Transmitter phenotype is a major determinant in the specificity of synapses formed by cholinergic neurons transplanted to the hippocampus

Embryonic habenular or striatal cholinergic tissues were transplanted to the hippocampal formation of adult rats. The connectivity of these grafts with the host hippocampal formation was analysed using acetylcholinesterase histochemistry and immunocytochemistry with a monoclonal antibody to choline acetyltransferase. Both graft types produced laminar arrangements of acetylcholinesterase-positive fibers in the hippocampal formation that closely resembled the native pattern of cholinergic innervation. In addition, graft-derived choline acetyltransferase-immunoreactive synapses were found in the host hippocampal formation. These synapses were formed on non-immunoreactive dendritic structures and were similar to the types of cholinergic synapses found in the hippocampal formation of normal animals. These data indicate that the cholinergic transmitter phenotype is a major determinant of whether a neuron will form typical cholinergic synapses with hippocampal targets.     

Neurotrophic effects of hippocampal target cells on developing septal cholinergic neurons in culture

The influence of hippocampal target cells on the development of cholinergic septal neurons was studied in rotation-mediated reaggregating cell cultures. Brain cells from 15-day-old mouse embryos were obtained from: septum, containing cholinergic cells which project to the hippocampus; hippocampus which contains target cells for the septal cholinergic neurons; and cerebellum, containing cells which are not targets for the septal cholinergic cells. The cells were then cultured for 3 weeks in a rotary incubator in the following combinations: septal cells alone; hippocampal cells alone; cerebellar cells alone; septal-hippocampal cells together; and septal-cerebellar cells together. After harvesting, fixation, and embedding, 50 micron sections were cut and processed for visualization of acetylcholinesterase activity. Sections from reaggregates containing either hippocampal or cerebellar cells alone contained only a few acetylcholinesterase-positive cells, but no positive fibers. Sections from septal-hippocampal coaggregates revealed a pattern of well-defined, fine-caliber acetylcholinesterase-positive fibers with extensive arborizations and varicosities suggesting axonal proliferation. In septal-cerebellar coaggregates, acetylcholinesterase-positive fibers appeared to be degenerating and distinct areas were observed which were essentially devoid of acetylcholinesterase fibers. In some experiments, either cerebellar or hippocampal cells were labeled with wheatgerm agglutinin-rhodamine prior to culture in order to identify these cells in the resulting reaggregates. Analysis of sections from these studies showed that acetylcholinesterase fibers were excluded from regions of coaggregates containing cerebellar cells, but were present in regions of coaggregates containing hippocampal cells. Finally, cell counts of acetylcholinesterase-positive cells in the various combinations revealed that these putative cholinergic neurons were significantly more numerous in septal-hippocampal coaggregates (271 +/- 19 per 10(6) septal cells added) than in septal reaggregates (38 +/- 6 per 10(6) septal cells added) or septal-cerebellar coaggregates (85 +/- 29 per 10(6) septal cells added). These results, taken together, suggest that hippocampal target cells influence the development and survival of cholinergic neurons.     

A primate model of Huntington's disease: cross-species implantation of striatal precursor cells to the excitotoxically lesioned baboon caudate-putamen

Summary Ibotenic acid was injected unilaterally into the baboon caudate-putamen (CP) to achieve a neural degeneration model in the primate, with a neuropathology similar to Huntington's disease. Four to six weeks later injections of cell suspensions of striatal precursor cells, obtained by dissection of the fetal rat striatal region (13–15 days gestational age), were made into the excitotoxically lesioned CP of 3 baboons immunosuppressed by Cyclosporin A. Morphological analysis indicated that in one of the baboons, which had the largest lesion of the CP and the shortest survival time (6 weeks after implantation), there was a surviving striatal implant. The implanted neurons grew in high densities in cellular aggregates within the host gliotic CP. These neurons had a neuronal size phenotypical for rat striatum, i.e. on average about a 25% smaller neuronal cell diameter than a similar population in the baboon caudate-putamen. Glial-fibrillary-acid-protein immunoreactivity was present on large astrocytes within the striatal implant, with a distinct border towards the lesion-induced astrogliosis of the host. Neuronal markers for acetylcholinesterase and Leu-enkephalin were distributed in a typical patchy manner in the striatal implants along with fiber staining for tyrosine-hydroxylase-like immunoreactivity (TH) possibly derived from afferent host dopaminergic axons. Some of these fibers in the implants came from intrinsic TH-positive neuronal somata, probably of neocortical fetal origin and transiently expressing the enzyme. In conclusion, the results indicate that neuronal replacement can be achieved by crossspecies implantation of fetal striatal precursor cells to the previously neuron depleted primate CP under immunosuppression but that the survival and growth of such implants may be variable and subject to unfavourable trophic conditions.     

