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New strategies have recently been developed where infusion of neurotrophic factors into the brain can rescue different populations of neurons. Infusion of nerve growth factor (NGF) has been used in combination with transplants of chromaffin tissue to the striatum in the rat model of Parkinson's disease as well as to patients suffering from Alzheimer's disease. In this study we have evaluated the distribution of recombinant human NGF (rhNGF) in different brain areas and evaluated morphological and electrophysiological effects after continuous infusion for 2 weeks of rhNGF (500 micrograms/ml) into the striatum of normal rats. One week after termination of rhNGF infusion, NGF levels in the infused striata were 10-fold increased while in contralateral striata normal levels were found. Extracellular recordings from striatal neurons revealed a significantly decreased spontaneous firing rate (0.76 +/- 0.07 Hz) in rats infused with rhNGF compared to vehicle-infused control animals (1.36 +/- 0.16 Hz). Local application of rhNGF during recordings showed no direct inhibitory effect of NGF on neuronal discharge rate. Immunohistochemistry, using antibodies against acetyl cholinesterase (AChE) and glial fibrillary acidic protein (GFAP), revealed a 38.7 +/- 7.0% increase in optical density of AChE immunoreactivity close to the NGF source and an increase in GFAP-positive profiles that was restricted close to the implanted dialysis fibre. In situ hybridization showed an increase in mRNAs for choline acetyltransferase, trkA, p75 and muscarinic m2 receptor in the large neurons of rhNGF-infused striatum. Messenger RNAs for m1 and m4 receptors in striatal neurons were not changed. Thus, chronic infusion of rhNGF into the striatum caused a cholinergic hyperinnervation and reduced spontaneous activity of striatal neurons.

Long-term survival of fetal ventral mesencephalic grafts implanted into dopamine-depleted rats was studied. There was a reduction in apomorphine-induced rotations, which reached a maximum 3 months post-grafting. Striatal neuronal discharge rate was increased on the intact side of the aged grafted animals when compared with young adult striatum. Ipsilateral to the lesion, proximal to the graft, where the dopamine nerve terminal density was high but still much lower than that seen on the intact side, the firing rate was significantly lower than that measured in the intact side of the aged host. In conclusion, the increased firing rate seen in striatum after dopamine depletion is normalized by ventral mesencephalic grafts and does not show the age-related increase seen in 2-year-old rats.

The search for trophic factors that can support injured dopaminergic neurons and can enhance dopaminergic graft survival and outgrowth for therapeutic uses in Parkinson's disease has lately focused on members of the transforming growth factor (TGF) beta super-family. In this paper we have studied the effects of a member of the TGB beta family, glial cell line-derived neurotrophic factor (GDNF), on immature and mature ventral mesencephalic tissue grafted to the anterior chamber of the eye. The results confirm that GDNF increases survival of TH-positive neurons and enhances TH-immunoreactive nerve fiber formation when the grafts are treated during their development. The distribution of nerve terminals is densest within the area of TH-immunoreactive neurons and at the surface of the grafts. However, there is no change in the number of calcium-binding protein (CaBP)-positive neurons, suggesting that the subpopulation of TH-positive neurons that is increased are the CaBP-negative neurons of the ventral tier of pars compacta. Terminals from those neurons form the striatal patches during normal development. When the grafts are treated with GDNF after maturation, no change in TH-positive cell survival is seen but an increase of nerve terminals is still found within the cell dense area of the graft. Potassium-evoked dopamine release, measured using in vivo chronoamperometry, revealed significantly increased extracellular overflow in transplants treated with GDNF during development. The dopamine uptake blocker nomifensine significantly increased the time for clearance of the released dopamine. These data suggest that GDNF treatment of immature grafts enhances survival of TH-positive neurons, which would have innervated the striatal patches, and also increases TH-immunoreactive nerve fiber formation and dopamine release. Furthermore, GDNF treatment of mature grafts also increases dopamine fiber formation within the TH-positive neuronal area, indicating that adult dopaminergic neurons are also responsive to this agent.

To define the importance of adoptive sensitization and duration of graft residence on transplant alloimmunization, behavioral and histochemical parameters were examined in unilaterally 6-OHDA-lesioned F344 rat hosts which received fetal ventral mesencephalic (VM) grafts from Wistar-Furth (WF) donors. In all animals which showed increased rotations after alloimmunization, increased numbers of T cell receptor (TcR) positive, CD8+ lymphocytes were detected in the grafts. In addition, an increased density of class I MHC antigens was seen in the graft and in the adjacent host brain. Lesser numbers of CD4+, CD11b+, and MHCII+ positive elements were also seen. Perivascular cuffing was often found in actively immunized animals. An increase in TcR+ and MHC class I+ elements was also seen in animals only adoptively immunized. The tyrosine hydroxylase positive graft area was also markedly reduced in actively immunized animals and the extent of reduction correlated with the number of cells used for immunization. These studies indicate that established allografts can evade rejection as long as host lymphocytes are not activated against graft alloantigens. In addition, increasing graft residence time in the host and adoptive immunization render the graft more susceptible to subsequent rejection.

In oculo transplantation provides a powerful tool to study development and gene expression of isolated brain regions. In this study we grafted striatal and mesencephalic brain tissue to the anterior eye chamber and allowed it to survive for 2 and 6 weeks. Striatal or mesencephalic pieces were either grafted alone (single grafts) or together in close connection (co-grafts). As a control normal adult untreated rats were analyzed at the striatal and hippocampal level. Using non-radioactive in situ hybridization with digoxigenin-labeled riboprobes we detected preprotachykinin-A mRNA, a neuropeptide marker for striatal neurons. We report that adult normal rats show a strong expression of preprotachykinin-A mRNA in the striatum, medial habenula and piriform cortex, verifying the specificity of the method. Mesencephalic in oculo grafts did not reveal any staining for preprotachykinin-A mRNA. In single striatal grafts only a very weak expression of preprotachykinin-A mRNA was found at both time points investigated. Co-grafts grown for 2 weeks were not different from single striatal grafts, however, when striatum was grown together with ventral mesencephalon for 6 weeks the level of preprotachykinin-A mRNA was strong and near normal adult levels. We conclude that the mesencephalic dopaminergic innervation to the striatum might be a potent stimulus to neurons expressing preprotachykinin-A mRNA.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, expresses potential effects on survival and outgrowth from dopaminergic neurons in ventral mesencephalon. In this study we have examined the expression of BDNF mRNA and its high affinity trkB receptor mRNA in the nigrostriatal system after grafting to the anterior chamber of the eye. The BDNF mRNA expression has been compared to the dopaminergic innervation of striatum as revealed by tyrosine hydroxylase (TH) immunohistochemistry and the development of D1 and D2 subtypes of the dopamine receptor mRNAs. Ventral mesencephalon and striatum anlage were either co-grafted or grafted alone and evaluated 2 weeks (immature grafts) or 6 weeks (mature grafts) after transplantation. In situ hybridization for BDNF revealed a positive signal over large neurons in the ventral mesencephalic grafts with an increased silver grain density in the mature grafts. The striatal grafts were negative for BDNF mRNA at both time points evaluated, but in situ hybridization for trkB truncated mRNA revealed increased silver grain density in both the ventral mesencephalic grafts and striatum, with a patchy appearance. The D1 and D2 mRNAs were expressed in a patchy pattern in the striatum both in single grafts and when co-implanted with ventral mesencephalon at both time points evaluated. Often the patches of D1 mRNA did not overlap with the D2 mRNA patches. TH-immunohistochemistry revealed positive neurons in all ventral mesencephalic grafts and a dense patchy innervation of the striatal co-grafts. In conclusion, the trkB truncated mRNA and the dopamine receptor mRNAs were expressed in the striatal graft independent of the contact to a ventral mesencephalic transplant and the dopaminergic input, and BDNF mRNA expression in the ventral mesencephalic transplants was independent of the contact to its striatal target.

