Neocortical transplants in the mammalian brain lack a blood-brain barrier to macromolecules.

In order to determine whether the blood-brain barrier was present in transplants of central nervous tissue, fetal neocortex, which already possesses blood-brain and blood-cerebrospinal fluid barriers to protein, was grafted into the undamaged fourth ventricle or directly into the neocortex of recipient rats. Horseradish peroxidase or a conjugated human immunoglobulin G-peroxidase molecule was systemically administered into the host. These proteins were detected within the cortical transplants within 2 minutes regardless of the age of the donor or postoperative time. At later times these compounds, which normally do not cross the blood-brain barrier, inundated the grafts and adjacent host brain and also entered the cerebrospinal fluid. Endogenous serum albumin detected immunocytochemically in untreated hosts had a comparable although less extensive distribution. Thus, transplants of fetal central nervous tissue have permanent barrier dysfunction, probably due to microvascular changes, and are not integrated physiologically within the host. Blood-borne compounds, either systemically administered or naturally occurring, which should never contact normal brain tissue, have direct access to these transplants and might affect neuronal function.

Circumventing the blood-brain barrier with autonomic ganglion transplants.

Superior cervical ganglia, whose vessels are fenestrated and permeable to protein tracers such as horseradish peroxidase, were transplanted to undamaged surfaces in the fourth ventricle of rat pup brains. Horseradish peroxidase, infused systemically into the host, was exuded from the graft's vessels into the graft's extracellular stroma within 1 minute. At later times the glycoprotein reached the extracellular clefts of adjacent brain tissue, the vessels of which appeared to retain their impermeability. The blood-brain barrier to horseradish peroxide was thus bypassed where the extracellular compartments of graft and brain became confluent. The graft of autonomic ganglia can serve as a portal through which peptides, hormones, and immunoglobulins may likewise enter the brain.

Permeability to blood-borne protein and 3HGABA in CNS tissue grafts. I. Intraventricular grafts.

In the present study, solid grafts of fetal CNS tissue from the rat neocortex, cerebellum, or ventral mesencephalon were placed into the lateral, III or IV ventricles of young adult hosts. Survival periods ranged from 2 days to 20 months. To study the permeability to protein and potential changes in the blood-brain barrier (BBB), macromolecules such as HRP, HRP-human serum albumin, and HRP-human IgG were administered intravascularly and circulated for periods between 3 minutes and 1 hour. Younger grafts were completely filled with the protein, even at 2 days, when the graft vasculature already contained host macrophages, whereas all older grafts showed variability in permeation with protein ingress initiating at the graft-host interface and subsequently diffusing through the extracellular spaces. Permeation was from several sources: permeable vessels of the circumventricular organs and the choroid plexus which grew into the grafts, the perivascular spaces surrounding these vessels, or from the normally impermeable vessels of the pia mater, which, because of their engulfment by the graft and subsequent angiogenesis, may have been rendered permanently leaky. Invading vessels were often "cuffed" by lymphocytic cells. Many grafts were only partially filled by the glycoprotein conjugates; ventral mesencephalic grafts allowed the least diffusion even when vascularized by choroidal vessels. Fenestrated vessels were not directly observed even though petechial leaks were evident and vessels indigenous to the CNS grafts retained BBB properties. To determine endogenous protein exudation, noninjected animals were immunocytochemically examined for rat serum albumin (RSA). The distribution of RSA mimicked that of the injected proteins at interface regions, although in most instances the entire graft was filled by a light, diffuse labeling suggesting a steady-state protein leakage over the life of the graft. When HRP was delivered intraventricularly, the intraventricular grafts were nearly filled with reaction product by 20 minutes. The depth of penetration in the grafts from the CSF interface was generally threefold greater than in normal brain. The increase in permeation suggests that solutes may flow through these grafts (out of or into the CSF) at an increased rate. Lastly the neurotransmitter tritiated gamma-aminobutyric acid (3HGABA) which does not cross the BBB was vascularly administered to hosts bearing neocortical grafts. These experiments not only confirmed the permeability in these grafts but showed that the blood-borne amino acid could be directly sequestered by grafted neurons or glia.(ABSTRACT TRUNCATED AT 400 WORDS)

Patterns of angiogenesis in neural transplant models: II. Fetal neocortical transplants.