Interleukin-6 improves the survival of mesencephalic catecholaminergic and septal cholinergic neurons from postnatal, two-week-old rats in cultures

Interleukin-6 (human recombinant) supported the survival of cultured mesencephalic, catecholaminergic and septal cholinergic neurons from postnatal, two-week-old (P13-P15) rats. Significantly, more catecholaminergic neurons, stained by monoclonal anti-tyrosine hydroxylase antibody, were found in cultures supplemented with interleukin-6 at a concentration of 5 ng/ml than in cultures not treated with interleukin-6. The optimal dose used was 50 ng/ml. The survival effect of interleukin-6 on postnatal rat, tyrosine hydroxylase-positive neurons was observed both in cultures using serum-containing and serum-free medium. Contents of dopamine and noradrenaline in cultures with interleukin-6 were also larger than in control cultures. Interleukin-6 also increased the survival of cultured embryonic (E17) rat midbrain tyrosine hydroxylase-positive neurons. The effect on these neurons was, however, smaller, and the optimal dose of interleukin-6 was nearly 5 ng/ml. Interleukin-6 also supported the survival of cultured postnatal (P13) rat septal cholinergic neurons, visualized by acetylcholinesterase staining. The concomitant addition of mouse nerve growth factor (100 ng/ml) and interleukin-6 (50 ng/ml) had a synergetic effect on the survival of acetylcholinesterase-positive neurons in culture. Our data suggest that the survival of cultured tyrosine hydroxylase-positive, mesencephalic, and acetylcholinesterase-positive, septal neurons from postnatal two-week-old rats was supported by interleukin-6, just as there was a different dose dependency of interleukin-6 on the cultured postnatal neurons compared with embryonic neurons.     

Guidance of acetylcholinesterase-containing fibres by target tissue in co-cultured brain slices

Slices of various brain regions were prepared from newborn and from 7-day old rats and co-cultured in different combinations. In the majority of co-cultures of septal and hippocampal slices, acetylcholinesterase-positive fibres originating in the septal nuclei invaded the adjacent hippocampal slice. A similar pattern of hippocampal ingrowth by acetylcholinesterase-positive fibres occurred with slices prepared from the nucleus basalis of Meynert and from spinal cord. Septal neurones also projected to cortical slices, an effect which even occurred in the presence of their natural target tissue. In contrast to these massive projections to brain areas which in situ receive cholinergic inputs, no significant acetylcholinesterase-positive fibre ingrowth was observed in tissues which lack major cholinergic afferents in situ (hypothalamus, substantia nigra and cerebellum). These results indicate that under our culture conditions, acetylcholinesterase-positive fibres selectively invade cholinergic target areas. This effect is independent of the brain area from which the cholinergic neurones were derived.     

Ultrastructural evidence for hippocampal target cell-mediated trophic effects on septal cholinergic neurons in reaggregating cell cultures

