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The regrowth of lesioned central acetylcholinesterase (AChE)-positive axons in the adult rat was studied in irides implanted to two different brain sites: in the caudal diencephalon and hippocampus, and in the hippocampal fimbria. At both implantation sites the cholinergic septo-hippocampal pathways were transected. At 2-4 weeks after lesion, newly formed, probably sprouting fibres could be followed in abundance from the lesioned proximal axon stumps into the iris transplant. Growth of newly formed AChE-positive fibres into the transplant was also observed from lesioned axons in the anterior thalamus, and to a minor extent also from the dorsal and ventral tegmental AChE-positive pathways and the habenulo-interpeduncular tract. The regrowth process of the sprouting AChE-positive, presumed cholinergic fibres into the iris target was studied in further detail in whole-mount preparations of the transplants. For this purpose the irides were removed from the brain, unfolded, spread out on microscope slides, and then stained for AChE. During the first 2-4 weeks after transplantation the sprouting central fibres grew out over large areas of the iris. The new fibres branched profusely into a terminal plexus that covered maximally about half of the iris surface, and in some areas the patterning of the regenerated central fibres mimicked closely that of the normal autonomic cholinergic innervation of the iris. In one series of experiments the AChE-staining was combined with fluorescence histochemical visualization of regenerated adrenergic fibres in the same specimens. In many areas there was a striking congruence in the distributional patterns of the regenerated central cholinergic and adrenergic fibres in the transplant. This indicates that - as in the normal iris - the sprouting cholinergic axons (primarily originating in the lesioned septo-hippocampal pathways) and adrenergic axons (primarily originating in the lesioned axons of the locus neurones) regenerate together along the deneravated Schwann cell sheaths. From a comparison between the central reinnervation process and the process of reinnervation of the iris by peripheral cholinergic axons after transplantation to the anterior eye chamber, it is concluded that the regenerative capacity of central cholinergic neurones (above all the septo-hippocampal system) is not much inferior to that of their peripheral counterparts when given similar growth conditions. Moreover, central cholinergic neurones seem partly able to replace the peripheral ones in the reinnervation of a denervated peripheral target.

The ability of embryonic hippocampal tissue to promote regeneration of cholinergic axons in the septohippocampal system has been studied in adult rats. Strips of embryonic hippocampus, taken from 7-40 mm rat fetuses, were implanted into a 2-3 mm wide cavity which completely transected the septal cholinergic axons innervating the intrinsic hippocampus. The ingrowth of cholinergic fibres into the denervated host hippocampal formation was monitored by measuring the activity of the enzyme, choline acetyltransferase (ChAT), and by acetylcholine esterase (AChE) histochemistry. The results demonstrated a gradual, partial return of both ChAT enzyme activity and AChE-positive fibres in the initially denervated hippocampal formation of the adult recipient. Time-course studies indicated that this ingrowth progressed from the implant into the rostral tip of the host hippocampus, and continued caudally to cover the entire dorsal hippocampus by 3-6 months postoperative. Although the regenerating AChE-positive fibres reached the hippocampal target in the recipient along abnormal routes, they reinnervated selectively the appropriate terminal areas within the host hippocampus and dentate gyrus, suggesting the presence of quite specific mechanisms to guide the regenerating axons back to their original targets. Lesions of the medial septum-diagonal band area of the host and horseradish peroxidase (HRP) injections into the host hippocampus, caudal to the implant, indicated that the origin of the regenerating axons was predominately from the ipsilateral ventral medial septum and diagonal band area of the host. The results provide evidence that axonal regeneration and reinnervation of a denervated target zone can be promoted by utilizing implants of embryonic CNS tissue to bridge a tissue defect between the target and the lesioned axonal stumps.

