Nerve growth factor-induced excitation of selected neurons in the brain which is blocked by a low-affinity receptor antibody.

We have investigated the electrophysiological effects of nerve growth factor (NGF) on single-neuron activity in central nervous system (CNS) grafts of septum, spinal cord, and hippocampus in oculo. NGF was found to have slow-onset, long-lasting excitatory effects on the spontaneous firing of neurons in septal grafts, while no such effects were found in neurons of either hippocampal or spinal cord grafts. Pretreatment with an antibody against the p75 low-affinity NGF receptor blocked the NGF-induced excitations. A second NGF application caused much stronger excitatory responses in sensitive neurons. Our data suggest that forebrain cholinergic neurons may be selectively sensitive to NGF also at the neurophysiological level, responding by excitations, and that NGF upregulates these responses within less than an hour.

The nerve growth factor receptor gene is expressed in both neuronal and non-neuronal tissues in the human fetus.

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.

Spatial distributions of cytoskeletal proteins and the nerve growth factor receptor in septal transplants in oculo: protection from abnormal immunoreactivity by hippocampal co-grafts.

Intraocular grafts of embryonic rat septum and co-grafts of septum plus hippocampus were studied with immunohistochemical markers after one and six months (short term) and 12 months (long term) of survival. Neurons in all the septal tissues expressed the epitope for the rat beta-nerve growth factor receptor in sections reacted with the monoclonal antibody 192-IgG. Stained fibers traversed the interface of the short and long term co-grafts and 192-IgG-positive processes were most prominent in the septum when combined with the hippocampal formation. In contrast, labeled processes were sparse and the perikarya of positive neurons appeared shrunken in the long term single septal transplants. Axon and dendrite profiles in the grafts were examined with antibodies that recognize the phosphorylated heavy neurofilament unit (RT97) and the high molecular weight microtubule-associated protein termed MAP 2, respectively. In the short term single and double grafts, characteristic arrays of RT97-positive processes defined the tissues and axonal tracts connecting the septum with the hippocampus. Typical immunostaining of the neuronal somas and the dendrite arbors were were outlined with the MAP 2 antibody. After one year in oculo, extensive changes in the patterns of axonal and dendritic immunoreactivity were noted in the isolated septal grafts. Abnormalities identified with the RT97 antibody included hypertrophied axons, short fragments of kinked axons and neurofilaments in the neuronal perikarya. The formation of circular "abnormal fiber aggregates" composed of densely packed abnormal and normal axonal processes were also distinctive in only the long term single septal transplants. In addition, a reduction in the density of dendrites and the presence of truncated arbors stained with the MAP 2 antibody suggested that regression of the dendrites had occurred. These spatial modifications in axonal and dendritic staining were not present in the septal portion of the combined preparations. In astrocytes, an increase in the antigenicity to glial fibrillary acidic protein paralleled the age of the transplant and was most extensive in the septal grafts. The results illustrate that intraocular co-grafts of hippocampus protect septal neurons and glial cells from abnormal changes in immunoreactivity to antibodies directed against cytoskeletal proteins and exemplify the long term supportive effects of the hippocampus on the morphology of septal neurons, including neurons that express the receptor for nerve growth factor.

Electrophysiologic effects of ethanol in human brain xenografts in oculo: antagonism by Ro15-4513.

Human cortex cerebri and cerebelli xenografts from first-trimester fetal tissue fragments were used to study the effects of ethanol on single human central neurons. Transplants were placed into the anterior eye chamber of athymic nude rats and allowed to develop for 3 to 11 months. Immunohistologic analysis revealed graft structures that stained positively for a number of neuronal, transmitter-related, glial and vascular markers. Superfusion of ethanol (EtOH) elicited a reversible and dose-dependent depression of action potential discharge. At least two populations of neurons could be identified--a more sensitive group with an EC50 of 3.0 mM and a less sensitive group with an EC50 of 22.4 mM. These EtOH levels are within the range eliciting behavioral signs of intoxication in humans. EtOH-induced depressions could be antagonized by administration of the benzodiazepine inverse agonist Ro 15-4513. This study represents the first demonstration, to our knowledge, of the electrophysiologic actions of EtOH on single neurons from human brain, and provides dose-response data collected with known concentrations of EtOH as well as evidence for the blockade of these EtOH effects by the Roche compound.