We have previously demonstrated at the light microscopic level that when embryonic day-15 septal neurons are co-cultured for 21 days with their target cells from the hippocampus, increased numbers of septal cholinergic neurons are present as compared with co-cultures employing cells from the non-target cerebellum. In addition, fine varicose axon-like cholinergic fibers are found to be associated with the hippocampal cells but not with cerebellar cells. We now provide ultrastructural evidence for hippocampal target cell-enhanced cholinergic neuronal survival, axonal proliferation, and synapse formation in this culture system. Dissociated cell suspensions from septal, hippocampal, and cerebellar areas were obtained from 15-day mouse embryos; and hippocampal and cerebellar cells were internally labeled with rhodamine-conjugated wheat germ agglutinin. Combinations of septal and hippocampal cells, and septal and cerebellar cells were allowed to reaggregate in rotation mediated culture for either 15 or 21 days. The reaggregates were then fixed, embedded, sectioned, and processed for acetylcholinesterase-positive acetylcholinesterase-positive cells and fibers, and under fluorescence to locate rhodamine-labeled cell populations. Representative reaggregate profiles were then re-embedded for electron microscopic examination. In both types of reaggregates, either labeled hippocampal target or cerebellar non-target cells segregated from the septal cells so that areas containing each of the respective cell populations could be studied. In sections of septal-hippocampal reaggregates from 15-day cultures, 571 out of 665 (85%) cholinergic neurons examined were intact, whereas 15% of the cells showed some ultrastructural features of degeneration. Similarly, at day 21, 297 out of 335 (88%) of the cholinergic neurons were intact. In sections of septal-cerebellar reaggregates from 15-day cultures, 473 out of 572 (83%) cholinergic neurons were intact. By day 21 of culture, however, only 15 out of 110 (14%) cholinergic neurons examined were intact from the septal-cerebellar reaggregates. In areas of septal-hippocampal reaggregates occupied by rhodamine-labeled hippocampal cells, profiles of acetylcholinesterase-labeled axons were identified, and synaptic specializations were observed between cholinergic terminals and dendrites as well as somata of hippocampal target cells. In contrast, areas of septal-cerebellar reaggregates occupied by rhodamine-labeled cerebellar cells were devoid of cholinergic fibers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Development of intrastriatal striatal grafts and their afferent innervation from the host.

The morphological maturation of cell suspension grafts of fetal striatal tissue (obtained from 14-15-day-old rat fetuses) was followed from two days to eight weeks after implantation into intact and ibotenic acid-lesioned striata of adult rats. The development of host afferent innervation of the grafts from the substantia nigra (tyrosine hydroxylase immunoreactive), mesencephalic raphe (serotonin immunoreactive), and the frontal cortex (anterogradely labelled with Phaseolus vulgaris leucoagglutinin) were revealed by immunohistochemistry. During the first weeks post-grafting, the striatal implants consisted of a mixture of mature- and immature-looking cell clusters. Grafts implanted into ibotenic acid-lesioned striatum grew rapidly (about five-fold) in volume over the first week. The areas of immature (probably proliferating) cells gradually disappeared, and by six to eight weeks the grafts had a fully mature appearance with patches of neurons which stained densely for DARPP-32 (i.e. were striatum-like) embedded within areas of essentially DARPP-32-negative (i.e. non-striatum-like) tissue. Peripheral clusters of grafted cells gradually intermingled with nearby areas of the surrounding lesioned host, and already by two to four days after implantation, coarse and densely immunoreactive host fibres from the substantia nigra, mesencephalic raphe and frontal cortex were present within the grafts. By four to five days the first DARPP-32-immunoreactive neurons appeared in patches within the mature portions of the grafts, and one to two days later the tyrosine hydroxylase-positive fibres began to sprout thin axons selectively within the DARPP-32-positive patches. Similarly, the serotonergic and cortical fibres in the grafts increased in number over the next two weeks, but they showed no preference for the DARPP-32-positive regions. Rich terminal networks were established by two to three weeks post-grafting, and by six to eight weeks the nigral, raphe and cortical afferents had reached terminal densities similar to those seen previously in long-term surviving grafts. Grafts implanted into dopamine-denervated hosts showed a normal morphological maturation of both DARPP-32-positive and -negative areas, although no tyrosine hydroxylase-positive innervation appeared within the grafts. Grafts implanted into non-lesioned striata did not grow beyond their initial size. The implanted cells showed less intermingling with the surrounding host striatum, thus resulting in sharply delineated graft-host borders. DARPP-32-positive patches developed, but they were smaller in size and generally present only in the most peripheral graft portions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Recovery of hippocampal cholinergic activity by transplantation of septal neurons in AF64A treated rats

Embryonic septal neurons were transplanted into the hippocampus of adult rats which had received lateral-ventricular administration of AF64A, a cholinergic neurotoxin, and the effects on hippocampal cholinergic activity were studied. One week after AF64A administration, we injected dissociated septal cell suspension into the dorsal hippocampus, unilaterally. About 3 months after the transplantation, acetylcholine (ACh)-rich septal grafts formed extensive acetylcholinesterase (AChE)-positive fibers into the host hippocampus, recovering choline acetyltransferase (ChAT) level only in the grafted side. These results indicate that septal implants can produce a partial recovery of the cholinergic activity in the chemically damaged hippocampus.     

