Martin L. Olsson

Forelimb akinesia in the rat Parkinson model: differential effects of dopamine agonists and nigral transplants as assessed by a new stepping test

Methods for the assessment of akinesia in the unilateral rat Parkinson model have so far been lacking. The experiments reported here evaluate the usefulness of a new “stepping test” to monitor forelimb akinesia in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the mesencephalic dopamine (DA) system, and to assess the ability of DA- receptor agonists and fetal DA neuron transplants to reverse these deficits. The 6-OHDA lesion induced marked and long-lasting impairments in the initiation of stepping movements with the contralateral paw. Systemic injections of low doses (chosen to be subthreshold for induction of rotation) of the mixed D1 and D2 receptor agonist apomorphine, the D1-selective agonist SKF 38393, and to a lesser extent also the D2-selective agonist quinpirole were effective in reversing these deficits. Similar effects was seen after a subrotational dose of L-dopa, whereas amphetamine had no effect. Fetal nigral transplants, implanted as multiple deposits in the ipsilateral caudate-putamen and substantia nigra, restored initiation of stepping to a similar degree as the DA agonists. Nigral grafts placed in substantia nigra alone were also effective, although the improvement was less pronounced. Apomorphine, at a dose effective in the lesion-only animals, had no additive effect in the grafted rats, whereas amphetamine appeared to further improve stepping in the rats with intranigral transplants. Identical experiments were performed on skilled forelimb use in the so- called staircase test. Interestingly, neither the DA agonist drugs nor the nigral transplants had any effects on the lesion induced deficits in this more complex task. The results show that forelimb stepping is a highly useful test to monitor lesion-/and transplant-induced changes in forelimb akinesia, a behavioral parameter that may be analogous to limb akinesia and gait problems seen in patients with Parkinsons disease.

A microtransplantation approach for cell suspension grafting in the rat parkinson model: A detailed account of the methodology

Shortcomings of current techniques used for the intracerebral transplantation of ventral mesencephalic dopamine neurons include low graft survival, high variability, considerable implantation trauma and suboptimal graft integration. In order to overcome these limitations, we have adopted a microtransplantation approach which allows precise and reproducible implantation of ventral mesencephalon cell suspensions at single or multiple sites with minimal trauma and improved survival and integration of the grafted neurons [Nikkhah et al. (1994) Brain Res. 633, 133-143]. The present study was undertaken to determine the influence of different grafting parameters as well as the time-course of development of micrografted dopaminergic neurons and to devise an optimal microtransplantation procedure in the rat Parkinson model, Rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway received four graft deposits of either 0.25, 0.5, 1.0 or 2.0 microliters along four injection tracts (150,000 cells/microliters) using either a glass capillary (o.d. 50-70 microns) or a regular cannula (o.d. 0.50 mm, metal cannula grafts). At one, two and 12 weeks postgrafting (capillary grafts) and at 12 weeks postgrafting (metal cannula grafts) dopamine neuron survival and graft volumes were measured and the implantation trauma assessed by glial fibrillary acidic protein expression. The results demonstrate that single deposits of 50,000-75,000 cells in 0.5 microliter, implanted with a glass capillary, provide the best environment both for dopaminergic and non-dopaminergic neuron survival. Grafts implanted with the glass capillary showed much weaker long-term glial fibrillary acidic protein expression along the injection tract and around the implants than was the case in grafts implanted with the thicker metal cannula. Optimal graft integration and minimal disturbances of host brain structures can reliably be achieved by small-sized implants (20,000-35,000 cells/deposit). Tyrosine hydroxylase-positive fiber outgrowth from micrografted dopaminergic neurons was seen not only in the surrounding caudate-putamen, but also along white matter tracts into the nucleus accumbens and the overlying cerebral cortex. Spreading of dopaminergic micrografts over multiple small deposits rather than increasing the volume of single grafts gave more extensive reinnervation of the entire host striatum. The micrografting technique provides a useful tool to improve graft-host interactions in the rat Parkinson model, and it allows more precise and reproducible quantitative studies on dopamine neuron survival and growth in intrastriatal ventral mesencephalon transplants. This technique should also be highly useful for the intracerebral implantation of cells derived from primary cultures or cell lines [Gage and Fisher (1991) Neuron 6, 1-12].

