Human neural stem cell-derived cholinergic neurons innervate muscle in motoneuron deficient adult rats.

Motoneuron damage occurs in spinal cord injury and amyotrophic lateral sclerosis. Current advances offer hope that human embryonic stem cells [Science 282 (1998) 1145] or neural stem cells (NSC) [Exp Neurol 161 (2000) 67; Exp Neurol 158 (1999) 265; J Neurosci Methods 85 (1998) 141; Proc Natl Acad Sci USA 97 (2000) 14720; Exp Neurol 156 (1999) 156 ] may be donors to replace lost motoneurons. Previously, we developed a priming procedure that produced cholinergic cells that resemble motoneurons from human NSCs grafted into adult rat spinal cord [Nat Neurosci 5 (2002a) 1271]. However, effective replacement therapy will ultimately rely on successful connection of new motoneurons with their muscle targets. In this study, we examined the potential of human fetal NSC transplantation to replace lost motoneurons in an animal model of chronic motoneuron deficiency (newborn sciatic axotomy) [J Comp Neurol 224 (1984) 252; J Neurobiol 23 (1992) 1231]. We found, for the first time, that human neural stem cell-derived motoneurons send axons that pass through ventral root and sciatic nerve to form neuromuscular junctions with their peripheral muscle targets. Furthermore, this new cholinergic innervation correlates with partial improvement of motor function.

Effects of fetal septal grafts on memory and learning performance with hippocampal acetylcholine and choline metabolism in fimbria transected rats.

Female Sprague-Dawley rats underwent aspirative lesion of the fimbria to produce septohippocampal disconnection. Two weeks after the lesion surgery, fetal septal grafts prepared from ventral forebrain of 13-15 days old fetuses of the same outbred strain were placed into the lesion cavity (grafted group). Three months after grafting, all rats were tested for spontaneous motor activity (SMA), step through passive avoidance (STPA) and in Morris' water maze (MWM). Six months after grafting, both basal and stimulated acetylcholine (ACh) and choline (Ch) release and their tissue levels were measured in ipsilateral hippocampal slices. Septohippocampal disconnection caused a significant impairment in Morris' water maze tasks, but did not alter spontaneous motor activity and step through passive avoidance. Fimbrial lesion, moreover, also declined both stimulated ACh release and tissue ACh levels in hippocampal slices. While lesion-induced change in Morris' water maze was ameliorated partially, declines in both stimulated ACh release and tissue ACh levels were raised to the control levels by fetal septal graft placed into the lesion cavity. These data show that grafted cholinergic neurons can work biochemically which may not result with a complete behavioral amelioration which is, in fact something more complex.

Intrahippocampal grafts containing cholinergic and serotonergic fetal neurons ameliorate spatial reference but not working memory in rats with fimbria-fornix/cingular bundle lesions.

Three-month-old Long-Evans female rats sustained aspirative lesions of the dorsal septohippocampal pathways and, 2 weeks later, received intrahippocampal suspension grafts containing cells from the mesencephalic raphe, cells from the medial septum and the diagonal band of Broca, or a mixture of both. Lesion-only and sham-operated rats were used as controls. All rats were tested for locomotor activity 1 week, 3 and 5 months after lesion surgery, for spatial working memory in a radial maze from 5 to 9 months, and for reference and working memory in a water tank during the 9th month after lesioning. Determination of hippocampal concentration of acetylcholine, noradrenaline, and serotonin was made after completion of behavioral testing. Compared to sham-operated rats, all rats with lesions, whether grafted or not, exhibited increased levels of locomotor activity and made more errors in the radial maze. The lesioned rats were also impaired in the probe trial (30 first seconds) of the water-tank test made according to a protocol requiring intact reference memory capabilities. While rats with septal or raphe grafts were also impaired, the rats with co-grafts showed performances not significantly different from those of sham-operated rats. With a protocol requiring intact working memory capabilities, all lesioned rats, whether grafted or not, were impaired in the water-tank test. In the dorsal hippocampus of lesion-only rats, the concentration of acetylcholine and serotonin was significantly reduced. In rats with septal grafts or co-grafts, the concentration of acetylcholine was close to normal, as was that of serotonin in rats with raphe grafts or co-grafts. These results confirm previous findings showing that co-grafts enabled the neurochemical properties of single grafts to be combined. Data from the water-tank test suggest that cholinergic and serotonergic hippocampal reinnervations by fetal cell grafts may induce partial recovery of spatial reference, but not working memory capabilities in rats.

Morris water maze analysis of 192-IgG-saporin-lesioned rats and porcine cholinergic transplants to the hippocampus.