Anatomical and behavioral comparison of unilateral dopamine-rich grafts implanted into the striatum of neonatal and adult rats.

The anatomical and functional characteristics of dopamine neuron-rich grafts implanted into rat pups were compared with those of identical grafts implanted into adult hosts. The host nigrostriatal dopaminergic pathway was unilaterally destroyed by an intrahypothalamic injection of 6-hydroxydopamine. This was followed five days later by the implantation of a cellular suspension obtained from rat embryonic mesencephali. Identical operations were carried out on adult and infant (PD3) rats. The survival rate of implanted tyrosine hydroxylase-positive cells was lower in the neonates. On the other hand, in the neonate hosts, surviving immunoreactive cells migrated extensively throughout the host striatum coursing preferentially below the corpus callosum and towards the subependymal zone. The structural integrity of the host parenchyma was well maintained after the neonatal implantation, in contrast to that observed in the adults. Despite a difference in the cell survival rate, there was no major difference in reinnervation density between the two types of host. The functional capacities of the implants were evaluated by measuring the rotational responses of the animals to dopaminergic agonists. The implants compensated the lesion-induced contralateral rotational response to the mixed agonist apomorphine and the D1 agonist SCH-38393 in both neonates and adults. However, the response to the D2 agonist LY-171555 was not significantly attenuated by the implant. The ipsilateral rotational response to amphetamine observed in the lesioned animals was also compensated and even reversed by the graft. It is concluded that with respect to rotational behavior, similar functional benefits were observed following adult stage or neonatal implantation, despite differences in their anatomical development.     

Enhanced graft survival in the hippocampus following selective denervation

The trophic effects of denervation on the survival of fetal cholinergic neuronal cell suspensions grafted to the hippocampal formation of the rat were assessed in the present study. Young adult female rats were injected with cell suspensions of neurons obtained from the fetal basal forebrain region into the hippocampal formation simultaneously with (or without) a fimbria-fornix transection, which removes the hippocampal cholinergic afferents. Four to six months later, one group of grafted animals was evaluated histochemically for: transplant volume; number of acetylcholinesterase-positive cells, and size of acetylcholinesterase-positive cells in the graft. A parallel study was conducted to determine the total number and size of the acetylcholinesterase-positive cells in the septal-diagonal band-substantia innominata complex of the adult rat, to match with the cell survival and growth in the grafts. A second group of grafted rats was taken in parallel for biochemical analysis of choline acetyltransferase activity in the grafted hippocampus. The transplant volume in the rats with fimbria-fornix transection was greater than twice the volume seen in animals without fimbria-fornix lesion. In addition, the number of acetylcholinesterase-positive cells in the transplant was twice as great in the denervated animals as in the non-denervated ones. However, the number of acetylcholinesterase-positive cells per mm3 of graft volume did not differ between the two groups, suggesting that the trophic effect of the denervation was not specific for the cholinergic neurons, but affected the entire grafted tissue. The hippocampal choline acetyltransferase activity of the animals that received the fimbria-fornix lesion simultaneously with transplantation was about three times higher than that of the rats that received grafts but no simultaneous fimbria-fornix transection. A control experiment with animals that received an aspirative lesion of the retrosplenial cortex, transecting the perforant path input, revealed no enhancing effect of hippocampal choline acetyltransferase activity over non-lesioned grafted animals. Thus, the denervation-enhancing effects of the fimbria-fornix lesion appear to be selective and not the result of a general wound-induced mechanism. These results strongly support the contention that neurotrophic factors are released as a result of denervation in the adult hippocampal formation, and that these neurotrophic factors can support survival and growth of central cholinergic neurons. However, the factors involved do not appear to be specific for the cholinergic neurons, but rather have their trophic effects on many types of cells.     

