Terrence W. Deacon

Transplanted xenogeneic neural cells in neurodegenerative disease models exhibit remarkable axonal target specificity and distinct growth patterns of glial and axonal fibres

Clinical trials are under way using fetal cells to repair damaged neuronal circuitry. However, little is known about how transplanted immature neurons can grow anatomically correct connections in the adult central nervous system (CNS). We transplanted embryonic porcine neural cells in vivo into adult rat brains with neuronal and axonal loss typical of Parkinsons or Huntingtons disease. Using complementary species-specific cellular markers, we found donor axons and CD44+ astroglial fibres in host white matter tracts up to 8 mm from CNS transplant sites, although only donor axons were capable of reaching correct gray matter target regions. This work demonstrates that adult host brain can orient growth of transplanted neurons and that there are differences in transplant donor glial and axonal growth patterns in cellular repair of the mature CNS.

Blastula-stage stem cells can differentiate into dopaminergic and serotonergic neurons after transplantation.

In order to assess the potential of embryonic stem cells to undergo neuronal differentiation in vivo, totipotent stem cells from mouse blastocysts (D3 and E14TG2a; previously expanded in the presence of leukemia inhibitory factor) were transplanted, with or without retinoic acid pretreatment, into adult mouse brain, adult lesioned rat brain, and into the mouse kidney capsule. Intracerebral grafts survived in 61% of cyclosporine immunosuppressed rats and 100% of mouse hosts, exhibited variable size and morphology, and both intracerebral and kidney capsule grafts developed large numbers of cells exhibiting neuronal morphology and immunoreactivity for neurofilament, neuron-specific enolase, tyrosine hydroxylase (TH), 5-hydroxytryptamine (5-HT), and cells immunoreactive for glial fibrillary acidic protein. Though graft size and histology were variable, typical grafts of 5-10 mm3 contained 10-20,000 TH+ neurons, whereas dopamine-beta-hydroxylase+ cells were rare. Most grafts also included nonneuronal regions. In intracerebral grafts, large numbers of astrocytes immunoreactive for glial fibrillary acidic protein were present. Both TH+ and 5-HT+ axons from intracerebral grafts grew into regions of the dopamine-lesioned host striatum. TH+ axons grew preferentially into striatal gray matter, while 5-HT+ axons showed no white/gray matter preference. These findings demonstrate that transplantation to the brain or kidney capsule can induce a significant fraction of totipotent embryonic stem cells to become putative dopaminergic or serotonergic neurons and that when transplanted to the brain these neurons are capable of innervating the adult host striatum.

Embryonic stem cells differentiated in vitro as a novel source of cells for transplantation

The controlled differentiation of mouse embryonic stem (ES) cells into near homogeneous populations of both neurons and skeletal muscle cells that can survive and function in vivo after transplantation is reported. We show that treatment of pluripotent ES cells with retinoic acid (RA) and dimethylsulfoxide (DMSO) induce differentiation of these cells into highly enriched populations of gamma-aminobutyric acid (GABA) expressing neurons and skeletal myoblasts, respectively. For neuronal differentiation, RA alone is sufficient to induce ES cells to differentiate into neuronal cells that show properties of postmitotic neurons both in vitro and in vivo. In vivo function of RA-induced neuronal cells was demonstrated by transplantation into the quinolinic acid lesioned striatum of rats (a rat model for Huntingtons disease), where cells integrated and survived for up to 6 wk. The response of embryonic stem cells to DMSO to form muscle was less dramatic than that observed for RA. DMSO-induced ES cells formed mixed populations of muscle cells composed of cardiac, smooth, and skeletal muscle instead of homogeneous populations of a single muscle cell type. To determine whether the response of ES cells to DMSO induction could be further controlled, ES cells were stably transfected with a gene coding for the muscle-specific regulatory factor, MyoD. When induced with DMSO, ES cells constitutively expressing high levels of MyoD differentiated exclusively into skeletal myoblasts (no cardiac or smooth muscle cells) that fused to form myotubes capable of spontaneous contraction. Thus, the specific muscle cell type formed was controlled by the expression of MyoD. These results provided evidence that the specific cell type formed (whether it be muscle, neuronal, or other cell types) can be controlled in vitro. Further, these results demonstrated that ES cells can provide a source of multiple differentiated cell types that can be used for transplantation.

