmacological and functional properties. By in situ hybridization, we studied the distribution of the mRNA for the a 4 subunit of nAChRs in brains of human 25-week old normal and fragile X fetuses. A strong hybridization signal was detected throughout the thalamus, cortex, pyramidal layer of the Ammon’s horn, and the granular layer of the dentate gyrus. Several other areas including the claustrum, caudate nucleus, putamen, globus pallidus, subthalamic nucleus, subiculum, entorhinal cortex, and Purkinje cell layer displayed a low to moderate radiosignal. With few exceptions, our data in the human brain agree those previously reported in the rat. Also, our data indicate that the a 4 subunit mRNA is produced early in the development, in the more differentiated cells, and in a site-specific manner. Additionally, the a 4 mRNA is produced in the brain of fragile X fetuses with the same pattern and same intensity than in the normal fetal brain suggesting that a 4 subunit mRNA production is not altered in the fragile X syndrome. High levels of a 4 subunit mRNA in human fetal brain support the hypothesis of a morphogenic role of nAChRs during the early CNS development. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Nicotinic acetylcholine receptor; a 4 Subunit; Human fetal brain; In situ hybridization; Anatomical localization; Fragile X syndrome

1. Introduction Transmission across neurons mainly depends upon the activation of neuroreceptor molecules which can be divided into two main classes: the ligand-gated channels and the metabotropic receptors. Ligand-gated channels have been shown to mediate fast transmission in the microsecond time scale whereas, given the second messenger processes triggered by their activation, metabotropic receptors are thought to be more important in the modulation of neuronal activity. In mammalian, ligand-gated channels can further be divided in two branches with those responsible for depolarization Žexcitation. of the neurons and those causing an hyperpolarization Žinhibition.. Neuronal nicotinic acetylcholine receptors ŽnAChRs. belong to the excitatory fast transmitting ligand-channel superfamily w7,18,30x. At present 11 genes coding for neuronal subunits have been identified in vertebrate genomes w19,21x. De) Corresponding author. [email protected]

Fax: q41-022-305-53-09; E-mail:

0169-328Xr98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 8 . 0 0 1 1 3 - 2

pending upon their structures and namely the presence of two adjacent cystein residues, neuronal subunits have been divided into a- and b-subclasses w30x. The functional neuronal nAChR results from the assembly of the a- and b-chains, with a putative stoichiometry of 2Ž a .:3Ž b . w3,8x. The a 4rb 2 receptor subtype is the major high affinity neuronal acetylcholine receptor for nicotine in the brain. In the central nervous system ŽCNS., the activation of pre- and postsynaptic nicotinic receptors stimulates the release of different neurotransmitters, such as acetylcholine, dopamine, or g-amino butyric acid. nAChRs are also involved in hormone secretion, motor function w14,29x, and brain functions such as memory and attention. They were also implicated in neurodegenerative disorders such as Alzheimer’s or Parkinson’s disease w15,20,33– 35,32,37,53,28x. Mutations in the gene encoding the a 4 subtype have been linked to a form of idiopathic partial epilepsy Žautosomal dominant nocturnal frontal lobe epilepsy. and polymorphisms of this gene have been associated with idiopathic generalized epilepsies Žchildhood and juvenile absence epilepsies, and juvenile myoclonic

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epilepsy. w22,49,50x. Anatomical, molecular, and genetic studies have suggested the implication of the a 7 neuronal nicotinic receptor in auditory gating deficit observed in schizophrenia w5,16,17,27,36x. Thus, further analysis of the specific distribution of nAChR subtypes in the human brain represent an important step to improve our understanding of pathophysiology of neurodegenerative disorders or brain alterations related to neurological disorders. Up to now, little is known about the distribution of the nAChR subunits in the human brain w1,2,9,41,42,47x. For instance, the distribution of a 4 subunit mRNA has been studied only in the adult cerebral neocortex w44,52x. As a first step, we investigated the regional and cellular distribution of the a 4 subunit mRNA by in situ hybridization in fetal human brains to further our knowledge of the a 4 subunit involvement in the cholinergic system.

