6N mice: A preliminary immunocytochemical study

Vol. 7, pp. 703-710, 1981. Printed in the U.S.A.

Brain Research Buktin,

Fetal Raphe and Hippocampal Transplants into Adult and Aged C57BL/6N Mice: A Preliminary Immunocytochemical Study E. C. AZMITIA

Anatomy

Department,

Mount Sinai School of Medicine, New York, NY 10029 M. J. PERLOW

3150 N. Lake Shore Drive, Chicago, IL 60657 M. J. BRENNAN Neurology Department,

New York University School of Medicine,

New York, NY 10016

AND J. M. LAUDER Anatomy

Department,

University of North Carolina at Chapel Hill, Chapel Hill, NC 27514

Received 2 October

1981

AZMJTIA, E. C., M. J. PERLOW, M. J. BRENNAN AND J. M. LAUDER. Fetal raphe and hippocampal transplants into adult and aged C57BLl6N mice: A preliminary immunocytochemical study. BRAIN RES. BULL. 7(6) 703-710. lggl.-Fetal mouse raphe and hippocampal tissue (from embryos with crown-rump length (CRL) 13-16 mm) was transplanted into adult and aged isogenetic mice to study the growth of serotonergic fibers between host and donor tissue. A specific antibody against serotonin (5HT) was used to immunocytochemically visualize S-HT containing cell bodies and fibers. Unilateral fetal transplants into the hippocampi of adult (4-6 mo.) or aged (24 mo.) mice matured and sent out processes which very densely innervated the transplant tissue itself and extended into the host hippocampus. The termination of these fibers was consistent with the known 5-HT-hippocampal lamination pattern in normal animals. Qualitative comparisons suggested that the density of outgrowth into adult hippocampus was greater than into aged hippocampus. Conversely, adult 5-HT neurons send sprouts into fetal hippocampal tissue transplanted into the lateral ventricle. Therefore, immunocytochemical procedures can be used to monitor outgrowth from the fetal tissue to the host and ingrowth from the adult host to the fetal tissue. Furthermore, the apparent normal S-HT lamination pattern produced by fetal raphe axons in adult hippocampus is consistent with reports that neuronal transplantation is effective in reversing the anatomical and behavioral deficits produced by homotypic denervation of a terminal field. Neuronal transplantation Mouse

Serotonin

Raphe nucleus

THE serotonin-producing neurons in the midbrain raphe nuclei of the adult mammalian brain undergo neurogenesis during early ontogeny [7,8]; their axons reach the hippocampus by gestation day 17 in the rat [16]. The plasticity of the S-HT-hippocampal system has been demonstrated following irradiation of the neonatal hippocampus [9] and after partial homotypic denervation in the adult hippocampus [l]. Furthermore, the plasticity can also be studied between tissues of different ages. The fetal raphe tissue transplanted to the adult brain survives for prolonged periods of time [lo, 13, 141 and innervates the host hippocampus [3]. In this latter study, the fetal 5-HT sprouts formed a termination pattern different from that produced during normal development. In the present study we used immunocytochemistry with

Copyright

@ 1981 ANKHO

International

Hippocampus

Aging

Immunocytochemistry

a specific antibody against serotonin (SHT) to label 5-HT containing cell bodies and fibers. Experiments were designed to define the growth and innervation pattern of fetal raphe neurons following transplantation into the brains of adult and aged mice; and to determine if host adult raphe serotonergic neurons can send processes into transplanted fetal hippocampus? Our immunocytochemical data show outgrowth of fetal serotonergic neurons into both adult and aged hippocampus, and ingrowth of adult serotonergic neurons into transplanted fetal hippocampus. METHOD