Human foetal dorsal root ganglia were grafted in place of native lumbar dorsal root ganglia in adult rat hosts. Between 4 weeks and 4 months later, the dorsal root entry zone (DREZ) in the grafted roots showed extensive peripheral outgrowth of astrocytic processes, in contrast to the normal 'smooth' interface between the peripheral and central nervous system compartments of the DREZ. Fibres originating from the grafted neurones and approaching the DREZ changed their direction of growth and entered the spinal cord through the pia by following blood vessels, grew into the grey matter and ramified there. These findings suggest that the DREZ astrocytes in vivo are non-permissive not only to mature peripheral regenerating axons, but also to growing axons from immature neurones.

Fetal ventral mesencephalic grafts have been used as a tool to counteract the symptoms of Parkinson's disease. In this study human fetal ventral mesencephalic xenografts were implanted into the lateral ventricle of unilaterally dopamine-depleted immunosuppressed rats. Rotational behavior elicited by low doses of apomorphine, host striatal dopamine receptor binding, and mRNA levels were investigated. Rotational behavior was reduced beginning 2 months after grafting. After 4 months only a small number of rotations, lasting approximately 30 min, were recorded. Seven months after transplantation, the rotational behavior was completely eleminated. Dopamine D2 receptor binding revealed significantly increased levels in sham-operated 6-hydroxydopamine- (6-OHDA) lesioned control striata. These increased levels decreased, and although still significantly higher at 4 months, normalized at the survival time of 7 months postgrafting. Regional differences were still obvious at 7 months in the dorsolateral quadrant of dorsal striatum. Dopamine D2 receptor mRNA revealed significantly increased levels in the lateral aspects of 6-OHDA-lesioned control striata, reversing by 4 months postgrafting. The D1 receptor binding revealed a moderately reduced signal in striata of lesioned animals. After grafting, this reduction became significantly lower than that seen in the control side, with a continous decrease over time. The same pattern was detected using in situ hybridization for dopamine D1 receptor mRNA, that is, moderate decreases after dopamine depletion and a significant decrease in the dorsomedial part of dorsal striatum 7 months postgrafting. Dopamine D3 receptor binding was increased after dopamine depletion, but reversed already by 4 months postgrafting. Taken together, human ventral mesencephalic xenografts are able to completely reverse apomorphine-induced rotational behavior, provided the grafts are left in vivo for a sufficient time. The increased striatal D2 receptors are reversed after grafting, but the human xenograft further suppressed the D1 receptor subtype both at binding and at mRNA levels. There was no strict correlation in the time courses of dopamine receptor changes and reduction of rotational behavior.

Trophic factors play an important role in the development of neurons and glia. In order to study the involvement of neurotrophins in human cortical development, human fetal parietal cortical tissue, obtained after early elective abortions, was transplanted to cortical cavities in immunosuppressed rats. Using in situ hybridization it was demonstrated that nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 mRNAs are expressed in developing human cortical xenografts. We conclude that neurotrophins may play a role in human cortical development and rat-derived astroglial cells could be involved in establishing reciprocal "permissive sites".

Glial cell line-derived neurotrophic factor (GDNF), a member of the transforming growth factor-beta superfamily, promotes the survival, morphological differentiation, and high-affinity dopamine (DA) uptake of cultured nigral DA neurons. In order to test potential methodology for peptide delivery in vivo, GDNF-containing fibrin glue balls (8 micrograms/ball) were incorporated with pieces of fetal ventral mesencephalon (E15) and transplanted into the anterior chambers of sympathetically denervated adult rats. Five weeks after grafting, the numbers of TH-positive neurons and the nerve fiber density were significantly higher in the ventral mesencephalic grafts treated with GDNF-containing glue balls than in those treated with vehicle. In addition, the laminin and GFAP immunoreactivities were similar between the two groups. These data support the concept that GDNF is a potent trophic factor for DA neurons in vivo and suggest that fibrin glue may provide a unique and safe means to permit prolonged delivery of trophic molecules to CNS tissues.

Grafting of human fetal tissue fragments has been used successfully in experimental and clinical trials. The development of techniques to store human fetal tissue fragments for longer time periods would allow to establish temporary tissue banks. We dissected several human cortical tissue fragments from one fetus and tested different storage conditions (cooling, freezing, culturing). After storage, the tissue fragments were transplanted into cavities in the cortex of host rats and the volume of the surviving grafts calculated. We report that human cortical tissue fragments grafted immediately after dissection (control group) or grafted after storage for 3 h in cryopreservation medium at room temperature survived grafting and resulted in graft sizes of 102 +/- 26 mm3 and 242 +/- 210 mm3, respectively, however, statistically not different. When the human cortical tissue fragments were slowly frozen and stored for 1 wk and/or when the fragments were cultured for 1 week in culture medium using a roller tube technique, grafts did not survive under our conditions. However, when the human cortical tissue fragments were stored for 1 week at +4 degrees C in cryopreservation medium, the graft size (48 +/- 24 mm3) was reduced but statistically not different from the control group. We conclude that human cortical tissue fragments can be stored at +4 degrees C for at least 1 wk without major loss of ability to survive grafting.

In the present study the hypothesis has been tested if the dopamine releasing drug D-amphetamine via volume transmission can, at least partly, restore dopamine communication in the dopaminergically denervated neostriatum of rats. The experimental model used, has been a unilateral 6-hydroxydopamine-induced degeneration of the nigrostriatal dopamine neurons, based on nigral microinjections of this neurotoxin. Studies on c-fos like immunoreactivity after systemic D-amphetamine treatment demonstrated a wide-spread appearance of c-fos like immunoreactive neuronal nuclear profiles within the neostriatum on both the unlesioned and denervated side. In the unlesioned neostriatum a peak density of c-fos like immunoreactive profiles was found within the central part of the neostriatum, while on the denervated side the distribution pattern of c-fos like immunoreactive profiles peaked medially and gradually declined in a lateral direction. The microdialysis experiments demonstrated, after systemic d-amphetamine treatment, a marked and sustained increase of extracellular dopamine levels in the neostriatum on the unlesioned side, while no increases in the extracellular dopamine levels were observed on the dopaminergically denervated neostriatum. In the electrophysiological experiments, systemic D-amphetamine treatment produced an inhibition of the neuronal activity on the denervated side which showed a significant increase in basal discharge rate compared with the recordings obtained from the striata on the unlesioned side. The present immunocytochemical microdialysis and electrophysiological analysis provides evidence that in the unilaterally markedly dopamine depleted neostriatum with clearcut signs of dopamine receptor supersensitivity (rotational behaviour results), dopamine transmission may be partly restored via systemic D-amphetamine treatment through the release of dopamine, predominantly from the unlesioned neostriatum, which may diffuse into the cerebrospinal fluid to reach the contralateral dopaminergically denervated neostriatum.