2. Materials and methods 2.1. Tissue preparation Tissue sections were taken from the brain of a 25-week old male fetus aborted because of a cardiac defect. In addition, we analyzed the brains of two other 25-week old fragile X fetuses: one female carrier of a full mutation on one of the two X chromosomes and one male carrier of a full mutation. These two other fetuses were obtained from medical abortion. The brains were frozen 2 h post-mortem using powdered dry-ice and stored at y808C until used. Tissue sections Ž14 m m. were cut on a cryostat at y208C and mounted onto slides previously coated with 2% 3-

aminopropyl-triethoxysilane in acetone. Sections were fixed for 20 min in 2% paraformaldehyde in 0.1 M phosphate buffer ŽpH 7.4., rinsed for 5 min in 1 M PBS Žphosphate-buffered saline., rinsed briefly in water and dehydrated in a series of increasing ethanol concentration Ž50, 75 and 100%.. Sections were then air-dried and stored at y808C. This procedure was used to preserve the mRNAs in fetal tissues. The access to human fetal material is in accordance with the Ethical Committee of Necker-Enfants Malades Hospital in Paris. 2.2. Hybridization probes Three oligonucleotide probes were synthesized and purified by Genset, France. They were 3X end-labeled with a35 S-dATP ŽNEN. using terminal deoxyribonucleotidyl transferase ŽLife Technology. at a specific activity of approximately 7 = 10 8 c.p.m.rmg. All probes were purified on biospin columns ŽBioRad. prior to use and stored at y208C in 40 mM DTT Ždithiothreitol.. The sequences of a 4 subunit oligonucleotides at positions 1890–1950; 1887–1933; 1271–1315 Žaccession number L35901. were used as probes. No homology with other nAChR subunits was found after data-base scanning ŽBlast Network Service.. The antisense sequences were: 5X-CAGTCCTCCTTCACCGAGAAGTCTGTGTCT TCGGCCTTCAGGTGGTCTGCAATGTACTGG-3X ; 5X-GGCCATCTTATGCATGGACTCGATGAGCCGC CGGCAATTGTCCTT-3X ; 5X-GAAGTCTGTGTCTTCGGCCTTCAGGTG GTCTGCAATGTACTGGACGC-3X .

Fig. 1. Autoradiographs of in situ hybridization with antisense a 4 subunit oligoprobe ŽA. and with sense a 4 oligoprobe ŽB. in coronal sections of the normal human fetal brain through the occipital cortex. Sections were exposed for 25 days. Scale bars s 1000 m m.

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The specificities of the oligonucleotide probes were checked by hybridization experiments using sense probe or adding unlabeled antisense probe in excess to the hybridization buffer Ž100-fold.. In both cases, no signal above background was detected ŽFig. 1.. 2.3. In situ hybridization and quantification Hybridization was performed by incubating the sections with a buffer containing 50% formamide, 4 = SSC Žstan-

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dard saline citrate., 1 = Denhardt’s solution, 0.25 mgrml yeast tRNA, 0.25 mgrml sheared salmon sperm DNA, 0.25 mgrml RNA poly A, 10% dextran sulfate, 100 mmol DTT and a35 S-dATP-labeled probes Ž6 = 10 6 c.p.m.rml.. A total of 100 m l of hybridization solution was added to each section. The slides were then covered with a parafilm coverslip and incubated in a humidified chamber at 438C for 20 h. The slides were washed twice in 1 = SSC containing 10 mmol DTT at 558C for 15 min each, then

Fig. 2. Autoradiographs showing the a 4 mRNA localization in coronal sections of the normal human fetal brain. Sections were exposed for 25 days. ŽA. frontal cortex, ŽB. neotriatum and claustrum, ŽC. thalamus, neostriatum, globus pallidus, and hippocampus, ŽD. temporal cortex and ŽE. hippocampus. Abbreviations: CA: Ammon’s horn, CL: claustrum, CPU: Caudate–putamen, DG: dentate gyrus, ENT: entorhinal cortex, GP: globus pallidus, HIP: hippocampus, IN: insular cortex, S: subiculum, THA: thalamus. Scale bars in A s 500 m m; in B and C s 2500 m m; in D and E s 1000 m m.

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C. Agulhon et al.r Molecular Brain Research 58 (1998) 123–131

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Fig. 4. Autoradiographs showing the a 4 mRNA localization in coronal sections of the normal fetus ŽA and B. and fragile X fetus ŽC and D. brains. Sections were exposed for 7 days. ŽA and C. Frontal cortex and ŽB and D. hippocampus and thalamus. Abbreviations: HIP: hippocampus, THA: thalamus. Scale bars in A and C s 2000 m m; in B and D s 3000 m m.