Adult (4-6 mo.) and aged (24 mo.) male C57BY6N mice

Inc.-0361-9230/81/120703-08$01.30/O

AZMITlA E7‘AL. were used (Charles River Breedings Labs). Isogenetic pregnant mice (1619 days of gestation) were killed by decapitation. The fetuses (CRL 13-18 mm) were removed with their placentae and placed in ice-cold oxygenated Ringer’s buffer. A surgical approach was developed for removal of the fetal raphe 5-HT nuclei. Under the dissecting microscope, the fetal tectum was removed using iris scissors. A mid-sagittal section of tissue (< 1 mm thick) between the mesencephalic and pontine flexures was dissected out using a #II scalpel blade. The strip was cut into small cubes (
(Fig. 2). The axons of these cells densely innervated the transplanted donor tissue as well as innervating to a lesser extent the host hippocampus. These fibers appeared to preferentially innervate the stratum lacunosum-moleculare of Ammon’s horn and the granular area (supra- and infragranular zones) of the dentate gyrus (Fig. 3). The total density of serotonergic fibers in these hippocampal regions was substantially increased compared to the corresponding nontransplanted contralateral areas. Transplants of fetal raphe tissue into the hippocampus of aged mice showed a similar intra-transplant maturation and hyperinnervation pattern. These fetal serotonergic fibers grew into the host (aged) hippocampus in a lamination pattern similar to that seen in an intact animal (Fig. 4). However, qualitative comparisons suggest that the outgrowth into aged host tissue was less than into adult host tissue. Fetul Hippocampal

Transplant

into Adult

Lateral

Ventricle

Fetal hippocampus transplanted into the lateral ventricle of adult mice survived without rejection for one month. Highly branched serotonergic fibers from the host brain grew into the transplanted tissue (Fig. 5). Although these fibers were most dense at the junction between the host and the fetal tissue, many of these 5-HT axons were observed to penetrate into the very center of the transplanted tissue. ABBREVIATIONS

Aq CA Caud cc DC DR E F G Hip LV M MLF MR NC P PO RA S T Thal TP

Cerebral Aqueduct Comu Ammonis Caudate Corpus Callosum Dentate Gyrus Dorsal Raphe Nucleus Ependymal Layer Fornix Granular Layer of Dentate Gyrus Hippocampus Lateral Ventricle Midbrain Flexture Medial Longitudinal Fasciculus Median Raphe Nucleus Neocortex Polymorphic Layer of Dentate Gyrus Pontine Flexture Raphe Nuclear Area Septum Tectum Thalamus Transplant

RESULTS DISCUSSION

Fetal Brain

The brains of fetal mice were immunocytochemically stained for 5-HT. The 5-HT neuronal cell bodies were seen primarily in the brainstem raphe from the mesencephalic flexure to the pontine flexure (Fig. 1). The cells were mainly biopolar and spindle shaped. Fetal Raphe

Transplants

to Adult

and Aged Hippocampus

Transplanted fetal serotonergic cells when injected into adult hippocampus matured into large, multipolar neurons

Previous workers have shown that damaged serotonergic axons can regenerate by sprouting [2, 11, 121. Removal of a homotypic afferent system induces intact undamaged serotonergic fibers to expand their territory of innervation by producing new collateral sprouts [l]. This sprouting response of undamaged (or regenerating) axons into a denervated area suggests a dynamic interaction between serotonergic fibers and their terminal field. When fetal raphe was transplanted into a denervated adult hippocampus, the outgrowth of 5-HT fibers preferentially terminated on the

NEURONAL

TRANSPLANTS

705

IN MICE

FIG. 1. Immunocytochemical localization of serotonergic cells in the brainstem of a C57BV6N mouse fetus (CRL 15 mm). The neuronal cell bodies extend from the mesencephalic flexure (M) to the pontine flexure (P). a. A low magnification view of mid-sagittal section. The dotted line outlines the area of highest concentration of serotonergic neurons. b. A higher magnification of the same area. The spindle shaped fetal neurons (arrows) are located near the ependymal layer.

outer molecular layer of the dentate gyrus, the zone vacated by the destroyed entorhinal afferents [3]. In our experiments, the transplants were injected into the intact host hippocampus with only a localized area of non-specific damage resulting from the transplantation procedure. The outgrowth of transplanted serotonergic axons, in our case, terminated in a normal laminar pattern. The existence of a trophic factor in the mature brain is suggested from the orientation of the fetal neuronal dendrites towards the host hippocampus, as well as by the axonal growth from the donor into the host hippocampus. We have observed that fetal transplants of either raphe or hippocam-

pal tissue promoted

inter-tissue growth of 5-HT fibers. The 5-I-IT innervation density after one month was always highest within the transplanted raphe tissue itself. Furthermore, the inter-tissue growth from fetal raphe tissue was greater into adult tissue than into aged tissue. The extent of the innervation of the aged brain by the fetal serotonergic axons was surprising in view of the substantial decrease in the sprouting of adrenergic fibers reported in the hippocampi of senescent rats 1151. This discrepancy underscores the importance of the age of the sprouting neurons when evaluating the potential of a terminal field to induce collateral sprouting. Furthermore, the increase in the extent of the innervation of the

FIG. 2. Immunocytochemical localization of the serotonergic cells and fibers in fetal tissue 30 days following transplantation into the adult hippocampus. a. A low magnitkation of the transplant located in the lateral hippocampus. b. A higher magnification of the same area (box) showing the hyperimtervation within the transplanted raphe tissue and the outgrowth of fibers into the host hippocampus. c. Oil immersion photograph of a single serotonergic neuron found within the transplant (box). The dendrites of the cell are oriented towards the host hippocampus.