The distribution of schizophrenia in families supports the hypothesis of heritable risk factors in schizophrenia, but there is as yet no identification of an inherited neurobiological defect. Human embryonic brain tissue fragments, derived from first trimester abortions, can be transplanted into rat hosts, where they continue neuronal development and are accessible for neurobiological investigation. Hippocampal transplants derived from three schizophrenic women and a larger series of normal women have been studied. If there are heritable neuronal defects associated with schizophrenia, a proportion of the transplants from schizophrenic women would be expected to carry these defects. The transplants from the first two schizophrenic women showed profound abnormalities in survival and growth, compared to the series of transplants from normal women. The transplants from the third schizophrenic woman showed normal growth and development, as well as typical histological and electrophysiological features. The data must be regarded as preliminary, because of the small number of subjects that have been studied. However, they are consistent with the transmission of a defect in neuronal development to some of the offspring of schizophrenic women, a possibility consistent with other studies of the pathogenesis of schizophrenia. The mechanism of the defect in development remains to be identified.

Fetal central nervous system transplants to the adult brain have been utilized to understand brain connectivity and as replacement therapy in Parkinson's disease (PD). Here we use fetal brain allografting in the rat unilaterally depleted of dopamine, a unilateral model of PD, and apomorphine-induced rotations as an index of graft functional status while peripherally manipulating the host's alloimmune status. This system allows the investigator to examine, dynamically, host-allograft interactions in the brain under differing states of alloimmunoreactivity without the need to biopsy or sacrifice the animal. In addition to this novel application, we established that brain allografts are differentially susceptible to immunologic attack depending upon the graft's duration of residence in the host brain. Increasing residence time increases graft 'rejectability' to peripheral allosensitization. Passive immunization also sensitizes the host to subsequent graft rejection. Lastly, simple host alloimmunocompetence is necessary but not sufficient to cause fetal graft 'rejection', defined as a return of apomorphine-induced rotations.

It has previously been shown that the chromaffin cells of the adrenal medulla respond to nerve growth factor (NGF) with neurite outgrowth and increased cell survival in tissue culture or after grafting. In the present study we evaluated the dose dependency in neurite outgrowth from chromaffin tissue to recombinant human NGF (rhNGF). Therefore, pieces of adrenal medullary tissue from adult rat were grafted into the anterior chamber of the eye of previously sympathectomized recepients. Survival time was 4 weeks. At grafting and at Days 7, 14, and 21 postgrafting, the eyes were injected with 5 microliters of rhNGF at concentrations of 10, 30, 60, 100, 150, and 200 micrograms/ml, or with a control solution. All grafts, including the controls, survived well and became vascularized. At the low doses of rhNGF, 10 and 30 micrograms/ml, a small area of the irides was reinnervated and the density of the nerve fiber network was low. The maximal response was obtained at 100 micrograms rhNGF/ml. Using larger concentrations of 150 and 200 micrograms rhNGF/ml, the density of the nerve fiber network did not change, but the reinnervated area of the irides was significantly decreased compared to the outgrowth seen in irides treated with 100 micrograms/ml. In conclusion, adult rat chromaffin tissue responds to rhNGF in a dose-dependent manner. However, at the highest doses used, the outgrowth area was suboptimal, although nerve fiber density was maximal. These results indicate that to obtain maximal effects, the dose of NGF is critical.

Human fetal parietal cortical tissue was transplanted to cortical cavities in immunosuppressed rats. Protoplasmic astrocytes in the human cortical grafts highly expressed human angiotensinogen mRNA as identified with 35S-labeled and digoxigenin-labeled riboprobes combined with immunohistochemistry for glial fibrillary acidic protein. Antibodies to human specific neurofilament protein 70 KD were used to characterize neurons in the graft and fiber outgrowth into the host brain. Immunohistochemistry revealed human angiotensinogen-like immunoreactivity in many small protoplasmic astrocytes and very few large neurons. These results demonstrate that human angiotensinogen mRNA and protein is synthesized in immature human glia. We assume that angiotensinogen is transformed into angiotensin peptides, which may participate in the regulation of growth processes. The results suggest that human angiotensinogen may play a role during human embryogenesis.

The human-to-rat xenograft approach offers possibilities to study aspects of primate cortex development and function without monkeys. Human fetal cortical tissue was grafted to prepared cortical cavities of immunosuppressed host rats. Fetal tissue fragments were collected after routine low-pressure vacuum aspiration abortions performed in the first trimester of gestation. Human derived neurons and human nerve fiber outgrowth were visualized by immunohistochemistry with antibodies against human neurofilament protein 70 kD (hNFP70). Ingrowth from rat host striatum or cortex into the grafts was analyzed by immunohistochemistry with antibodies against tyrosine hydroxylase. Astrocytes were evaluated by immunohistochemistry with antibodies against glial fibrillary acidic protein. The grafts grew into different sizes (1-10 mm in diameter) and contained large numbers of hNFP70-positive nerve fibers. All grafts gave rise to outgrowth of hNFP70-positive fibers into the host with partly a cortical layering; layers III and IV received a majority of the human fibers. In several cases, the graft-derived nerve fibers entered the host brain at restricted areas, while there was no crossing over of nerve fibers at the rest of the graft-host interface. Tyrosine hydroxylase-positive fibers were usually not abundant in the grafts. Interestingly, cases of massive ingrowth occurred from host striatum into the graft in a pattern suggesting "permissive sites" at the graft-host interface in the same way as outgrowth from graft to host was found. Additionally, tyrosine hydroxylase-immunoreactive fibers from host cortex were found to grow into the transplant. Glial fibrillary acidic protein immunoreactivity was increased at the interfaces between graft and host cortex or host striatum. Immunohistochemistry using antibodies against rat IgG indicated the presence of rat IgG within the grafts, and in bordering areas of host brain, possibly indicating a defective graft-host barrier. Taken together, these results show that human cortical tissue pieces grafted to cortical cavities of immunosuppressed rats survive grafting and develop, and that reciprocal nerve fiber growth between grafts and hosts occur. Human cortical neurons can grow into the rat host brain in a pattern which is partly determined by host cortical architecture.