Emulation of seizure induced brain damage in neural tissue transplants to the anterior chamber of the eye.

We have developed a model system in which the mechanisms of neuronal damage due to hyperexcitation can be studied in isolation and where extended observation periods can be used. Substantia nigra pars reticulata (SNPR) develops a hypermetabolic necrosis following status epilepticus (Nevander et al. 1985; Auer et al. 1986). We transplanted rat fetal nigral area alone or together with fetal frontal neocortex to the anterior chamber of the eye in adult rats. Following 3 months of transplant maturation the hosts were subjected to status epilepticus for 60 min. In single nigral transplants no sign of structural damage was found. In the double transplants of frontal cortex and the substantia nigra a tissue necrosis had developed in the nigral part. This was demonstrated by a total loss of glial fibrillary acidic protein (GFA) immunoreactivity within a circumscribed necrotic region in the nigral part of the double transplant. Such a loss of GFA immunofluorescence had also developed in the host SNPR, as we have earlier shown (Eriksdotter-Nilsson et al. 1987). Thus, intraocular brain tissue transplants provide a unique model for studies on the development of neuronal damage and functional dependence between different neuronal structures for the development of such damage.

Structural and neurochemical effects in mouse cerebellum following neonatal methylazoxymethanol and 6-hydroxydopamine treatment.

The effects of neonatal treatment with the antimitotic agent methylazoxymethanol and the catecholamine neurotoxin 6-hydroxydopamine on cerebellar morphology and monoamine innervation in the N.M.R.I. mouse has been studied. Methylazoxymethanol (25 mg/kg s.c.) treatment induced a cerebellar weight reduction of 40% as observed in the adult stage, while other CNS regions analysed were unaffected. An obvious atrophy of the cerebellar cortex was found, with an irregular distribution of the Purkinje cells, while Bergmann glia fibers deviated from their normal radial configuration and showed a tendency to form clusters. A 65% increase of tyrosine hydroxylase immunoreactive fiber density was found in the cerebellar cortex and 3H-5-hydroxytryptamine in vitro synaptosomal uptake was increased by 55%. Noradrenaline and 5-hydroxytryptamine concentrations in the cerebellum increased by 50 and 30%, respectively, whereas the total content of both neurotransmitters in cerebellum was approximately unchanged after methylazoxymethanol treatment. A significant reduction in total cerebellar in vitro binding of 3H-WB-4101 and 3H-dihydroalprenolol was also found, indicating compensatory receptor alterations following methylazoxymethanol treatment. The effect of combined treatment of methylazoxymethanol and the neurotoxin 6-hydroxydopamine (50 mg/kg s.c., day 1) showed a very pronounced reduction of noradrenaline concentration in cortex cerebri, while the noradrenaline concentration in cerebellum was increased by 185% and the tyrosine hydroxylase immunoreactive fiber density by 125%, indicating an additional relative hyperinnervation of cerebellar noradrenaline fiber due to a "pruning effect" of the 6-hydroxydopamine treatment. The results imply a relatively rigid development of terminal arborization of central nervous system monoamine neurons, relatively independent of neuronal and glial arrangement in the target area.

Growth of brain tissue grafts is dependent upon host age.