The regeneration of the septal cholinergic system in adult rats has been studied in animals bearing transplants of hippocampus taken from 20-40 mm rat fetuses (approximately 17-21 days of gestation). The septal axons located within the fimbria and the dorsal fornix were lesioned and a cavity was prepared at the rostral end of the hippocampus. The embryonic tissue was placed adjacent to the severed end of the fornix-fimbria. The time-course of ingrowth of cholinergic fibers into the transplant was monitored by acetylcholine esterase (AChE) histochemistry and the determination of the levels of choline acetyltransferase (ChAT). Both methods indicate that there is a progressive ingrowth into the transplant of cholinergic fibers up to 3 months after transplantation. The newly-formed AChE-positive fibers in the transplant remain beyond one year after transplantation and are thus presumably permanent. Both horseradish peroxidase (HRP) injections into the implant and radiofrequency lesions of the septal-diagonal band area indicate that the principal source of these fibers is the AChE-positive neurons of the medial septum and the nucleus of the diagonal band which normally form the septohippocampal cholinergic projection. The results suggest: (1) that implants of a normal embryonic target tissue can promote axonal regeneration in mature neurons of the mammalian central nervous system; (2) that some neurons in the adult mammalian CNS retain at least part of their embryonic capacity to generate axons and recognize specific postsynaptic targets in developing CNS tissue; and (3) that this host-implant interaction can result in the formation of quite specific innervation patterns in the implanted target tissue.

Rats with a unilateral transplant of embryonic substantia nigra, placed in a cortical cavity overlying the caudate-putamen, were compared with control animals on a range of behavioral tests following bilateral 6-OHDA lesions of the ascending dopaminergic nigrostriatal pathway. Tests designed to reveal behavioural asymmetry--such as spontaneous, tail-pinch and amphetamine-induced rotation, sensorimotor orientation, and side preference in a T-maze--revealed that the rats with bilateral 6-OHDA lesions and a unilateral transplant are similar to unilaterally lesioned animals with one intact nigrostriatal pathway. Both transplanted and bilaterally lesioned control rats became spontaneously akinetic after the second 6-OHDA lesion. This akinesia could be reversed by a low dose of amphetamine (0.5 mg/kg) in the transplanted but not in the non-transplanted control rats. The attenuated effects of apomorphine and L-DOPA on activity and rotation suggest that the nigral transplant produced a partial reversal of receptor supersensitivity following the 6-OHDA lesion on the same side as the transplant. However, other effects of the bilateral 6-OHDA lesion, including the development of aphagia, adipsia and akinesia, were not reversed by the presence of the transplant. The transplants were shown by fluorescence histochemistry to have densely reinnervated the dorsal parts of the denervated caudateputamen on the side ipsilateral to the transplant. The results show that intracortical nigral grafts reinnervating parts of the dorsal caudate-putamen can reverse some, but not all, functional impairments associated with bilateral destruction of the nigrostriatal pathway.

Pieces of embryonic mesencephalic tissue rich in dopamine and cholecystokinin immunoreactive neurones were grafted to the dorsal surface of the caudate-putamen of adult host rats subjected to unilateral dopamine depleting lesions. After 3 months, neuronal survival in the graft and fibre outgrowth into the host brain were studied by tyrosine hydroxylase and cholecystokinin immunohistochemistry, both in serial sections and by elution and restaining of the same sections. Both dopamine- and cholecystokinin-containing neurones as well as neurons containing both compounds survived the transplantation process. The ratio of neurones in which dopamine and cholecystokinin-like immunoreactivity occurred independently and in coexistence was similar in the grafts to that seen in the intact ventral mesencephalon. This suggests that the grafted cells maintain and express at least some of their normal chemical characteristics in the ectopic cortical location. Only those fibres which contained tyrosine hydroxylase but apparently lacked the cholecystokinin-like peptide showed extensive reinnervation of the host neostriatum. The cholecystokinin-positive fibres were found in a narrow zone immediately adjoining the graft. These results indicate that the dopaminergic reinnervation of the denervated neostriatum is preferentially carried out by the population of grafted mesencephalic dopamine neurones apparently lacking the cholecystokinin-like peptide. This suggests the presence of growth regulating mechanisms in the denervated neostriatum which selectively favour the ingrowth of fibres from the appropriate dopaminergic neuronal subset. The transplantation technique may therefore provide a powerful tool for the study of neurone-target interactions in the establishment of neuronal connections, and of the possible role of peptidergic coexistence in the development and organization of monoaminergic pathways and their innervation patterns.