twice in 0.5 = SSC containing 10 mmol DTT at 558C for 15 min each, and finally in 0.5 = SSC containing 10 mM DTT at room temperature for 15 min. The sections were then dehydrated in a graded series of ethanol concentrations and exposed with Amersham Betamax X-ray film. For higher microscopic resolution, the slides were then dipped in NTB2 photographic emulsion diluted with water Ž1:1., stored in the dark for 8 weeks at 48C, and subsequently developed in Kodak D-19, fixed, counterstained with toluidine blue, coverslipped with Eukitt, and analyzed by microscopy. Photomicrographs were taken on Kodak T-MAX ŽASA 100. films. Film optical densities in several representative brain areas of each brain studied were measured by a computerassisted analyzer ŽSAMBA, TITN Alcatel, France. w23x. Optical densities ŽO.D.. were in the range in which the radioactivity of the 3 H microscales shown a near-linear relation. The absolute quantification of the content of mRNA in each relevant area, was determined by subtracting non-specific labeling from total labeling. Arbitrarily, scores were assigned to the labeling of the anatomical structures as follows: q Žlow intensity: O.D.- 10., qq Žintermediate intensity: 10 - O.D.- 20., and qqq Žhigh intensity: 20 - O.D.- 30.. The cell density was estimated using reticule, in five sections of each brain areas. 3. Results The three antisense oligonucleotidic probes used gave the same labeling pattern in the fetal brain sections while

an excess of unlabeled antisense probes or corresponding sense probes gave no hybridization signal ŽFig. 1.. It was therefore assumed that the hybridization signal detected in autoradiographs most likely reflects the in situ expression of the a 4 subunit mRNA. As illustrated in Figs. 1–4 and Table 1, a 4 mRNA expression was widely distributed through the fetal brains with higher expression in the thalamus, cerebral cortex, pyramidal layer of Ammon’s horn, and the granular layer of the dentate gyrus. Weaker hybridization signal was detected in the claustrum, striatum, globus pallidus, subthalamic nucleus, entorhinal cortex, subiculum, and cerebellum. Moreover, a 4 mRNA is produced with the same pattern and with similar intensity in the two brains of fragile X fetuses as well as in the control fetal brain ŽFig. 4 and Table 1.. In the cerebral cortex, the intensity of the labeling varied in different cortical areas. In the frontal, parietal, and occipital cortex, the outer and inner layers were more strongly labeled than the other layers ŽFig. 1AFig. 2A,CFig. 4A,C., whereas the temporal and insular cortex had a strong staining of an intermediate layer and a faintly staining of the inner layer ŽFig. 2B–D.. Due to the early stages of development, layers could not be characterized in more detail, but it is clear that the layers with higher cellular density present the higher labeling, as observed on bright field exposures. Moderate labeling was observed in the caudate nucleus, putamen, globus pallidus, and subthalamic nucleus ŽFig. 2B,C.. The heterogeneous distribution of staining can be attributed to the different cellular densities in these struc-

Fig. 3. Cellular localization of the a 4 subunit mRNA in the normal human fetal brain. Bright-field microphotographs of the thalamus ŽA., the pyramidal cell layer of CA1 region ŽB., the granular cell layer of dentate gyrus ŽC. and the Purkinje cells of cerebellum ŽD.. Sections were exposed for 8 weeks. Black grains in the photomicrographs show the localization of a 4 subunit mRNA. Arrows indicate the granular cell of the dentate gyrus and Purkinje cells of the cerebellum. Abbreviations: ML: molecular layer, PL: Purkinje cell layer, GL: granular layer. Scale bars in A, B, C, and D s 25 m m.

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Table 1 Absolute quantification of the content of the a 4 subunit mRNA in the normal and fragile X human fetal brains Brain structures

a 4 labeling

Cortex Claustrum Caudate nucleus Putamen Globus pallidus Subthalamic nucleus Thalamus Entorhinal cortex Subiculum Granular cells of dentate gyrus Pyramidal cells of Ammon’s horn Purkinje cells of cerebellum

qqq qq qq qq q qq qqq qq qq qqq qqq qq

Scores were attributed as follows: q Žlow intensity: O.D.-10., qq Žintermediate intensity: 10 -O.D.- 20., and qqq Žhigh intensity: 20 -O.D.- 30..

tures rather than to distributions in the particular subpopulation of cells. The thalamus was most strongly labeled, particularly the large cells ŽFig. 2CFig. 3AFig. 4B,D.. It is difficult to differentiate thalamic nuclei at this stage but labeling was stronger in the lateral posterior and dorsomedial areas than in other thalamic areas presenting weaker cell densities. In the hippocampus, the entorhinal cortex, subiculum, granular layer of the dentate gyrus, and the pyramidal layer of the Ammon’s horn were labeled. The strongest hybridization signals were in the granular cells of the dentate gyrus and the pyramidal cells of the Ammon’s horn Žareas with high cellular density. while the signal detected in the subiculum and entorhinal cortex were weaker Žareas with weaker cellular density. ŽFig. 2C,EFig. 3B,CFig. 4B,D.. In the cerebellum, the Purkinje cells and the dentate nucleus showed an intermediate labeling ŽFig. 3D.. Analysis of emulsion-dipped and toluidine blue counter-stained sections suggests that, according to their localization and histological characteristics, most of the labeled cells are neurons in all brain areas examined. No signal was detected in periventricular germinal cells.