Increase of striatal methionin enkephalin content following lesion of the nigrostriatal dopaminergic pathway in adult rats and reversal following the implantation of embryonic dopaminergic neurons: a quantitative immunohistochemical analysis

The aim of the present study was to test whether intrastriatal implants of embryonic dopaminergic neurons are able to normalize the lesion-induced dysfunction of striatal enkephalinergic neurons, one of the major output systems of the striatum. The ascending dopaminergic pathway of adult rats was unilaterally lesioned. Three weeks later a cell suspension obtained from the mesencephali of ED14 rat embryos was implanted into the denervated striatum and striatal methionin enkephalin immunostaining was quantified six months later by the use of an image analyser. Methionin enkephalin immunostaining was unevenly distributed in the striatum of control animals. Besides the classical patch/matrix pattern, a mediolateral gradient was also present and, moreover, immunostaining decreased towards caudal levels. Seven months after the lesion of the nigrostriatal dopaminergic pathway, methionin enkephalin immunostaining was found to be increased in the denervated striatum by about 50%. However, relative increases were more sustained in the areas where basal methionin enkephalin immunostaining were lowest, i.e. the lateral striatum and posterior striatal areas. This resulted in an attenuation of the global gradients seen in the normal striatum. Increased immunostaining was also found in the ipsilateral globus pallidus. The implantation, into the denervated striatum, of embryonic dopaminergic neurons led to a reversal of the lesion-induced increase of striatal and pallidal methionin enkephalin immunostaining six months later. Moreover, this reversal resulted in an overshoot, as the level of immunostaining in the graft-bearing striatum was found to be lower than the levels found in the normal striatum. It is concluded that grafts of embryonic dopaminergic neurons can normalize the function of one of the major output systems of the striatum and, through it, influence more distant targets of this structure. This suggests a physiological basis for the behavioral effects observed previously with such grafts.     

Embryonic striatal grafts restore neuronal activity of the globus pallidus in a rodent model of Huntington's disease

It has been demonstrated in rats that embryonic striatal grafts placed in the excitotoxically lesioned striatum establish neuronal connections with the host globus pallidus. In order to determine whether the morphologically verified connections between the grafts and host are functional, the present study investigated the effects of embryonic striatal grafts on changes in the neuronal activity of the globus pallidus in rats with quinolinic acid-induced striatal lesions. The activity of pallidal neurons was determined by use of quantitative cytochrome oxidase histochemistry and an electrophysiological technique. Striatal lesions induced an increase in both the cytochrome oxidase activity and the spontaneous firing rate of the globus pallidus ipsilateral to the lesions. Grafts derived from the lateral ganglionic eminence, but not the medial ganglionic eminence, reversed the lesion-induced increase in the cytochrome oxidase activity of the globus pallidus with concomitant reduction of apomorphine-induced rotational asymmetry. The lateral ganglionic eminence grafts also attenuate the increase in the firing rate of pallidal neurons in rats with striatal lesions. The present results provide evidence that striatal lesions lead to the loss of a tonic inhibitory input to the globus pallidus with consequent increase in the activity of pallidal neurons, and that intrastriatal striatal grafts reverse the altered activity of pallidal neurons. The findings strongly suggest that embryonic striatal grafts functionally repair the damaged striatopallidal pathway.     

Characterization of angiogenesis and inflammation surrounding ePTFE implanted on the epicardium

The response of epicardial tissue to the implantation of expanded polytetrafluoroethylene (ePTFE) was evaluated and compared with identical material implanted within subcutaneous and adipose tissues. These two tissue environments were selected for comparison with epicardial implants because they represent tissue often involved in device implantation. Discs of ePTFE (6 mm) were implanted into three different tissue sites in Sprague-Dawley rats. At 5 weeks, polymers and surrounding tissues were harvested and processed for light microscopy. General histology and histochemistry data indicated all polymers to be well incorporated with new tissue. Subcutaneous implants were covered by a dense fibrous capsule (55-70 microm). Epicardial and adipose implants had no fibrous capsule and a significantly greater number of microvessels (arterioles, capillaries, and venules) within the surrounding tissues compared with subcutaneous implants. An increased level of inflammation was also observed around epicardial implants compared with the other implants. Additionally, the new vasculature surrounding epicardially implanted ePTFE revealed an altered microvessel density and vessel type distribution compared with normal (control) epicardium. These results suggest that epicardial tissue responds to implanted ePTFE with a robust inflammatory response that may support the formation of a new microvasculature that is uniquely different from the native epicardial microvasculature.     