Adults rats were lesioned with 192-IgG-saporin, an immunotoxin that targets cholinergic neurons in the basal forebrain expressing the low-affinity nerve growth factor receptor (p75). One month later, rats received E30-35 porcine cholinergic neurons bilaterally into the hippocampus, and were tested in the Morris water maze and the passive avoidance task 4.5-6 months after transplantation (in two experiments, rats were retested in the water maze) followed by histological and cellular analyses. The 192-IgG-saporin-lesioned animals displayed clear cognitive deficits in the Morris water maze. In all experiments the lesioned animals had spatial probe deficits on day 5 testing. A large variance was found among the transplanted animals, with individual animals exhibiting improved performance, but little overall improvement when compared to lesion-alone animals as a group. The relationships between behavioral performance and graft cholinergic factors were established by histological analyses. Grafted animals exhibited an increase in cholinergic innervation of the dentate gyrus (DG) region of the dorsal hippocampus when compared to lesion-alone animals. There was a significant correlation between the level of cholinergic innervation in the dentate gyrus and spatial navigation performance (latency and spatial probe) in the Morris water maze task. These data provide evidence of memory and spatial deficits following cholinergic denervation, and of target-specific growth of xenogeneic cholinergic neurons into the hippocampus. The lack of a clear treatment (transplant) effect in the behavioral measures leads us to believe that functional restoration of cognitive function would require cholinergic reinnervation of both the hippocampus and the neocortex in this 192-IgG-saporin animal model.

Suppression of kindling epileptogenesis in rats by intrahippocampal cholinergic grafts.

Selective immunolesioning of the basal forebrain cholinergic system by 192 IgG-saporin, which leads to a dramatic loss of the cholinergic innervation in cortical and hippocampal regions, facilitates the development of hippocampal kindling in rats. The aim of the present study was to explore whether grafted cholinergic neurones are able to reverse the lesion-induced increase of seizure susceptibility. Intraventricular 192 IgG-saporin was administered to rats which 3 weeks later were implanted with rat embryonic, acetylcholine-rich septal-diagonal band tissue ('cholinergic grafts') or cortical tissue/vehicle ('sham grafts') bilaterally into the hippocampal formation. After 3 months, the grafted animals as well as non-lesioned control rats were subjected to daily hippocampal kindling stimulations. In the animals with cholinergic grafts, which had reinnervated the hippocampus and dentate gyrus bilaterally, there was a marked suppression of the development of seizures as compared with the hyperexcitable, sham-grafted rats. This effect was significantly correlated to the density of the graft-derived cholinergic innervation of the host hippocampal formation. The kindling rate in the rats with cholinergic grafts was similar to that in non-lesioned controls. These results provide further evidence that the intrinsic basal forebrain cholinergic system dampens kindling epileptogenesis and demonstrate that this function can be exerted also by grafted cholinergic neurones.

Nitric oxide synthase in ventral forebrain grafts and in early ventral forebrain development.

Embryonic ventral forebrain (VFB) grafts to cortex contain neurons that synthesize acetylcholine and partially ameliorate behavioral deficits caused by excitotoxic damage to the nucleus basalis magnocelullaris in rats. An additional neurotransmitter, nitric oxide (NO), is synthesized by a subset of cholinergic neurons in rat ventral forebrain. If this neurotransmitter is expressed also by grafted cholinergic neurons (which include the embryonic medial septum and diagonal band), its functional contribution should be considered. Six to twelve months after transplantation of embryonic VFB tissue rats were sacrificed. Brain tissue was processed either for in situ hybridization of nNOS and neuropeptide Y (NPY) or for immunohistochemistry of choline acetyltransferase (ChAT) and neuronal nitric oxide synthase (nNOS). Quantification of messenger ribonucleic acid (mRNA) for nNOS was performed with radioactively labeled probes (silver grains were counted) and a preliminary comparison was made of graft sections to sections of the ventral forebrain of developing rats. Plots of silver grain counts against cell size revealed similar patterns in the grafts and in the ventral forebrain of developing rats. The rates of expression of mRNA for nNOS in the grafts were intermediate between those of the ventral forebrain of postnatal day 19 and those of postnatal day 12. Double immunohistochemical labeling revealed that 45.87 + 8.26% of cells expressing ChAT also expressed nNOS in the grafts, significantly higher than 33.16 + 3.9% which was the rate of co-expression observed in the adult ventral forebrain. This study suggests that possible contribution of NO to graft-associated modulation of behavior should be examined.

Intraretrosplenial cortical grafts of fetal cholinergic neurons and the restoration of spatial memory function.