4. Discussion This is the first anatomical description of a 4 subunit mRNA distribution in the human fetal brain. With few exceptions mentioned below, our observations agree previous studies reported in brains of prenatal rats and extend studies in human adult brains w6,10,9,38,41–44,51,56x. 4.1. Cortex Our data, shown differential a 4 labeling according to the cortical areas. Similarly, nicotine binding studies in the human adult neocortex have demonstrated distinct labeling

patterns in the various cortical areas. Indeed, nicotine binding is concentrated in the outer and inner layers of the striate cortex, the outer two layers of the motor cortex, and the upper middle layer of the cingulate cortex w9x. The nAChRs are also common in layers IIrIII and IV of the human adult temporal and occipital cortices w43x. However, two studies of the human adult precentral cortex Žarea 4. and frontal cortex using rat cRNA probes indicated that the a 4 mRNA is present throughout the entire cortex, without differences in intensity w44,52x. This discrepancy may be explained by either a different a 4 mRNA distribution between fetal and adult cortex or by differences in the probes used. Accordingly, the homology may not be sufficient between the rat and the human a 4 sequences for specific labeling. In addition, the RNA probes may not be specific enough in the case of a multigenic family and may explain the homogeneous labeling in all layers of the adult cortex. Our findings are consistent with those of a study in the prenatal rat brain w56x showing different intensities of a 4 mRNA labeling in different cortical layers at E17 to E19 stages: the labeling being more abundant in inner layers than in outer layers. Similar levels and patterns of a 4 mRNA are present from the first postnatal day to the first postnatal week in the rat and thereafter decline and remain low w6x. In addition, in the adult rat, a 4 transcripts are expressed in all layers, throughout the isocortex with different intensities depending on the layers w51x. Thus, the studies in rat and our data in human fetuses suggest a layer- and cortex-specific production of the a 4 mRNA at prenatal as well as at adult stages. 4.2. Claustrum The a 4 labeling observed in the human fetal claustrum is consistent with data in the prenatal rat brain, which demonstrated that a 4 mRNA and b 2 mRNA are produced in the claustrum w51,56x. In adult rat brain, also a 4 mRNA and b 2 mRNA are produced in the claustrum, and in human adult brain nicotine binding was found in this area w38x. These finding strongly suggest that nAChRs are produced in claustrum, at fetal as well as at adult developmental stages. This receptor may be involved in visual attention, a function for which the claustrum is thought to be important w25x. 4.3. Striatum, globus pallidus and subthalamic nucleus Here, a 4 hybridization signals were found in the caudate nucleus, putamen, globus pallidus, and subthalamic nucleus of the human fetal brain. The presence of a 4 mRNA in the human fetal striatum was not described before. Only studies report strong nicotine binding in the human adult striatum w38,39,41,42x. Together, these observations suggest that nAChRs are present at human fetal as

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well as at adult stages in these brain areas. On the contrary, a 4 mRNA was not detected in the prenatal rat striatum in two studies w51,56x, suggesting differences in specific expression of a 4 subunit in the striatum between rodents and humans. 4.4. Thalamus Our findings indicated that a 4 subunit mRNA was present in the neurons of the thalamus early in development. This is the first a 4 in situ hybridization study performed in the human thalamus and our data are in agreement with those of Zoli et al. w56x in the rat nervous system. This previous study has demonstrated a high level of a 4 mRNA in the thalamus at pre- and postnatal stages. b 2 mRNA is also produced during these stages and this distribution pattern suggests that a 4rb 2 receptors may be functional in the rat thalamus during the early development of the CNS. Similar a 4 mRNA expression and nicotine binding patterns occur throughout the thalamus at postnatal day 1 and in the adult rat brain w6,51x, supporting the high levels of nicotine binding observed in the adult human thalamus, particularly in the lateral geniculate nucleus w38,39,41,42x and in the dorsomedial nucleus w38x. The early expression of the a 4 subunit may indicate a function in the neurogenesis of thalamic nuclei that occurs before birth. The high density of nicotinic receptors in the thalamus suggests their implication in the thalamic control of sensory input and selective attention w9x. 4.5. Hippocampus Our data indicated a strong labeling for the a 4 mRNA in the entorhinal cortex, subiculum, granular cell layer of the dentate gyrus, and pyramidal layer of the Ammon’s horn. These results are consistent with those of Court et al. w10x in preterm human fetuses Ž22–27-week pregnancy. showing that the levels of nicotine binding in the hippocampal CA1 region and the entorhinal cortex are higher than at any other time of development w9x. This binding remains high until adulthood in the entorhinal cortex, subicular cortex, the stratum lacunosum molecular in the CA1, CA2, and low in the granular cell layer of the dentate gyrus but decreases in all other hippocampal regions w9x. Zoli et al. w56x reported weak labeling for a 4 mRNA in the rat hippocampal formation Žgranular layer of the dentate gyrus, and pyramidal layer of the Ammon’s horn. at the E17 to E19 stages and at the birth day. Moderate quantity of b 2 mRNA is coexpressed in the same regions, suggesting the existence of few a 4rb 2 nAChRs in these areas, at these stages. Together, these observations support the hypothesis previously suggested w9x that nAChRs are involved in the cortical and thalamic innervations of the entorhinal cortex and subiculum.