Septal cholinergic neurons from postnatal rat can survive in the dissociate culture conditions in the presence of nerve growth factor

The survival effect by nerve growth factor (NGF) on the cholinergic neurons of postnatal rat septal neurons in culture was examined. When the septal neurons from 10 to 12-day-old rats were cultured without NGF, the activities of choline acetyltransferase gradually decreased during the period of cultivation. The addition of NGF to the culture prevented the decline of activities. And, the number of acetylcholinesterase-positive neurons in culture with NGF was found to be more than that without NGF, after 5 days in culture. These results suggest that NGF promotes the survival of septal cholinergic neurons from postnatal rats in culture.     

Characteristics of brain injury-derived neurotrophic peptide-binding sites on rat brain synaptosomes and neurons in culture

Brain injury-derived neurotrophic peptide is the fragmental 13-mer peptide of the novel neurotrophic factor which was extracted and purified from Sponge Gelform made of gelatin implanted at the mechanically-induced injury site in neonatal rat brains. Brain injury-derived neurotrophic peptide supports survival of septal cholinergic and mesencephalic dopaminergic neurons in culture, and rescues hippocampal neurons in culture from glutamate neurotoxicity. Here we studied the binding characteristics of brain injury-derived neurotrophic peptide to synaptosomes from normal adult rat brains and neurons in culture from neonatal rat brains. [125I]Asp-[Tyr11]-brain injury-derived neurotrophic peptide binding to rat brain synaptosomes was specific and saturable. Equilibrium binding studies revealed that [125I]Asp-[Tyr11]-brain injury-derived neurotrophic peptide bound to 1.1 pmol/mg protein with a Kd (dissociation constant) of 0.17 microM in hippocampal synaptosomes and to 2.0 pmol/mg protein with a Kd of 0.38 microM in septal synaptosomes. [125I]Asp-[Tyr11]-brain injury-derived neurotrophic peptide could bind to a subpopulation of hippocampal neurons in culture from embryonic rat brains. Affinity cross-linking with the carboxyl-reactive cross-linking reagent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide-HCl and [125I]Asp-[Tyr11]-brain injury-derived neurotrophic peptide produced radiolabeled bands corresponding to 100,000, 50,000 and 40,000 mol. wt molecules on hippocampal neurons in culture. These results suggest that the 13-mer sequence of brain injury-derived neurotrophic peptide plays a crucial role in expressing the neurotrophic properties of the factor.     

Neuronal activity of the septal pacemaker of theta rhythm under the influence of stimulation and blockade of the median raphe nucleus in the awake rabbit

The control of theta rhythm in neuronal activity of the medial septal area and hippocampal electroencephalogram by the brainstem structures was investigated in waking rabbits. In the first series of experiments stimulating electrodes were implanted into the midbrain reticular formation and median raphe nucleus. The standard frequency of theta-bursts in medial septal area neurons and in the electroencephalogram was uniformly and chronically decreased in all rabbits with electrodes implanted into the median raphe nucleus (4.7 +/- 0.5 Hz versus 5.2 +/- 0.19 Hz in animals without electrodes in median raphe nucleus). Weak electrical stimulation of the median raphe nucleus resulted in additional decrease of theta expression in the medial septal area neurons and its disappearance from the hippocampal electroencephalogram, where it was substituted by delta-waves and spindles. Stimulation of the reticular formation had the opposite effect, with an increase in theta frequency, regularity and expression in medial septal area neuronal activity and hippocampal electroencephalogram. In the second series of experiments reversible functional blockade of the median raphe nucleus by local microinjection of lidocaine was performed. This resulted in expression of theta-bursts in an additional group of medial septal area neurons, an increase in theta-burst frequency (by 0.5-2 Hz) and regularity with concomitant changes in the electroencephalogram. The effects of sensory stimuli on the background of increased theta activity were suppressed or significantly decreased. It is concluded that, in accordance with the data of other authors, the median raphe nucleus can be regarded as a functional antagonist of the reticular formation, powerfully suppressing theta-bursts of the medial septal area neurons and hippocampal theta rhythm. It is suggested that, in combination with the theta-enhancing influences of reticular formation, the median raphe nucleus may participate in termination of attention, its switching to other stimuli and stabilization of the effects of learning.     

