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Intracranial transplantation and survival of tuberomammillary histaminergic neurons
Neuroscience Vol. 64, No. 1, pp. 61 70, 1995
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Pergamon
0306-4522(94)00371-8
Elsevier Science Ltd Copyright © 1994 IBRO Printed in Great Britain. All rights reserved 0306-4522/95 $9.50 + 0.00
I N T R A C R A N I A L T R A N S P L A N T A T I O N AND SURVIVAL OF T U B E R O M A M M I L L A R Y H I S T A M I N E R G I C N E U R O N S H. B E R G M A N , * t ~ J. I. N A G Y § and A. C. G R A N H O L M : ~ tDepartment of Cell Biology, University of Link6ping, Link6ping, Sweden :~Department of Basic Science, University of Colorado School of Dentistry, Denver, CO 80262, U.S.A. §Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada Abstract--Investigations were undertaken to determine whether fetal histaminergic neurons in the tuberomammillary nucleus of the posterior hypothalamus survive intracranial transplantation to adult hosts. Two methods of transplantation were utilized. Grafts were placed either into the delayed cavity of a fimbria-fornix lesion or directly into the hippocampus using stereotaxic techniques. The tissue was taken from rat fetuses at embryonic days 16-17 and grafted into adult rats of either the Sprague-Dawley or the Fischer 344 strain. Routine histology and immunohistochemistry were used to evaluate the grafts. All transplants to Sprague-Dawley rats showed signs of rejection, while no signs of rejection were seen in any of the Fischer 344 rats. Transplants placed directly into the delayed fimbria-fornix cavity did not grow as well or contain as many surviving neurons as the intraparenchymal grafts. The largest number of surviving histamine-positive neurons was obtained with grafts of posterolateral blocks of hypothalamus from fetal day 17 placed directly into the CAI region of the rostral hippocampal formation of Fischer 344 hosts. Histamine-immunoreactive cell bodies with neuritic outgrowth were found in all Fischer 344 rats that received hypothalamic grafts. Cell bodies exhibited histamine immunoreactivity evenly throughout the cytoplasm and had morphological characteristics resembling histaminergic neurons in situ. Axonal outgrowth extended throughout the grafted hypothalamic tissue, and was sometimes seen in the host hippocampal tissue as well. It is concluded that fetal histaminergic neurons survive transplantation to the adult hippocampal formation, and that this allograft procedure can supplement current strategies to investigate the function of histaminergic tuberomamillary neurons in the central nervous system.
The existence of magnocellular neurons in the tuberal region of the hypothalamus has been known for several decades) ° These neurons in the tuberomammillary nucleus (TM) were subsequently found to be histaminergic, 43 and were described as consisting of five subgroups, located medially, laterally and ventrally. 2°'26,35'44 The target areas of histaminergic neurons in the rodent brain have been mapped using antibodies directed against conjugated histamine 27'37 or the histamine-synthesizing enzyme histidine decarboxylase. 12'22'41'42 The ontogenesis of histaminergic neurons appears to be distinctly different from that of the catecholaminergic neurons, 21 but closely related to the development of serotonergic neurons.l'4° Their neurogenesis in the rat begins at embryonic day (E) 13, peaks on E l 6 and is completed by El8. 31 Histaminergic neurons with well developed dendrites and axons extending considerable distances from cell bodies were shown to survive in hypothalamic explants from neonatal rats for up to nine weeks in v i t r o . 32 It has also been demonstrated that
explanted histaminergic neurons can innervate coexplanted hippocampal tissue in v i t r o . 19 However, long-term studies of histaminergic plasticity and axonal growth properties have not been performed to date. Biochemical and immunohistochemical data has demonstrated that T M histaminergic neurons innervate all areas of the hippocampal formation with moderate densities of histamine-positive axons, and a large proportion of the histidine decarboxylase was reported to be concentrated in synaptosomal fractions indicating localizations in axons and terminals in this brain region. 2,29 Approximately 60% of these histaminergic projections were suggested to enter the hippocampal formation via a dorsal pathway consisting of the fimbria-fornix and cingulum, and the remainder via a ventral route through the amygdaloid area. 2 The same routes of hippocampal innervation have been described for other monoaminergic neuro n s ) 8 Since the anatomy and physiology of the hippocampal formation are well known, and the afferent and efferent connections can be relatively easily manipulated by, for example, surgical fimbriafornix transection,~l this brain region has often served as an innervation target for studies of neuronal plasticity. Bilateral fimbria-fornix transection results in a partial deafferentation of the hippocampal formation, producing specific spatial memory impair-
*To whom correspondence should be addressed, at Denver. ADA, adenosine deaminase; BSA, bovine serum albumin; E, embryonic day; GFAP, glial fibrillary acidic protein; PB, phosphate buffer; PBS, phosphatebuffered saline; TH, tyrosine hydroxylase; TM, tuberomammillary nucleus.
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m e n t in rats, 24 a n d t r a n s p l a n t a t i o n o f fetal n e u r o n s to lesioned h i p p o c a m p i has b e e n utilized to investigate the role o f specific t r a n s m i t t e r s y s t e m s in these m n e m o n i c f u n c t i o n s , s'u'33 Recently, we have r e p o r t e d successful t r a n s p l a n t a t i o n o f h y p o t h a l a m i c tissue into the a n t e r i o r c h a m b e r o f the eye in rats. ~7 T h e grafts d o u b l e d their size in oculo, a n d c o n t a i n e d n u m e r o u s h i s t a m i n e r g i c n e u r o n s w h i c h were able to i n n e r v a t e the h i p p o c a m p a l p o r t i o n o f a d o u b l e graft in oculo, l e a d i n g to a significant i n n e r v a t i o n d e n s i t y with time. ~7 E l e c t r o p h y s i o l o g i c a l studies i n d i c a t e t h a t g r a f t e d h i s t a m i n e r g i c n e u r o n s in the a n t e r i o r eye chamber exhibit physiological and pharmacological p r o p e r t i e s similar to t h o s e in situ. 3 In t h e p r e s e n t study, we i n v e s t i g a t e d the ability o f h i s t a m i n e r g i c n e u r o n s to survive t r a n s p l a n t a t i o n to f i m b r i a - f o r n i x lesioned h i p p o c a m p u s in the rat. T h e specific aims were: (i) to establish w h e t h e r h i s t a m i n e r gic n e u r o n s c o u l d survive t r a n s p l a n t a t i o n intracranially; (ii) to define the o p t i m a l d o n o r stage a n d d o n o r area for t r a n s p l a n t a t i o n ; a n d (iii) to d e t e r m i n e whether axons from grafted histaminergic neurons w o u l d c r o s s the graft h o s t b o r d e r a n d i n n e r v a t e s u r r o u n d i n g h o s t brain. EXPERIMENTAL PROCEDURES