Growth of grafts of cortex cerebri, hippocampus, septum and cerebellum in oculo were significantly reduced in 16--17-month-old hosts as compared to growth in 3-month-old and 1.5-month-old rat hosts. (Host age is given as the age of the recipients at the time of grafting.) This growth difference was less pronounced in locus coeruleus grafts. The vascular network (as observed with laminin immunofluorescence) in cortex cerebri, hippocampus, cerebellum and septum grafts in 16--17-month-old hosts was abnormal with few thick-walled vessels in clusters as compared to the more 'normal' vascularization found in 1.5-month-old hosts with a high number of thin-walled blood vessels evenly distributed throughout the grafts. Grafts in the oldests hosts were markedly more gliotic than grafts in 1.5- and 3-month-old hosts as evaluated using immunofluorescence with antibodies against glial fibrillary acidic protein. Neurofilament immunoreactivity in the grafts seemed not to be influenced by host age. When a second cortex cerebri or hippocampus graft was placed into contact with a previously grafted locus coeruleus graft, the second graft grew less well in 16--17-month-old hosts as compared to 1.5-month-old hosts. When cortex cerebri was added to a previously grafted cortex cerebri graft, the second graft in both 16--17- and 3-month-old hosts grew to larger sizes than the corresponding single cortex grafts, although the growth differences between the two groups of hosts described above were still maintained. Thus, cortex grafts in 16--17-month-old hosts still have the ability to become trophically stimulated. The vascularization of the second graft in both groups was almost normalized and the gliotic reaction was less pronounced in the second grafts in both groups as compared to the single cortex grafts. In conclusion, the present results indicate that host age affects growth and morphology of intraocular single grafts from several brain regions. Using double grafts of cortex cerebri it was shown that grafts in 16-17-month-old hosts still had the capacity to become trophically stimulated. Data on brain transplants in older hosts are important in view of clinical possibilities to use transplantation strategies to counteract the symptoms of neurodegenerative diseases, which usually occur in old patients.

Morphological and electrophysiological studies of human hippocampal transplants in the anterior eye chamber of athymic nude rats.

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.

Multiple changes in noradrenergic mechanisms in the coeruleo-hippocampal pathway during aging. Structural and functional correlates in intraocular double grafts.

Age-related changes of the coeruleo-hippocampal noradrenergic system were investigated using intraocular double transplants. Pieces of fetal hippocampus were grafted into the anterior chamber of the eye and placed into contact with previously inserted locus coeruleus grafts. Ages of both transplants and hosts were varied to enable studies of intrinsic versus extrinsic determinants of aging in an isolated neuronal circuit. Four different experimental groups, with the approximate age in months of grafts/hosts at the time of recording given in parentheses, were studied; young grafts in the eyes of young hosts (3/7), young grafts in the eyes of old hosts (3/23), mature transplants in adult host rats (8/12) and aged transplants in the eyes of aged rats (21/25). Extracellular recordings from the hippocampal part of the double grafts were performed. Superfusion with alpha-adrenergic antagonists and the alpha 2-agonist clonidine elicited significant increases in the discharge rate of the grafted hippocampal neurons in all groups except the aged transplants in the aged hosts (21/25), where a small excitation was elicited with clonidine and no effect at all was seen with alpha-adrenergic antagonists. The host age did not seem to be important since young transplants in the old hosts (3/23) showed a similar increase in discharge rate as transplants in the young and adult hosts. Tyrosine hydroxylase immunohistochemistry and high-performance liquid chromatography revealed that hippocampal transplants remaining in oculo for a minimum of 6-10 months became permanently hyperinnervated by noradrenergic fibers from the locus coeruleus grafts. The density of noradrenergic fibers was significantly lower in young transplants.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-related alterations in noradrenergic input to the hippocampal formation: structural and functional studies in intraocular transplants.