The ability of mature dopaminergic neurons derived from ventral mesencephalon to re-initiate growth after making contact with a non-innervated target was studied using the intra-ocular grafting model. Foetal ventral mesencephalic tissue or brain stem including the locus coeruleus area was grafted to the anterior chamber of the eye. Two weeks, 6 weeks or 1 year after the first implantation, foetal striatal tissue was placed in contact with the nigral graft or grafted alone. The size of the transplants was measured through the cornea. The final size of the striatal grafts was significantly larger when placed alone than when co-grafted with 1-year-old or 6-week-old dopaminergic grafts. Striatum grafted together with 2-week-old nigra was larger than when grafted adjacent to mature substantia nigra, but not significantly so. Nerve fibre outgrowth into the iris from the nigral transplants did not increase after maturation, but the re-innervated area of the host iris progressively increased around the locus coeruleus grafts. Ingrowth of tyrosine hydroxylase (TH) immunoreactive nerve fibres into the striatal grafts was studied 6 weeks after the second implantation. TH immunohistochemistry revealed innervation of the striatal piece in all cases, except for the group where striatum alone was grafted. With the short survival time for co-grafts of 6 weeks, TH-positive nerve fibres innervated a larger volume, had a patchy appearance and the density was higher in striatum grafted to 2 week-old nigral transplants than that seen in striatal transplants grafted to mature nigral grafts. The patchy pattern of TH-immunoreactive nerve terminals was also seen in striatum co-grafted with 6-week-old or 1-year-old nigral transplants. No difference in striatal innervation volume was detected between those latter two groups. When striatum was implanted adjacent to mature ventral mesencephalon and grown together for 6 months--the longer survival time--the same dense TH-positive innervation as seen in striatum co-grafted with immature nigral tissue at the shorter survival time was found. Additionally, the nigral part of the co-grafts showed increased TH-immunoreactive nerve fibre density. In conclusion, dopaminergic neurites from mature ventral mesencephalic transplants can re-initiate growth if placed in contact with non-innervated striatal tissue. The nigral grafts do not progressively re-innervate the host iris, while locus coeruleus grafts do. The intra-ocular grafting model can be used to study the in vivo effects of trophic factors on mature dopaminergic neurons.

The ability of grafted fetal ventral mesencephalic dopaminergic (DAergic) neuroblasts to reinnervate the unilaterally DA denervated rat striatum and improve motoric asymmetry has been well documented in several laboratories. The importance of host target specificity, and catecholamine (CA) neurotransmitter species, in the ability of grafts to ameliorate rotational responses to apomorphine and to affect electrophysiological characteristics of striatal neurons has not been systematically studied. We unilaterally lesioned Sprague-Dawley rats with 6-hydroxydopamine (6-OHDA) and verified the lesions using apomorphine (0.05 mg/kg, s.c.)-induced rotational behavior. Some of the animals subsequently received, intrastriatally, either DA neuroblasts from ventral mesencephalon that normally innervate the striatum, or from arcuate nucleus that do not. Additionally, two other groups were included that received either a CAergic graft from the noradrenergic nucleus locus coeruleus or a graft of cerebral cortex, which normally projects to the striatum but does not contain CAergic neurons. Only the fetal ventral mesencephalic grafts were able to reduce apomorphine-induced rotations and normalize striatal cell firing rates; striatal cell firing rates with ventral mesencephalic grafts were 1.43 Hz +/- 0.22, with arcuate nucleus grafts were 6.03 +/- 0.73, with locus coeruleus grafts were 4.71 +/- 0.74, and with cerebral cortex grafts were 4.36 +/- 0.45. Moreover, only the ventral mesencephalic grafts produced a dense tyrosine hydroxylase (TH)-immunoreactive nerve terminal network in the striatum; in contrast, the arcuate nucleus grafts did not reinnervate the striatum. In locus coeruleus grafted striata, few very long TH-positive axons were seen. We thus conclude that target specificity and neurotransmitter type are critically important in the ability of a graft to functionally reinnervate the 6-OHDA denervated striatum.

The potential role of glial cell line-derived neurotrophic factor (GDNF) as a trophic molecule for midbrain dopamine neurons was examined using two different approaches: in situ hybridization and intraocular transplantation. The presence of mRNA for GDNF was noted in striatal and ventral limbic dopaminergic target areas in the developing (E20-P7) rat, but not the adult rat. Signals were also found in nondopaminergic areas during maturation, such as the cerebellar anlage, spinal cord, and thalamus. Lesions of the nigrostriatal pathway in neonatal or adult rats, using 6-hydroxydopamine injected into the medial forebrain bundle, did not elicit upregulation of mRNA for GDNF. Grafts of fetal ventral mesencephalon in the anterior eye chamber were exposed to repeated injections of GDNF, which elicited a marked and dose-dependent increase in transplant volume. A low (0.1 microgram/eye) and high (1 microgram/eye) dose of GDNF both led to a somewhat larger mean area of dopamine fiber outgrowth into host irides. In the transplants, cell counts of tyrosine hydroxylase (TH)-immunoreactive neurons revealed a doubling of cell numbers in the low-dose group and about four times as many cells in the high-GDNF-dose group compared to controls. Moreover, the density of TH-immunoreactive nerve fibers was markedly and significantly higher in transplants treated with the high GDNF dose. Since the volumes of these transplants were also larger, the total amount of both TH-positive cells and TH-positive nerve fibers was many-fold greater in the high-GDNF group than that in the controls. Taken together, these data support the concept that GDNF functions as a dopaminotrophic factor in vivo.

In the unilateral 6-hydroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease, controversy exists concerning the use of apomorphine- or D-amphetamine-induced rotations as reliable indicators of nigrostriatal dopamine depletion. Our objective was to evaluate which, if either, drug-induced behavior is more predictive of the extent of nigrostriatal dopamine depletion. Fischer 344 and Sprague-Dawley rats were unilaterally injected with 9 micrograms/4 microliters/4 min 6-hydroxydopamine into the medial forebrain bundle. The animals were behaviorally tested with apomorphine (0.05 mg/kg, s.c.) and D-amphetamine (5.0 mg/kg, s.c.). Following testing, the brains were removed and the right and left striata, substantia nigra and ventral tegmental area were dissected free and quickly frozen at -70 degrees C for analysis of catecholamine content by high performance liquid chromatography coupled with electrochemical detection. Our results indicate that an animal which has greater than a 90% depletion of dopamine in the striatum might not rotate substantially on apomorphine, without a concomitant depletion of > 50% of the DA content in the corresponding substantia nigra. No correlations were seen involving depletions of the ventral tegmental area and the extent of the lesions to the striatum. Submaximally lesioned (75-90% depleted) rats were found to rotate on D-amphetamine but not on apomorphine. In addition, control rats that did not receive lesions were often seen to rotate extensively on D-amphetamine. We therefore conclude that maximal lesions of the striatum and substantia nigra are required to generate rotations demonstrable with low dose apomorphine but not with D-amphetamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Members of the fibroblast growth factor (FGF) family have earlier been shown to exert potent trophic effects on cells of both the central and peripheral nervous system. The presence of FGF-1 and -2 (FGF-1, acidic FGF; FGF-2, basic FGF) has recently been demonstrated in the dopaminergic cells of substantia nigra in rat and FGF-2 has been shown to be able to increase survival and promote neurite outgrowth of cultured mesencephalic neurons. In the presence study, we have investigated possible trophic effects of FGF-1 and FGF-2 on developing rat ventral mesencephalon of different fetal stages by utilizing the in vivo method of intraocular transplantation to sympathetically denervated hosts. Survival and growth of developing grafts after growth factor treatment was followed in oculo. The Falck-Hillarp technique was used for evaluation of catecholaminergic fiber outgrowth into the host iris in whole-mount preparations. FGF-2 significantly increased the volume of the mesencephalic grafts when compared to grafts treated with the vehicle alone. The mean volume of FGF-1-treated grafts was larger than that of control grafts, but this difference was not statistically significant. FGF-1 significantly increased the area of outgrowth of dopaminergic fibers into the host iris without a corresponding increase in the number of dopaminergic neurons, as evaluated by TH immunohistochemistry. FGF-2 had no effect on dopaminergic fiber outgrowth on grafted E14 ventral mesencephalon but it did have a significant effect on fiber outgrowth from E15 and E16 grafts. Moreover, the FGF-2 treated E16 grafts contained a larger number of dopaminergic neurons as compared to controls.(ABSTRACT TRUNCATED AT 250 WORDS)