Vascular integration between transplanted fetal CNS tissues and host brain is essential for long-term transplant survival. This study compares the time course and mechanism of vascularization in allografts of fetal cerebral cortex inserted either into the fourth ventricle or directly into the parietal cortex or hippocampus of perinatal rats. Recipient animals were administered 3H-thymidine after various postoperative time periods. The tissues were processed for light microscopic autoradiography to determine the temporal pattern of endothelial proliferation at the graft sites. Correlative electron microscopy depicted the morphological changes in transplant vasculature. Some recipients were prelabelled with 3H-thymidine prior to transplantation to determine if host vessels invaded the grafts; conversely, some donor tissues were prelabelled in utero to ascertain if the intrinsic vascular anlagen survived. Intraventricular transplants contained patent vessels, probably originating from the host pia mater, as early as 24 hours postoperative. Intraparenchymal transplants had patent vessels by 72 hours and a more complete network by 5 days. Prelabelling experiments and ultrastructural observations demonstrated that adjacent host pial vessels became incorporated into the perimeter of the intraventricular transplants and later grew centrally into the grafts. Intraparenchymal transplants also contained host vessels that exhibited a similar growth pattern. Intrinsic graft vessels remained viable and continued their development, and presumably anastomosed with the ingrowing host vasculature. Temporal labelling studies revealed that both vessel populations attained their highest proliferative rates within 72 hours after transplantation. This study demonstrates that the vasculature which develops within both intraventricular and intraparenchymal fetal CNS transplants is chimeric, consisting of intrinsic fetal vasculature and proliferating host vessels. The mechanism of transplant vascularization may be significant with regard to astrocytic, immunological, or blood-brain-barrier characteristics at these transplantation sites.

Immunocytochemical expression of the blood-brain barrier glucose transporter (GLUT-1) in neural transplants and brain wounds.

The present study examined the immunocytochemical expression of the blood-brain barrier glucose transporter (GLUT-1) in a series of fetal neocortical transplants, autonomic tissue transplants, and stab wounds to the rat brain. GLUT-1 is one of a family of different glucose transporters and is found exclusively on barrier-type endothelial cells. In the brain it is responsible for the regulated facilitative diffusion of glucose across the blood-brain barrier. This investigation is the first to determine if this important molecule is altered during the process of angiogenesis that occurs following neural transplantation procedures or direct brain injury. Beginning in late fetal brain, e.g., E18 and continuing into maturity, GLUT-1 was strongly and exclusively expressed on normal cerebral vessels. In solid fetal central nervous system (CNS) transplants up to around 3 weeks postoperative, GLUT-1 was only weakly expressed, particularly as exemplified by colloidal gold immunostaining when compared with the host. At later times examined, up to 15 months postoperative, GLUT-1 immunoexpression was comparable with the normal adjacent brain. In autonomic tissue transplants, where the vessels do not have a blood-brain barrier, as expected, GLUT-1 was not expressed. In stab wounds, at 1 week there was extensive gliosis, and the injured vessels appeared fragmented and collapsed but still expressed GLUT-1, although to a somewhat lesser extent than normal brain. Between 3 and 6 weeks, GLUT-1 was expressed on tortuous vessels and in apparently fibrillar processes in the wound vicinity with a similar pattern to astrocyte (GFAP) reactivity. These results suggest the occurrence of a down-regulation of GLUT-1 in early transplants, perhaps related to reduced glycolytic activity or transient ischemia, or possibly due to the utilization of alternative energy sources. That GLUT-1 expression was not entirely lost in stab wounds to the mature brain suggests that the protein may be more labile in fetal or perinatal brain than in the adult and may not be affected by direct injury. Coupled with previous transplantation studies that have shown reduced neuronal glycolysis and potential barrier alterations, the reduction of GLUT-1 activity within nearly the identical time frame could indicate a relatively early critical period in cellular metabolism following transplantation of CNS tissue.

