Basic fibroblast growth factor increases dopaminergic graft survival and function in a rat model of Parkinson's disease.

The clinical use of fetal neural grafts as an intracerebral source of dopamine for patients with Parkinson's disease has met with limited success. Since basic fibroblast growth factor (bFGF) enhances the survival and growth of dopaminergic neurons in vitro, we explored whether cells genetically modified to produce bFGF would improve the functional efficacy of dopaminergic neurons implanted into rats with experimental Parkinson's disease. Results show that bFGF-producing cells grafted together with fetal dopamine neurons have potent growth-promoting effects on the implanted neurons in vivo. Moreover, rats implanted with such co-grafts display the most pronounced behavioural improvements post-grafting. These findings not only provide insight into the function of bFGF in situ, but also suggest an approach for enhancing the survival and function of dopamine neurons grafted into the damaged brain.

Cone inputs to ganglion cells in hereditary retinal degeneration.

The photoreceptor layer degenerated, but cone nuclei apparently devoid of outer segments were retained in retinas of aged rats of the Royal College of Surgeons strain from which optic tract activity was recorded. Measures of sensitivity showed these single axons of retinal ganglion cells to have photopic spectral responses. Cone remnants containing a cone pigment may be the photoreceptive elements in these retinas.

Survival and function of intrastriatally grafted primary fibroblasts genetically modified to produce L-dopa.

A combination of gene transfer and intracerebral grafting may provide a powerful technique for examining the role of discrete substances in the development or functioning of the brain. In the present study, primary fibroblasts obtained from a skin biopsy from inbred Fischer rats were used as donor cells for genetic modification and grafting. When grafted to the striatum of Fischer rats with a prior 6-hydroxydopamine lesion, primary fibroblasts containing a transgene for either tyrosine hydroxylase (TH) or beta-galactosidase survived for 10 weeks and continued to express the transgene. TH synthesized by the implanted fibroblasts appeared to convert tyrosine to L-dopa actively, as observed in vitro, and to affect the host brain, as assessed through a behavioral measurement. These results suggest that primary fibroblasts genetically altered to express TH have the capacity to deliver L-dopa locally to the striatum in quantities sufficient to compensate partially for the loss of intrinsic striatal dopaminergic input.

Genetically modified cells: applications for intracerebral grafting.

Grafting cells to the CNS is a useful approach to address fundamental and clinical issues in neurobiology. Recently, a hybrid technique - the genetic modification of cells followed by intracerebral implantation - has emerged, which may potentially enhance the power of CNS grafting. However, several methodological considerations need to be addressed to test the reliability of this new approach. Progress in the gene transfer-grafting technique has implications for expanding the range of issues and problems that may be addressed in both the basic science and clinical arenas.

In vivo and ex vivo gene transfer to the brain.

The use of gene transfer techniques to express novel proteins within different cellular populations has provided insights into the function and plasticity of the brain. Recently, this technique has been successfully used to explore physiological processes within the CNS and to intervene in neurodegenerative disease and cancer. Progress in manipulating transgene products in vivo and in achieving cell-specific targeting of genetic material offers promise for enhancing the usefulness of this technique and its therapeutic potential for treating human disorders of the CNS.

Intracerebral grafting in the dopaminergic system: issues and controversy.

A review of work in the dopaminergic system reveals both progress and controversy. More than 100 papers on intracerebral grafting were published last year. Several groups have published the clinical outcome of fetal substantia nigra implants in Parkinsonian patients and studies suggest that sprouting of dopaminergic fibers in response to grafting procedures ameliorates the behavioral deficits of dopamine-depleted animals. Trophic factors for dopamine neurons have also been identified. In addition, genetically modified cells continue to be developed as an alternative method for delivering molecules to the brain. The speed with which neural grafting has become a therapeutic procedure in Parkinsonian patients continues to stimulate debate.

Cholinergic strategies for Alzheimer's disease.

