Reformation of the nigrostriatal pathway by fetal dopaminergic micrografts into the substantia nigra is critically dependent on the age of the host.

The aim of this study was to determine whether the growth of axons along the nigrostriatal pathway from fetal dopamine cells, transplanted into the substantia nigra of young postnatal 6-OHDA-lesioned rats, is dependent on the age of the host brain. Neonatal rats were lesioned bilaterally by intraventricular injection of 6-OHDA at postnatal day 1 (P1) and received grafts of E14 ventral mesencephalon at day 3 (group P3), day 10 (group P10), or day 20 (group P20) into the right substantia nigra. One lesioned group was left untransplanted. Six months after surgery the animals were subjected to analysis of drug-induced rotation following injection of amphetamine, apomorphine, a D1 agonist (SKF38393), or a D2 agonist (Quinpirole). Animals transplanted intranigrally at day 3 and day 10 showed a strong amphetamine-induced rotational bias toward the side contralateral to the transplant. Animals transplanted into substantia nigra at P20, like the lesioned control animals, showed no rotational bias. Apomorphine and selective D1 and D2 agonists induced ipsilateral turning behavior in the P3 and P10 group, but not in the P20 or the lesion control groups. Immunofluorescence histochemistry in combination with retrograde axonal tracing, using FluoroGold injection into the ipsilateral caudate-putamen showed colocalization of tyrosine hydroxylase and FluoroGold in large numbers of transplanted neurons in the animals transplanted at postnatal day 3 and postnatal day 10, which was not observed in the group P20. The lesion control group showed a 90% complete lesion of the TH-positive cells in the substantia nigra while largely sparing the neurons in the ventral tegmental area. The results indicate that intranigral grafts can be placed accurately and survive well within the substantia nigra region at various time points during postnatal development. Furthermore, embryonic dopamine neurons have the ability to extend axons along the nigrostriatal pathway and reconnect with the dopamine-depleted striatum when transplanted at postnatal day 3 and postnatal day 10, but not at postnatal day 20.

Long-term survival of fetal porcine lateral ganglionic eminence cells in the hippocampus of rats.

Embryonic porcine brain tissue from the lateral ganglionic eminence was transplanted into the adult rat hippocampus to determine whether fetal striatal cells could survive, differentiate, and integrate in a heterotopic site. The hippocampus, a common site of epileptic seizure activity, was chosen to determine if fetal striatal cells could supply inhibitory GABAergic neurons that may serve to block seizures. Cells were either implanted with a single deposit using a standard metal cannula or by five smaller disseminated deposits with a glass micropipette. At 20-24 weeks, animals immunosuppressed with cyclosporin showed long-term survival of porcine cells in the adult hippocampus. Analysis by immunohistochemistry and in situ hybridization showed that the grafts contained glial and neuronal cell types, including GABAergic neurons within graft core and networks of porcine neuronal fibers extending from the graft into the host parenchyma. In addition, a marker of porcine presynaptic terminals, synaptobrevin, was abundant within the grafts and was found associated with hippocampal structures and cell layers suggesting functional integration of grafted cells within the host. The survival of xenografts in the hippocampus and potential integration of inhibitory components provides evidence that these grafts may serve as an internal negative feedback mechanism to quench epileptiform activity.

Cerebellar precursors transplanted to the neonatal dentate gyrus express features characteristic of hippocampal neurons.

During the development of the CNS, a salient issue is whether neuronal phenotype is defined by the lineage or by the environment of precursor cells. Transplants permit these two possibilities to be tested, as cell fate can be examined in a new location. Dissociated cerebellar cells from newborn rats treated with tritiated thymidine or from NSE-lacZ transgenic mice were grafted into the dentate gyrus of the developing hippocampus. Implanted cells integrated into the granule cell layer, which contains the cell bodies of host granule neurons. Immunohistochemistry showed that grafted cells in the granule cell layer, like the host hippocampal granule neurons, were calbindin positive and upregulated FOS in a seizure paradigm. Electron microscopic analysis also showed that cells grafted to the dentate gyrus share features with host dentate neurons. These assays indicate that transplanted cerebellar cells acquired morphological and antigenic features characteristic of hippocampal neurons. These results show that metencephalic precursors are capable of differentiating in response to signals in the telencephalon, suggesting that the environment controls the regional fate of neuronal precursor cells during neurogenesis.