Intrinsic versus extrinsic determinants of age-related alterations in hippocampal noradrenergic transmission were investigated using intraocular allografts in rats. Three groups of animals were examined: young hippocampal transplants in young hosts, old transplants in old hosts and young transplants in old hosts. Postsynaptic sensitivity to noradrenaline (NA) was measured by extracellular recordings of spontaneous activity and superfusion with known concentrations of catecholamines in the anterior chamber of the eye. Hill plots demonstrated that the dose-response relationships of NA-induced depressions were linear and parallel in the 3 groups. Aged hippocampal grafts displayed a highly significant subsensitivity to NA of one order of magnitude. The EC50 for this group was 203.1 microM as compared to 29.2 in young grafts. Young intraocular grafts in old hosts responded similarly to transplants in young hosts, with an EC50 of 32.4 microM for the depressant actions of NA. Collaterals of the host iris sympathetic ground plexus invaded the hippocampal grafts. The density of this noradrenergic innervation was estimated by immunohistochemistry for tyrosine hydroxylase. A slightly increased density and fluorescence intensity of the noradrenergic fibers were observed in the old transplants as compared to the young transplants in young and old hosts. This was correlated with a significantly (P less than 0.01) increased content of NA in old transplants, as measured with high performance liquid chromatography. The old transplants also contained a large number of autofluorescent lipofuchsin granules, which were absent in the young transplants, regardless of the recipient age. Taken together, these results suggest the existence of alterations in pre- as well as postsynaptic noradrenergic mechanisms in the aging hippocampus. These changes were dependent on transplant age rather than host age, thus suggesting an involvement of intrinsic rather than extrinsic determinants in this model system.

Nerve growth factor can influence growth of cortex cerebri and hippocampus: evidence from intraocular grafts.

The effects of nerve growth factor and antiserum against nerve growth factor on cortical cholinergic projection areas in the central nervous system and cerebellum were evaluated using intraocular grafts of cortex cerebri, hippocampus and cerebellum in rat hosts receiving injections into the anterior chamber of the eye of nerve growth factor (at transplantation, 5 and 10 days after transplantation) or antiserum to nerve growth factor (every 5 days). The controls received cytochrome c or preimmune serum. Growth of grafts was followed by repeated observations directly through the cornea of the host using a stereomicroscope. Nerve growth factor-treated grafts of cortex cerebri and hippocampus grew significantly smaller as compared to the corresponding control grafts. In one experiment, growth of cytochrome c and saline-treated cortex cerebri was compared and no difference in growth was found. Growth of nerve growth factor-treated cerebellar grafts did not differ significantly from growth of cytochrome c-treated grafts. Morphological analysis using Nissl-staining, antibodies to glial acidic fibrillary protein to evaluate the degree of gliosis and antiserum to neurofilament as a neuronal marker did not reveal any marked differences between nerve growth factor- and cytochrome c-treated grafts. Cortical grafts receiving anti-nerve growth factor antiserum by injection or by immunizing host rats against nerve growth factor showed similar growth to the controls. Similarly, grafts of fetal hippocampus to rats immunized with nerve growth factor were not significantly different from grafts to host rats immunized with cytochrome c. We conclude that exogenous nerve growth factor affects the development of grafted cortex cerebri and hippocampus. The fact that these cortical areas stop growing earlier in the presence of nerve growth factor without the grafts showing evidence of disturbed glial or neuronal populations compared to control grafts indicates that nerve growth factor acts to induce overall/premature differentiation and maturation. The mechanism for this whether or not it is receptor-mediated and which cells are primarily affected by nerve growth factor is not yet known.

Testicular atrophy and loss of nerve growth factor-immunoreactive germ cell line in rats exposed to n-hexane and a protective effect of simultaneous exposure to toluene or xylene.

Testicular and germ cell line morphology in rats were studied 2 weeks, 10 months and 14 months after cessation of a 61-day inhalation exposure to 1000 ppm n-hexane. Androgen biosynthetic capacity of testis, testosterone blood concentration, vas deferens morphology and noradrenaline (NA) concentration, epididymal sperm morphology, and fertility were also studied. Severe testicular atrophy involving the seminiferous tubules with loss of the nerve growth factor (NGF) immunoreactive germ cell line was found. Total loss of the germ cell line was found in a fraction of animals up to 14 months post-exposure, indicating permanent testicular damage. No impairment of androgen synthesis or androgen dependent accessory organs was observed. Simultaneous administration of 1000 ppm n-hexane and 1000 ppm toluene, or 1000 ppm n-hexane and 1000 ppm xylene, did not cause germ cell line alterations or testicular atrophy. Toluene and xylene were thus found to protect from n-hexane induced testicular atrophy.