Alzheimer's disease is a devastating degenerative disorder of the central nervous system that results in gradual deterioration of cognitive function and severe alteration of personality. Degeneration of neurons in the nucleus basalis Meynert, the origin of the major cholinergic projections to the neocortex, occurs early in the course of the disease, and is correlated with the cognitive decline. This link between cholinergic dysfunction in the basal-cortical system and cognitive deficits has focused scientific efforts on developing tools to elucidate the neurobiological role of the cholinergic system in cognition and to develop therapeutic interventions in the disorder. An important step in understanding the mechanisms underlying cognitive dysfunction has been the development of in vivo rodent models that mimic some of the features of Alzheimer's disease. Acute excitotoxic or immunotoxic lesions of the nucleus basalis in rodents have revealed a role of the basal-cortical system in attention, learning and memory. More recent advances in developing mouse gene technology offer newer models to systematically examine the underlying neuropathological cascade leading to dysfunctions in mnemonic processing. Using in vivo rodent models, several cholinergic enhancement strategies have been tested and proven to be effective in alleviating lesion-induced cognitive deficits, including neuropharmacological approaches (acetylcholinesterase inhibitors), neurotrophic factor administration (nerve growth factor), and transplantation of cholinergic-enriched fetal grafts. Successful results have also been obtained using ex vivo gene transfer to deliver nerve growth factor or acetylcholine to compromised regions of the basal-cortical system. Gene therapy may be of particular interest for clinical applications, because this approach provides a method for topographically restricted and selective delivery of therapeutic genes and their products to afflicted areas of the brain. Advanced techniques in molecular biology (e.g., exogenous regulatable gene transfer) and newly developed tools of modern neuroscience (e.g., neural precursor cells) will be important contributions for deciphering the biological bases of neuronal degeneration and for refining therapeutic strategies for Alzheimer's disease.

Preservation of retinal structure in aged pigmented mice.

The effects on the retina of advancing age were studied in pigmented mouse strain (C57BL/6J). The mice range in age from 65 days to 1000 days, an age well beyond the mean life span of the population (850 days). The thickness of the neuronal and plexiform layers and the planimetric density and size of the component neurons were assessed in both central (200-500 micrometers from the optic disc) and peripheral (within 200 micrometers of the retinal margin) areas. In addition, the overall size of the retina was determined by measuring its length along the horizontal meridian. Although retinas of albino rodents degenerate extensively during aging [10, 18, 31, 32, 40], in the retinas of pigmented mice neither the central nor the peripheral locus showed either marked thinning of the retinal layers or neuronal loss with advancing age. We suggest that previous findings of severe retinal degeneration in albino rodents during aging can be attributed to their lack of pigment and that pigmented animals offer a more suitable animal model for normal retinal aging.

Cytotoxic aldehydes as possible markers for childhood cancer.

Concentrations of 22 known aldehydes (byproducts of lipid peroxidation), 5 acyloins, free and total carnitine and acylcarnitines were measured in plasma and urine obtained from pediatric patients with various forms of cancer before any treatment, and following treatment with doxorubicin or daunorubicin. Aldehydes, before the initiation of chemotherapy, were significantly elevated in cancer patients compared to controls. Aldehydes such as hexanal, heptanal, and malondialdehyde were strikingly higher in samples from cancer patients, while trans 4-cis-4-decenal was the prominent aldehyde in the blood of controls. In addition, in each form of cancer the pattern of aldehydes appeared to be unique when compared to controls, or to others forms of cancer. In cancer patients receiving chemotherapy there was a general trend toward a reduction 24 h after both the first and after the fifth doxorubicin dose. These changes however were not significant statistically due to large inter-patient variation. Free and total plasma carnitine levels remained in the normal range, and there were no abnormal acylcarnitines detected in urine. Possible hypotheses to explain the elevations in aldehydes, and the reasons for the changed aldehyde profiles in different forms of cancer are discussed.