The retrosplenial cortex (RSC) receives cholinergic afferent fibers from the medial septal nucleus and diagonal band of Broca (DBB) by way of the cingulate bundle and the fornix. Bilateral lesions of both the cingulate and fornix pathways result in a complete depletion of cholinergic input to the RSC. In the present study we have examined the effects of transplanting cholinergic neurons from fetal rat pups to the RSC of adult rats following lesions of the cingulate bundle and fornix. The animals with lesions exhibited severe spatial memory impairments with a complete loss of extrinsic cholinergic afferents to the RSC. Animals with intraretrosplenial cortical transplants exhibited significant improvements in learning and memory performance as revealed by decreased escape latencies in spatial reference memory tests, increased numbers of platform crossings in spatial navigation tests, and a higher percentage of correct choices in a spatial working memory task. These improvements appeared to be cholinergically mediated because atropine administration significantly disrupted spatial navigation performance. The survival of the transplanted cholinergic neurons and their innervation of the RSC were characterized using a monoclonal antibody to choline acetyltransferase (ChAT). The staining of graft-derived ChAT-positive fibers also revealed a pattern of innervation that mimicked that of the cholinergic input in normal animals. These results indicate that intraretrosplenial cortical transplants of cholinergic neurons can rectify spatial memory deficits produced by the loss of intrinsic cholinergic afferents from the medial septal nucleus.

Autotransplantation of peripheral cholinergic neurons into the brains of Alzheimer model rats.

Current hypotheses regarding Alzheimer's disease implicate cholinergic function. In this study, peripheral cholinergic neurons in the vagal nodosal ganglion were transplanted into the brains of Alzheimer model rats. Eighteen Sprague-Dawley strain rats were divided into three groups: 1) unoperated control rats, 2) rats that had undergone bilateral destruction of the nucleus basalis of Meynert (NBM) (Alzheimer model), and 3) the transplantation group in which the vagal nodosal ganglion was transplanted into the cerebral neocortex one week after the bilateral destruction of the Meynert nucleus. Seven weeks after the transplantation rat behaviour was assessed using psychological tests (spontaneous activity, passive avoidance response and the Hebb-Williams maze test). The Alzheimer model rats had a statistically significant increase in spontaneous activity in comparison with controls (P less than 0.01). The transplant rats showed some amelioration of this abnormal increase in spontaneous activity observed in the Alzheimer model rats. All of the control rats showed conditioned passive avoidance responses, while only one Alzheimer model rat retained is shocked-conditions behaviour before 24 hours (P less than 0.01). Three of the six transplanted rats showed complete improvement in the passive avoidance response test. In the Hebb-Williams maze test, the rats with NMB lesions made more errors than the control rats. The transplanted rats had a lower number of errors than NBM-lesioned rats but still more than the controls. Histological examination revealed many cholinergic cells in the transplanted tissue, especially in the area adjacent to the cerebral cortical surface. The present results indicate that autotransplantation of peripheral cholinergic cells ameliorates abnormal behaviour in Alzheimer model rats.

Modulation by muscarine and opioid receptors of acetylcholine release in slices from striato-striatal grafts in the rat.

The accumulation of tritium during incubation with [3H]choline and the subsequent efflux of tritium were studied in striatal slices from non-operated rats, in striatal slices from animals which had received a contralateral striatal ibotenic acid lesion, and in slices from striato-striatal suspension grafts, 16-31 weeks after implantation into previously lesioned striata. In graft slices, the accumulation of tritium as well as the overflow of tritium evoked by electrical stimulation (360 pulses, 3 Hz) was much smaller than in slices from non-operated controls. The muscarine receptor agonist oxotremorine (0.1-1 micromol/l) inhibited the stimulation-evoked overflow, and this effect was blocked by the muscarine receptor antagonists atropine (0.1 micromol/l) and pirenzepine (1 micromol/l) in all experimental groups to the same extent. The delta-receptor selective opioid peptide [D-Pen2, D-Pen5]enkephalin (0.3 micromol/l) inhibited [3H]acetylcholine release in all groups, although its effect was smaller in grafts than in normal tissue. The preferential mu-receptor agonist [D-Ala2,N-methyl-Phe4,Gly-ol5]enkephalin also reduced [3H]acetylcholine release in all groups, but only at the high concentration of 10 micromols/l. The effect of both drugs was antagonized by naloxone (1 micromol/l). The preferential kappa-receptor agonist ethylketocyclazocine enhanced the stimulation-evoked overflow in non-operated animals, an effect abolished by naloxone and also by sulpiride. In grafts, ethylketocyclazocine caused no change. It is concluded that acetylcholine release in striato-striatal grafts can be modulated by muscarine autoreceptors and by opioid delta receptors. The enhancement by kappa-receptor activation of [3H]acetylcholine release in non-operated striata depends on a dopaminergic input to the cholinergic cells which does not exist in grafts.