Lesions, dissections and transplantation A total of 14 Sprague-Dawley and 18 Fischer 344 rats (170-230g females, Harlan Laboratories) were used as recipients for intracranial hypothalamic grafts. The animals were anesthetized with chloral hydrate (0.3 g/kg i.p.) and placed in a stereotaxic frame. An incision was made in the skin overlying the skull, and a suction lesion of the cortex and underlying fimbria-fornix was performed through a burr hole at the following stereotaxic coordinates: bregma - 0 . 5 to - 1.5 mm, lateral 0-4 mm and 5 mm deep. Gelfoam was placed in the cavity and the lesion site was left to heal for one to two weeks. In four of the 18 Fischer 344 hosts, bilateral fimbria fornix lesions were performed. Unilateral grafting permitted the contralateral side to serve as a lesioned control. Furthermore, two additional animals received fimbri~fornix lesions and no grafts. Pieces of the posterior hypothalamus (Fig. 1A) were dissected from Sprague Dawley or Fischer 344 fetuses of El6 or El7. The pieces were freed from the pia mater and kept in ice-cold Ringer solution until transplantation. The first set of experiments involved intracavity (11 animals) or intraparenchymal (three animals) transplantation into Sprague-Dawley hosts. The animals were anesthetized with chloral hydrate (0.3 g/kg i.p.) and placed in a stereotaxic frame. For intra-
A
ventral E17
B
cavity transplants, the original burr hole was uncovered, and pieces of hypothalamic tissue (1 × 1 2 mm) were placed into the lesion cavity, and covered with gelfoam (Fig. I B). For the intraparenchymal transplants, pieces of fetal hypothalamic tissue (1 x 1-2 mm) were implanted stereotaxically with a modified hypodermic needle with an insert from a spinal needle. This intracranial transplantation method was developed by Str6mberg and collaborators. 39 The tissue pieces were inserted directly into the CA1 area of the rostral hippocampal formation, caudal to the prior fimbria fornix lesion (Fig. 1C). Stereotaxic coordinates for hippocampal implants were: bregma - 2 . 5 m m , 1.5mm lateral and 3 3.5 mm deep. The second set of experiments involved intracavity (four animals) or intraparenchymal (14 animals) transplantation into Fischer 344 hosts. The transplants into Fischer 344 hosts were divided into a short-term group, with survival times of two to eight weeks, and a long-term group, which was left for five months postgrafting. Immunohistochemistry Host rats were anesthetized with chloral hydrate (0.3 g/kg i.p.), and perfused transcardially with 100ml ice-cold 0.9% NaCI solution for 2 min, followed by 200 ml ice-cold 4% l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide28 in phosphate-buffered saline (PBS) for 20 min. Brains were postfixed in 4% carbodiimide and 4% paraformaldehyde in PBS overnight and transferred to 30% sucrose in PBS for at least 16h. Cryostat sections were collected from each brain for routine histology. Sections were mounted on chrome-alum-coated slides, stained with Toluidine Blue or Cresyl Violet, dehydrated and mounted in Eukitt and examined by light microscopy (Nikon Optiphot). For immunohistochemistry, 3 0 # m cryostat sections were collected and placed in tissue trays on a shaker table. The sections were rinsed in dilution media (0.05% Triton X-100 in 0.1 M phosphate buffer, PB) for 6 x 10min and treated with 0.3% H202 in dilution medium for 20 min to inhibit residual endogenous peroxidase. Sections were rinsed in dilution medium for 3 x 10 min, followed by incubation with normal goat serum (3%) and bovine serum albumin (BSA; 2%) in dilution medium for 1 h to block background staining. The antibodies were diluted in PB with 1% normal goat serum, 1% BSA and 0.3% Triton X-100, and sections were incubated for 48-72h at room temperature. Antibodies used in these experiments were directed against histamine (1:2000, Incstar Corp.)fl 8 adenosine deaminase (ADA; 1:2000), 25 glial fibrillary acidic protein (GFAP; 1:1000) 8 and tyrosine hydroxylase (TH; 1:1000, Eugene Tech.). After the incubation, the sections were rinsed for 6 x 10 min in dilution medium and incubated with biotinylated goat anti-rabbit in 1% normal goat serum and 1% BSA in dilution medium for 1 h, after which the sections were rinsed for 6 × 10 min. Sections were incubated in the ABC 'Elite' substrate (Vector Labs) for 75 min using the same solution as for the second antibody, followed by rinsing in PB for 3 x 10 min. Finally, the sections were developed with a metal-intensified 3,3'-diaminobenzidine reaction for
bregma-l,3mm
C
bregma-2,6mm
Fig. 1. Schematic illustration showing the dissection of lateral hypothalamic tissue from fetal donors (A, arrows) and placement of hypothalamic transplants into a cavity created after a fimbria fornix lesion (B, arrow) or directly into the CA1 of the rostral hippocampal formation (C, arrow).
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A
Fig. 2. Routine histological staining of sections from a Fischer 344 host with an intracavity transplant (A, B), and an intracavity transplant in a Sprague-Dawley host (C, D). Note the lack of general growth of the intracavity transplant (A), as compared to the transplants shown in Fig. 3. However, at higher magnifications, the intracavity transplant in the Fischer 344 host contained neurons and glial cells, and no obvious signs of rejection (B). The Sprague-Dawley transplants exhibited numerous signs of rejection, and the transplanted tissue (TP) was often restricted to small remnants already at eight weeks postgrafting (C). In the close-up (D), accumulations of leucocytes as well as degenerating cell debris is obvious. Arrows in A signify the location of transplanted tissue, and arrows in C demonstrate the border between host brain (HB) and the cavity (CAV). Scale bars = 500 #m (A); 200 #m (C); 100/tm (B, D).
5-15min in a solution with 0.05% diaminobenzidine, 0.3 ppm nickel ammonium sulfate and 0.I ppm H202 in PB, followed by rinsing for 4 x 10 min in PB. Sections were dehydrated and mounted on glass slides with Eukitt and analysed by light microscopy. RESULTS
Routine histology Both with intracavity and intraparenchymal transplants, Sprague-Dawley hosts contained remnants of hypothalamic tissue eight weeks following transplantation, but all transplants in these hosts exhibited significant signs of rejection (Fig. 2C, D), and only small portions of the grafts were attached to the host brain tissue. Large accumulations of lymphocytes and macrophages were observed throughout the transplanted tissue (Fig. 2D). Furthermore, well developed neurons with intact dendrites and axons were rarely seen in Sprague-Dawley hosts, which instead contained cell debris, degenerating neurons and large areas of vacuolization. In the Fischer 344
hosts, on the other hand, leucocyte accumulations were rare, and all transplants, both to intraparenchyreal and intracavity sites, contained what appeared to be healthy neurons and glial cells. The intracavity transplants to Fischer 344 hosts grew less, and appeared to become less integrated with the host brain (Fig. 2A, B) than the intraparenchymal transplants (Fig. 3A-C). Even after longer time periods, the intracavity transplants never grew to fill the cavity entirely (Figs I B, 2A). The intraparenchyrnal transplantation to Fischer 344 hosts thus appeared to be the most successful site and host for transplantation. These transplants became very well integrated with host brain tissue (Fig. 3A-C), and contained tissue similar in general morphology to the ventral hypothalamus in situ.
