Ulf Stenevi

The organization of tubero-hypophyseal and reticulo-infundibular catecholamine neuron systems in the rat brain

Summary The topography of the median eminence-pituitary catecholamine innervation has been studied with respect to the origin, course and termination of its different components. Thirteen types of electrolytic or mechanical lesions were placed in selected areas of the basal telencephalon, thalamus, hypothalamus (particularly the ventral periventricular zone) and brain stem of female rats. The brains, including the median eminence-pituitary region, were prepared by the Falck-Hillarp method for histochemical localization of cellular monoamines. There are reticulo-infundibular noradrenaline neurons with cell bodies located in the lower brain stem and axon traversing the medial forebrain bundle to innervate the internal and subependymal layers of the median eminence and the stalk. This projection is partially crossed and partially uncrossed. The noradrenergic innervation of the neuro-intermediate lobe is probably exclusively of peripheral, sympathetic origin, and confined to larger vessels of the neural lobe and to the vascular border zone to the pars intermedia. The remaining terminals in the median eminence-pituitary region arise from dopamine-containing cell bodies in the arcuate nucleus and the adjacent periventricular zone above the arcuate nucleus. These constitute a tubero-hypophyseal system which can be separated into 3 components. One arises from the most rostral zone of the arcuate nucleus and innervates the entire pars intermedia. A second group originates in a small group of cells immediately caudal to the first and innervates the neural lobe. The third group is an arcuato-infundibular system innervating the external layer of the median eminence and the stalk and, probably, the internal and subependymal layers too.

Septal transplants restore maze learning in rats with fornix-fimbria lesions.

In a study of the capacity of neural grafts to promote functional recovery in rats with fimbria-fornix lesions, 5 groups of rats were studied behaviourally and with acetylcholinesterase (AChE) histochemistry: (1) sham-operated controls; (2) bilateral fimbria-fornix lesions; (3) bilateral lesions plus bilateral solid embryonic septal grafts to the lesion cavity; (4) bilateral lesions plus bilateral embryonic septal suspension injections into the hippocampus; and (5) bilateral lesions plus bilateral solid embryonic locus coeruleus grafts to the lesion cavity. Seven months were allowed for growth of the grafts and reinnervation of the host hippocampus prior to behavioural testing. The control rats were able to rapidly learn a rewarded alternation task, while the performance of animals with bilateral fimbria-fornix lesions alone remained at a chance level. Both types of septal grafts (rich in cholinergic neurones) but not the locus coeruleus grafts (rich in noradrenergic neurones) reversed the impairment. Behavioural recovery correlated significantly with AChE-positive fibre ingrowth from the grafts into the denervated host hippocampus. However, the septal grafts did not ameliorate the lesion-induced disturbances in spontaneous activity or spontaneous alternation. Thus, the observed behavioural recovery appears specific to the conditioned alternation task and dependent upon cholinergic reinnervation of the hippocampus.

Plastic changes in the adrenergic innervation of the rat septal area in response to denervation

Abstract The adrenergic innervation of the rat septal area was studied using the Falck-Hillarp histofluorescence method in normal animals and in ones in which the septum had been denervated by a unilateral hippocampal lesion. In the normal septum adrenergic terminals are found in all of the principal septal nuclei with the heaviest innervation apparent in the lateral septal nucleus, the nucleus accumbens and the interstitial nucleus of the stria terminalis. A smaller but significant innervation is also present in the medial septal nucleus, nucleus of the diagonal band, posterior septal nucleus and the hippocampal rudiment. Following a total, unilateral transection of the hippocampal fimbria, there is a marked increase in the adrenergic innervation of those septal nuclei that receive a significant projection from the hippocampus. This first appears between 8 and 15 days after operation, becomes marked by 30–60 days, and persists for at least 100 days after the septal denervation. The increase in the number of adrenergic terminals is initially found in the caudal part of the lateral septal nucleus and it subsequently is evident through most of the lateral septal nucleus, the medial septal nucleus and the posterior septal nucleus. No changes were noted in the other nuclei of the septal area. Concomitant with the histochemically demonstrable increase in adrenergic innervation, there is a biochemically detectable increase in the norepinephrine content of the septum. The source of the adrenergic innervation in both the normal and denervated septum appears to be axons traversing the medial forebrain bundle since section of this tract produces a substantial reduction in the adrenergic innervation of the septal nuclei. It is concluded that the increase in norepinephrine-containing terminals produced by removal of the non-adrenergic hippocampal afferents is due to the formation of new terminals which have sprouted from intact norepinephrine-containing axons normally innervating the septal nuclei.