The nicotinic cholinergic antagonist alpha-bungarotoxin (alpha-BT) binds throughout the rat hippocampal formation. The binding is displaceable by d-tubocurarine. The most heavily labeled cells are GABA-containing interneurons in the dentate and in Ammon's horn. These neurons have several different morphologies and contain several neuropeptides. alpha-BT-labeled interneurons in the dentate are small cells between the granular and molecular layers that often contain neuropeptide Y. alpha-BT-labeled interneurons in CA1 are medium-sized interneurons, occasionally found in stratum pyramidale, but more often found in stratum radiatum and stratum lacunosum moleculare. These neurons often contain cholecystokinin. The largest alpha-BT-labeled interneurons are found in CA3, in both stratum radiatum and stratum lucidum. These neurons are multipolar and frequently are autofluorescent. They often contain somatostatin or cholecystokinin. These large interneurons have been found to receive medial septal innervation and may also have projections that provide inhibitory feedback directly to the medial septal nucleus. The cholinergic innervation of the hippocampus from the medial septal nucleus is under the trophic regulation of NGF and brain-derived neurotrophic factor, even in adult life. Expression of mRNA for both these factors is increased in CA3 and the dentate after intraventricular administration of alpha-BT, but not after administration of the muscarinic antagonist atropine. alpha-BT-sensitive cholinergic receptors on inhibitory interneurons may be critical to medial septal regulation of the hippocampal activity, including the habituation of response to sensory input.

Human fetal brain tissue was obtained from first-trimester elective abortions of two women who also had schizophrenia. Portions of the embryonic hippocampus or cerebral cortex were transplanted into the anterior eye chamber of immunologically compromised athymic nude rats. In this environment, embryonic brain tissue derived from normal women generally continues organotypic growth and development for many months. Although initial survival after transplantation was normal, the tissue derived from schizophrenic women manifested less robust growth. However, cells in the transplants showed typical neuronal differentiation, with development of different neuronal types, such as pyramidal cells, granule cells, and gamma-aminobutyric acid (GABA)-containing interneurons. Rhythmic electrical activity was also observed, indicative of some local synaptic organization. The presence of messenger RNA (mRNA) for brain-derived neuronotrophic factor (BDNF) was observed using in situ hybridization. The reason for the decreased rate of growth of these transplants remains unknown and the significance of the finding cannot be assessed from only two fetuses. However, these preliminary findings suggest that fetal transplants may be a useful model system for the detection of developmental pathogenic processes in the expression and transmission of schizophrenia.

Two patients with advanced Parkinson's disease were followed for 6 months before, and 18 months after, receiving stereotaxic grafts of fetal mesencephalic tissue from aborted human fetuses. Parameters studied included a series of standardized tests of movement, response to levodopa, electrophysiological recording of the motor readiness potential, and positron emission tomography (PET) with ligands based upon levodopa and upon the dopamine reuptake inhibitor nomifensine. The patients each received stereotaxic implantation of ventral mesencephalic tissue containing midbrain dopamine neurons from aborted human fetuses of 8 to 10 weeks gestational age into the caudate and putamen of one hemisphere. Throughout their 18-month course, the patients were treated with cyclosporine, azathioprine, and glucocorticoids to minimize the risk of graft rejection. There were no significant complications from the procedure, but there was also no major change in their assessment of impairment on the Hoehn and Yahr scale. However, significant changes were observed in clinical, electrophysiological, and PET measures. Changes in these parameters, apparent at 6 months postoperatively, were described in detail in a previous report. The purpose of this present report is to provide follow-up data from the subsequent year with an emphasis on longitudinal evaluation methodology. Standardized clinical testing showed a small but long-term improvement in the first of the two patients. Following the operation, she was able to walk in "off" periods, which she had not been able to do preoperatively. This improvement was accompanied by increased walking speed and reduction in the time necessary to perform a series of pronation and supination movements using both hands. Although these improvements have continued throughout the postoperative period, they have not alleviated her basic neurological impairment. The second patient showed similar improvement during the first 6 months; she then reverted to her preoperative status at the end of the 18-month follow-up period. The electrophysiological recordings were consistent with the clinical findings. Both patients had significant changes in the motor readiness (bereitschafts) potential amplitude, which was greatest 5 to 7 months postoperation. The amplitude of the potential declined subsequently for both patients, but remained significantly elevated over the preoperative baseline for patient 1. The analysis of the PET scans was somewhat compromised by technical problems in the preoperative scans. However, they are also consistent with the clinical data. In comparisons of the operated and the unoperated sides, fluoro-dopa showed increased uptake in the caudate nucleus of patient 1 at 6 months and at 13 months.(ABSTRACT TRUNCATED AT 400 WORDS)

Human fetal mesencephalic tissue was grafted to rats with unilateral lesions of the nigrostriatal pathway. The animals were immunosuppressed with cyclosporine A. Grafts were placed either into the lateral ventricle ipsilateral to the lesion or in the cingulate cortex above corpus callosum. The grafts and newly formed fibers were visualized by immunohistochemistry with antibodies against tyrosine hydroxylase (TH) and the human Thy-1 glycoprotein. TH-positive fibers covered the total volume of striatum when the graft was placed either in the ventricle or in the cortex. When the transplant was located in the ventricle, TH-positive cells migrated from the graft into host striatum. No cell migration was seen into any other areas than striatum. Cortex and septum were sparsely reinnervated by the graft, but not to a density higher than that normally seen. Globus pallidus was totally devoid of TH-positive fibers. When the graft was placed in cingulate cortex, fiber bundles penetrated through corpus callosum into either striatum, to arborize in its dorsal parts, or followed the medial side of the lateral ventricle to ventral limbic areas, where a fiber network also was formed. Human specific Thy-1-immunohistochemistry revealed positivity only on the lesioned side. These data suggest that dopamine neurons in human mesencephalic tissue, grafted to the rat brain, can migrate specifically into host striatum. Furthermore, TH-positive fiber outgrowth occurred only into dopamine denervated areas of the host, avoiding areas that are normally not innervated by nigral neurons, but also able to reach distant target cells.

Electrophysiological recordings were performed on caudate neurons in rats with dopamine (DA) depleted striatum in combination with pertussis toxin (PT) lesions. Pertussis toxin inactivates the G protein coupled to D2 receptors. DA depletions were performed by unilateral injections of 6-hydroxydopamine (6-OHDA). After the 6-OHDA lesion, rats were challenged with low doses of apomorphine. When a double peak rotational pattern was stable over repeated rotational tests, PT was injected into striatum ipsilateral to the DA depleted side. Two days after the PT injections extracellular recordings with local applications of the D1 agonist SKF 38393 and the D2 agonist N-0437 were performed. Spontaneous firing rates, measured before drug application, were elevated in animals with both 6-OHDA and 6-OHDA/PT combination of lesions. In rats with only 6-OHDA lesions, a supersensitivity to N-0437 was observed, while no significant change in response to the D1 agonist was detected. Recordings from caudate neurons in rats with a combination of 6-OHDA and PT resulted in no response to the D2 agonist. However, a subsensitivity to the D1 agonist was detected and only 60% of neurons were inhibited by SKF 38393. Taken together, these data suggest an interaction between the D1 and D2 receptors, which is revealed only after an upregulation of the D2 receptors and subsequent blockade of D2 mediated effects.