Immunohistochemistry Histamine-immunoreactive cell bodies were found in all grafts transplanted to Fischer 344 hosts, but in only three of the 14 transplants to Sprague Dawley
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hosts. The histamine-positive cell bodies appeared to be located only in one portion of the grafted tissue, and in this portion five to 30 cell bodies were found per section. The cell bodies appeared mostly scattered throughout the neuropil (Fig. 4), or sometimes in small groups (Fig. 6A). The histamine immunoreactivity appeared evenly throughout the cell body, dendrites and axons (Figs 5A-C, 6A, B), The histamine-immunoreactive neurons contained one to three primary dendrites (Figs 5A, B, 6A, B), which branched extensively in the vicinity of the cell bodies (Figs 5A, 6B). The neurons extended varicose, thin axons (Fig. 5C) over long distances in the grafted hypothalamic tissue. Eight weeks postgrafting, the histamine-positive fibers had an uneven distribution, with parts of the grafts more densely innervated than others, while the five month transplants had a more
al.
even and dense innervation of histamine-positive neurites (Fig. 4). At eight weeks, very few or no fibers crossed the graft-host border. However, in the longterm group, sparse fibers were seen to traverse the graft-host border and enter the host brain (Fig. 7C). In the four animals which were bilaterally fimbriafornix lesioned, the non-grafted side contained no visible histamine-positive fibers in the rostral hippocampal formation or dentate gyrus (Fig. 7A). However, the host brain on the transplanted side exhibited a few histamine-immunoreactive, thin, varicose fibers extending into the CA1 as well as the dentate gyrus (Fig. 7B). Intraparenchymal as well as intracavity transplants to Fischer 344 hosts also contained numerous ADApositive cell bodies, dendrites and axons (Fig. 6C). These cells resembled the histamine-immunoreactive
Fig. 3. Routine histological staining of sections from Fischer 344 hosts carrying intraparenchymal hypothalmic transplants. (A) An overview photomicrograph with the transplant delineated by arrowheads. (B) A photomontage of another intraparenchymal transplant in a Fischer 344 host (transplant, TP). (C) A close-up from an intraparenchymal transplant (TP) and the graft-host border (arrowheads). Note that the transplanted tissue often extends along the needle tract into the cerebral cortex of the host (A, B). Scale bars = 500#m (A); 200#m (B); 100~m (C).
Fig. 4. P h o t o m o n t a g e of a parenchymal hypothalamic graft (TP). The section was incubated with antibodies against histamine. Note histamine-immunoreactive cell bodies (arrows) and dense plexi of fibers throughout the graft. The graft was examined after a host survival time of five months. Arrowheads outline the g r a f t ~ o s t border. Scale bar = 200/lm.
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cells in terms of dendritic branching and size, and were located in the same area of the transplants as the histamine-positive cell bodies on adjacent sections. TH-immunoreactive cell bodies and/or fibers could sometimes be found in transplants from donor stage El6, and particularly in cases where larger mediolateral strips of hypothalamic tissue were dissected for grafting (Fig. 6D). They were located in small groups and extended dendrites and axons, having a similar morphological appearance to those of ADA (Fig. 6C) and histamine-immunoreactive neurons (Fig. 6A, B). TH-positive neurons were rarely seen in dissections from El 7, or when only lateral hypothalamic tissue was used. The slightly larger overall size of the hypothalamic area at El7, as compared to El6, allowed for a more selective dissection of the lateral portion of this brain region (see Fig. IA). All transplants exhibited a slight gliosis at the border between graft and host tissues, as demonstrated by GFAP immunoreactivity (Fig. 7D). However, a continuous glial barrier was never seen in the graft host junction. Astrocytes in the transplants (Fig. 7D) had a distribution and density similar to that seen in the TM region in situ (shown in Fig. 7E).
DISCUSSION
The present results demonstrate survival and growth of fetal histaminergic neurons transplanted into a delayed cavity or directly into the rostral hippocampus of adult rats. While multiple signs of rejection of grafts were observed in all Sprague Dawley hosts, such signs were absent in Fischer 344 hosts, even at five months postgrafting. Histaminergic neurons with dendritic arborizations were found in all transplants in Fischer 344 hosts. Histaminepositive axons innervated the surrounding grafted hypothalamic tissue, resulting in a significant innervation density of the grafted tissue with time. Some fibers were also seen traversing the graft-host border and innervating the surrounding host brain. Transplants appeared to grow best, and become most integrated when implanted directly into the CAI region of the rostral hippocampus. It has been suggested that allografts between outbred donor and host of the same strain will become rejected shortly after transplantation.7 Previous work by us and o t h e r s t5'16'23'39 has demonstrated successful transplantation of fetal brain tissue to outbred
Fig. 5. Details of histamine immunoreactivity in three different intraparenchymal transplants to Fischer 344 hosts. Note thick branching dendrites (A) in the same transplant as depicted in Fig. 4. B and C show close-ups of histamine-positiveneurons in intraparenchymal grafts. Note the varicose, thin axons emerging from cells and extending for long distances in the tissue (C). Scale bars - 100/~m (A, C); 50/~m (B).
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Fig. 6. Small group of histamine-immunoreactive neurons in an intraparenchymal Fischer 344 graft (A), and a close-up of a histamine-immunoreactive cell body (B) with at least two primary dendrites (D) and several secondary and tertiary dendrites. C demonstrates a small group of ADA-immunoreactive neurons in an intracranial hypothalamic graft, and D demonstrates TH-positive neurons and fibers in the same transplant as in C. Scale bars = 50 #m (A, C, D); 25 #m (B).
Sprague-Dawley rats without any signs of rejection, even at long time periods after transplantation. Successful allografts to outbred hosts could result from rapid formation of a functional b l o o d - b r a i n barrier from vessels within the grafted tissue. 7 However, it is well known that the medioventral hypothalamus is part of the so-called "circumventricular organs" areas that are excluded from the normal b l o o d brain barrier, j8 Intravenous injection of horseradish peroxidase results in a transport of reaction material into the neuropil of the median eminence, 6 which is a region of the hypothalamus most likely included in the transplants in the present study. In a recent study, we have demonstrated that intraocular hypothalamic transplants develop a patchy b l o o d brain barrier, as evidenced with antibodies directed against a specific rat b l o o d - b r a i n barrier protein. 4 These previous studies could explain our findings that all transplants into the outbred S p r a g u ~ D a w l e y strain exhibited rejection signs, while the transplants into the more inbred Fischer 344 strain did not, even after five months postgrafting. We therefore suggest that an inbred strain is essential for successful transplantation of ventral hypothalamic tissue intracra-
nially, since this region seems to be susceptible to rejection, possibly due to a deficient blood-brain barrier. Histamine-immunoreactive neurons were found in all transplants in Fischer 344 hosts. In contrast, only a minority of the transplants to Sprague-Dawley hosts contained viable histamine-immunoreactive neurons. The cell bodies and processes of the grafted neurons exhibited morphological features resembling mature histaminergic neurons in situ, t2"29 in terms of staining properties and dendritic branching. A continued development of histaminergic neurons has previously been demonstrated also in hypothalamic transplants in o c u l o . 17 C o m p a r e d with the total number of histaminergic T M neurons in these nuclei in situ, 2°'44 few histaminergic neurons appeared to have survived transplantation to the intracranial cavity or hippocampal formation in the present study. This low number of surviving neurons could depend on dissection techniques or fetal donor stage. It is possible that fetal neurons from the lateral, relatively small T M subnucleus 44 were the only histaminergic neurons included in the dissections, Future experiments, with detailed evaluation of donor stage and area, as well
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as transplantation of cell suspensions, should reveal the most advantageous techniques for harvesting of fetal histaminergic neurons. The fimbria-fornix transections were found to result in a total disappearance of histamine-immunoreactive fibers from the rostral hippocampal formation. It is likely that the 60% of histamine content in the hippocampus originating in the dorsal fimbria-fornix pathway 2 is the portion that innervates the rostral hippocampus. Even though other biogenic amines, such as the noradrenergic neurons of the brainstem locus coeruleus, will completely reinnervate the deafferented hippocampus when
transplanted to a fimbria-fornix lesioned host, 5 this was not the case with the histaminergic neurons in the present study. However, the low innervation density from transplants into the surrounding host hippocampal formation is not surprising, in view of the relatively sparse histaminergic innervation of hippocampal regions in the intact animal. 29 Furthermore, the relatively low number of neurons transplanted in the present study might not have been sufficient for host brain innervation. The histaminergic neurons of the T M in the rat have been shown to contain a number of other neuroactive substances and enzyme markers, includ-
Fig. 7. Micrographs illustrating a portion of the hippocampal formation from the control side of a bilaterally fimbria-fornix lesioned rat, exhibiting no histamine-immunoreactive fibers (A), and from the transplanted side with a few histamine-immunoreactive fibers extending significant distances into the hippocampal tissue (B). In C, multiple histamine-immunoreactive fibers are seen leaving an intracranial transplant (TP), and growing into the host brain (HB). Arrows illustrate the graft-host border. D demonstrates GFAP immunoreactivity in an intracranial transplant (TP) and the host brain surrounding it (HB). Note that the astrocytes do not appear to form a dense or continuous glial barrier in the graft-host border (arrows). E demonstrates GFAP immunoreactivity in the lateral TM region in situ, Scale bars = 100/~m.