The lateral ganglionic eminence is the origin of cells committed to striatal phenotypes: neural transplantation and developmental evidence

In order to determine whether the lateral ganglionic eminence (LGE) of the fetal telencephalon is the primary source of striatal precursors in striatal transplants and tissue cultures, cells derived exclusively from the LGE of fetal rat brains were transplanted into the quinolinic-acid-lesioned striatum of adult rats. After 2-3 months they produced grafts that were almost entirely AChE-positive as well as DARPP-32-, TH-, and calbindin-immunoreactive. The grafts were integrated into the host striatum so that host corticofugal fiber tracts interdigitated with graft tissues similar to the way they penetrate the gray matter of the normal striatum. Fast Blue dye injected into the ipsilateral globus pallidus of LGE grafted produced retrogradely labeled neurons within the grafts, but Fluorogold dye injected into the ipsilateral substantia nigra did not. In a separate experiment using DARPP-32-immunohistochemstry as a striatal marker, fetal (E16) and neonatal (P2) rat brains showed DARPP-32 immunoreactivity in the LGE but not in the adjacent medial ganglionic eminence (MGE). In summary, both fetal LGE cells and LGE grafts express specific striatal markers, and LGE grafts integrate into the host striatum and innervate the major striatal efferent target within the host brain. These data suggest that the LGE is the origin of cells committed to striatal phenotypes in the developing brain.

Xenotransplantation of Porcine Fetal Ventral Mesencephalon in a Rat Model of Parkinson's Disease: Functional Recovery and Graft Morphology

Neurotransplantation of human fetal dopamine (DA) neurons is currently being investigated as a therapeutic modality for Parkinsons disease (PD). However, the practical limitations of human fetal transplantation indicate a need for alternative methodologies. Using the 6-hydroxydopamine rat model of PD, we transplanted dopaminergic neurons derived from Embryonic Day 27 porcine fetuses into the denervated striatum of cyclosporine-A (CyA)-treated or non-CyA-treated rats. Functional recovery was assessed by amphetamine-induced rotation, and graft survival and morphology were analyzed using neuronal and glial immunostaining as well as in situ hybridization with a porcine repeat element DNA probe. A significant, sustained reduction in amphetamine-induced rotational asymmetry was present in the CyA-treated rats whereas the non-CyA-treated rats showed a transient behavioral recovery. The degree of rotational recovery was highly correlated to the number of surviving transplanted porcine dopaminergic neurons. TH+ neuronal survival and graft volume were significantly greater in the CyA-treated group as compared to the non-CyA group. By donor-specific neuronal and glial immunostaining as well as donor-specific DNA labeling, we demonstrate that porcine fetal neuroblasts are able to survive in the adult brain of immunosuppressed rats, mediate functional recovery, and extensively reinnervate the host striatum. These findings suggest that porcine DA neurons may be a suitable alternative to the use of human fetal tissue in neurotransplantation for PD.

Increased proportion of acetylcholinesterase-rich zones and improved morphological integration in host striatum of fetal grafts derived from the lateral but not the medial ganglionic eminence

Fetal striatal grafts are found to have a modular organization revealed by acetylcholinesterase (AChE) histochemistry. The AChE-rich zones represent the only portions of these grafts that are anatomically and functionally integrated into the host brain. In this study, the medial and lateral ganglionic eminences (MGEs and LGEs) were selectively dissected from the basal telencephalon of embryonic-day-14 (E14) rat fetuses to compare their relative contributions to the AChE-rich fraction of intrastriatal grafts. Separate cell suspensions prepared from either eminence were stereotaxically implanted into excitotoxically lesioned neostriatum of adult rats. Eight weeks after transplantation, grafts of the MGE were compared with those of the LGE with respect to the proportion of AChE-rich zones, graft size, graft morphology, and afferent dopaminergic innervation as revealed by tyrosine hydroxylase (TH) immunostaining. The mean AChE-rich fraction in LGE grafts (87%±4%) was markedly greater than the AChE-rich fraction in MGE grafts (25%±10%). The LGE grafts were also morphologically better incorporated into the lesioned host striatum, partially reconstituting the striatal morphology. There was no statistically significant difference in graft size between the two groups. The AChE-rich LGE grafts were TH immunoreactive, whereas the AChE-poor MGE grafts were not. We conclude that grafts derived exclusively from the fetal LGE reconstitute the striatal morphology and consist almost entirely of AChE-rich zones.