Patterns of angiogenesis in neural transplant models: I. Autonomic tissue transplants.

Functional vascular connections must form rapidly to prevent ischemic damage to grafted neural tissues. The temporal sequence by which transplant circulation is re-established provides information about the angiogenic capacity of either intact or damaged CNS blood vessels. This study compares the time course and mechanism of vascular reperfusion in allografts of superior cervical ganglia or adrenal medulla inserted either into the fourth ventricle or directly into the parietal cortex of perinatal rats. Tritiated thymidine was administered to recipients to determine angiogenic patterns at various postoperative time periods. After processing for light microscopic autoradiography, host and graft endothelial labelling indices were determined in order to establish the temporal sequence and location of vascular proliferation. Correlative electron microscopy depicted the morphological changes in transplant vasculature. Some recipients were prelabelled with 3H thymidine prior to transplantation to determine if host vessels invaded the grafts. Intraventricular graft vessels initially collapsed but sustained minimal ischemic damage and were completely reperfused by 24 hours postoperative. Adjacent intact host vessels attained peak 3H thymidine incorporation at 20 hours. Intrinsic graft vessels were radioactively labelled only after 48 hours. Intraparenchymal transplants surrounded by minimal trauma exhibited a similar temporal sequence of reperfusion and host endothelial proliferation. Intrinsic graft vessels in intraparenchymal grafts sustained more severe damage. With increased trauma, a concomitant delay in graft reperfusion time was observed. Grafts within prelabelled hosts rarely contained any labelled endothelium, indicating that anastomotic connections were made between original, intrinsic graft vessels and nearby host vascular sprouts. This study demonstrates that mature autonomic tissue stimulates the growth of adjacent host vessels when transplanted to undamaged brain surfaces. The anastomosis of nascent host vessels with pre-existing graft vessels is responsible for the rapid re-establishment of circulation within the transplants. A similar mechanism occurs within intraparenchymal grafts, although the rapidity of reperfusion appears to be predicated on the amount of trauma present at the graft site.

Alterations of the blood-brain barrier after transplantation of autonomic ganglia into the mammalian central nervous system.

Autonomic (superior cervical) ganglia, the vessels of which are freely permeable to macromolecules, from mature rat donors (allografts or autografts) were transplanted to different sites in the central nervous system (CNS). Minimal trauma was caused by grafts into the IVth ventricle while grafts to intraparenchymal locations such as cerebral cortex and spinal cord were necessarily traumatic and produced glial scarring. Postoperative periods were between 4 weeks and 30 months. A potentially significant aspect of neural transplantation is the functional vascular connections between host and graft. It is highly likely that grafting procedures alter the blood-brain barrier (BBB) in the recipient brain. In order to determine permanent BBB changes, the glycoprotein horseradish peroxidase (HRP) (M.W. 40,000) was injected intravascularly for circulation periods ranging between 50 seconds and 90 minutes. Protein exudation was monitored by using the chromogens DAB and the highly sensitive TMB. All autonomic ganglia transplants, regardless of postoperative or HRP circulation times, were permeable to the injected protein; no qualitative differences were found between allografts and autografts. The blood-borne protein traversed the autonomic graft and infiltrated into the host brain for distances between 200 micron in intraparenchymal grafts to over 1 mm in intraventricular grafts; a smaller exudate was found in the intraparenchymal model than in the intraventricular site probably due to glial scarring that impeded the protein movement in the interstitial spaces. Significantly, TMB demonstrated that the systemic protein entered the cerebrospinal fluid. HRP was detected on the ventricular floor and in the perivascular spaces of the microvasculature. Transplantation of an autonomic ganglion into the brain provides a biological portal that bypasses normal barriers to macromolecules. The vascular and extracellular confluences between host and graft could provide direct access for systematically administered substances to enter brain regions where they, normally, would be excluded.

Electron microscopic observations of nodes of Ranvier in the external cuneate nucleus.