Distribution and immunohistochemical localization of GDNF protein in selected neural and non-neural tissues of rats during development and changes in unilateral 6-hydroxydopamine lesions

The tissue distribution of glial cell line-derived neurotrophic factor (GDNF) during development and changes in GDNF levels by unilateral 6-hydroxydopamine lesions were investigated in rats using a newly established enzyme immunoassay system and by immunohistochemistry. The detection limit of the assay was 0.3 pg/0.2 ml and the system recognized glycosylated mature GDNF. Concentrations of GDNF were relatively high in the kidney and testis during the embryonic and neonatal periods, respectively, and decreased with age. In the striatum, hippocampus and brain stem, GDNF reached a maximal level at around postnatal day 14. However, brain levels were generally lower than those in non-neural tissues. In the CNS, GDNF immunoreactivity was observed in striatal neurons, pyramidal neurons in the hippocampus and the Vth layer of the cortex, large neurons in the diagonal band and brain stem, and spinal motor neurons. It was also evident in several non-neural, tissue-specific cells, such as cells in the renal collecting ducts and distal tubules, and testicular Sertoli cells. Destruction of nigral dopaminergic neurons by 6-hydroxydopamine enhanced the levels of striatal GDNF protein, with apparent involvement of astrocytes. These results suggest that GDNF is normally synthesized in neurons, but may also be produced by astroglial cells in damaged brains.     

Alterations in excitatory and GABAergic inhibitory connections in hippocampal transplants

Solid pieces of embryonic hippocampal tissue were implanted in a cavity formed by aspiration of the fimbria-fornix and the overlying cingulate cortex in adult rats. Six to 8 months after the transplantation, chronic recording electrodes were implanted into the graft and the host hippocampi for the recording of electroencephalogram and unit activity in the freely moving animal. Irregularly occurring sharp waves or electroencephalogram spikes and concurrent synchronous discharge of large groups of neurons dominated the electrical activity of the grafts, in contrast to the situation in normal animals. Light microscopy and GABA immunocytochemistry in the grafts revealed that the three major cell types of the hippocampal formation, i.e. pyramidal neurons, dentate granule cells and GABA-immunoreactive interneurons were present in the hippocampal grafts. At the ultrastructural level, however, significant alterations in connectivity were observed. The most striking finding was the absence or sparse occurrence of synapses on the axon initial segments of pyramidal neurons. The axon initial segments are normally densely covered by GABAergic synapses derived from a specialized type of interneuron, the chandelier or axo-axonic cell. On the other hand, numerous GABA-immunoreactive terminals were found in synaptic contact with somata of pyramidal neurons, suggesting that other types of GABAergic interneurons and their efferent connections may have developed in a normal manner. The cell bodies of pyramidal neurons received, in addition, several asymmetric synapses from GABA-negative terminals. These presumably excitatory synapses are not present on the somata of pyramidal cells in the normally developing hippocampus. We hypothesize that the somatic excitatory synapses originate, at least in part, from the axon collaterals of the neighbouring pyramidal cells in the graft. We suggest that the hyperexcitability of the neuronal circuitry within the graft is due to reduced inhibition (lack of axo-axonic synapses) coupled with increased collateral excitation of the pyramidal neurons.     