NGF treatment promotes development of basal forebrain tissue grafts in the anterior chamber of the eye.

The effects of nerve growth factor (NGF) on developing central cholinergic neurons were studied using intraocular grafts of rat fetal (E17) basal forebrain tissue. Prior to grafting, grafts were incubated in NGF or saline. Transplants were allowed to mature for six weeks, receiving weekly intraocular injections of NGF or saline. Measurements of NGF levels in oculo after one single injection showed that NGF slowly decreases in the anterior chamber fluid, and after one week, low but significant levels were still present in the eye. Following pretreatment with diisopropylfluorophosphate (DFP), the cholinergic neurons in the grafts were analyzed using three morphological markers: antibodies to cholineacetyltransferase (ChAT), antibodies to acetylcholinesterase (AChE Ab) and acetylcholinesterase histochemistry (AChE). The transplants grew well and became vascularized within the first week. The growth of the NGF-treated basal forebrain grafts was significantly enhanced as compared to the growth of the saline-treated grafts evaluated with repeated stereomicroscopical observations directly through the cornea of the ether-anaesthetized hosts. The NGF-treated grafts contained almost twice as many cholinergic neurons seen with all the cholinergic markers used, as the saline-treated grafts. However, there was no difference in cholinergic cell density between the two groups. The morphology and size of an individual cholinergic neuron was similar in the two groups. The fiber density as evaluated with AChE-immunohistochemistry did not change after NGF-treatment. The DFP-treatment did not seem to affect the AChE-immunoreactivity since an extensive fiber network was found, whereas almost no fibers were seen using conventional AChE histochemistry. We have demonstrated that in oculo transplantation of basal forebrain is a useful model for examining in vivo effects of NGF on central cholinergic function. The marked volume increase of NGF-treated grafts and the unchanged density of cholinergic cells and terminals suggests, that NGF increases the survival of not only developing cholinergic neurons, but possibly other non-cholinergic neurons and non-neuronal cells as well. These results support the notion that NGF acts as a neurotrophic factor on cholinergic and possibly non-cholinergic cells in the central nervous system.

Toluene exposure during maturation of intraocular brain tissue transplants: alterations of host and graft cerebellar Purkinje neuron function and sensitivity to norepinephrine.

The effects of chronic toluene exposure on central neurons were examined using syngeneic grafts into the anterior chamber of the eye. Young adult albino rats with intraocular brain transplants inhaled toluene (1000 ppm) for 9 weeks starting at the time of transplantation, or from Week 8 to 17 after the graft was placed in oculo. Control animals were exposed to room air during the same intervals. Toluene treatment during development did not affect general growth or morphology of any of the brain areas examined. The distribution of neurofilament or glial fibrillary acidic protein immunoreactivity was similar in the experimental group and control group as well. Extracellular recordings of cerebellar Purkinje neurons showed a significantly reduced spontaneous firing rate, of 15-25%, both in intraocular transplants and in cerebellum in situ in toluene exposed animals. Postsynaptic sensitivity of intraocular and in situ Purkinje neurons to norepinephrine (NE) was evaluated. Purkinje neurons in transplants exposed to toluene during development were markedly supersensitive to superfused NE as compared to controls, while neither Purkinje neurons in mature cerebellar grafts nor cerebellum in situ showed any effects of the toluene treatment on NE sensitivity. The tissue content of NE in transplants exposed to toluene during maturation, evaluated with high-performance liquid chromatography coupled with electrochemical detection, was greater than that in the control grafts. Moreover, the content of free (3-methoxy-4-hydroxyphenyl) ethylene glycol (MHPG) was increased in both transplant and host cerebellum after toluene exposure. Taken together, these data indicate that toluene exposure during development of cerebellar grafts in oculo causes changes in postsynaptic noradrenergic sensitivity as well as decreased spontaneous activity of Purkinje neurons. Toluene exposure of adult cerebellum in situ or in oculo appears to decrease the Purkinje neuron discharge rate and increase NE turnover, but has no marked effect on postsynaptic NE sensitivity.