Dopaminergic microtransplants into the substantia nigra of neonatal rats with bilateral 6-OHDA lesions. II. Transplant-induced behavioral recovery.

Transplants of fetal ventral mesencephalic (VM) dopamine neurons implanted into the substantia nigra in 6-hydroxy-dopamine (6-OHDA)-lesioned neonatal pups establish axonal connections with the denervated caudate putamen (Nikkhah et al., 1995). In the present study, we have explored the functional capabilities of these animals after they reached adulthood on a battery of spontaneous and drug-induced behavioral tasks. The results demonstrate that unilateral intranigral VM grafts in bilaterally lesioned neonates induce a marked bias in spontaneous- and stress-induced rotation contralateral to the implant not present in the lesion-only controls. Amphetamine and apomorphine induced vigorous contra- and ipsilateral rotation, respectively. Moreover, grafted animals achieved 75% of the performance level in contralateral skilled forelimb use when compared to normal controls, which was significantly above lesion-only animals (50% of normal). Spontaneous nocturnal locomotor activity was elevated 2.2-fold in the grafted animals. Sensorimotor orientation and disengage behavior was spared by the neonatal dopamine lesion and unaffected by the grafts. The level of functional restoration seen in the present study was more extensive than reported previously in neonatally 6-OHDA-lesioned rats where the VM grafts were implanted ectopically into the striatum. However, functional recovery remained incomplete also after intranigral graft placement compared to normal intact animals. The present approach should provide a new promising avenue for the continued exploration of the mechanisms involved in functional recovery and structural repair in the damaged nigrostriatal system.

Dopaminergic microtransplants into the substantia nigra of neonatal rats with bilateral 6-OHDA lesions. I. Evidence for anatomical reconstruction of the nigrostriatal pathway.

Reconstruction of the nigrostriatal pathway by long axon growth derived from dopamine-rich ventral mesencephalic (VM) transplants grafted into the substantia nigra may enhance their functional integration as compared to VM grafts implanted ectopically into the striatum. Here we report on a novel approach by which fetal VM grafts are implanted unilaterally into the substantia nigra (SN) of 6-hydroxydopamine (6-OHDA)-lesioned neonatal pups at postnatal day 3 (P3) using a microtransplantation technique. The results demonstrate that homotopically placed dopaminergic neurons survive and integrate well into the previously 6-OHDA-lesioned neonatal SN region. Moreover, the tyrosine hydroxylase (TH)-positive neurons extended axons rostrally along the white matter tract of the internal capsule closely following the course of the original nigrostriatal pathway. The graft reestablished a TH-positive axon terminal network in the ipsilateral caudate-putamen, with the highest density in the medial and central parts. Retrograde labeling with Fluoro-Gold from the host striatum demonstrated that most of the transplant neurons giving rise to the graft-derived fiber outgrowth were TH-positive, but revealed also a small proportion of projecting neurons which were TH-negative. Amphetamine-induced striatal Fos expression was normalized in the caudate-putamen ipsilateral to the intranigral VM grafts, showing hyperexpression in some areas of the striatum, and the apomorphine-induced Fos expression seen in the 6-OHDA-lesioned animals was completely reversed on the grafted side. These findings indicate that the graft-derived dopaminergic reinnervation of the striatum is functional. The microtransplantation strategy may provide new avenues for the exploration of morphological and functional integration of fetal dopamine neurons in the nigrostriatal system and give new insights into the mechanisms controlling long-distance axon growth in the brain.

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].

Intranigral fetal dopamine grafts induce behavioral compensation in the rat Parkinson model.