Development of synaptic arrays in the inner plexiform layer of neonatal mouse retina.

Retinas from mice of the C57BL/6 strain were sampled at frequent intervals from birth to postnatal day 33 to determine the numerical density of conventional and ribbon synapses within the inner plexiform layer (IPL) as a function of time. Synaptic arrays of the IPL were formed in three phases. During Phase I, from day 3 to day 10, conventional synapses were produced at a mean rate of 0.44 synapses/1,000 micrometer3/hour, but no ribbons were seen. During Phase II, from day 11 to day 15, ribbons formed at a rate of 0.38 ribbons/1,000 micrometer3/hour and conventional synapses were produced at a rate of 1.15 synapses/1,000 micrometer3/hour. Phase III began at day 15, the approximate time of eye opening in these animals, and was characterized by a sharp reduction in the rate of production of both ribbons and conventional synapses. During this phase ribbons achieved a final mean density of 113 ribbons/1,000 micrometer3 and conventionals achieved a final mean density of 250 synapses/1,000 micrometer3. Serial appeared in Phase II but remained at low densities.

Retinal synaptic arrays: continuing development in the adult goldfish.

We report a light- and electron-microscopic examination of the inner plexiform layer of the central retina of young (c. 1 year) and old (3-4 year) goldfish. There were no new neurons added to this region during the growth period. Nonetheless, there were substantially more synapses (per cell, per mm2, or per degree 2) in the older retinas. This result is discussed in the contexts of retina function and neural development.

Hippocampal grafts of acetylcholine-producing cells are sufficient to improve behavioural performance following a unilateral fimbria-fornix lesion.

Lesions of the septohippocampal pathway produce cognitive deficits that are partially attenuated by grafts of cholinergic-rich tissue into denervated target regions or by systemic administration of cholinomimetic drugs. In the present study, fibroblasts engineered to produce acetylcholine were used to test the hypothesis that restoration of hippocampal acetylcholine in rats with septohippocampal lesions is sufficient to improve cognitive processing post-damage. Rats received unilateral grafts of acetylcholine-producing or control fibroblasts into the hippocampus immediately prior to an aspirative lesion of the ipsilateral fimbria-fornix. Some rats with fimbria-fornix lesions were implanted with acetylcholine-producing or control fibroblasts into the neocortex, another major target of the basal forebrain cholinergic system, to determine if the site of acetylcholine delivery to the damaged brain is critical for functional recovery. Rats were tested in a hidden platform water maze task, a cued water maze task and activity chambers between one and three weeks post-grafting. Compared to unoperated controls, rats with fimbria fornix lesions only were significantly impaired in hidden platform water maze performance. Hippocampal grafts of acetylcholine-producing cells reduced lesion-induced deficits in the water maze, whereas hippocampal control grafts and cortical grafts of either cell type were without effect. Locomotor activity and cued water maze performance were unaffected by the lesion or the implants. Taken together, these data indicate that water maze deficits produced by fimbria fornix lesions, which disrupt a number of hippocampal neurotransmitter systems, can be attenuated by target specific replacement of acetylcholine in the hippocampus and that this recovery occurs in the absence of circuitry repair.

Electrophysiological characteristics of cells within mesencephalon suspension grafts.