Peripheral nerve "bridges" demonstrate the ability to facilitate axonal growth and regenerate adult and fetal central nervous system tissue. The purpose of this study was to determine if co-grafted peripheral nerve tissue could enhance the ability of fetal dopamine (DA) cell transplants to reinnervate host striatum that had been denervated unilaterally. Male Fisher-344 rats were unilaterally lesioned with 6-hydroxydopamine to eliminate the nigrostriatal DA pathway. A total of 31 rats demonstrated a pattern of rotation indicative of a greater than 98% depletion in DA. Rats were kept as nongrafted controls (n = 6), grafted with sciatic nerve (PN) minces (n = 6), grafted with fetal ventral mesencephalon (VM; n = 10), or co-grafted with VM and PN minces (n = 9). All groups were then tested for changes in apomorphine-induced rotational behavior. The PN control group showed no significant differences in rotation when compared to pregrafting levels and to the lesioned nongrafted group. Both the VM-grafted group and the VM-PN co-grafted group showed significant (P less than 0.01, one-way ANOVA) decreases in rotations beginning at 1.5 weeks postgrafting. There was a progressive decrease in rotations up to 12 weeks, the last test point examined. Interestingly, the co-graft group revealed a significantly greater decrease in rotation (P less than 0.05) than the VM group beginning at 5 weeks and continuing out to the 12-week test point. Histological and immunocytochemical studies showed good survival of both PN and VM grafts. The augmented recovery could not be accounted for by increased DA cell survival or host brain DA reinnervation in the co-graft group. Taken together, these findings suggest that PN tissue enhances the ability of fetal VM grafts to reinnervate host brain.

Solid pieces of human fetal mesencephalic tissue were grafted to the lateral ventricle adjacent to dopamine-depleted striata of rats immunosuppressed with cyclosporin A. Apomorphine-induced rotations were performed before and at monthly intervals after grafting. Reductions in rotations were seen at 2 months post-grafting and these reductions progressively increased. Spontaneously active dopaminergic cells were found within the grafts using extracellular single-unit recording techniques. Recordings of striatal cells ipsilateral to the graft revealed "normal" firing rates compared to those of neurons in the control striatum. In response to the local application of the dopamine antagonist cis-flupenthixol, both the dopaminergic and striatal neurons showed dose-dependent excitations. Potassium-evoked releases of electroactive species ipsilateral to the fetal human graft, measured using high-speed in vivo electrochemistry, revealed response amplitudes that were similar to control striatum when an electrode was placed adjacent to the graft; distal to the graft the responses showed smaller amplitudes but prolonged time courses. Much greater levels of dopamine and serotonin were detected in the grafts, compared to in normal rat substantia nigra, as measured with HPLC coupled to a 16-channel electrochemical array detector. Immunocytochemical studies using antibodies against tyrosine hydroxylase (TH), revealed not only TH-positive cells within the graft, but also a few positive neurons that migrated into the host striatum. Numerous TH-immunoreactive fibers penetrated into striatum and reinnervated its total volume. Taken together, these data suggest that intraventricular graft placement may be a highly efficacious technique for studying fetal brain tissues in terms of maturation, reinnervation, and function.

Experimental studies in rodents show that beta-nerve growth factor can increase the survival, neurite outgrowth, and functional effect of grafts of adrenal chromaffin cells to the basal ganglia. We, therefore, have begun to investigate whether treatment with nerve growth factor might also increase the functional effect of autografts of adrenal medullary tissue in patients with Parkinson's disease. Previous studies have shown that stereotactic implantation of adrenal tissue pieces produces a transient functional improvement that lasts for a few months. This report describes a trial of grafting of adrenal chromaffin tissue into the putamen, supported by infusion of nerve growth factor. The patient is a 63-year-old woman with a 19-year history of Parkinson's disease, now complicated by on-off phenomena and drug-induced hyperkinesia, despite optimized medical management. The left adrenal gland was removed, and the medulla was dissected into 1- to 2-mm3 pieces in a solution containing nerve growth factor purified from mouse submandibular gland. Pieces were implanted in six tracts 3 to 4 mm from a previously placed cannula in the left putamen. Through the cannula, nerve growth factor was infused for 23 days for a total dose of 3.3 mg. Clinical assessment consisted of global ratings for rigidity and/or hypokinesia and for drug-induced hyperkinesia. Measures of gait and fine-motor control were also made. The motor readiness potential and auditory evoked potentials were recorded.(ABSTRACT TRUNCATED AT 250 WORDS)

In situ hybridization was used to study expression of beta-nerve growth factor receptor (NGF-R) mRNA in the early human fetus. In 8- to 12-week old fetuses, high labelling was found over motoneurons along the entire length of the lateral motor column. High levels of NGF-R mRNA were also seen over most developing nerve cell bodies in both the dorsomedial and ventrolateral part of the dorsal root ganglia. Lower, but clearly specific labelling was detected over a subpopulation of cells in Auerbach's plexus in the intestines. Evidence for a non-neuronal expression of NGF-R mRNA came from labelling over a subpopulation of cells in glomeruli of the kidney in a 12-week old human embryo. Myoblasts in skeletal muscle anlagen were labelled as well as cells along peripheral nerve. The widespread expression of NGF-R mRNA in the human fetus suggests that the NGF-R is important for development of a variety of different tissues of both neuronal and non-neuronal origin.

While human fetal xenografts placed into immunocompromised animal hosts have been shown to survive and grow, their ability to function and influence the host tissue has not been fully examined. Therefore, we implanted grafts of human fetal mesencephalic tissue intracranially into rats with unilateral 6-hydroxydopamine lesions of their nigrostriatal dopaminergic innervation and tested the rats behaviorally for reductions in apomorphine-induced rotations. The purpose of this study was to test the ability of these grafts to provide a functional reinnervation by comparing the behavioral changes with the morphology and presence of electrophysiologically active dopaminergic neurons within the graft and with firing rates of host striatal neurons. Adult Sprague-Dawley rats that had been unilaterally lesioned and that showed a stable two peak pattern of apomorphine-induced rotations received grafts of human fetal mesencephalic tissue placed directly into the lesioned striatum. These rats were then further tested each month for five months for reductions in their turning behavior. At 5 to 6 months postgrafting, electrophysiological recordings were made of cells within the graft and within the host striatum. The rats were then examined immunohistochemically to evaluate graft survival and extent of reinnervation of the host tissue. The rats receiving mesencephalic dopaminergic grafts demonstrated a 79% reduction in their apomorphine-induced rotations. Electrophysiological recordings revealed spontaneously active dopaminergic neurons within the graft as well as host striatal cell firing rates consistent with those of dopamine-innervated cells. Furthermore, immunohistochemical studies confirmed graft survival and revealed marked fiber outgrowth from the graft into and throughout the striatum. Taken together these findings provide evidence that grafts of human fetal mesencephalic tissue are able to produce behavioral improvements in lesioned animals which are associated with the presence of dopaminergic neurons within the graft and are consistent with normal host striatal cell activity levels.