Transplantation of peripheral cholinergic neurons into Alzheimer model rat brain.

The effect of transplantation of peripheral cholinergic neurons was examined in rats with a lesion of the nucleus basalis Meynert (NBM). The rats with an NBM lesion showed abnormal increase of spontaneous activity, disturbance of memory retention, and disturbance of learning acquisition. In contrast, the rats which had received transplantation of cholinergic neurons into the cerebral cortex displayed amelioration of abnormal behavior produced by the destruction of the NBM. Morphological study (acetylcholinesterase) clearly showed survival of many transplanted cholinergic cells. The present study suggests that autotransplantation of peripheral cholinergic cells may be a possible therapy for Alzheimer's disease.

Transplantation of septal cholinergic neurons to the hippocampus improves memory impairments of spatial learning in rats treated with AF64A.

Embryonic septal neurons were transplanted into damaged hippocampus in adult rats which had received lateral ventricular administration of AF64A, a cholinergic neurotoxin. About 3 months after transplantation, the rats with bilateral septal grafts showed significant improvement in the radial maze and T-maze tasks. Many ingrowths of acetylcholinesterase (AChE)-positive fibers originating from the grafts were observed in the hippocampus of the rats which showed good performance in these learning tasks. These results indicate that transplantation of septal cholinergic neurons into the AF64A-treated hippocampus may induce at least partial recovery in learning tasks believed to involve the hippocampus.

NGF treatment promotes development of basal forebrain tissue grafts in the anterior chamber of the eye.

The effects of nerve growth factor (NGF) on developing central cholinergic neurons were studied using intraocular grafts of rat fetal (E17) basal forebrain tissue. Prior to grafting, grafts were incubated in NGF or saline. Transplants were allowed to mature for six weeks, receiving weekly intraocular injections of NGF or saline. Measurements of NGF levels in oculo after one single injection showed that NGF slowly decreases in the anterior chamber fluid, and after one week, low but significant levels were still present in the eye. Following pretreatment with diisopropylfluorophosphate (DFP), the cholinergic neurons in the grafts were analyzed using three morphological markers: antibodies to cholineacetyltransferase (ChAT), antibodies to acetylcholinesterase (AChE Ab) and acetylcholinesterase histochemistry (AChE). The transplants grew well and became vascularized within the first week. The growth of the NGF-treated basal forebrain grafts was significantly enhanced as compared to the growth of the saline-treated grafts evaluated with repeated stereomicroscopical observations directly through the cornea of the ether-anaesthetized hosts. The NGF-treated grafts contained almost twice as many cholinergic neurons seen with all the cholinergic markers used, as the saline-treated grafts. However, there was no difference in cholinergic cell density between the two groups. The morphology and size of an individual cholinergic neuron was similar in the two groups. The fiber density as evaluated with AChE-immunohistochemistry did not change after NGF-treatment. The DFP-treatment did not seem to affect the AChE-immunoreactivity since an extensive fiber network was found, whereas almost no fibers were seen using conventional AChE histochemistry. We have demonstrated that in oculo transplantation of basal forebrain is a useful model for examining in vivo effects of NGF on central cholinergic function. The marked volume increase of NGF-treated grafts and the unchanged density of cholinergic cells and terminals suggests, that NGF increases the survival of not only developing cholinergic neurons, but possibly other non-cholinergic neurons and non-neuronal cells as well. These results support the notion that NGF acts as a neurotrophic factor on cholinergic and possibly non-cholinergic cells in the central nervous system.

Transplantation of fetal and early postnatal rat septal cholinergic neurons cultured in serum-free and serum-containing medium with nerve growth factor.

Fetal rat (E17-E19) septal neurons were cultured in a defined, serum-free medium for 6-8 days with or without nerve growth factor (NGF) and transplanted into the hippocampus or the surrounding ventricle of 28 adult rats denervated of its septal input by a fimbria-fornix transection. The cholinergic septal neurons, which were visualized by acetylcholinesterase (AChE) histochemistry, always survived in transplantation to the adult brains from nearly pure neuronal cultures. Although choline acetyltransferase (ChAT) activity of septal neurons in culture was greatly increased (5.59-fold) by the addition of NGF to the defined medium, this ChAT induction appeared to have little effect on the subsequent survival or growth of the septal neurons after transplantation. These results demonstrate that survival of cultured fetal septal cholinergic neurons following transplantation is not dependent upon the presence of NGF or serum- or glia-derived factors during the preliminary culture. Postnatal rat (P4) septal neurons cultured for 5 days in serum-containing medium with NGF were also successfully transplanted in one of 3 cases.