During the course of an investigation of the synaptic organization of the external cuneate nucleus (ECN) in the Sprague-Dawley albino rat, the ultrastructural morphology of nodes of Ranvier in the neuropil has been studied. The majority of nodes observed have the basic morphological features of conventional central nodes but there is individual variation with regard to length, surface area and cytoplasmic organelles. In addition, nodes with multiple myelinated branches are observed. Some nodes of Ranvier were observed to form specialized synaptic boutons. Two types of nodal synaptic boutons were present; a simple type and a complex type. Simple nodal boutons were observed more frequently. These nodes usually synapse upon a single dendritic element; the portion of the node opposite the presynaptic area has a morphology similar to conventional nodes. Complex nodal boutons are of greater dimension than simple nodal boutons and are usually in contact with several neuronal elements. They may be presynaptic to dendritic shafts or spines and are occasionally observed to be postsynaptic to small axonic profiles, a synaptic relationship which, until this report, has not been demonstrated in the central nervous system (CNS). The possible functional significance of these observations is discussed.

Synaptic organization of the external cuneate nucleus in the rat.

The normal synaptic organization of the rat external cuneate nucleus (ECN) has been investigated. The characteristic feature of the ECN neuropil is complex synaptic arrangements termed synaptic glomeruli. These involve both axo-dendritic and axo-axonic synapses and usually are isolated from the surrounding neuropil by lamellar glial sheaths. Glial isolation of synaptic glomeruli is conspicuous near capillaries. Central glomerular bouton profiles vary in size (2-9 micron diam) and configuration. They form asymmetrical synapses onto dendritic shafts and spines and occasionally receive symmetrical synapses from en passant bouton profiles. Other forms of axo-dendritic synapses not associated with synaptic glomeruli are observed. The type frequently observed is formed by one or more boutons (0.5 4.0 micron diam) synapsing onto a dendrite or dendritic spine. "Isolated" axo-dendritic synapses are surrounded by glial lamellae. Elongated "giant" boutons make multiple synaptic contacts along a dendritic shaft. Serial axo-axonic synapses are found mainly in caudal regions of the nucleus. Axo-somatic synapses are formed by small boutons (0.3.2.0 micron diam) contacting medium (16-24 micron) and small (9-14 micron) neurons. The least frequently observed bouton type in the rat ECN contains numerous granular vesicles, 80-100 nm in diameter. These boutons may contact dendrites, neuronal somas or other boutons.

Patterns of degeneration in the external cuneate nucleus after multiple dorsal rhizotomies.

Unilateral, intradural dorsal rhizotomies (C3-Cs) were performed on adult rats to study the patterns of synaptic organization of ascending dorsal root fibers in the external cuneate nucleus (ECN). Animals were permitted to survive for periods of time ranging from 3 hours to 12 days. Sham-operated animals presented a morphology indistinguishable from that of normal, unoperated animals. In rhizotomized animals, degeneration was observed ipsilaterally at all survival periods. After postoperative survivals of 3 to 14 hours some terminal boutons displayed clumping and diminution in numbers of synaptic vesicles and, in addition, degeneration myelinated axons were observed at this time. There was considerable degeneration in the neuropil between 24 and 48 hours postoperative. Two forms of degeneration occurred in axons and terminal boutons with comparable frequency: electron lucent degeneration and electron opaque degeneration. Reactive phagocytic glial cells contained degenerated masses, lipoid droplets, lysosome-like structures and myelin fragments. After postoperative survivals of four to six days, lucent and opaque degenerating terminals were less numerous. Neurofilamentous degeneration was observed only occasionally. Unaltered synaptic membrane specializations were present and were usually abutted by glia. At 12 days postoperative, synaptic glomeruli and serial synapses were not seen. Invaginating dendritic spines were rarely seen. Bouton populations that remained unualtered were: small (0.3-3.0 micron) boutons that contact dendritic shafts and somata, nodal synaptic boutons and boutons containing granular vesicles (80-100 nm).

Functional fetal nigral grafts in a patient with Parkinson's disease: chemoanatomic, ultrastructural, and metabolic studies.