The neural circuitry underlying primate calls and human language

There are significant structural and functional differences between primate calls and human speech. In addition, these two forms of vocal communication appear to largely depend on nonhomologous brain structures. However, an analysis of the underlying axonal circuitry of these brain systems suggests that there are significant interrelationships between them, both in functional and in evolutionary terms. Based on both primate neuroanatomical studies and humanin vivo mapping studies it is argued that the ventral prefrontal area is the critical link, both functionally and anatomically between these distinct vocal systems. A model of human brain evolution with respect to language is proposed in which limbic-midbrain vocalization circuits became progressively subordinated to the activity of prefrontal-midbrain and frontalmotor circuits for regulating facial gesture, skilled oral food manipulation, and conditional association learning. Quantitative and developmental data are used to suggest that this resulted from the relative enlargement of prefrontal areas and the consequences this has on the relative proportions of different corticomidbrain and diencephalic-midbrain projections. Although humans exhibit a significantly reduced call repertoire, it is argued that the display-vocalization circuits that play the central role in all other primate communication have neither been eliminated, supplanted nor suppressed by language systems. They have instead become integrated into the more distributed language circuits and play a ubiquitous though subordinate role in all normal language processes.

Fallacies of progression in theories of brain-size evolution

The tacit assumption that relative enlargement and differentiation of brains reflect a progressive evolutionary trend toward greater intelligence is a major impediment to the study of brain evolution. Theories that purport to establish a linear scale for this presumed correlation between brain size and intelligence are undermined by the absence of an unbiased allometric baseline for estimating differences in encephalization, by the incompatibility of allometric analyses at different taxonomic levels, by the nonlinearity of the ‘criterion of subtraction” used to partition the somatic and cognitive components of encephalization, and by the failure to independently demonstrate any cognitive basis for the regularity of brain/body allometry. Analyzing deviations from brain/body allometric trends in terms of “encephalization” obfuscates the complementarity between brain and body size and ignores selection on body size, which probably determines most deviations. By failing to analyze the effects of allometry at many levels of structure, comparative anatomists have mistaken methodological artifacts for progressive evolutionary trends. Many structural changes, which are assumed to demonstrate progression of brain structure from primitive to advanced forms, are the results of allometric processes. Increased brain size turns out to have some previously unappreciated functional disadvantages.

Specific axon guidance factors persist in the adult brain as demonstrated by pig neuroblasts transplanted to the rat.

The presence and specificity of axon guidance cues in the mature brain were examined by transplanting several types of xenogeneic neural cells from fetal pig brains into adult rat brains with selective neuronal loss. Committed neuronal phenotypes from cortical, mesencephalic and striatal fetal regions were implanted in homotopic or ectopic central nervous system locations. Using specific neurofilament and neural markers, axonal target selection by transplanted fetal neurons was determined throughout the central nervous system. Different types of donor neurons grew axons specifically to appropriate adjacent and distant host brain regions from ectopic or homotopic brain implantation sites and independent of the pattern of prior selective neuronal loss. Since the fetal donor neurons could orient axonal growth towards their normal synaptic termination zones, it shows that the adult brain also elaborates highly specific signals for axon guidance. These results obtained by xenotransplantation also demonstrate that the adult brain exhibits a latent potential for long-distance axon guidance that is evolutionarily conserved. These and related studies indicate that the necessary processes for connection of specific neurocircuitry also exist in the adult central nervous system, if axonal growth inhibition is overcome.

Cortical connections of the inferior arcuate sulcus cortex in the macaque brain

Injections of the retrograde/anterograde tracers Wheat Germ Agglutinin-Horseradish peroxidase (WGA-HRP) into the cortex along the banks of the inferior limb of the arcuate sulcus in the cortex of 4 macaque monkeys (Macaca fascicularis) were used to investigate its cortico-cortical connections. All injections produced transported label within the sulcus principalis, the ventral lateral prefrontal cortex, the anterior cingulate sulcus and the dorsal insular cortex. The distribution of label within each of these areas differed slightly depending on the injection site. Injections along the caudal bank of the inferior arcuate sulcus label premotor, supplementary motor, and precentral motor areas but produce relatively sparse prefrontal labeling. Posteriorly label is transported to the inferior parietal cortex and the dorsal opercular bank of the Sylvian fissure. Injections along the rostral bank of the sulcus do not label motor areas but produce labeling in dorsal, lateral and orbital prefrontal areas, and in cortex along the ventral bank of the superior branch of the arcuate sulcus. Posteriorly label is transported to cortical areas in the superior temporal gyrus including the dorsal bank of the superior temporal sulcus. The more dorsal rostral bank injection produced both superior temporal and some sparse inferior parietal labeling and the more ventral rostral bank injection produced extensive superior temporal labeling but no parietal labeling. No labeling was ever seen in cortex ventral to the fundus of the superior temporal sulcus. Although other auditory recipient prefrontal areas have been reported, this is the first demonstration of a region chiefly devoted to auditory connections within the ventral frontal cortex. Its adjacency to areas associated with vocal muscle movement, and its connections to midline cortical areas associated with vocal functions in both primates and humans may provide important clues to the organization of Brocas language area.