Intracranial transplantation ing G A B A , ~3 galanin 22 a n d A D A . 3°'34-36 It was f o u n d here t h a t t r a n s p l a n t e d h y p o t h a l a m i c T M n e u r o n s are able to m a i n t a i n their n o r m a l c o n t e n t of at least one o f these markers, namely A D A . 25 The presence o f A D A - i m m u n o r e a c t i v e cell bodies a n d neurites in grafts suggested a close relationship between hist a m i n e a n d A D A in grafts, but their co-localization will require c o n f i r m a t i o n by double labeling techniques. It would be o f interest to investigate whether the grafted n e u r o n s c o n t a i n o t h e r neuroactive substances n o r m a l l y present in T M neurons, such as G A B A or galanin, a n d w h e t h e r the expression o f these substances is d e p e n d e n t o n the site of host t r a n s p l a n t a t i o n , thereby reflecting m o d u l a t i o n of expression by the target of innervation. A n t i b o d i e s directed against T H indicated the degree to which d o p a m i n e r g i c n e u r o n s o f the h y p o t h a l a m i c nuclei 14 were included in the dissection. In some grafts from d o n o r stage E l 6 , when mediolateral strips were dissected, groups o f TH-positive cell bodies could be f o u n d in the grafts. TH-positive cell bodies were rarely seen in t r a n s p l a n t s of d o n o r stage E l 7 , where more lateral h y p o t h a l a m i c tissues were obtained. D e v e l o p m e n t a l studies have d e m o n -
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strated t h a t TH-positive d o p a m i n e r g i c n e u r o n s of the arcuate nucleus first a p p e a r in the midline of the h y p o t h a l a m u s at E 1 5 - E 1 6 in the rat. 9 These findings suggest t h a t inclusion of d o p a m i n e r g i c n e u r o n s in the dissection can be avoided by c h o o s i n g a m o r e lateral piece o f h y p o t h a l a m i c tissue, which can be accomplished by using fetuses o f a later fetal stage. The m a i n finding o f the present study was that histaminergic n e u r o n s could survive t r a n s p l a n t a t i o n a n d c o n t i n u e m a t u r a t i o n within intracranial hypothalamic grafts. T a k e n together, these results illustrate a useful a p p r o a c h for studies o f histaminergic n e u r o n a l plasticity a n d connectivity within the central nervous system. Acknowledgements--We thank Drs lngrid Str6mberg and John Hudson for expert technical help with the lesions and transplantation, and Ms Lorie Gottshalk for administrative assistance. Thanks are also due to Dr Doris Dahl for providing the GFAP antibody. The present work was supported by the Swedish MRC, grant 8650, and USPHS grant no. MH49661. H. Bergman was supported by scholarships from the Swedish MRC and the Blanceflor Foundation.
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21. Kinnunen A. and Panula P. (1991) Histamine and tyrosine hydroxytase in developing rat brain. Agents Actions 33, 108 111. 22. K6hler C., Ericson H., Watanabe T., Polak J., Palay S. L., Palay V. and Chan-Palay V. (1986) Galanin immunoreactivity in hypothalamic histamine neurons: further evidence for multiple chemical messengers in the tuberomamillary nucleus. J. comp. Neurol. 250, 58~4. 23. McAllister J. P. II, Walker P. D., Zemanick M. C., Weber A. B., Kaplan L. I. and Reynolds M. A. (1985) Morphology of embryonic neostriatal cell suspensions transplanted into adult neostriata. Devl Brain Res. 23, 282--286. 24. Morris R. (1984) Development of a water-maze procedure for studying learning in the rat. J. Neurosci. Meth. 11, 47 60. 25. Nagy J. I., LaBella L. A., Buss M. and Daddona P. E. (1984) lmmunohistochemistry of adenosine deaminase: implications for adenosine neurotransmission. Science 224, 166 168. 26. Oishi R., Nishibori M. and Saeki K. (1983) Regional distribution of histamine and tele-methylhistamine in the rat, mouse and guinea-pig brain. Brain Res. 280, 172 175. 27. Panula P., Yang H.-Y. T. and Costa E. (1984) Histamine-containing neurons in the rat hypothalamus. Proe. natn. Acad. Sei. U.S.A. 8, 2572 2576. 28. Panula P., H/ipp61/i O., Airaksinen M. S., Auvinen S. and Virkam/iki A. (1988) Carbodiimide as a tissue fixative in histamine immunohistochemistry and its application to developmental neurobiology. J. Histochem. Cytochem. 36, 259-270. 29. Panula P., Pirvola U., Auvinen S. and Airaksinen M. S. (1989) Histamine-immunoreactive nerve fibers in the rat brain. Neuroscience 28, 585~10. 30. Patel B. T., Tudball N., Wada H. and Watanabe T. (1986) Adenosine deaminase and histidine decarboxylase coexist in certain neurons of the rat brain. Neurosci. Lett. 63, 185-189. 31. Reiner P. B., Semba K., Fibigei H. C. and McGeer E. G. (1988) Ontogeny of histidine-decarboxylase-immunoreactive neurons in the tuberomammillary nucleus of the rat hypothalamus: time of origin and development of transmitter phenotype. J. comp. Neurol. 276, 304 311. 32. Reiner P. B., Heimrich B., Keller F. and Haas H. L. (1988) Organotypic culture of central histamine neurons. Brain Res. 442, 166 170. 33. Richter-Levin G. and Segal M. (1991) The effects of serotonin depletion and raphe grafts on hippocampal electrophysiology and behavior. J. Neurosci. 11, 1585 1596. 34. Senba E., Dadonna P. E., Watanabe T., Wu J.-Y. and Nagy J. 1. (1985) Coexistence of adenosine deaminase, histidine decarboxylase, and glutamate decarboxylase in hypothalamic neurons of the rat. J. Neurosci. 5, 3393 3402. 35. Staines W. A., Dadonna P. E. and Nagy J. I. (1987) The organization and hypothalamic projections of the tuberomammillary nucleus in the rat: an immunohistochemical study of adenosine deaminase-positive neurons and fibers. Neuroscience 23, 571 596. 36. Staines Wm. A., Yamamoto T., Daddona P. E. and Nagy J. I. (1987) The hypothalamus receives major projections from the tuberomammillary nucleus in rat. Neurosci. Lett. 76, 257 262. 37. Steinbusch H. W. M. and Mulder A. H. (1984) Localization and projections of histamine immunoreactive neurons in the central nervous system of the rat. In Handbook o f Chemical Neuroanatomy. Classical Transmitters and Transmitter Receptors in the CNS (eds Bj6rklund A., H6kfelt T. and Kuhar M. J.), Vol. 3, pp. 126 140. Elsevier, Amsterdam. 38. Storm-Mathisen J. and Guldberg H. C. (1974) 5-Hydroxytryptamine and noradrenaline in the hippocampal region: effects of transection of afferent pathways on endogenous levels, high affinity uptake and some transmitter-related enzymes. J. Neurochem. 