Neural transplantation in experimental Parkinsonism has so far focused on the ectopic placement of fetal ventral mesencephalic (VM) neurons into the dopamine-denervated caudate-putamen. VM grafts are effective in restoring dopamine neurotransmission in the grafted caudate-putamen and in partial amelioration of behavioral deficits. Recent pharmacological and physiological data have provided strong evidence that dopamine released from dendrites of the substantia nigra pars compacta (SNc) neurons within the pars reticulata (SNr) plays an important role in the regulation of the basal ganglia output pathways. Using a novel microtransplantation approach, multiple small cell suspension grafts (250 nl) derived from the VM of E14 rat embryos were implanted into the SNr of unilaterally 6-hydroxydopamine-lesioned rats. Behavioral changes in drug-induced rotation asymmetry were monitored for up to 14 weeks postgrafting, followed by a quantitative assessment and correlation of tyrosine hydroxylase (TH)-positive cell survival. The reduction in rotational asymmetry caused by the intranigral VM grafts was 64% for SKF 38393 (D1 agonist), 54% for apomorphine (mixed D1 and D2 agonist), and 67% for quinpirole (D2 agonist) when compared to a control spinal cord graft group. By contrast, amphetamine-induced rotation was completely unaffected. The correlation between number of TH-positive cells and behavioral compensation was highest for the D1 agonist (R = -0.729), though clear-cut also for the mixed D1/D2 agonist apomorphine (R = -0.664) and the D2 agonist quinpirole (R = -0.642). Favorable morphological features of the VM micrografts included extensive migration of the dopaminergic neurons into the host SNr and the formation of dense patches of dendrite-like TH-positive terminal networks within the SNr. The results demonstrate a novel pattern of behavioral recovery induced by intranigral VM transplants in the rat Parkinson model. This may have important implications for the understanding of how the nigrostriatal dopamine system influences motor control in the basal ganglia as well as for the development of optimal transplantation strategies in Parkinson's disease.

Improved graft survival and striatal reinnervation by microtransplantation of fetal nigral cell suspensions in the rat Parkinson model.

A microtransplantation approach has been used in order to achieve more complete reinnervation of the dopamine denervated rat striatum by fetal nigral cell suspensions injected into multiple striatal sites. A total of 450,000 cells, obtained from the ventral mesencephalon of embryonic day 14 rat fetuses, were implanted either in the conventional way as two 1.8-microliters deposits centrally in the head of the caudate-putamen ('Macro grafts'), or as eighteen 0.2-microliter deposits disseminated over six needle penetrations in the same area using a 50-70 microns glass capillary tip ('Micro grafts'). Non-grafted lesioned rats served as controls. Dopamine neuron survival (as assessed by tyrosine hydroxylase immunohistochemistry at 4 months after transplantation) was 2.8-fold greater in the Micro grafts as compared to the Macro grafts. Striatal dopamine tissue levels (determined in a separate group of rats) was increased 2.5-fold in the head of the caudate-putamen (from 12.5% of normal in the Macro graft group to 30% of normal in the Micro graft group). Consistent with this, the overall graft-derived tyrosine hydroxylase positive fiber outgrowth was more extensive in the Micro graft group and covered larger areas of the previously denervated caudate-putamen. The results show that distribution of the fetal nigral tissue in multiple small deposits provides for increased dopamine neuron survival, probably because of a closer contact between the implanted cells and the surrounding host striatal tissue in the small-sized graft deposits. Less bleeding and necrosis at the implantation site may also have contributed to this effect. The present microtransplantation procedure is an efficient means to increase overall dopamine neuron survival and to achieve more complete reinnervation of the denervated striatum in the rat Parkinson model. It also substantially increased the reproducibility of DA graft survival between animals.

A hypothermic miniaturized stereotaxic instrument for surgery in newborn rats.

We describe a simple, scaled-down instrument which enables accurate, reproducible stereotaxic placements into specific sites in the brain of the newborn rat. The instrument is specially designed for the administration of long-term hypothermia, yet permits the use of alternative methods of anesthesia. The design of the head-stabilizing mechanism allows head positioning to be finely adjusted to achieve precise horizontal and vertical zero planes. This adaptability also allows the device to accommodate a large range of animal sizes and levels of maturity. Furthermore, the apparatus can be fitted onto a conventional adult stereotaxic frame or used by itself in combination with a free-standing manipulator. As a model preparation, we describe a procedure for stereotaxic surgery in the post-natal day (P1) rat. The versatility of the instrument has permitted successful stereotaxic surgery in adolescent as well as neonatal rats, newborn and adult mice, and newborn hamsters.

Region-specific differentiation of the hippocampal stem cell line HiB5 upon implantation into the developing mammalian brain.