A patient with Parkinson's disease received bilateral fetal human nigral implants from six donors aged 6.5 to 9 weeks post-conception. Eighteen months following a post-operative clinical course characterized by marked improvement in clinical function, this patient died from events unrelated to the grafting procedure. Post-mortem histological analyses revealed the presence of viable grafts in all 12 implant sites, each containing a heterogeneous population of neurons and glia. Approximately 210,146 implanted tyrosine hydroxylase-immunoreactive (TH-ir) neurons were found. A greater number of TH-ir grafted neurons were observed in the right (128,162) than the left (81,905) putamen. Grafted TH-ir neurons were organized in an organotypic fashion. These cells provided extensive TH-ir and dopamine transporter-ir innervation to the host striatum which occurred in a patch-matrix fashion. Quantitative evaluations revealed that fetal nigral grafts reinnervated 53% and 28% of the post-commissural putamen on the right and left side, respectively. Grafts on the left side innervated a lesser area of the striatum, but optical density measurements were similar on both sides. There was no evidence that the implants induced sprouting of host TH-ir systems. Electron microscopic analyses revealed axo-dendritic and occasional axo-axonic synapses between graft and host. In contrast, axo-somatic synapses were not observed. In situ hybridization for TH mRNA revealed intensely hybridized grafted neurons which far exceeded TH mRNA expression within residual host nigral cells. In addition, gamma-amino butyric acid (GABA)-ergic neurons were observed within the graft that formed a dense local neuropil which was confined to the implant site. Serotonergic neurons were not observed within the graft. Cytochrome oxidase activity was increased bilaterally within the grafted post-commissural putamen, suggesting increased metabolic activity. In this regard, a doubling of cytochrome oxidase activity was observed within the grafted post-commissural putamen bilaterally relative to the non-grafted anterior putamen. The grafts were hypovascular relative to the surrounding striatum and host substantia nigra. Blood vessels within the graft stained intensely for GLUT-1, suggesting that this marker of blood--brain barrier function is present within human nigral allografts. Taken together, these data indicate that fetal nigral neurons can survive transplantation, functionally reinnervate the host putamen, establish synaptic contacts with host neurons, and sustain many of the morphological and functional characteristics of normal nigral neurons following grafting into a patient with PD.

Grafts of EGF-responsive neural stem cells derived from GFAP-hNGF transgenic mice: trophic and tropic effects in a rodent model of Huntington's disease.

The present study examined whether implants of epidermal growth factor (EGF)-responsive stems cells derived from transgenic mice in which the glial fibrillary acid protein (GFAP) promoter directs the expression of human nerve growth factor (hNGF) could prevent the degeneration of striatal neurons in a rodent model of Huntington's disease (HD). Rats received intrastriatal transplants of GFAP-hNGF stem cells or control stem cells followed 9 days later by an intrastriatal injection of quinolinic acid (QA). Nissl stains revealed large striatal lesions in rats receiving control grafts, which, on average, encompassed 12.78 mm3. The size of the lesion was significantly reduced (1.92 mm3) in rats receiving lesions and GFAP-hNGF transplants. Rats receiving QA lesions and GFAP-hNGF-secreting grafts stem cell grafts displayed a sparing of striatal neurons immunoreactive (ir) for glutamic acid decarboxylase, choline acetyltransferase, and neurons histochemically positive for nicotinamide adenosine diphosphate. Intrastriatal GFAP-hNGF-secreting implants also induced a robust sprouting of cholinergic fibers from subjacent basal forebrain neurons. The lesioned striatum in control-grafted animals displayed numerous p75 neurotrophin-ir (p75NTR) astrocytes, which enveloped host vasculature. In rats receiving GFAP-hNGF-secreting stem cell grafts, the astroglial staining pattern was absent. By using a mouse-specific probe, stem cells were identified in all animals. These data indicate that cellular delivery of hNGF by genetic modification of stem cells can prevent the degeneration of vulnerable striatal neural populations, including those destined to die in a rodent model of HD, and supports the emerging concept that this technology may be a valuable therapeutic strategy for patients suffering from this disease.

Angiogenic and astroglial responses to vascular endothelial growth factor administration in adult rat brain.