Regional incorporation and site-specific differentiation of striatal precursors transplanted to the embryonic forebrain ventricle

The developmental potential of neural progenitors derived from the E13.5-E14 lateral or medial ganglionic eminences (LGE and MGE, respectively) or the E12 ventral mesencephalon (VM) was examined in cross-species transplantation model. After injection into the E15 rat forebrain ventricle, mouse LGE progenitors (unlike those of the MGE or VM) were consistently integrated into the host striatum, expressing neurochemical phenotypes and axonal projections characteristic of striatal projection neurons. Additionally, both LGE and MGE precursors displayed widespread incorporation into distinct forebrain and midbrain structures, whereas the more caudally derived VM cells were largely confined to midbrain structures. These results suggest that many LGE precursors are positionally specified for striatal incorporation, while a portion also possess greater potential reflected in more widespread integration following intraventricular injection.

Bacterial glycosidases for the production of universal red blood cells.

Enzymatic removal of blood group ABO antigens to develop universal red blood cells (RBCs) was a pioneering vision originally proposed more than 25 years ago. Although the feasibility of this approach was demonstrated in clinical trials for group B RBCs, a major obstacle in translating this technology to clinical practice has been the lack of efficient glycosidase enzymes. Here we report two bacterial glycosidase gene families that provide enzymes capable of efficient removal of A and B antigens at neutral pH with low consumption of recombinant enzymes. The crystal structure of a member of the α-N-acetylgalactosaminidase family reveals an unusual catalytic mechanism involving NAD+. The enzymatic conversion processes we describe hold promise for achieving the goal of producing universal RBCs, which would improve the blood supply while enhancing the safety of clinical transfusions.

Early specification of striatal projection neurons and interneuronal subtypes in the lateral and medial ganglionic eminence

The striatum is thought to be generated from two transient swellings in the ventral telencephalon, the lateral and medial ganglionic eminences, present at mid-stages of embryonic rat development. We have studied the relative contribution of these structures to the specific generation of striatal neuronal subtypes such as projection neurons and cholinergic and somatostatin-containing interneurons at an early stage and a mid stage in striatal neurogenesis. Dissociated progenitors isolated from the embryonic day 12.5 and embryonic day 15.5 rat lateral ganglionic eminence grafted into the previously ibotenic acid lesioned adult striatum, produce grafts containing extensive numbers of neurons expressing messenger RNA for the striatal projection neuron marker, DARPP-32, whereas grafts of the embryonic day 12.5 and embryonic day 15.5 medial ganglionic eminences do not. While preprosomatostatin messenger RNA-expressing neurons were observed in grafts from each of the lateral ganglionic eminence and medial ganglionic eminence at both embryonic day 12.5 and embryonic day 15.5, choline acetyltransferase messenger RNA-expressing cholinergic neurons were largely found in grafts derived from the embryonic day 12.5 medial ganglionic eminence. These results suggest that the neuronal diversity of the adult striatum may derive both from the lateral ganglionic eminence, providing DARPP-32-expressing projection neurons as well as somatostatin-containing interneurons, and the early stage medial ganglionic eminence specifically contributing the cholinergic interneurons.

Projection neurons in fetal striatal transplants are predominantly derived from the lateral ganglionic eminence

In the present study, we have characterized aspects of integration, growth and phenotypic differentiation of embryonic grafts derived from the selective dissection of either the lateral or medial portion of the ganglionic eminences of the rodent forebrain. Donor tissues were derived from embryonic day 15 rat, or embryonic day 14 mouse embryos, and injected, as single cell suspensions into the striatum or substantia nigra of adult rats previously subjected to an intrastriatal ibotenic acid lesion. Two to six weeks following grafting, immunocytochemical detection of DARPP-32, the 32,000 mol. wt dopamine- and cyclic AMP-regulated phosphoprotein, was used to identify areas with a striatum-like phenotype within both the intrastriatal and the intranigral grafts. It was thus revealed that all the lateral ganglionic eminence grafts, irrespective of their placement, were dominated by striatum-like tissue (up to 90% of the total graft volume), while the medial ganglionic eminence transplants were only sparsely positive (< 10% of the total graft volume). These striatum-like regions of the grafts were selectively innervated by tyrosine hydroxylase immunopositive fibres from the host substantia nigra. Furthermore, axons derived from the lateral ganglionic eminence mouse grafts placed in the striatum, as detected by the mouse-specific neuronal marker M6, showed a more extensive and directed outgrowth towards the globus pallidus when compared to fibres emanating from the medial ganglionic eminence grafts. Mouse lateral and medial ganglionic eminence grafts placed into the substantia nigra exhibited similar fibre outgrowth patterns; both types of grafts thus innervated the substantia nigra-pars reticulata and extended axons into the cerebral peduncle. These results show that DARPP-32-positive striatal projection neurons are derived, for the most part, from the lateral ganglionic eminence and that the restricted lateral ganglionic eminence dissection provides a more optimal source of striatal tissue for grafting in the rat Huntington model.