22, 793 803. 39. Str6mberg I., Johnson S., Hoffer B. and Olson L. (1985) Reinnervation of dopamine-denervated striatum by substantia nigra transplants. Immunocytochemical and electrophysiological correlates. Neuroscience 14, 981 998. 40. Vanhala A., Yamatodani A. and Panula P. (1993) Synthesis of histamine in developing rat brain. Soc. Neurosci. Abstr. 19, 452:3. 41. Watanabe T., Taguchi Y., Hayashi H., Tanaka J., Shiosaka S., Tohyama M., Kubota H., Terano Y. and Wada H. (1983) Evidence for the presence of a histaminergic neuron system in the rat brain: an immunocytochemical analysis. Neurosci. Lett. 39, 249 254. 42. Watanabe T., Taguchi Y., Shiosaka S., Tanaka J., Kubota H., Terano Y., Tohyama M. and Wada H. (1984) Distribution of the histaminergic neuron system in the central nervous system of rats: a fluorescent immunohistochemical analysis with histidine decarboxylase as a marker. Brain Res. 295, 13 25. 43. Wilcox B. J. and Seybold V. S. (1982) Localization of neuronal histamine in rat brain. Neurosci, Lett. 29, 105 110. 44. Yamatodani A., Inagaki N., Panula P., Itowi N., Watanabe T. and Wada H. (1991) Structure and functions of the histaminergic neuron system. In Handbook of Experimental Pharmacology. Histamine and Histamine Agonists (ed. Uvn/is B.), Vol. 97, pp. 243 283. Springer, Heidelberg. (Accepted 13 May 1994)
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I N T R A C R A N I A L T R A N S P L A N T A T I O N AND SURVIVAL OF T U B E R O M A M M I L L A R Y H I S T A M I N E R G I C N E U R O N S H. B E R G M A N , * t ~ J. I. N A G Y § and A. C. G R A N H O L M : ~ tDepartment of Cell Biology, University of Link6ping, Link6ping, Sweden :~Department of Basic Science, University of Colorado School of Dentistry, Denver, CO 80262, U.S.A. §Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada Abstract--Investigations were undertaken to determine whether fetal histaminergic neurons in the tuberomammillary nucleus of the posterior hypothalamus survive intracranial transplantation to adult hosts. Two methods of transplantation were utilized. Grafts were placed either into the delayed cavity of a fimbria-fornix lesion or directly into the hippocampus using stereotaxic techniques. The tissue was taken from rat fetuses at embryonic days 16-17 and grafted into adult rats of either the Sprague-Dawley or the Fischer 344 strain. Routine histology and immunohistochemistry were used to evaluate the grafts. All transplants to Sprague-Dawley rats showed signs of rejection, while no signs of rejection were seen in any of the Fischer 344 rats. Transplants placed directly into the delayed fimbria-fornix cavity did not grow as well or contain as many surviving neurons as the intraparenchymal grafts. The largest number of surviving histamine-positive neurons was obtained with grafts of posterolateral blocks of hypothalamus from fetal day 17 placed directly into the CAI region of the rostral hippocampal formation of Fischer 344 hosts. Histamine-immunoreactive cell bodies with neuritic outgrowth were found in all Fischer 344 rats that received hypothalamic grafts. Cell bodies exhibited histamine immunoreactivity evenly throughout the cytoplasm and had morphological characteristics resembling histaminergic neurons in situ. Axonal outgrowth extended throughout the grafted hypothalamic tissue, and was sometimes seen in the host hippocampal tissue as well. It is concluded that fetal histaminergic neurons survive transplantation to the adult hippocampal formation, and that this allograft procedure can supplement current strategies to investigate the function of histaminergic tuberomamillary neurons in the central nervous system.
The existence of magnocellular neurons in the tuberal region of the hypothalamus has been known for several decades) ° These neurons in the tuberomammillary nucleus (TM) were subsequently found to be histaminergic, 43 and were described as consisting of five subgroups, located medially, laterally and ventrally. 2°'26,35'44 The target areas of histaminergic neurons in the rodent brain have been mapped using antibodies directed against conjugated histamine 27'37 or the histamine-synthesizing enzyme histidine decarboxylase. 12'22'41'42 The ontogenesis of histaminergic neurons appears to be distinctly different from that of the catecholaminergic neurons, 21 but closely related to the development of serotonergic neurons.l'4° Their neurogenesis in the rat begins at embryonic day (E) 13, peaks on E l 6 and is completed by El8. 31 Histaminergic neurons with well developed dendrites and axons extending considerable distances from cell bodies were shown to survive in hypothalamic explants from neonatal rats for up to nine weeks in v i t r o . 32 It has also been demonstrated that
explanted histaminergic neurons can innervate coexplanted hippocampal tissue in v i t r o . 19 However, long-term studies of histaminergic plasticity and axonal growth properties have not been performed to date. Biochemical and immunohistochemical data has demonstrated that T M histaminergic neurons innervate all areas of the hippocampal formation with moderate densities of histamine-positive axons, and a large proportion of the histidine decarboxylase was reported to be concentrated in synaptosomal fractions indicating localizations in axons and terminals in this brain region. 2,29 Approximately 60% of these histaminergic projections were suggested to enter the hippocampal formation via a dorsal pathway consisting of the fimbria-fornix and cingulum, and the remainder via a ventral route through the amygdaloid area. 2 The same routes of hippocampal innervation have been described for other monoaminergic neuro n s ) 8 Since the anatomy and physiology of the hippocampal formation are well known, and the afferent and efferent connections can be relatively easily manipulated by, for example, surgical fimbriafornix transection,~l this brain region has often served as an innervation target for studies of neuronal plasticity. Bilateral fimbria-fornix transection results in a partial deafferentation of the hippocampal formation, producing specific spatial memory impair-
*To whom correspondence should be addressed, at Denver. ADA, adenosine deaminase; BSA, bovine serum albumin; E, embryonic day; GFAP, glial fibrillary acidic protein; PB, phosphate buffer; PBS, phosphatebuffered saline; TH, tyrosine hydroxylase; TM, tuberomammillary nucleus.