Both spontaneous and evoked extracellular electrophysiological activity of neurons within fetal mesencephalon suspension grafts to the dopamine-depleted striatum of rats were examined. In some cases, extracellular recording was combined with intracellular labeling to identify recorded neurons. Grafted rats displaying a complete cessation of ipsilateral rotations following amphetamine administration were examined at post-implantation time intervals of two, four, five, eight and nine months. Four separate classes of neurons were distinguished within the transplanted striatum based on electrophysiological properties. The first of these groups, the type I cells, appeared to be non-grafted striatal neurons. When spontaneously active, these striatal-like cells fired bursts of action potentials separated by periods of decreased activity. Evoked responses in these cells were characteristic of striatal cells. Type I cells which were intracellularly labeled were found outside the grafts and displayed the characteristic morphology of the medium spiny neuron of the neostriatum. The other three cell classes displayed electrophysiological properties similar to neurons recorded in situ within the reticular formation, substantia nigra pars compacta and substantia nigra pars reticulata. Neurons from these three groups which were labeled with an intracellular marker were found to lie within the suspension grafts. The spontaneous activity of the pars compacta dopaminergic-like neurons was predominantly irregular, with some cells also firing in a regular or pacemaker-like pattern. Infrequently, irregular firing dopaminergic-like neurons displayed episodes of doublet bursting. Many of the grafted neurons responded to electrical stimulation of prefrontal cortex and striatum, indicating that the graft was receiving functional inputs from host neurons. Comparison of the firing rate and pattern of grafted neurons to in situ mesencephalic neurons as a function of time following grafting suggested that the grafted neurons and/or the neuronal circuitry is slowly developing within the host environment. A prolonged time-course for the maturation of the graft may be reflected in the time required to achieve improvements in some behavioral deficits following transplantation. However, the relatively rapid recovery of drug-induced rotational asymmetry following grafting suggests that this form of recovery may not require mature functioning of the grafted neurons.

Petit mal epilepsy and parkinsonian tremor: hypothesis of a common pacemaker.

Rhythmic oscillation in neuronal systems may serve physiological purposes or may interfere with normal functions of the brain. In disorders of petit mal epilepsy and parkinsonian tremor, centrally and peripherally observable rhythmic patterns are due to network oscillations of thalamocortical cells. This article reviews the afferent mechanisms that might be critically involved in controlling the ionic conductances of thalamic neurons in the behaving organism. We propose that during active behavior the subcortical aminergic and cholinergic inputs to the thalamus act as anti-burst and anti-oscillation mechanisms. We suggest further that the thalamopetal GABAergic inputs (pars reticulata of substantia nigra, entopeduncular nucleus, pallidum) are burst- and oscillation-promoting systems, whose output is controlled by the striatum. Experimental or disease-related decrease of the striatal dopamine levels is hypothesized to increase the efficacy of the GABAergic burst-promoting systems resulting in rhythmic network oscillation of thalamocortical neurons during rest. The recognition of the overlapping neuronal mechanisms in petit mal epilepsy and parkinsonian tremor, and the multistage control of thalamic oscillation suggests that drugs effectively used in petit mal attacks may be effective in levodopa-refractory parkinsonian tremor, and conversely, epileptic patients may benefit from drugs acting on the extrapyramidal system.

Interplexiform cell of the mouse retina: a Golgi demonstration.

A neuron of the inner nuclear layer (INL) with some processes extending to the outer plexiform layer (OPL) and others to the inner plexiform layer (IPL) was discovered near the posterior pole of the mouse retina. The neuron's location and appearance are similar to the interplexiform cells of several other species. The relatively recent recognition of this cell type along with its characteristic of infrequent staining by the Golgi technique make the extent of its distribution among species uncertain. With each demonstration in a new species, the existence of the interplexiform cell as a sixth neuronal element of all vertebrate retinas becomes more assured.

Synaptic lamellae of the photoreceptors of pearl and wild-type mice.

The retina of the pearl mutant mouse, C57BL/6J pe/pe, exhibits reduced light sensitivity in the dark-adapted condition (Balkema and Pinto, J Neurophysiol 48:968, 1982). The authors searched for an anatomic correlate in the retina which could relate to the functional deficit. Electron microscopic mosaics of the outer plexiform layer of light- and dark-adapted pearl and wild-type mice were analyzed. The numerical density and length of the photoreceptor synaptic lamellae showed these parameters to be indistinguishable in wild-type and pearl retinas under conditions of both light- and dark-adaptation. Light-adapted pearl retinas exhibited some rod spherules that contained structurally modified synaptic lamellae with bulbous thickenings and adjacent electron-dense bodies. These lamellar modifications were neither apparent in the light-adapted, wild-type retinas, nor in the dark-adapted retinas of either genotype. Pronase application to ultrathin sections hydrolyzed synaptic lamellae, bulbous thickenings and electron dense bodies.