Connectivity of striatal grafts implanted into the ibotenic acid-lesioned striatum--II. Cortical afferents

Grafts of fetal striatal tissue, implanted into the ibotenic acid-lesioned caudate-putamen in adult recipient rats, have previously been shown to receive extensive afferent inputs from various subcortical areas in the host. In the present study, the formation of graft afferents from the host neocortex has been analysed, 3-12 months after transplantation, by means of the anterograde axonal tracer Phaseolus vulgaris leucoagglutinin, iontophoretically injected at multiple sites into the host frontal cortex, in combination with intra-transplant injections of the fluorescent retrograde tracer, Fluoro-Gold. From the cortical injection sites, fibres labelled with Phaseolus vulgaris leucoagglutinin could be traced through the corpus callosum, along the myelinated fascicles of the internal capsule, into the host caudate-putamen. The labelled axons passed in large numbers across the host-graft border zone to form a rich terminal plexus that covered large parts of the grafted tissue, in close association with the grafted neurons. The labelled corticostriatal terminal network was dense, particularly in the peripheral portions of the grafts, although it did not reach the density seen in the spared portions of the host caudate-putamen. Injections of Fluoro-Gold into the depth of the striatal grafts labelled large numbers of neurons in the frontoparietal cortical regions. Consistent with previous findings, labelled neurons were also found, ipsilateral to the graft, in the intralaminar nuclear complex of the thalamus, basolateral amygdala, substantia nigra and dorsal raphe. The host neocortical neurons labelled by Fluoro-Gold injections into the graft had the same laminar and regional distribution as the neurons labelled from the homotopic area of the intact striatum, indicating that the cortical afferents to the graft were derived from those corticostriatal neurons which projected to the area of the ibotenic acid-lesioned caudate-putamen into which the graft had been placed. The number of Fluoro-Gold-labelled cortical neurons ipsilateral to the graft reached in the best cases about 1/3 of the number labelled by identical Fluoro-Gold injections into the intact caudate-putamen. Labelled neurons occurred, with a similar distribution, also in the contralateral frontoparietal cortex, but they were considerably fewer in number. The results demonstrate that fetal striatal tissue, implanted into the previously neuron-depleted adult caudate-putamen, receives innervations from all normal principal cortical and subcortical striatal afferent systems, thus pointing to a remarkable capacity of the implanted fetal striatal neurons to become anatomically integrated into the lesioned host striatal circuitry.     

Correspondence between the dopamine islands and striosomes of the mammalian striatum

During the development of the mammalian striatum, the early-forming dopamine innervation is broken up into macroscopic patches called "dopamine islands". These express high tyrosine hydroxylase-like immunoreactivity and are also rich in acetylcholinesterase activity. The mature striatum has prominent macroscopic compartments called "striosomes" that were first characterized by their low acetylcholinesterase activity and since have been related to heterogeneities in striatal input-output organizations. This report describes two sets of experiments designed to determine the relationship between the dopamine islands and the striosomes. The distributions of striatal tyrosine hydroxylase-like immunoreactivity and acetylcholinesterase activity were first compared in a series of kittens and young cats ranging in age from 1-228 postnatal days. During this time, the pattern of tyrosine hydroxylase-like immunoreactivity changed from islandic (patchy) to diffuse, and the pattern of acetylcholinesterase staining changed from one of acetylcholinesterase-rich patches to one of acetylcholinesterase-poor striosomes. The dopamine islands were in register with the acetylcholinesterase-poor patches at early developmental stages and at later stages the islands matched striosomes. These observations establish a correspondence between the dopamine islands and striosomes and demonstrate that the acetylcholinesterase-rich patches of the immature caudate nucleus become the acetylcholinesterase-poor striosomes of the adult. In a second set of experiments, cat fetuses were exposed to [3H]thymidine at embryonic days 22-29 in order to label the clustered subpopulations of striatal neurons known from previous experiments to lie in striosomes [Graybiel and Hickey (1982) Proc. natn. Acad. Sci. U.S.A. 79, 198-202]. The [3H]thymidine-labeled brains were examined at late fetal (embryonic days 50-52), early postnatal (days 1-21) and later postnatal (days 62-199) ages. The clusters of [3H]thymidine-labeled neurons were aligned with tyrosine hydroxylase-rich, acetylcholinesterase-rich patches early in development, and with acetylcholinesterase-poor striosomes at later stages. There were marked dorsoventral differences in the intensity of tyrosine hydroxylase-like immunoreactivity in the dopamine islands and this was confirmed in neonatal rats. A "dorsal islandic system" was defined as having crisp, highly immunoreactive islands; ventrally, regions of low and medium tyrosine hydroxylase-like immunoreactivity formed a mosaic.(ABSTRACT TRUNCATED AT 400 WORDS)