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m e n t in rats, 24 a n d t r a n s p l a n t a t i o n o f fetal n e u r o n s to lesioned h i p p o c a m p i has b e e n utilized to investigate the role o f specific t r a n s m i t t e r s y s t e m s in these m n e m o n i c f u n c t i o n s , s'u'33 Recently, we have r e p o r t e d successful t r a n s p l a n t a t i o n o f h y p o t h a l a m i c tissue into the a n t e r i o r c h a m b e r o f the eye in rats. ~7 T h e grafts d o u b l e d their size in oculo, a n d c o n t a i n e d n u m e r o u s h i s t a m i n e r g i c n e u r o n s w h i c h were able to i n n e r v a t e the h i p p o c a m p a l p o r t i o n o f a d o u b l e graft in oculo, l e a d i n g to a significant i n n e r v a t i o n d e n s i t y with time. ~7 E l e c t r o p h y s i o l o g i c a l studies i n d i c a t e t h a t g r a f t e d h i s t a m i n e r g i c n e u r o n s in the a n t e r i o r eye chamber exhibit physiological and pharmacological p r o p e r t i e s similar to t h o s e in situ. 3 In t h e p r e s e n t study, we i n v e s t i g a t e d the ability o f h i s t a m i n e r g i c n e u r o n s to survive t r a n s p l a n t a t i o n to f i m b r i a - f o r n i x lesioned h i p p o c a m p u s in the rat. T h e specific aims were: (i) to establish w h e t h e r h i s t a m i n e r gic n e u r o n s c o u l d survive t r a n s p l a n t a t i o n intracranially; (ii) to define the o p t i m a l d o n o r stage a n d d o n o r area for t r a n s p l a n t a t i o n ; a n d (iii) to d e t e r m i n e whether axons from grafted histaminergic neurons w o u l d c r o s s the graft h o s t b o r d e r a n d i n n e r v a t e s u r r o u n d i n g h o s t brain. EXPERIMENTAL PROCEDURES
Lesions, dissections and transplantation A total of 14 Sprague-Dawley and 18 Fischer 344 rats (170-230g females, Harlan Laboratories) were used as recipients for intracranial hypothalamic grafts. The animals were anesthetized with chloral hydrate (0.3 g/kg i.p.) and placed in a stereotaxic frame. An incision was made in the skin overlying the skull, and a suction lesion of the cortex and underlying fimbria-fornix was performed through a burr hole at the following stereotaxic coordinates: bregma - 0 . 5 to - 1.5 mm, lateral 0-4 mm and 5 mm deep. Gelfoam was placed in the cavity and the lesion site was left to heal for one to two weeks. In four of the 18 Fischer 344 hosts, bilateral fimbria fornix lesions were performed. Unilateral grafting permitted the contralateral side to serve as a lesioned control. Furthermore, two additional animals received fimbri~fornix lesions and no grafts. Pieces of the posterior hypothalamus (Fig. 1A) were dissected from Sprague Dawley or Fischer 344 fetuses of El6 or El7. The pieces were freed from the pia mater and kept in ice-cold Ringer solution until transplantation. The first set of experiments involved intracavity (11 animals) or intraparenchymal (three animals) transplantation into Sprague-Dawley hosts. The animals were anesthetized with chloral hydrate (0.3 g/kg i.p.) and placed in a stereotaxic frame. For intra-
A
ventral E17
B
cavity transplants, the original burr hole was uncovered, and pieces of hypothalamic tissue (1 × 1 2 mm) were placed into the lesion cavity, and covered with gelfoam (Fig. I B). For the intraparenchymal transplants, pieces of fetal hypothalamic tissue (1 x 1-2 mm) were implanted stereotaxically with a modified hypodermic needle with an insert from a spinal needle. This intracranial transplantation method was developed by Str6mberg and collaborators. 39 The tissue pieces were inserted directly into the CA1 area of the rostral hippocampal formation, caudal to the prior fimbria fornix lesion (Fig. 1C). Stereotaxic coordinates for hippocampal implants were: bregma - 2 . 5 m m , 1.5mm lateral and 3 3.5 mm deep. The second set of experiments involved intracavity (four animals) or intraparenchymal (14 animals) transplantation into Fischer 344 hosts. The transplants into Fischer 344 hosts were divided into a short-term group, with survival times of two to eight weeks, and a long-term group, which was left for five months postgrafting. Immunohistochemistry Host rats were anesthetized with chloral hydrate (0.3 g/kg i.p.), and perfused transcardially with 100ml ice-cold 0.9% NaCI solution for 2 min, followed by 200 ml ice-cold 4% l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide28 in phosphate-buffered saline (PBS) for 20 min. Brains were postfixed in 4% carbodiimide and 4% paraformaldehyde in PBS overnight and transferred to 30% sucrose in PBS for at least 16h. Cryostat sections were collected from each brain for routine histology. Sections were mounted on chrome-alum-coated slides, stained with Toluidine Blue or Cresyl Violet, dehydrated and mounted in Eukitt and examined by light microscopy (Nikon Optiphot). For immunohistochemistry, 3 0 # m cryostat sections were collected and placed in tissue trays on a shaker table. The sections were rinsed in dilution media (0.05% Triton X-100 in 0.1 M phosphate buffer, PB) for 6 x 10min and treated with 0.3% H202 in dilution medium for 20 min to inhibit residual endogenous peroxidase. Sections were rinsed in dilution medium for 3 x 10 min, followed by incubation with normal goat serum (3%) and bovine serum albumin (BSA; 2%) in dilution medium for 1 h to block background staining. The antibodies were diluted in PB with 1% normal goat serum, 1% BSA and 0.3% Triton X-100, and sections were incubated for 48-72h at room temperature. Antibodies used in these experiments were directed against histamine (1:2000, Incstar Corp.)fl 8 adenosine deaminase (ADA; 1:2000), 25 glial fibrillary acidic protein (GFAP; 1:1000) 8 and tyrosine hydroxylase (TH; 1:1000, Eugene Tech.). After the incubation, the sections were rinsed for 6 x 10 min in dilution medium and incubated with biotinylated goat anti-rabbit in 1% normal goat serum and 1% BSA in dilution medium for 1 h, after which the sections were rinsed for 6 × 10 min. Sections were incubated in the ABC 'Elite' substrate (Vector Labs) for 75 min using the same solution as for the second antibody, followed by rinsing in PB for 3 x 10 min. Finally, the sections were developed with a metal-intensified 3,3'-diaminobenzidine reaction for
bregma-l,3mm
C
bregma-2,6mm
Fig. 1. Schematic illustration showing the dissection of lateral hypothalamic tissue from fetal donors (A, arrows) and placement of hypothalamic transplants into a cavity created after a fimbria fornix lesion (B, arrow) or directly into the CA1 of the rostral hippocampal formation (C, arrow).