Transplantation of embryonic ventral forebrain grafts to the neocortex of rats with bilateral lesions of nucleus basalis magnocellularis ameliorates a lesion-induced deficit in spatial memory.

Embryonic ventral forebrain grafts containing developing cholinergic cells were transplanted to the neocortex of rats with bilateral quisqualic acid lesions of the nucleus basalis magnocellularis. A lesion-induced deficit on performance of a spatial alternation test of memory was reduced by such transplants. When the same animals were treated with the acetylcholinesterase inhibitor physostigmine (0.05 mg/kg), however, performance on the behavioral task was not further promoted, and therefore, under these conditions, the cholinergic cortical transplants appear not to be subject to modulation by anticholinesterase drugs.

Human fetal basal forebrain neurons grafted to the denervated rat hippocampus produce an organotypic cholinergic reinnervation pattern.

The septal/diagonal band (SDB) area, obtained from a 9- to 10-week-old aborted human fetus, was grafted to the hippocampal formation of adult, immunosuppressed rats subjected to an aspirative lesion of the fimbria-fornix. Nineteen weeks after transplantation, microscopical analysis revealed large, partly acetylcholinesterase (AChE)-positive grafts in the hippocampus in 3 of the 5 recipients. The AChE-positive grafts gave rise to a reinnervation of the host hippocampus and an AChE-positive lamination of the different hippocampal subfields with the same characteristics as the normal septum-derived innervation. Immunological evaluation of host sera revealed that all rats were immunized by the graft. This indicates that grafted human cholinergic SDB neurons can respond to or interact with factors that regulate and guide the innervation of the rat hippocampus.

Transplants of cholinergic septal explants reinnervate adult rodent hippocampus.

Embryonic septal-basal forebrain tissue was grown in explant culture for 1, 4 or 5 days prior to transplantation to the hippocampus of adult rats denervated of its septal input by a fornix/fimbria transection. The explant transplants were compared with transplants of septal cell suspensions. Analysis of fiber ingrowth by acetylcholinesterase (AChE) histochemistry at various timepoints post-transplantation shows little difference in the developmental time course or extent of cholinergic axon ingrowth into the host hippocampus between the suspension transplants and the explant transplants. Septal explants in culture for 5 days were as effective as those in culture for one day prior to transplantation in providing cholinergic reinnervation to the host hippocampus. Studies using antibodies to choline acetyltransferase (ChAT) and the fluorescent retrograde tracer, fast blue, confirmed that the AChE-positive cells in the transplants were cholinergic and that the innervation to the host hippocampus came from the transplanted cholinergic neurons. Thus the results demonstrate that embryonic cholinergic septal neurons can be maintained in short-term culture prior to transplantation without adversely affecting their ability to innervate an appropriate CNS target in vivo.

Cholinergic neurones acquire adrenergic neurotransmitters when transplanted into an embryo.

During development, cells become progressively restricted, until they reach their final phenotype. Differentiation was originally thought to be irreversible, but phenotypic plasticity has been observed in a variety of cell types, for example sympathetic neurones, the limb blastema and some glial cell types. A detailed description of the individual steps that lead to expression or reversal of phenotype is essential to understand the molecular events underlying cell differentiation. We examined whether ciliary neurones acquire adrenergic properties when exposed to a permissive embryonic environment. Cholinergic neurones were selectively labelled with a retrogradely transported marker and injected into chick embryos during active neural crest migration. Four to five days after injection, some of the labelled neurones were found in 'adrenergic sites' and had developed catecholamine histofluorescence. The cells had thus accumulated adrenergic neurotransmitters even after differentiation into cholinergic neurones. This result shows that neurotransmitter plasticity occurs in cholinergic neurones and suggests that the neurotransmitter phenotype can be modified by the embryonic environment.

Cholinergic ventral forebrain grafts into the neocortex improve passive avoidance memory in a rat model of Alzheimer disease.

The memory dysfunction of Alzheimer disease has been associated with a cortical cholinergic deficiency and loss of cholinergic neurons of the nucleus basalis of Meynert. This cholinergic component of Alzheimer disease can be modeled in the rat by ibotenic acid lesions of the cholinergic nucleus basalis magnocellularis. The memory impairment caused by such unilateral lesions, as reflected in passive avoidance behavior, is reversed by grafts into the deafferented neocortex of embryonic neurons of the cholinergic ventral forebrain, but not by grafts of noncholinergic hippocampal cells.