Age-related changes in cerebellar noradrenergic pre- and postsynaptic mechanisms: intrinsic vs extrinsic determinants evaluated with brain grafts in oculo.

Intrinsic versus extrinsic determinants of changes in cerebellar noradrenergic transmission during senescence in the rat were measured using homologous cerebellar grafts in oculo. Postsynaptic sensitivity of Purkinje neurons to catecholamines was determined by perfusing the anterior eye chamber with known concentrations of norepinephrine (NE) dissolved in a balanced salt solution. NE elicited a dose-dependent slowing of spontaneous Purkinje neuron discharge in both young (3-6 months) and aged (20-22 months) cerebellar grafts. Hill plots demonstrated that the dose-response relationships in both age-groups were linear and parallel to one another. Aged transplant Purkinje neurons manifested a marked and highly significant subsensitivity to NE with an EC50 of 583 microM, as compared with an EC50 of only 15.9 microM in the young grafts. Young grafts in 15-21-month-old hosts manifested an EC50 of 20 microM for the depressant actions of NE. Collaterals of host iris sympathetic fibers innervate the grafts. Activity of these fibers can be reflexly altered by changing illumination of the retina. The dynamics of presynaptic NE release from these fibers was evaluated using in vivo electrochemistry with Nafion-coated graphite epoxy capillary electrodes, which are highly selective for the monoamine neurotransmitters. As illumination of the ipsilateral retina is increased, the release of catecholamine in the cerebellar graft decreases. A mean change in the extracellular electroactive species of 4.2 +/- 0.6 microM was found in young cerebellar grafts. Equivalent stimuli induced a mean change of 2.3 +/- 0.8 microM in aged grafts. However, this diminished release was not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutamic acid decarboxylase- and peptide-immunoreactive neurons in cortex cerebri following development in isolation: evidence of homotypic and disturbed patterns in intraocular grafts.

Fetal parietal cerebral cortex was transplanted to the anterior eye chamber of adult Sprague-Dawley rats. After two to three months the grafts, with or without colchicine treatment, were subjected to immunohistochemical analysis using antibodies against cholecystokinin (CCK), somatostatin (SOM), neuropeptide tyrosine (NPY), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI) and the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD). Cerebral cortex in situ of untreated and colchicine-treated rats was always analyzed in parallel. A dense plexus of CCK-immunoreactive fibers was distributed in all parts of the transplants, and after colchicine treatment a large number of CCK-positive cells was observed. These cells were markedly increased in number as compared to normal cortical tissue in colchicine-pretreated rats. The amount of NPY-immunoreactive cells was also markedly increased, whereas somatostatin-positive cells were found in numbers similar to those seen in cortex in situ. In the grafts only a few VIP- and PHI-positive fibers were seen with a few VIP-positive cell bodies, but no clearly discernible PHI-positive cells. A very dense plexus of GAD-positive fibers with an even distribution throughout the grafts was observed. Cortex in situ exhibited a lower density of GAD-immunoreactive fibers. Even after colchicine treatment the number of GAD-positive cells in the grafts was low. Using double-staining techniques, it was found that most of the few GAD-positive cells in the grafts were also NPY-positive, SOM-positive or, to a minor extent, CCK-positive. The present results demonstrate that several peptides and transmitter markers are expressed in cortical grafts in oculo, but marked differences in their expression can be observed in cortical tissue that has developed in isolation. Thus, the intraocular cortex graft, alone and in combination with other brain areas, should provide a useful model in which to study factors that regulate brain development.

Sustained seizures cause circumscribed cerebral changes in glial fibrillary acidic protein, neurofilament and laminin immunofluorescence.