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A
Fig. 2. Routine histological staining of sections from a Fischer 344 host with an intracavity transplant (A, B), and an intracavity transplant in a Sprague-Dawley host (C, D). Note the lack of general growth of the intracavity transplant (A), as compared to the transplants shown in Fig. 3. However, at higher magnifications, the intracavity transplant in the Fischer 344 host contained neurons and glial cells, and no obvious signs of rejection (B). The Sprague-Dawley transplants exhibited numerous signs of rejection, and the transplanted tissue (TP) was often restricted to small remnants already at eight weeks postgrafting (C). In the close-up (D), accumulations of leucocytes as well as degenerating cell debris is obvious. Arrows in A signify the location of transplanted tissue, and arrows in C demonstrate the border between host brain (HB) and the cavity (CAV). Scale bars = 500 #m (A); 200 #m (C); 100/tm (B, D).
5-15min in a solution with 0.05% diaminobenzidine, 0.3 ppm nickel ammonium sulfate and 0.I ppm H202 in PB, followed by rinsing for 4 x 10 min in PB. Sections were dehydrated and mounted on glass slides with Eukitt and analysed by light microscopy. RESULTS
Routine histology Both with intracavity and intraparenchymal transplants, Sprague-Dawley hosts contained remnants of hypothalamic tissue eight weeks following transplantation, but all transplants in these hosts exhibited significant signs of rejection (Fig. 2C, D), and only small portions of the grafts were attached to the host brain tissue. Large accumulations of lymphocytes and macrophages were observed throughout the transplanted tissue (Fig. 2D). Furthermore, well developed neurons with intact dendrites and axons were rarely seen in Sprague-Dawley hosts, which instead contained cell debris, degenerating neurons and large areas of vacuolization. In the Fischer 344
hosts, on the other hand, leucocyte accumulations were rare, and all transplants, both to intraparenchyreal and intracavity sites, contained what appeared to be healthy neurons and glial cells. The intracavity transplants to Fischer 344 hosts grew less, and appeared to become less integrated with the host brain (Fig. 2A, B) than the intraparenchymal transplants (Fig. 3A-C). Even after longer time periods, the intracavity transplants never grew to fill the cavity entirely (Figs I B, 2A). The intraparenchyrnal transplantation to Fischer 344 hosts thus appeared to be the most successful site and host for transplantation. These transplants became very well integrated with host brain tissue (Fig. 3A-C), and contained tissue similar in general morphology to the ventral hypothalamus in situ.
Immunohistochemistry Histamine-immunoreactive cell bodies were found in all grafts transplanted to Fischer 344 hosts, but in only three of the 14 transplants to Sprague Dawley
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hosts. The histamine-positive cell bodies appeared to be located only in one portion of the grafted tissue, and in this portion five to 30 cell bodies were found per section. The cell bodies appeared mostly scattered throughout the neuropil (Fig. 4), or sometimes in small groups (Fig. 6A). The histamine immunoreactivity appeared evenly throughout the cell body, dendrites and axons (Figs 5A-C, 6A, B), The histamine-immunoreactive neurons contained one to three primary dendrites (Figs 5A, B, 6A, B), which branched extensively in the vicinity of the cell bodies (Figs 5A, 6B). The neurons extended varicose, thin axons (Fig. 5C) over long distances in the grafted hypothalamic tissue. Eight weeks postgrafting, the histamine-positive fibers had an uneven distribution, with parts of the grafts more densely innervated than others, while the five month transplants had a more
al.
even and dense innervation of histamine-positive neurites (Fig. 4). At eight weeks, very few or no fibers crossed the graft-host border. However, in the longterm group, sparse fibers were seen to traverse the graft-host border and enter the host brain (Fig. 7C). In the four animals which were bilaterally fimbriafornix lesioned, the non-grafted side contained no visible histamine-positive fibers in the rostral hippocampal formation or dentate gyrus (Fig. 7A). However, the host brain on the transplanted side exhibited a few histamine-immunoreactive, thin, varicose fibers extending into the CA1 as well as the dentate gyrus (Fig. 7B). Intraparenchymal as well as intracavity transplants to Fischer 344 hosts also contained numerous ADApositive cell bodies, dendrites and axons (Fig. 6C). These cells resembled the histamine-immunoreactive
Fig. 3. Routine histological staining of sections from Fischer 344 hosts carrying intraparenchymal hypothalmic transplants. (A) An overview photomicrograph with the transplant delineated by arrowheads. (B) A photomontage of another intraparenchymal transplant in a Fischer 344 host (transplant, TP). (C) A close-up from an intraparenchymal transplant (TP) and the graft-host border (arrowheads). Note that the transplanted tissue often extends along the needle tract into the cerebral cortex of the host (A, B). Scale bars = 500#m (A); 200#m (B); 100~m (C).
Fig. 4. P h o t o m o n t a g e of a parenchymal hypothalamic graft (TP). The section was incubated with antibodies against histamine. Note histamine-immunoreactive cell bodies (arrows) and dense plexi of fibers throughout the graft. The graft was examined after a host survival time of five months. Arrowheads outline the g r a f t ~ o s t border. Scale bar = 200/lm.
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cells in terms of dendritic branching and size, and were located in the same area of the transplants as the histamine-positive cell bodies on adjacent sections. TH-immunoreactive cell bodies and/or fibers could sometimes be found in transplants from donor stage El6, and particularly in cases where larger mediolateral strips of hypothalamic tissue were dissected for grafting (Fig. 6D). They were located in small groups and extended dendrites and axons, having a similar morphological appearance to those of ADA (Fig. 6C) and histamine-immunoreactive neurons (Fig. 6A, B). TH-positive neurons were rarely seen in dissections from El 7, or when only lateral hypothalamic tissue was used. The slightly larger overall size of the hypothalamic area at El7, as compared to El6, allowed for a more selective dissection of the lateral portion of this brain region (see Fig. IA). All transplants exhibited a slight gliosis at the border between graft and host tissues, as demonstrated by GFAP immunoreactivity (Fig. 7D). However, a continuous glial barrier was never seen in the graft host junction. Astrocytes in the transplants (Fig. 7D) had a distribution and density similar to that seen in the TM region in situ (shown in Fig. 7E).
DISCUSSION
The present results demonstrate survival and growth of fetal histaminergic neurons transplanted into a delayed cavity or directly into the rostral hippocampus of adult rats. While multiple signs of rejection of grafts were observed in all Sprague Dawley hosts, such signs were absent in Fischer 344 hosts, even at five months postgrafting. Histaminergic neurons with dendritic arborizations were found in all transplants in Fischer 344 hosts. Histaminepositive axons innervated the surrounding grafted hypothalamic tissue, resulting in a significant innervation density of the grafted tissue with time. Some fibers were also seen traversing the graft-host border and innervating the surrounding host brain. Transplants appeared to grow best, and become most integrated when implanted directly into the CAI region of the rostral hippocampus. It has been suggested that allografts between outbred donor and host of the same strain will become rejected shortly after transplantation.7 Previous work by us and o t h e r s t5'16'23'39 has demonstrated successful transplantation of fetal brain tissue to outbred
Fig. 5. Details of histamine immunoreactivity in three different intraparenchymal transplants to Fischer 344 hosts. Note thick branching dendrites (A) in the same transplant as depicted in Fig. 4. B and C show close-ups of histamine-positiveneurons in intraparenchymal grafts. Note the varicose, thin axons emerging from cells and extending for long distances in the tissue (C). Scale bars - 100/~m (A, C); 50/~m (B).