Previous studies have shown that transplants of embryonic substantia nigra, which reinnervate the dorsal neostriatum, can compensate for certain, but not all, behavioural deficits induced by unilateral or bilateral destruction of the nigrostriatal dopamine pathways in adult rats. The present study shows the nigral transplants which are placed in the lateral cortex so as to reinnervate ventral and lateral parts of the neostriatum, give an entirely different pattern of behavioural recovery. Thus, the laterally placed grafts were more efficient than the previous dorsally placed ones in compensating for the sensorimotor asymmetry in unilaterally lesioned animals, and the akinesia seen after bilateral lesions. Conversely, the drug-induced motor asymmetry which was completely abolished by the dorsal grafts was not significantly affected in the present animals. These results support the idea of topographic heterogeneity with respect to striatal functions, and suggest that the technique can be used as a tool for more detailed analysis of the functional organization of the meso-telencephalic dopamine systems and the functional heterogeneity of the dopaminergically innervated striatal-based forebrain regions.

The axons of the septohippocampal pathway reach the hippocampal formation via the fimbria, dorsal fornix and supracallosal striae. Complete lesions of these pathways denervate most of the hippocampal formation but a small, residual innervation occurs via a different ventral route. The cells of origin for this additional route for cholinergic innervation of the hippocampal formation have been identified in the present study with a combined technique using a fluorescent retrograde tracer and acetylcholinesterase histochemistry. These cells were localized to the diagonal band of Broca. In a subsequent experiment it was demonstrated, by destroying these cells unilaterally, that they were responsible to a large extent for the cholinergic compensatory collateral sprouting in the hippocampus that has previously been shown to develop following complete fimbria-fornix transection.

Selective lesions of discrete aminergic and cholinergic afferent pathways to the hippocampal formation have been studied as an experimental model of behavioral recovery after partial hippocampal deafferentation in adult rats. The noradrenergic, serotoninergic and cholinergic afferents reach the hippocampal formation along three routes: a supracallosal pathway, a subcallosal pathway (along the fimbria-fornix), and a ventral pathway (along the ansa lenticularis and the ventral amygdaloid pathway). Bi-lateral lesion of the supracallosal pathway resulted in significant reductions in biochemical markers of the hippocampal noradrenergic, serotoninergic and cholinergic innervations. Within 6 weeks following lesion these markers were found to have recovered to levels close to normal. A more substantial deafferentation, obtained by a bilateral lesion of both dorsal pathways, resulted in a more profound initial decrease in the biochemical marker levels, and a less complete recovery in the long-term animals. Concomitant with the initial phase of deafferentation, rats with lesions of one or both of the dorsal afferent pathways exhibited an equally profound behavioral deficit in acquisition of a forced choice alternation learning task in a T-maze. However, while rats subjected to the combined supracallosal-fimbria-fornix lesion remained impaired, the ones with lesions of the supracallosal pathway alone exhibited a significant recovery of the capacity to perform the maze task. The correlation between behavioral recovery and recovery of the biochemical markers in the supracallosal pathway-lesioned group was significant for the serotoninergic innervation of the hippocampus. The present results suggest that reinnervation of the partially deafferented hippocampus through compensatory collateral sprouting is a valuable new experimental model system within which to analyze mechanisms underlying functional recovery after lesions in the CNS.

Transplants of the embryonic locus coeruleus (LC) region were implanted into the circuity of the hippocampal formation in adult rats in which the normal adrenergic afferents to the hippocampus had been removed. The growth of new adrenergic axons from the implant in the denervated hippocampus was followed for 1-14 months after surgery by means of fluorescence histochemistry, and the function of the implant-hippocampal connections was tested electrophysiologically after 2-3 months survival. In the successful cases the entire hippocampal formation was reinnervated from the LC implant within 3-6 months after operation, and the newly formed innervation still persisted unchanged by 14 months. The reinnervation was equally effective irrespective of the route by which the axons entered the hippocampus, i.e. along the lesioned fornix-fimbria or along a retrosplenial route. The pattern formed by the ingrowing LC axons mimicked to a large extent that of the normal LC afferents. Little growth was seen into denervated terminal fields of the commissural, septal or entorhinal afferents, pointing to a preference of the ingrowing LC fibers for the areas normally innervated by adrenergic afferents. In the electrophysiological experiments, stimulation of the LC implants caused (in 20 out of 29 cells monitored) an inhibition of the spontaneous activity of neurons in the host hippocampus. This inhibition had a relatively long latency and a long duration, similar to that observed after stimulation of the innate LC in the intact rat. As in the normal rat, the inhibitory responses were blocked by systemic or local application of the beta-adrenergic receptor blockers propranolol or sotalol. It is concluded that the adult rat brain is capable of carrying out all steps involved in correct functional reinnervation of a denervated region. Moreover, the implant-hippocampal preparation should be a highly suitable model system for functional studies of a central noradrenergic connection.

