Strands of embryonic mesencephalic tissue show greater dopamine neuron survival and better behavioral improvement than cell suspensions after transplantation in parkinsonian rats.

The success of embryonic neural transplants as a treatment for patients with Parkinson's disease has been limited by poor survival of transplanted dopamine neurons. To see if a new partially intact tissue preparation method improves survival, we have developed a technique for extruding embryonic tissue into strands. We expected this method to reduce cell damage and improve transplant survival as well as provide improved tissue delivery. We have compared transplants of tissue strands with mechanically dispersed suspensions of embryonic day 15 rat ventral mesencephalon. Tissue from ventral mesencephalon was transplanted into a single site in dopamine denervated striatum of unilateral 6-hydroxydopamine (6-OHDA) lesioned rats. To evaluate the effects of striatal cografts and growth factors on dopamine cell survival, dispersed mesencephalic cells were cotransplanted with dispersed striatal cells. Another group had dispersed mesencephalic cells cotransplanted with striatal cells incubated in the cold for 2 h with glial cell line-derived neurotrophic factor (GDNF, 100 ng/ml), insulin-like growth factor-I (IGF-I, 1500 ng/ml), and basic fibroblast growth factor (bFGF, 150 ng/ml). Behavioral improvement was assessed monthly by changes in methamphetamine-induced rotational behavior. Animals were sacrificed after 3 months, and dopamine neurons were identified by tyrosine hydroxylase (TH) immunohistochemistry. Transplants of tissue strands produced better dopamine neuron survival and led to more robust behavioral restoration than did cell suspensions even when suspensions were supported with cografts of striatal cells or pretreatment with growth factors.

Growth factors improve immediate survival of embryonic dopamine neurons after transplantation into rats.

Embryonic dopamine neurons survive poorly after transplant into models of Parkinson's disease, possibly due to programmed cell death (apoptosis). Apoptosis in cultured dopamine neurons can be reduced by growth factors such as glial cell line-derived neurotrophic factor (GDNF) or a combination of insulin-like growth factor-I (IGF-I) and basic fibroblast growth factor (bFGF). To improve the survival of dopamine neurons in grafts, strands of E15 rat ventral mesencephalon were pretreated with a combination of GDNF, IGF-I, and bFGF and then transplanted into 6-hydroxydopamine-lesioned rats. In control animals, only 32% of dopamine neuron profiles survived the first 24 h after transplant. Growth factor pretreatment increased survival to 49% on day 1. Growth factors reduced the apoptotic rate of transplanted cells, just as they had in the previous in vitro experiments. Apoptotic nuclear morphology was observed in the transplanted dopamine neurons. We conclude that the majority of transplanted dopamine neurons die in grafts within the first 24 h after transplant, most likely by an apoptotic mechanism. Prevention of apoptosis with anti-apoptotic agents may improve the viability of dopamine neurons grafted for Parkinson's disease.

Inhibition of proliferation and expression of T-antigen in SV40 large T-antigen gene-induced immortalized cells following transplantations.

Rat dopamine-producing nerve cells (1RB3AN27) and rat parotid acinar cells (2RSG) were immortalized by insertion of simian virus 40 (SV40) large T-antigen gene (LTa). Both of these cells divided and produced nuclear LTa in vitro. In order to assess the relationship between cell proliferation and expression of LTa in vivo, immortalized dopamine-producing nerve cells and parotid cells were grafted into the striatum and parotid gland of adult Sprague-Dawley rats, respectively. Grafted cells exhibited nuclear LTa at 1 day but not at 7 and 30 days after transplantation. At 30 days after transplantation, no tumor was found, and there was no evidence of cell division as determined by H and E staining. When the striatal areas containing the grafts were cultured, these cells did not express LTa at 4 days after plating; however, after 3 weeks, when most host cells were eliminated, the cultured grafted cells expressed LTa. After 3 months of culturing, only cells exhibiting LTa were present. These cells had the same morphology and divided with the same doubling time as 1RB3AN27 cells before grafting. Results suggest the presence of a LTa-inhibiting factor in vivo, and support the hypothesis that the expression of LTa is directly linked with proliferation of immortalized cells.

Characterization and transplantation of two neuronal cell lines with dopaminergic properties.

Immortalized rat mesencephalic cells (1RB3AN27) produced dopamine (DA) at a level that was higher than produced by undifferentiated or differentiated murine neuroblastoma cells (NBP2) in culture. Treatment of 1RB3AN27 and NBP2 cells with a cAMP stimulating agent increased tyrosine hydroxylase (TH) activity and the intensity of immunostaining for the DA transporter protein (DAT). 1RB3AN27 cells were labelled with primary antibodies to neuron specific enolase (NSE) and nestin and exhibited very little or no labeling with anti-glial fibrillary acidic protein (GFAP). 1RB3AN27 cells exhibited beta- and alpha-adrenoreceptors, and prostaglandin E1 receptors, all of which were linked to adenylate cyclase (AC). Dopamine receptor (D1) and cholinergic muscarinic receptors linked to AC were not detectable. The levels of PKC alpha and PKC beta isoforms were higher than those of PKC gamma and PKC delta in 1RB3AN27 cells. The 1RB3AN27 cells were more effective in reducing the rate of methamphetamine-induced turning in rats with unilateral 6-OHDA lesion of the nigrostriatal system than differentiated NBP2 cells. The grafted 1RB3AN27 were viable as determined by DiI labelling, but they did not divide and did not produce T-antigen protein; however, when these grafted cells were cultured in vitro, they resumed production of T-antigen and proliferated after the primary glia cells and neurons of host brain died due to maturation and subsequent degeneration. Examination of H&E stained sections of the grafted sites revealed no evidence of infiltration of inflammatory cells in the grafted area suggesting that these cells were not immunogenic. They also did not form tumors.