The effects of exogenous vascular endothelial growth factor (VEGF) on angiogenesis, blood-brain barrier permeability and astroglial proliferation in the adult rat CNS in situ were investigated. Recombinant human VEGF(165) (25 or 50 ng/ml) was delivered for up to 1 week using either intracerebral osmotic minipumps or less traumatic subdural gelatin sponge placement. By 3 days, VEGF delivery caused significantly increased cerebral angiogenesis (25 ng/ml was most effective) in both experimental models when compared to saline controls; VEGF infusion resulted in a 100% increase in an index of vascular proliferation, and gelatin sponge delivery produced a 65% increase. The blood-brain barrier hallmark endothelial glucose transporter-1 was not present in nascent vascular sprouts. Infusion of VEGF produced extensive protein leakage that persisted after saline-induced permeability was mostly resolved, while gelatin sponge administration caused milder barrier dysfunction. Administration of the angiogenic factor had unexpected proliferative effects on astroglia in both models, resulting in an 80-85% increase in mitotically active astroglia when compared to controls. Immunohistochemical results and semi-quantitative reverse transcriptase-polymerase chain reaction indicated that the VEGF receptors flk-1 and flt-1 were up-regulated in response to the infusion trauma; flt-1 was localized to reactive astroglia, while flk-1 was expressed in vascular endothelium but predominantly in neuronal somata and processes adjacent to the delivery site. mRNA for the VEGF(121), VEGF(165) and VEGF(188) isoforms was also increased after delivery of the recombinant protein. These data show that VEGF application has substantial proliferative effects on CNS endothelium and astroglia and causes up-regulation of its own message. Flt-1 and flk-1 receptor mRNAs and proteins are up-regulated in both vascular and non-vascular cell types following infusion trauma. From these results we suggest that administered VEGF has heretofore unanticipated pleiotrophic effects in the adult CNS.

Vascular, glial and neuronal effects of vascular endothelial growth factor in mesencephalic explant cultures.

Vascular endothelial growth factor is a highly conserved, heparin-binding protein which mediates a number of critical developmental processes in both vertebrates and invertebrates, including angiogenesis, vasculogenesis and hematopoiesis. We employed an organotypic rat explant model (produced from embryonic day 17 fetuses) to assess the effects of vascular endothelial growth factor on brain microvasculature in general and the ventral midbrain specifically. Immunohistochemistry using antisera to rat endothelial cell antigen and laminin demonstrated a robust, dose-dependent effect of vascular endothelial growth factor, resulting in increased vessel neogenesis, branching and lumen size by three days in vitro. This effect was blocked by addition of an anti-vascular endothelial growth factor antibody. At higher doses of vascular endothelial growth factor, the effect was attenuated, though a statistically significant increase in both astrocyte, and neuronal density was observed using antisera to glial and neuronal markers. Tyrosine hydroxylase-immunoreactive (i.e. dopaminergic) neurons, particularly, exhibited increased survival in response to vascular endothelial growth factor application. Vascular endothelial growth factor had a mitogenic effect on endothelial cells and astrocytes, but not dopaminergic neurons, as demonstrated by the addition of [3H]thymidine to the cultures 2 h after the cultures were established. Similarly, results of a radioreceptor assay indicated that specific vascular endothelial growth factor binding sites were present on blood vessels and astrocytes, and were up-regulated by exposure to vascular endothelial growth factor. We conclude that, in explants of the ventral mesencephalon, exogenously applied vascular endothelial growth factor is mitogenic for endothelial cells and astrocytes, and promotes growth/survival of neurons in general and dopaminergic neurons in particular.

An innovative coating technique for light and electron microscopic autoradiography.