Proliferating precursors to the distinct cell types constituting the mammalian brain can be identified by the presence of the nestin intermediate filament. We report the establishment of a nestin-positive cell line, HiB5, from embryonic precursor cells to the rat hippocampus. Since it was immortalized using the temperature-sensitive allele tsA58 of SV40 large T antigen, these cells grow continuously at 33 degrees C, but not at 39 degrees C, the body temperature of rodents. To test their developmental capacity, HiB5 cells were implanted into both the neonatal hippocampus and cerebellum. The cells integrated into the host tissue and acquired morphologies characteristic of the neurons and glial cells found at the implant site. HiB5 cells might thus be useful in characterizing the signals regulating cell type determination in the mammalian brain.

Effect of cocaine and lidocaine on the development of kindled seizures.

The effect of a subconvulsive dose of cocaine or lidocaine on the development of kindling was studied in male Long-Evans rats. Animals were divided into three groups and kindled by daily electrical stimulation of the pyriform cortex. Fifteen minutes before each stimulation each animal received an intraperitoneal injection of either saline, 20 mg/kg cocaine hydrochloride, or 20 mg/kg lidocaine hydrochloride. Following kindling the drug treatment was discontinued and the transfer of kindling to a nondrug state was assessed by test stimulations given 2, 6, and 48 days after the last day of kindling. Both cocaine and lidocaine dramatically accelerated the development of kindling. Furthermore, the duration of clonus at kindling criterion was significantly longer in lidocaine-treated animals than in animals treated with saline, and the onset of clonus in cocaine-treated animals occurred significantly sooner after stimulation. However, this performance did not transfer fully to the nondrug state, with some animals failing to exhibit clonus. Among those animals exhibiting clonus at nondrug tests, afterdischarge duration was significantly higher in cocaine-treated than in saline-treated animals, but clonus duration was no longer elevated in lidocaine-treated animals, and the latency to clonus rose dramatically in animals previously treated with either cocaine or lidocaine. These results indicate that a subconvulsive dose of cocaine or lidocaine can facilitate the development of kindling when the drug is active at the time of electrical stimulation, apparently by means of the local anesthetic action shared by the two drugs. The kindling produced in this fashion is not entirely equivalent to kindling produced by electrical stimulation alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Observations on choline acetyltransferase containing structures in the CD-1 mouse brain.

Central cholinergic structures within the CD-1 mouse were evaluated by immunohistochemical visualization of choline acetyltransferase (ChAT) using the monoclonal antibody AB8. Rostrally, cholinergic neurons were seen within the neostriatum, medial septal nucleus (Ch1), ventral (Ch2) and horizontal (Ch3) limb nuclei and nucleus basalis-substantia innominata complex (Ch4). Caudally, cholinergic neurons were seen in the cuneiformis-pedunculopontine nuclei (Ch5), lateral dorsal tegmental (Ch6) and parabigeminal (Ch8) nuclei as well as the medial habenular nucleus and cranial motor nuclei. Additional cholinergic perikarya were found in the hippocampus and cerebral cortex. ChAT stained fibers were observed in the cerebral cortex and in many fiber fascicles.

Effects of single daily doses of a pyridil-2-tetrahydrothiophene derivative (40749 RP) on 24 hour H+ activity, nocturnal acid output, gastrin and pepsinogen I profiles in duodenal ulcer patients.

40749 RP is a pyridil-2-tetrahydrothiophene derivative, belonging to a new class of gastric antisecretory drugs. We compared its effects on gastric secretion with cimetidine. Intragastric acidity, nocturnal acid output, gastrin and pepsinogen-I profiles were measured in patients with duodenal ulcer in clinical remission. A single dose of 100 mg 40749 RP reduced median 24 h gastric acidity as effectively as cimetidine 1000 mg given as four divided doses, 0.63 vs 1.6 mmol/l. Continued treatment with 40749 RP for 10 days reduced the median 24 h gastric acidity even further, to 0.006 mmol/l (p less than 0.001) and significantly increased fasting concentrations of gastrin and pepsinogen-I (p = 0.02). The incremental gastrin secretion to a standard meal was significantly increased after 10 days treatment with 40749 RP when compared with the first day of 40749 RP, or with cimetidine. These results show that 40749 RP exerts a powerful inhibitory effect on gastric acid secretion after a single 100 mg dose, and that this inhibitory effect increases with continued administration.