Behavioral and neurochemical asymmetries following unilateral trephination of the rat skull: is this control operation always appropriate?

The present results are from rats that were intended as sham-operated controls in a study of unilateral lesion of the cortical barrel fields. These animals received a trephine hole through the skull, centered over the barrel fields of one hemisphere. Unexpectedly, they showed time-dependent behavioral and neurochemical asymmetries: 1 + 4 days after unilateral skull trephination they scanned an open field mainly with the contralateral vibrissae. Thereafter (days 7 + 14), scanning recovered to symmetry; however, an ipsilateral asymmetry was induced now by challenge with the dopamine receptor agonist apomorphine. At the same time period after skull trephination, an asymmetry of thigmotactic swimming had developed, with more thigmotactic swimming ipsilateral to the side of skull trephination. Neurochemically, there were indications for changes in neostriatal dopamine metabolism because the tissue levels of dopamine and dihydroxyphenylacetic acid were lower on the ipsilateral side in animals killed 6-16 days after trephination. The time courses of behavioral and neurochemical asymmetries after unilateral skull trephination paralleled those seen following unilateral barrel cortex lesion or unilateral removal of the corresponding contralateral vibrissae; however, without exception, the asymmetries after trephination were in the opposite direction than after cortex lesion or vibrissae removal. The possible mechanisms by which skull trephination might have affected behavior and neurochemistry are discussed, especially with respect to the vibrissae-barrel cortex system and the basal ganglia. Because trephination of the skull is routinely employed, both as a control procedure and for CNS manipulation, these results may have important implications for the design of future experiments.

Behavioral and neurochemical indices of barrel cortex-basal ganglia interaction.

Previous experiments from our laboratory have shown a wide variety of time-dependent lateralized changes in behavior and nigrostriatal function following unilateral manipulation of the mystacial vibrissae of rats. The present experiment investigated the effects of unilateral radiofrequency lesion of the cortical vibrissae representation (the barrel fields) in light of these results. We measured lateralized changes in behavior as well as tissue monoamines in neostriatum and substantia nigra, between 1 and 16 days post-lesion. Short-term asymmetries in exploratory behavior (thigmotactic scanning) and neostriatal serotonin metabolism that lasted up to day 6 were seen. In substantia nigra, time-related asymmetries in dopamine concentrations were found with higher ipsilateral values on day 3 and higher contralateral values on day 6. After day 6, the animals had recovered from these acute effects and thereafter, neostriatal dopamine metabolism became asymmetrical. Also during this time, they showed a directional bias in spontaneous and apomorphine-induced turning. Finally, neostriatal serotonin was bilaterally elevated on day 16. These results parallel some of the effects previously seen following unilateral removal of the vibrissae, indicating that the barrel cortex is a critical link in the functional interaction between the vibrissae and basal ganglia.

Differences in human and monkey sensitivity to acoustic cues underlying voicing contrasts.

Humans and monkeys were compared in their differential sensitivity to various acoustic cues underlying voicing contrasts specified by voice-onset time (VOT) in utterance-initial stop consonants. A low-uncertainty repeating standard AX procedure and positive-reinforcement operant conditioning techniques were used to measure difference limens (DLs) along a VOT continuum from--70 ms (prevoiced/ba/) to 0 ms (/ba/) to + 70 ms (/pa/). For all contrasts tested, human sensitivity was more acute than that of monkeys. For voicing lag, which spans a phonemic contrast in English, human DLs for a/ba/(standard)-to-/pa/ (target) continuum averaged 8.3 ms compared to 17 ms for monkeys. Human DLs for a/pa/-to-/ba/ continuum averaged 11 ms compared to 25 ms for monkeys. Larger species differences occurred for voicing lead, which is phonemically nondistinctive in English. Human DLs for a /ba/-to-prevoiced/ba/ continuum averaged 8.2 ms and were four times lower than monkeys (35 ms). Monkeys did not reliably discriminate prevoiced /ba/-to-/ba/, whereas humans DLs averaged 18 ms. The effects of eliminating cues in the English VOT contrasts were also examined. Removal of the aspiration noise in /pa/ greatly increased the DLs and reaction times for both humans and monkeys, but straightening out the F1 transition in /ba/ had only minor effects. Results suggest that quantitative differences in sensitivity should be considered when using monkeys to model the psychoacoustic level of human speech perception.