We describe a modified nuclear emulsion coating technique for both electron and light microscopic autoradiography. We propose that by reversing the application of formvar film so that it adheres to and covers thin sections placed on grids, we have developed a technically accessible methodology that produces optimal conditions for the tracing of specific nuclear activity. A smooth, continuous base is formed over the sections on which a monolayer of evenly packed silver halide crystals can be applied by dip-coating. The same principle is applied to pre-stained 1-micron plastic sections of glass slides. We suggest that the application of formvar film over thin sections does not impede or interfere with the exposure of the emulsion by the labeled tissue. On the contrary, it virtually eliminates contamination and background radiation, enhancing the specificity and quality of resolution at even low magnifications. This technical modification, which facilitates the application of the emulsion, could render electron microscopic autoradiography a routine laboratory procedure, allowing for easily reproducible results and quantitative evaluation. At the light microscopic level, this technique prevents chemical fogging caused by certain stains, and thus allows routine pre-staining before coating with emulsion.

Localization of receptors for bombesin-like peptides in the rat brain.

BN-like peptides and receptors are present in discrete areas of the mammalian brain. By radioimmunoassay, endogenous BN/GRP, neuromedin B, and ranatensin-like peptides are present in the rat brain. High-to-moderate concentrations of BN/GRP are present in the rat hypothalamus and thalamus, whereas moderate-to-high densities of neuromedin B and ranatensin-like peptides are present in the olfactory bulb and hippocampus, as well as in the hypothalamus and thalamus. While the distribution of neuromedin B and ranatensin-like peptides appears similar, it is distinct from that of BN/GRP. When released from CNS neurons, these peptides may interact with receptors for BN-like peptides. BN, GRP, ranatensin, and neuromedin B inhibit specific [125I-Tyr4]BN binding with high affinity. By use of in vitro autoradiographic techniques to detect binding of [125I-Tyr4]BN to receptors for BN-like peptides, high grain densities were found in the olfactory bulb and tubercle, the nucleus accumbens, the suprachiasmatic and paraventricular nucleus of the hypothalamus, the central medial and paraventricular thalamic nuclei, the hippocampus, the dentate gyrus, and the amygdala of the rat brain. Some of these receptors may be biologically active and mediate the biological effects of BN-like peptides. For example, when BN is directly injected into the nucleus accumbens, pronounced grooming results and the effects caused by BN are reversed by spantide and [D-Phe12]BN. Thus, the putative BN receptor antagonists may serve as useful agents to investigate the biological significance of BN-like peptides in the CNS.

Diminished expression of microtubule-associated protein (MAP-2) and beta-tubulin as a putative marker for ischemic injury in neocortical transplants.

The present study examined the immunoexpression of the neuronal cytoskeletal proteins, MAP-2 and beta-tubulin within a timed series of rat fetal neocortical transplants. beta-tubulin is a major component of microtubules and MAP-2 regulates the assembly and stability of neuronal microtubules and is a major site for the phosphorylation cAMP dependent protein kinase in neurons. Both proteins are strongly expressed in the soma and dendrites of normal neurons. MAP-2 has been shown to be a sensitive marker for ischemia in neurons and is downregulated in this form of injury. Immunoexpression of both MAP-2 and beta-tubulin in grafted cortical neurons was markedly reduced when compared to age-matched or even perinatal specimens at all post-operative times. Dendritic staining was confined to random, thin processes with no laminar patterns and staining within somata was very weak. In some specimens, somatic expression was increased and dendrites were more robustly stained when a portion of the graft was juxtaposed to a fiber tract even though in other regions of the same graft there was very weak immunostaining. The present results corroborate previous studies of cortical transplants indicating an immature structure and metabolism, and it is suggested here that the primary factor is a sublethal form of ischemic injury. Another possibility for the relative paucity of cytoskeletal protein expression could be that transplanted neurons undergo a new developmental scheme (neodevelopment) that is brought about by truncated migration patterns and abnormal synaptic connections.

Cytoskeletal protein immunoexpression in fetal neural grafts: distribution of phosphorylated and nonphosphorylated neurofilament protein and microtubule-associated protein 2 (MAP-2).