Adrenal medullary tissue from adult rats was dissociated into cell suspensions and injected into the anterior chamber of the eye, where the cells were made to attach to the previously sympathectomized irides with the use of fibronectin. Short- and long-term survival of the chromaffin cells was examined in whole mounts of irides using Falck-Hillarp fluorescence histochemistry or indirect immunohistochemistry with antibodies against adrenaline and dopamine-beta-hydroxylase (DBH). After 6 days in oculo all cells were immunoreactive for adrenaline; almost none displayed processes even if beta-nerve growth factor (NGF) was given at grafting. One month after weekly intraocular injections of NGF, many cells were surrounded by nerve fiber networks and all cells were DBH-immunoreactive. Eight months postgrafting and 7 months after the last injection of NGF almost the entire iris was reinnervated and resembled a normal, sympathetically innervated iris. Both at 1 and 8 months, chromaffin cells, ganglion cells and transitional cell forms (chromaffin cells transforming towards ganglion-like cells) were found in irides from the NGF-treated eyes. The number of ganglion cells was remarkably increased with time by NGF, while the number of chromaffin cells decreased compared to controls. A single treatment with NGF at grafting had no marked effects as examined up to 3 months; at this time there was a certain outgrowth of nerve terminals, which, however, was not as pronounced as 1 month after repeated NGF injections. In conclusion, it is shown that some cells in a chromaffin cell suspension attach to the iris, transform to ganglion cells after an induction with exogenous NGF, and reinnervate the sympathetically denervated iris. Such cells remain ganglion-like in character and continue to form processes even after cessation of exogenous NGF treatment.

By using stereotaxic surgical techniques, ventral mesencephalic tissues from aborted human fetuses of 8 to 10 weeks' gestational age were implanted unilaterally into the striata in two patients with advanced Parkinson's disease. The patients were treated with a cyclosporine, azathioprine, and steroid regimen to minimize the risk for graft rejection. They were examined for 6 months preoperatively and 6 months postoperatively and continued to receive the same doses of antiparkinsonian medication. There were no significant postoperative complications. No major therapeutic effect from the operation was observed. However, in the clinical tests, both patients showed small but significant increases of movement speed for repeated pronation-supination, fist clenching, and foot lifting. The rate of walking also increased in the one patient tested. For both patients, there was an initial worsening postoperatively, followed by improvement vs preoperative performance at 1 to 3 months. Both patients also showed significant improvement in the magnitude of response to a single dose of levodopa (L-dopa), but there was no increase in the duration of drug action. The motor readiness potential increased in both patients postoperatively, primarily over the operated hemisphere. Neurophysiological measurements also showed a more rapid performance of simple and complex arm and hand movements on the side contralateral to transplantation in one patient at 5 months postoperatively. Positron emission tomography demonstrated no increased uptake of 6-L-(18F)-fluorodopa in the transplanted striatum at 5 and 6 months. Taken together, these results suggest that the fetal nigral implants may have provided a modest improvement in motor function, consistent with the presence of small surviving grafts. Although our results support further scientific experimentation with transplantation in Parkinson's disease, widespread clinical trials with this procedure are probably not warranted at this time.

Nerve growth factor (NGF) is the best-characterized neurotrophic substance and has recently been shown to influence cholinergic neurons in the basal forebrain. Hippocampus and neocortex, the primary targets for these central neurons, have further been found to contain high levels of NGF. Using enzyme immunoassay and acetylcholine esterase (AChE) histochemistry we have now studied the levels of NGF and AChE after a specific cholinergic lesion, transection of the fimbria fornix comprising the major cholinergic input to hippocampus. Fimbriectomy in adult rats led to a marked decrease in AChE-positive nerve terminals in both hippocampus and neocortex 10 days later, and to an increase (40%) of NGF protein concentration in hippocampus. thirty days after surgery, NGF had returned to control levels and remained there after 90 days. Removal of superior cervical ganglion did not alter the results. The density of cholinergic terminals in both hippocampus and neocortex increased with time (90 days) after fimbrial transection. The results support the idea that cholinergic neurons in the basal forebrain take up and retrogradely transport NGF from their target areas and suggest that transection of the pathway interrupts the transport thereby increasing NGF distal to the lesion. The return of NGF to control levels probably reflects reestablished cholinergic contracts in the hippocampus.

Rotational behavior was studied in rats with unilateral 6-hydroxydopamine-induced degeneration of the nigrostriatal dopamine system, following intrastriatal grafting of pieces of the adrenal medulla or nervus opticus. The rats were placed in rotometers immediately after the operation. No rotational response was seen in animals implanted with nervus opticus. Rats that received one whole adrenal medullary gland, divided into 4 pieces, showed a strong rotational response with a peak after 100 min and a duration of 400 min. Adrenal medulla-induced rotations were dose-dependent: when rats were grafted with 2, 4 or 8 pieces (where 4 pieces equals one whole adrenal medulla) into 2 sites, 2 pieces induced about half the amount of rotation as 4 pieces, while 8 pieces caused a higher total number of rotations with the 100 min peak approximately doubled compared with rats that had received 4 pieces. The coordinates for the implantation site were also important determinants of the rotational behavior: 4 implantation sites were tested and it was shown that the most central site in the caudate caused the highest total amount of rotations. The rotational behavior could be blocked in a dose-dependent manner by the dopamine-receptor antagonists haloperidol and cis-flupenthixol given immediately after grafting. It is concluded that implantation of chromaffin tissue from the adrenal medulla into the unilaterally 6-hydroxydopamine-denervated striatum causes highly reproducible acute rotational responses that vary with the amount and location of the implanted tissue and that can be blocked by neuroleptics. Thus the experiments permit screening of areas within neostriatum that produce different rotational responses to chromaffin implants.(ABSTRACT TRUNCATED AT 250 WORDS)

Rats with unilateral 6-hydroxydopamine-induced degeneration of the left nigrostriatal dopamine system were given intrastriatal implants of one cortex-free adrenal medulla divided into 4 pieces. Two pieces were placed in the center of the anterior part of the denervated caudate and two pieces in a more posterior position in lateral caudate. The distribution of catecholamines (CA) in grafts and host brain was studied 2, 100 and 400 min after grafting by HPLC-electrochemistry and Falck-Hillarp fluorescence histochemistry combined with computer-aided image analysis. Two minutes after implantation the chromaffin tissue grafts contained large amounts of adrenaline (A) and noradrenaline (NA) and small amounts of dopamine (DA). The chromaffin cells had a relatively normal fluorescence histochemical appearance. From the grafts, CA had spread into the surrounding host brain tissue where high levels of A and NA and low levels of DA were now found in the denervated host striatum. Fluorescence histochemistry and image analysis showed the CA to have spread 1-1.5 mm in all directions from the grafts. The CA concentrations decreased almost linearly with increasing distance from the grafts. At 100 min after implantation approximately a third of the chromaffin cells were still strongly fluorescent while the rest of the cells were very weakly fluorescent or non-fluorescent. The amounts of A, NA and DA in the host brain had decreased considerably, while the size of the fluorescent halo around the grafts had not diminished. At 400 min after grafting, only scattered cells in the chromaffin implants were strongly fluorescent and the surrounding host striatum contained low amounts of CA. It is concluded that intrastriatal adrenal medullary implants acutely release or leak large amounts of CA into surrounding host brain tissue. Taken together with results from the accompanying paper these data show that the grafts can maintain CA levels in host striatum high enough to elicit strong rotational responses during approximately 200 min.