Incorporation and glial differentiation of mouse EGF-responsive neural progenitor cells after transplantation into the embryonic rat brain.

In vitro, epidermal growth factor (EGF)-responsive neural progenitor cells exhibit multipotent properties and can differentiate into both neurons and glia. Using an in utero xenotransplantation approach we examined the developmental potential of EGF-responsive cells derived from E14 mouse ganglionic eminences, cortical primordium, and ventral mesencephalon, after injection into the E15 rat forebrain ventricle. Cell cultures were established from control mice or from mice carrying the lacZ transgene under control of the promoters for nestin, glial fibrillary acidic protein (GFAP), or myelin basic protein (MBP). The grafted cells, visualized with mouse-specific markers or staining for the reporter gene product, displayed widespread incorporation into distinct forebrain and midbrain structures and differentiated predominantly into glial cells. The patterns of incorporation of cells from all three regions were very similar without preference for the homotopic brain areas. These results suggest that EGF-responsive progenitor cells can respond to host derived environmental cues, differentiate into cells with glial-like features, and become integrated in the developing recipient brain.

Polymorphism and recombination events at the ABO locus: a major challenge for genomic ABO blood grouping strategies

. The blood group ABO gene codes for a glycosyltransferase that adds the ultimate monosaccharide to a glycoconjugate and forms the A or B blood group specific antigen. The DNA structure of the three major alleles of the human blood group ABO system was first described in 1990. This review describes the subsequent developments, including the increasing number of variants of these common alleles and the underlying mutations thought to be responsible for the occurrence of some of the weak subgroups of blood group A and B. Several inactive (O) alleles are also now known. Our knowledge of the DNA sequence of the normal A and B alleles and of the rare and intriguing cisAB and B(A) phenotypes has resulted in plausible explanations for these.

ADP Acting on P2Y13 Receptors Is a Negative Feedback Pathway for ATP Release From Human Red Blood Cells

Red blood cells may regulate tissue circulation and O2 delivery by releasing the vasodilator ATP in response to hypoxia. When released extracellularly, ATP is rapidly degraded to ADP in the circulation by ectonucleotidases. In this study, we show that ADP acting on P2Y13 receptors on red blood cells serves as a negative feedback pathway for the inhibition of ATP release. mRNA of the ADP receptor P2Y13 was highly expressed in human red blood cells and reticulocytes. The stable ADP analogue 2-MeSADP decreased ATP release from red blood cells by inhibition of cAMP. The P2Y12 and P2Y13 receptor antagonist AR-C67085 (30 &mgr;mol/L), but not the P2Y1 blocker MRS2179, inhibited the effects of 2-MeSADP. At doses where AR-C67085 only blocks P2Y12 (100 nmol/L), it had no effect. AR-C67085 and the nucleotidase apyrase increased cAMP per se, indicating a constant cAMP inhibitory effect of endogenous extracellular ADP. 2-MeSADP reduced plasma ATP concentrations in an in vivo pig model. Our results indicate that the ATP degradation product ADP inhibits ATP release by acting on the red blood cell P2Y13 receptor. This negative feedback system could be important in the control of plasma ATP levels and tissue circulation.

A Rapid and Simple ABO Genotype Screening Method Using a Novel B/O2 versus A/O2 Discriminating Nucleotide Substitution at the ABO Locus

An ABO genotype screening method discriminating the common alleles A1, A2, B, O1 and O2 at the ABO locus was made possible by the discovery of a novel nucleotide substitution (G1096A) present only in B and O2 alleles. A rapid and reliable single‐tube approach using multiplex PCR with four primers amplifying exons 6 and 7 of the ABO genes followed by simultaneous addition of two restriction enzymes was developed and validated in a population of 150 Swedish blood donors. This technique is the most cost‐efficient and informative ABO genotyping method reported to date.