The present study examined the immunocytochemical expression of important cytoskeletal proteins within the neurons of an extended series of neocortical grafts and smaller group of ventral mesencephalic (nigral) grafts. Using antibodies that were directed at all three neurofilament (NF) epitopes, NF-L, NF-M, and NF-H, we attempted to determine whether these neurons would have an altered cytoskeletal profile following the stress of transplantation, because previous studies have shown such changes following ischemia or direct brain injury. We studied phosphorylated NF protein, which is found predominantly in axons, nonphosphorylated NF protein, which is found predominantly in the somata-dendritic compartment, and MAP-2, a specific microtubule marker that is localized exclusively in the somato-dendritic compartment. The results show that in all neocortical grafts examined, both phosphorylated and nonphosphorylated NF immunoexpression was significantly downregulated and appeared only in relatively few axons and somatic profiles, respectively, even though there were numerous Nissl-stained neuronal profiles in the grafts. There was no particular pattern to the immunopositive profiles. At later times occasional neuronal profiles were positive for phosphorylated NF protein, suggesting a reaction to cellular injury. In contrast to neocortical grafts, the cytoskeletal profiles of MAP-2 and phosphorylated NF protein in nigral grafts appeared very similar to age-matched control although the nonphosphorylated NF protein expression did appear somewhat lessened at 1-2 mo postoperative. Because cytoskeletal proteins play important roles in neuronal size, shape, and structural stability, they may subserve key cellular issues in neural grafting. These results show a significant loss of cytoskeletal protein expression in neocortical grafts that does not occur in nigral grafts. These results suggest that fetal neurons from different brain regions (i.e., graft source) may respond differently to the grafting procedure insofar as their cytoskeletal makeup is concerned. In addition, a potential lack of appropriate growth substrates or synaptic contacts may also produce cytoskeletal alterations. As such, the cytoskeletal protein profiles in central nervous system (CNS) grafts may be useful markers for functional performance, perhaps reflecting a degree of cellular injury.

Developmental expression of calcium-binding protein-containing neurons in neocortical transplants.

The present study examined the development of calcium binding protein-containing neurons in a timed series of fetal neocortical transplants. The immunoexpression of parvalbumin and calbindin, which are subpopulations of GABAergic neurons, have been widely studied in normal development and in disease and injury states. Because of their purported resistance to oxidative injury by their ability to buffer Ca++ influx, these neurons have been particularly studied following ischemia. Because it is likely that oxidative stress is associated with the grafting procedure, we sought to determine if these neurons displayed enhanced survival characteristics. Normally, parvalbumin and calbindin represent about 5-10% of cortical neurons. Within 2-4 wk after grafting the expression of both proteins increased markedly in that a relatively larger number of neurons (27% for parvalbumin) were immunopositive. This increase was transitory, however, and by 4 mo and beyond, confocal microscopic data showed a reduction of over 50% of parvalbumin (+) neurons and processes. Calbindin (+) processes showed a qualitative change in that they were smaller with less terminal branching. Electron microscopy confirmed a substantial reduction in parvalbumin synaptic contacts. Interestingly, in older grafts, remaining parvalbumin neurons were those that were strongly NSE (+) suggesting a link between normal metabolism and Ca++ buffering in grafted neurons. It is possible that in early grafts certain neuronal populations transiently upregulated calcium binding proteins as a defensive mechanism against Ca++ influx associated with oxidative stress. Over time, however, following physiological normalization within grafts, the calcium binding protein (+) neurons are diminished, possibly due to lack of appropriate afferent input to the interneuronal pool.

An improved autoradiographic coating technique for neurohistopathological stains and electron microscopy.

We describe modifications and improvements to our first report of a new nuclear emulsion coating technique for both light and electron microscopic autoradiography. Although this technique was originally designed to facilitate electron microscopic autoradiography, the methodology also allows pre-staining of plastic-embedded tissue sections prior to coating the slides with nuclear emulsion for light microscopic autoradiography. We now demonstrate that paraffin sections can be autoradiographically processed after being subjected to a combination of immunocytochemical reactions and special neuroanatomical strains. Parlodion film has been found to be more resistant to temperature changes and less prone to contamination than Formvar film. The shape of the double adhesive tape is an important aspect of the electron microscopic technique; it has been modified to minimize contamination and facilitate the removal of the grids from the glass slide. These technical adjustments facilitate the application of the nuclear emulsion and increase radionuclide specificity, thus expanding the investigative horizons of light and electron microscopic autoradiography.