Human fetal hippocampal tissue from normal women was obtained following elective abortion in the 8th to the 11th week of gestation. The hippocampal tissue was transplanted to the anterior chamber of the eye of adult athymic nude rats, where it was allowed to develop for up to 9 months before histological and electrophysiological evaluation. The transplants were revascularized from the host iris and many grew extensively in oculo. Large neurons were present in all transplants. Immunohistochemical studies revealed glutamic acid decarboxylase-containing terminals and clusters of gamma-aminobutyric acid-positive nerve cell bodies within the transplants, as well as scattered tyrosine hydroxylase-positive and acetylcholinesterase-containing fibers. Single neurons recorded extracellularly from transplants 4-9 months in oculo showed a slow spontaneous discharge, with both complex and single action potentials. Stimulation of the transplant surface evoked a small initial wave followed by a larger and longer-lasting field potential, similar to that seen in hippocampus in situ. A conditioning-testing paradigm was used to evaluate the presence of inhibitory circuitry in the hippocampal transplants. Significant suppression of the evoked test response was seen with interstimulus intervals ranging from 20 to 500 ms. Superfusion of enkephalin (100-300 nM) or penicillin (1600 U/ml) increased slow-wave activity, as did tetanic electrical stimulation. These treatments appeared to generate ictal-like activity, which in some cases persisted as interictal spikes. Illumination of the retina also increased neuronal activity, presumably by reflex activation of cholinergic afferents from the parasympathetic innervation of the iris. Taken together, our data suggest that fragments of hippocampus from aborted first trimester human fetuses, grafted to the eye chamber of rodent hosts, develop many organotypic histological and physiological features. This preparation may provide a unique means for the study of neurobiological properties of human brain in both normal and disease states.

This study evaluates the production of adrenergic nerve fibers by adrenal medullary tissue of the adult rat grafted to the anterior chamber of the eye of adult recipients. The chromaffin grafts attach to and become vascularized by the host iris. They decrease in size intraocularly during the first 3 weeks. This decrease is somewhat counteracted by sympathetic denervation of the host iris, and better counteracted by sympathetic denervation and addition of nerve growth factor (NGF, given at grafting and 1 and 2 weeks after grafting). Outgrowth of adrenergic nerve fibers from the grafts into the host iris was studied in wholemount preparations by use of the Falck-Hillarp technique 3 weeks after grafting. The innervated area of the host iris was approximately doubled in the chronically sympathectomized group and doubled again in the chronically sympathectomized NGF-supplemented group. Chronic sympathetic denervation had no effect on density of outgrowing nerves, whereas addition of NGF more than doubled nerve density. Since sympathetic denervation causes a slight elevation of NGF activity in the iris, the present experiments are taken as evidence that the level of NGF in the iris regulates formation of nerve fibers by adrenal medullary tissue grafts from adult rats.

The iris and choroid membrane of the adult rat contain nerve fibers expressing immunoreactivity to the neuropeptide galanin. The density and distribution of galanin-positive nerve fibers varied from iris to iris and, particularly, among animals. Smooth, non-terminal axons were seen running in nerve bundles consisting of otherwise negative fibers. From the choroid membrane these bundles reached the iris via the ciliary body. Axons were frequently seen to branch giving rise to a sparse system of varicose, single fibers in the dilator plate and sphincter area. Galanin-positive fibers were sometimes also seen outlining blood vessels. Capsaicin, in a dose that causes permanent depletion of substance P- and cholecystokinin-immunoreactive fibers in the iris, caused no change in amount of galanin-positive fibers. Removal of the superior cervical ganglion caused a rapid and pronounced increase in the number of galanin-immunoreactive nerve fibers. Similarly, removal of the ciliary ganglion appeared to increase galanin immunoreactivity, while removal of the pterygopalatine ganglion was less effective. Lesioning of the trigeminal ganglion caused a disappearance of galanin immunoreactivity. The sympathectomy-induced increase was counteracted by capsaicin. Galanin-positive nerve cell bodies were present in both the superior cervical and the trigeminal ganglia. In the superior cervical ganglion, immunoreactive galanin did not seem to coexist with neuropeptide Y-positive cells; in the trigeminal ganglion, some galanin-positive cells also contained calcitonin gene-related peptide (CGRP) immunoreactivity, while most cells did not. In the iris, double-staining suggested that CGRP and galanin immunoreactivities were contained in different fiber populations.(ABSTRACT TRUNCATED AT 250 WORDS)

Human fetal mesencephalic tissue obtained from elective first-trimester abortions was grafted to 6-hydroxydopamine-denervated striatum of athymic (nude) rats. After 3-6 months, the transplants were evaluated by light and electron microscopy using antibodies against tryosine hydroxylase (TH), human specific Thy-1 (Thy-1), 5-hydroxytryptamine (5-HT), and laminin. In vivo chronoamperometric studies of K+-induced release of electroactive species were done prior to the histochemical evaluations. At the light microscopical level, Thy-1-immunoreactivity was evenly distributed throughout the entire transplants. Thy-1-immunoreactive nerve fibers were observed radiating from the graft into the host striatum. In sections that were double-stained with antibodies against Thy-1 and TH, such nerve fibers contained both markers. Also 5-HT-immunoreactive cells were found in the grafts with processes both in the grafts and radiating into host neuropil. Laminin immunohistochemistry showed an even distribution of capillaries in the graft with less density than in host brain, suggesting immaturity of graft tissue. At the ultrastructural level, TH-immunoreactive axons made symmetric contacts with unlabeled dendritic shafts and dendritic spines within the host brain. A few asymmetric contacts with TH-immunoreactive axons were seen. 5-HT-immunoreactive terminals made both symmetric and asymmetric contacts with unlabeled dendritic shafts and spines. In vivo chronoamperometry using local application of K+ revealed average signals that were lower on the transplanted side than in control striatum. However, close to the grafts significant amounts of the K+-evoked signal amplitudes were as large as 1.3 microM, and the ratio of the reduction to oxidation currents suggested release of a mixture of dopamine and 5-HT. Taken together, this study shows that human fetal mesencephalic tissue pieces survive grafting into nude rats, develop normal vascularization, and express coexistence of TH- and Thy-1-immunoreactivity. Human TH- and 5-HT-immunoreactive nerve fibers form synapses in host striatum and release monoamine neurotransmitters.

Human fetal substantia nigra tissue, obtained following therapeutic termination of first trimester pregnancies, was grafted to cavities overlying the striatum in ciclosporin-treated rats whose nigrostriatal dopamine system had been removed unilaterally by 6-hydroxydopamine. Tyrosine hydroxylase (TH) immunocytochemistry revealed large numbers of surviving human substantia nigra neurons that matured and formed TH-positive nerve fibers reinnervating the host rat striatum. Apomorphine-induced rotational behavior in grafted animals was reduced by 70-80% in optimal cases 3-5 months after grafting. Thus human fetal dopamine neurons can correct functional deficits in dopamine-denervated rat hosts.