The cholinergic and adrenergic afferents innervating the hippocampal formation in the rat reach the target region via three distinctly separate routes, two dorsal and one ventral one. Partial deafferentation of the hippocampus obtained by destruction of the dorsal routes (through the fimbria-fornix and the supracallosal striae) resulted in removal of 90% and 60% of the cholinergic and adrenergic innervations, respectively, within one month. By 6-10 months after lesion, the remaining cholinergic and adrenergic inputs, reaching the target via the ventral route, had expanded more than two-fold, resulting in a significant recovery in the original cholinergic and adrenergic innervation patterns. Because of its slow and protracted time-course and its ability to re-establish innervation also in initially denervated areas, this compensatory collateral sprouting phenomenon may be of particular interest for the understanding of the long-term, protracted functional recovery that is seen both after experimental brain lesions as well as in patients with severe brain injuries.

A brief review of the capacity of neural tissue transplants to reinnervate the deafferented hippocampus and repair functional deficits induced by the lesion. The techniques for transplantation of solid pieces of embryonic septum, locus coeruleus or raphe nuclei, or tissue suspensions of embryonic septum, to the adult rat hippocampus are described. Such grafts manifest good long-term survival, provide a good reinnervation of the hippocampus that is histochemically and biochemically appropriate and specific, can establish ultrastructural synaptic contacts with the host, and are electrophysiologically active. Rats with septal grafts manifest recovery of the capacity to learn certain aspects of radial 8-arm maze. T-maze alternation and Morris water-maze tasks. Rats with locus coeruleus grafts manifest an amelioration of lesion-induced hyperactivity. It is concluded that neural tissue transplantation provides a powerful new tool in the study of the functional organization of the hippocampus and its various neurotransmitter-specific afferent systems.

Implants of embryonic hippocampus, placed into a cavity transecting the septo-hippocampal pathway in adult rats, have previously been shown to promote the regeneration of the lesional septo-hippocampal axons across the cavity. The present study provides electrophysiological evidence that these regenerating acetylcholinesterase-containing axons are able to re-establish atropine-sensitive excitatory synaptic connections in the grafted hippocampus as well as in the dorsal part of the initially denervated host hippocampus, thus demonstrating that intracerebral tissue implants can promote true, functional regeneration across a major tissue defect in the adult mammalian CNS.

Adult rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway were tested for amphetamine-induced rotational asymmetry. In animals with functional deficits a fetal mesencephalic transplant was placed intracranially over the superior colliculus within the caudal end of a 2- to 3-cm-long heterologous sciatic nerve segment laid longitudinally on the skull. Two months later the rostral tip of the peripheral nerve graft was cut and inserted through a burr-hole into the denervated striatum. Animals were tested monthly for 5 months for rotational asymmetry and selected rats were sacrificed for histochemical examination. It was shown that the use of a peripheral nerve segment as a bridge between a distant neuronal transplant and a selected region of the adult host brain resulted in the growth of monoaminergic axons into the denervated striatum from the extracerebrally located grafted neurons. The nerve bridge was cut extracranially in rats whose rotational asymmetry had decreased towards normal levels to determine if this behaviour was dependent on axons reaching the striatum through the graft. Each of these animals exhibited an increase in rotational asymmetry one and three weeks after the transection of the nerve. These anatomical and functional observations suggest that the decrease in rotational asymmetry observed in these animals is related to the growth of long axons from the implanted mesencephalic neurons into the denervated host's striatum which traversed the entire peripheral nerve segment.

The growth of new axonal sprouts was studied from transected, ascending noradrenergic axons into transplants of iris tissue in the caudal hypothalamus of the rat. A single intraventricular injection of nerve growth factor, given at the time of axonal damage, resulted in an increased formation and growth of new noradrenaline sprouts 7 days later. The effect seemed to be proportional to the administered dose of nerve growth factor.