Sustained experimental seizures in rats have previously been shown to cause an extensive necrosis in pars reticulata of substantia nigra (SNPR) and globus pallidus (GP). In the present paper we have studied the effects of hexafluorodiethyl ether-induced seizures on the immunoreactivity seen with antibodies directed against glial fibrillary acidic protein, GFA, used to visualize astrocytes, antibodies to the glycoprotein laminin as a marker for blood vessel walls and neurofilament (NF) antibodies to monitor neuronal disturbances. Already 12 h after a 20-min seizure period a reduction in GFA immunofluorescence intensity was observed in SNPR. After 3 days, marked lesions were noted in SNPR and GP as seen with cresyl violet staining. The lesions contained almost no GFA-positive structures. In the proximity of the lesions, an increase in GFA-immunoreactivity was noted. Such an increase, although less pronounced, was also seen in the major projection areas of SNPR. Two months post-seizure, the gliotic reaction had disappeared, and only a thin and elongated gliotic scar was observed. In spite of the development of a profound central necrosis especially evident in SNPR, both laminin- and NF-immunoreactivity was slightly increased within the lesioned areas. NF-immunoreactivity was also increased in the superior colliculus and in the reticular formation. Two months post-experiment NF-immunofluorescence was normalized but the former lesion sites showed signs of hypervascularization. We conclude that hexafluorodiethyl ether-induced 20-min seizures lead to rapid, localized glial and neuronal changes in the rat brain as evidenced by GFA and NF immunohistochemistry, while the vascular network remains intact.

Laminin immunohistochemistry: a simple method to visualize and quantitate vascular structures in the mammalian brain.

Immunohistochemistry using antiserum against the basement membrane glycoprotein laminin, was shown to be an excellent marker for brain blood vessels. Throughout the brain of mice, rats, guinea pigs, monkeys and humans, the basement membrane of the vascular structures were strongly laminin-positive. The neuropil itself was laminin-negative, whereas a positive reaction was observed in the meaninges. When the laminin antiserum was preabsorbed with its proper antigen, no specific fluorescence was observed. Using India ink perfusion as a comparative method, it was found that probably all vascular structures were also visualized with laminin immunohistochemistry. Laminin immunofluorescence was found well-suited for computer-assisted quantitative image analysis of brain vascularity. As expected in the periphery, the basement membrane of many other structures except blood vessels such as endoneurium, epithelium and smooth muscle cells were laminin-positive. Although the vascular network was also strongly laminin-immunoreactive, it was difficult to differentiate between blood vessels and non-vascular structures in the periphery as compared to the central nervous system. In conclusion, laminin immunohistochemistry has proven to be a simple, useful and specific method to study vascular structures in the central nervous system and an excellent alternative to more conventional and laborious methods such as perfusion with India ink.

Growth and development of intraocular fetal cortex cerebri grafts in rats of different ages.

Cortex cerebri pieces from fetal donors were homologously and bilaterally grafted to the anterior chamber of the eye of 1.5-, 3.0- and 7.5-month-old rats. Repeated stereoscopic in vivo measurements revealed that the grafts in the young group grew to a size twice as large as those in the older two groups. The degree of gliosis was studied immunohistochemically using antibodies against glial fibrillary acidic protein. Both grafts to young and to older hosts were clearly gliotic as compared to normal cerebral cortex. However, the gliosis was significantly more pronounced in grafts to 3.0- and 7.5-month-old hosts as compared to grafts to 1.5-month-old hosts. The vascular component was evaluated using antibodies against laminin. We found laminin immunofluorescence to be an excellent marker of brain tissue vascularization, particularly at the capillary level, revealing the entire capillary tree and endothelial budding. The density of the vascular plexus and the average thickness of the capillaries of cortex cerebri grafted to the youngest recipients was remarkably similar to normal cerebral cortex. In marked contrast, grafts to the older hosts had a clearly pathological vascular network characterized by few, thick-walled blood vessels and very few normal-looking capillaries. We conclude that host age factors profoundly influence development and growth of intraocular brain tissue grafts.