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4.6. Cerebellum Our data shown that a 4 mRNA was present in the Purkinje cell layer and the dentate nucleus in the human fetal cerebellum. The external granular cell layer and the dentate nucleus in the human fetuses Ž23–39 weeks gestation. have higher levels of nicotine binding than those of adults and there is no apparent nicotine binding in Purkinje cells in either fetuses or young adults w12x. This suggests a presynaptic location for the a 4 subunit in the Purkinje cells which is consistent with in vitro studies showing that nAChRs may function at presynaptic locations in the adult rat cerebellar cortex w26x. Moreover, the level of cerebellar choline acetyltransferase ŽChAT. activity is at its highest during the human preterm period and is first detected in Purkinje cells w11,12x. High levels of nicotinic receptors associated with high ChAT activity in the human fetal cerebellum are consistent with strong cholinergic innervation and increased intensity of cholinergic transmission at this stage of development. In the cerebellum, an area of brain with relatively low cholinergic activity in the adult, this transmitter system may have a neurotrophic role during the development. In 25-week pregnancy old human fetuses, the Purkinje cells are in an active phase of synaptogenesis w55x and it is possible that this peak of cholinergic activity is involved in synaptogenesis. Our data are consistent with findings in preterm rats showing prenatal production of a 4 mRNA in Purkinje cells w56x. Our study confirms and specifies a 4 mRNA production in the fetal human brain. Anatomical differences suggest a specific role for nicotinic receptors containing the a 4 subunit in different structures of the brain. Additionally, the a 4 mRNA is produced in the two brains of fragile X fetuses with the same pattern and with the same density than in the normal fetal brain, suggesting that a 4 mRNA production and stability are not altered in the fragile X syndrome. Cholinergic neurons are among the first to differentiate in the CNS w56x and are thought to be involved in subsequent differentiation of other neuronal populations w4,40,46,45x. Less is known about the development of cholinergic innervation. However, markers of cholinergic innervation, such as acetylcholine levels w9x, ChAT and acetylcholinesterase show very high activities in the human fetal brain w12,13x, suggesting that the limited number of prenatal cholinergic terminals may have higher activity than those of the adult w54x. Furthermore, it was reported that prenatal or neonatal in vivo manipulations of acetylcholine systems and nAChRs decrease the cortical cell number, and retard the maturation of cortical cholinergic systems w31,48x. This biochemical and morphological evidence suggest that, besides its classical neurotransmitter role, acetylcholine and the nAChRs may play a key role as a trophic factor during the early stages of pre- and postnatal life and in the development of cortical synapses w24,27x. However, the maintenance of high levels of nicotine bind-

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ing sites in the immediate postnatal period in some brain areas, such as the entorhinal cortex and frontal cortex, suggests continuing involvement of the nicotine receptors in the plasticity of these brain regions into adulthood. Our data, documenting high levels of nAChR a 4 subunit mRNA in the human fetal brain, support the notion of a trophic role for nAChRs in the prenatal period. It is becoming clear that nicotinic receptors are probably involved in the morphogenesis, plasticity, and in the control of neuronal transmission Žboth pre- and postsynaptic.. This suggests that further studies of the a 4 subunit and other nAChR subunits expressions, may increase our understanding of cholinergic systems and their involvement in neurological and psychiatric disorders.

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Acknowledgements We thank Dr. Marc Abitbol for providing tissues, Dr. Olivier Robin and Waltraut Rudolph for their assistance, and Dr. Mehdi Tafti for critical reading of the manuscript. This work was supported by the Swiss National Priority Program PNR38 No. 4038-044050.

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