Light exposure can reduce selectively or abolish the C-wave of the albino rat electroretinogram.

The ERG (electroretinogram) of the albino rat is reported to lack a c-wave. Observations of our own suggested that the conditions of light-rearing are important. Consequently, the authors recorded c-waves in two groups of albino rats. One group was reared from birth in dim illumination (dark-reared) and the other in 12/12 cyclic light (light-reared). Rats were tested after birth from 22 days to about 1 year. All dark-reared animals had a c-wave. Rats reared in cyclic light typically had no detectable c-wave. Physiologic and anatomic evidence suggests this consistent difference, c-waves present in dark-reared animals but absent or diminished in light-reared animals, is probably not due to extensive light induced retinal damage. No consistent differences between the two groups were seen in a- or b-wave thresholds, a- or b-wave intensity-response functions, and in the time-course of b-wave dark adaptation.

The vascular activity of some isoflavone metabolites: implications for a cardioprotective role.

Legume-derived isoflavones such as genistein, diadzein and equol have been associated with a reduction in risk of cardiovascular disease. In the current study, we explore the vascular activity of several isoflavone metabolites namely dihydrodaidzein, cis and trans-tetrahydrodaidzein and dehydroequol for potential cardioprotective properties. Rat isolated aortic rings were used. 17beta-oestradiol, equol, and all four of the metabolites studied significantly antagonized contractile responses to noradrenaline. The direct vasodilatory action of these compounds were examined and in contrast to 17beta-oestradiol, the vasodilatory effect of which was demonstrated to be endothelium independent, the dilatory action of all four compounds could be inhibited by endothelium denudation. Further, the dilatory action of both dihydrodaidzein and cis-tetrahydrodaidzein were inhibited by the nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine (NOLA), by the soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and by 40 mM KCl. Dilatory responses to dehydroequol and trans-tetrahydrodaidzein, on the other hand, were inhibited by 40 mM KCL but not by NOLA nor ODQ. Finally, we examined the protective potential of these compounds in inhibiting endothelium damage by oxidized low density lipoprotein (ox-LDL). Trans-tetrahydrodaidzein was at least 10 fold more potent than 17beta-oestradiol in protecting against ox-LDL induced damage. We conclude that the isoflavone metabolites, dihydrodaidzein, cis- and trans-tetrahydrodaidzein and dehydroequol, may potentially represent a novel series of cardioprotective therapeutics.

Engineered cells: a promising therapeutic approach for neural disease.

The transplantation technique has been invaluable for studying the CNS. Recently, the use of genetically modified cells for CNS grafting has further increased the utility of this technique. Studies conducted during the past 10 years have shown that a variety of genes can be successfully expressed in both neural and non-neural populations. Depending on the cell type used for gene transfer, engineered cells survive well within the CNS and continue to synthesize engineered products. In addition to providing insights into CNS development and plasticity, genetically modified cells have revealed the therapeutic role of different factors in neural disease. Cells engineered to produce growth factors have been shown to prevent and/or minimize neural degeneration following an experimental damage while the intracerebral transplantation of cells genetically modified to produce neurotransmit-ters have successfully reversed behavioral impairments of animals with experimental Parkinson's disease or Alzheimer's disease. Recent results with engineered cells transplanted into the brain of non-human primates suggest the potential of engineered cells for human therapy. Work with encapsulation techniques to isolate engineered cells from a host brain offers one of several approaches for ensuring the safety of genetically modified cells grafted into the CNS. Identifying factors that influence the survival and gene expression of engineered cells following transplantation will enhance the usefulness of these cells for studying and repairing the CNS.