FIG. 3. Immunocytochemical localization of S-HT neuronal processes 30 days following transplantation of fetal raphe into adult hippocampus. COmParison between the t~splanted and control sides of the same adult brain. a. A dark-field photograph of the transplanted raphe in the host hippocampus. The fetal fibers can be seen growing into the lacunosum-moleculare of Ammon’s horn and into the polymorphic layer of the dentate gyrus. b. A dark-field photograph of the non-transplanted opposite side of the same brain. The normal density and lamination pattern of the serotonergic fibers can be seen. Note the relatively sparse innervation of the stratum radiatum of CA3 area on the non-transplanted side. c. Oil immersion photograph showing the increased innervation of the dentate gytus on the transplanted side of the brain. The serotonergic fibers are highly varicose and can be seen next to and within the granular layer of the host hippocampus, The polymorphic layer is especially dense with immunoreactive fibers. d. Oil immersion photograph showing the non-transplanted opposite side of the same host brain.

lsm

FIG. 4. Immunocytochemical localization of the serotonergic cells and fibers in fetal raphe 30 days after transplantation into the hippocampus of an aged mouse (24 mo.). a. A low magnification view of the transplant located adjacent to the lateral hippocampus. b. A higher magnification view showing the increased innervation of the transplanted raphe and the outgrowth of fibers into the host aged hippocampus (box). The degree of outgrowth into the host hippocampus is less than noted in the adult (Fig. 2). c. Oil immersion photograph of a group of serotonergic neurons found within the transplant (box). The neurons are highly branched and are larger than observed in the fetal brain (Fig. I).

a

709 NEURONAL

TRANSPLANTS

IN MICE

FIG. 5. ~~unocyt~hemi~ localization of host (adult) serotonergic fibers innervating the fetal (CRL IS mm) hippocampal transplant 30 days after transplantation. a. Low magnification photograph showing the location of the transplanted hippocampus. The fetal tissue is embedded in the septum-fomix. b. Higher magnification of the same area with dark-field iUum~~tion showing serotonergic fibers within and around the transplant. c. Oil immersion photograph of the same area (box). The serotonergic fibers which have innervated the fetal transplant are very highly branched and varicose.

710

AZMITIA ET AL.

host hippocampus as compared to the number of fibers seen in the intact non-transplanted hippocampus raises the interesting point that the hippocampus is not normally saturated with 5-HT fibers in either the fetal, adult, or aged brain. The equilibrium established between brainstem and cortex during development by serotonergic axons within the hippocampus can be substantially shifted by introduction of fetal raphe or hippocampal tissue. The present studies demonstrate that transplanted fetal 5-HT neurons can mature and send sprouts into intact host brains. The apparent normal 5-HT lamination pattern produced by fetal raphe axons in adult hippocampus is consistent with reports that neuronal transplantation can effectively reverse the anatomical and behavioral deficits produced by homotypic denervation of the terminal field [4, 141. The ability of the host adult 5-HT neurons to send sprouts into fetal hippocampus demonstrates that the plasticity seen in response

to denervation

in the adult brain [I] is also present in the intact brain, and should therefore be considered a growth response induced by the terminal field on the afferent fiber system. Furthermore, in our studies, the fetal hippocampus not only survived in the lateral ventricle of an adult host, but also received tierent fibers from the host 5-HT system. Serotonergic fibers may thus assist in the integration of transplanted cortical tissue into the substance of the host brain [6]. Whether these cortical transplants can functionally replace lost cortical tissue remains to be determined. ACKNOWLEDGEMENT

These studies were supported by a grant from the National Science Foundation (BNS-79-06474). We gratefully acknowledge the technical assistance capably provided by Verne11 Daniels, and photographic expertise of Patrick Cannon.

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