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Fig. 6. Small group of histamine-immunoreactive neurons in an intraparenchymal Fischer 344 graft (A), and a close-up of a histamine-immunoreactive cell body (B) with at least two primary dendrites (D) and several secondary and tertiary dendrites. C demonstrates a small group of ADA-immunoreactive neurons in an intracranial hypothalamic graft, and D demonstrates TH-positive neurons and fibers in the same transplant as in C. Scale bars = 50 #m (A, C, D); 25 #m (B).
Sprague-Dawley rats without any signs of rejection, even at long time periods after transplantation. Successful allografts to outbred hosts could result from rapid formation of a functional b l o o d - b r a i n barrier from vessels within the grafted tissue. 7 However, it is well known that the medioventral hypothalamus is part of the so-called "circumventricular organs" areas that are excluded from the normal b l o o d brain barrier, j8 Intravenous injection of horseradish peroxidase results in a transport of reaction material into the neuropil of the median eminence, 6 which is a region of the hypothalamus most likely included in the transplants in the present study. In a recent study, we have demonstrated that intraocular hypothalamic transplants develop a patchy b l o o d brain barrier, as evidenced with antibodies directed against a specific rat b l o o d - b r a i n barrier protein. 4 These previous studies could explain our findings that all transplants into the outbred S p r a g u ~ D a w l e y strain exhibited rejection signs, while the transplants into the more inbred Fischer 344 strain did not, even after five months postgrafting. We therefore suggest that an inbred strain is essential for successful transplantation of ventral hypothalamic tissue intracra-
nially, since this region seems to be susceptible to rejection, possibly due to a deficient blood-brain barrier. Histamine-immunoreactive neurons were found in all transplants in Fischer 344 hosts. In contrast, only a minority of the transplants to Sprague-Dawley hosts contained viable histamine-immunoreactive neurons. The cell bodies and processes of the grafted neurons exhibited morphological features resembling mature histaminergic neurons in situ, t2"29 in terms of staining properties and dendritic branching. A continued development of histaminergic neurons has previously been demonstrated also in hypothalamic transplants in o c u l o . 17 C o m p a r e d with the total number of histaminergic T M neurons in these nuclei in situ, 2°'44 few histaminergic neurons appeared to have survived transplantation to the intracranial cavity or hippocampal formation in the present study. This low number of surviving neurons could depend on dissection techniques or fetal donor stage. It is possible that fetal neurons from the lateral, relatively small T M subnucleus 44 were the only histaminergic neurons included in the dissections, Future experiments, with detailed evaluation of donor stage and area, as well
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as transplantation of cell suspensions, should reveal the most advantageous techniques for harvesting of fetal histaminergic neurons. The fimbria-fornix transections were found to result in a total disappearance of histamine-immunoreactive fibers from the rostral hippocampal formation. It is likely that the 60% of histamine content in the hippocampus originating in the dorsal fimbria-fornix pathway 2 is the portion that innervates the rostral hippocampus. Even though other biogenic amines, such as the noradrenergic neurons of the brainstem locus coeruleus, will completely reinnervate the deafferented hippocampus when
transplanted to a fimbria-fornix lesioned host, 5 this was not the case with the histaminergic neurons in the present study. However, the low innervation density from transplants into the surrounding host hippocampal formation is not surprising, in view of the relatively sparse histaminergic innervation of hippocampal regions in the intact animal. 29 Furthermore, the relatively low number of neurons transplanted in the present study might not have been sufficient for host brain innervation. The histaminergic neurons of the T M in the rat have been shown to contain a number of other neuroactive substances and enzyme markers, includ-
Fig. 7. Micrographs illustrating a portion of the hippocampal formation from the control side of a bilaterally fimbria-fornix lesioned rat, exhibiting no histamine-immunoreactive fibers (A), and from the transplanted side with a few histamine-immunoreactive fibers extending significant distances into the hippocampal tissue (B). In C, multiple histamine-immunoreactive fibers are seen leaving an intracranial transplant (TP), and growing into the host brain (HB). Arrows illustrate the graft-host border. D demonstrates GFAP immunoreactivity in an intracranial transplant (TP) and the host brain surrounding it (HB). Note that the astrocytes do not appear to form a dense or continuous glial barrier in the graft-host border (arrows). E demonstrates GFAP immunoreactivity in the lateral TM region in situ, Scale bars = 100/~m.
Intracranial transplantation ing G A B A , ~3 galanin 22 a n d A D A . 3°'34-36 It was f o u n d here t h a t t r a n s p l a n t e d h y p o t h a l a m i c T M n e u r o n s are able to m a i n t a i n their n o r m a l c o n t e n t of at least one o f these markers, namely A D A . 25 The presence o f A D A - i m m u n o r e a c t i v e cell bodies a n d neurites in grafts suggested a close relationship between hist a m i n e a n d A D A in grafts, but their co-localization will require c o n f i r m a t i o n by double labeling techniques. It would be o f interest to investigate whether the grafted n e u r o n s c o n t a i n o t h e r neuroactive substances n o r m a l l y present in T M neurons, such as G A B A or galanin, a n d w h e t h e r the expression o f these substances is d e p e n d e n t o n the site of host t r a n s p l a n t a t i o n , thereby reflecting m o d u l a t i o n of expression by the target of innervation. A n t i b o d i e s directed against T H indicated the degree to which d o p a m i n e r g i c n e u r o n s o f the h y p o t h a l a m i c nuclei 14 were included in the dissection. In some grafts from d o n o r stage E l 6 , when mediolateral strips were dissected, groups o f TH-positive cell bodies could be f o u n d in the grafts. TH-positive cell bodies were rarely seen in t r a n s p l a n t s of d o n o r stage E l 7 , where more lateral h y p o t h a l a m i c tissues were obtained. D e v e l o p m e n t a l studies have d e m o n -
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strated t h a t TH-positive d o p a m i n e r g i c n e u r o n s of the arcuate nucleus first a p p e a r in the midline of the h y p o t h a l a m u s at E 1 5 - E 1 6 in the rat. 9 These findings suggest t h a t inclusion of d o p a m i n e r g i c n e u r o n s in the dissection can be avoided by c h o o s i n g a m o r e lateral piece o f h y p o t h a l a m i c tissue, which can be accomplished by using fetuses o f a later fetal stage. The m a i n finding o f the present study was that histaminergic n e u r o n s could survive t r a n s p l a n t a t i o n a n d c o n t i n u e m a t u r a t i o n within intracranial hypothalamic grafts. T a k e n together, these results illustrate a useful a p p r o a c h for studies o f histaminergic n e u r o n a l plasticity a n d connectivity within the central nervous system. Acknowledgements--We thank Drs lngrid Str6mberg and John Hudson for expert technical help with the lesions and transplantation, and Ms Lorie Gottshalk for administrative assistance. Thanks are also due to Dr Doris Dahl for providing the GFAP antibody. The present work was supported by the Swedish MRC, grant 8650, and USPHS grant no. MH49661. H. Bergman was supported by scholarships from the Swedish MRC and the Blanceflor Foundation.
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