Reinnervation of the partially deafferented hippocampus by compensatory collateral sprouting from spared cholinergic and noradrenergic afferents

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.

Reformation of the severed septohippocampal cholinergic pathway in the adult rat by transplanted septal neurons.

SummaryTransplants containing developing cholinergic neurons were obtained from the septum-diagonal band area of rat fetuses and were implanted into a lesion of the septohippocampal cholinergic pathway or into a cavity of the occipital cortex in adult recipient rats. The growth of new cholinergic fibres from the implant into the hippocampal formation was followed with choline acetyltransferase (ChAT) determinations and acetylcholine esterase (AChE) histochemistry. A fimbrial lesion alone, transecting the septohippocampal pathway, caused an almost complete cholinergic denervation of the hippocampal formation that persisted throughout the five month experimental period. A septal transplant implanted into the cavity of the fimbrial lesion restored a new AChE-positive innervation pattern in the hippocampus and the dentate gyrus that closely mimicked the original innervation removed by the lesion. In parallel, there was a progressive recovery in the ChAT levels, starting in the septal end, and progressing in a temporal direction. A new cholinergic fibre supply could be established in the hippocampal formation also along an abnormal route, i.e. from the transplants implanted into a cavity in the occipital cortex (involving also the dorsal part of the entorhinal cortex). Provided the hippocampus previously had been denervated of its normal cholinergic innervation, a partly normal AChE-positive terminal pattern was thus re-established also from this abnormal position. If, on the other hand, the cholinergic afferents were left intact, the ingrowing fibres were restricted mainly to the outer portion of the dentate molecular layer, i.e. the terminal zone of the lesioned entorhinal perforant path fibres. This suggests that the growth of the sprouting AChE-positive fibres into the normal cholinergic terminal fields was blocked by the presence of an intact cholinergic innervation. It is concluded that regrowing cholinergic axons can be guided over large distances within the hippocampal formation, and that their patterning within the terminal fields is very precisely regulated by mechanisms released by deafferentation.

Functional reinnervation of the neostriatum in the adult rat by use of intraparenchymal grafting of dissociated cell suspensions from the substantia nigra

SummaryDissociated cell suspensions were prepared from the substantia nigra of 15–17 day-old rat embryos and grafted via an intraparenchymal injection into the depth of the neostriatum of adult recipient rats. The survival and fibre outgrowth of the dopamine-containing neurones in the implants were studied by fluorescence histochemistry, and the functional capacity of the grafts was monitored by repeated testing of the amphetamine-induced turning behaviour of the implanted rats.Before transplantation the target neostriatum of the recipient rats was denervated of its normal dopaminergic innervation by an injection of 6-hydroxydopamine into the ipsilateral nigrostriatal dopamine pathway. The completeness of the denervation was ascertained by measurement of the intensity of the amphetamine-induced turning response. After injection of the dissociated cells large numbers of dopamine-containing neurones were found in clusters at the site of injection as well as scattered in the apparently intact neostriatal tissue up to a distance of about 0.5 mm from the site of injection. Extensive dopamine-containing fibre networks had developed around the implant. These newly formed fibres, which were most abundant around the cell clusters at the injection site, extended in a loose network into large areas of the initially denervated caudate-putamen. In all animals with surviving dopamine neurones the amphetamine-induced turning response was reduced, and in the most extensively reinnervated cases even reversed, within 3–5 weeks after transplantation. This strongly suggests that the implanted dopamine neurones are capable of restoring dopaminergic neurotransmission in the denervated neostriatum, probably via reinnervation of the denervated neostriatal tissue.The use of dissociated brain tissue preparations thus permits reliable intraparenchymal grafting of neurones to plausibly any desired site within the central nervous system, and should open entirely new possibilities for investigation of neuronal growth dynamics and functional reconstruction of damaged brain circuits, perhaps even in brains of larger mammals.

Functional Activity of Substantia Nigra Grafts Reinnervating the Striatum: Neurotransmitter Metabolism and [14C]2-Deoxy-d-glucose Autoradiography

Abstract: Dopaminergic innervation of the caudate nucleus in adult rats can be partially restored by the grafting of embryonic substantia nigra into the overlying parietal cortex with concomitant compensation of certain behavioral abnormalities. In this study the function of such grafts was investigated neurochemically by quantification of transmitter metabolism and glucose utilization in the reinnervated target. Rats with unilateral 6‐hydroxydopamine lesions of the nigrostriatal bundle received a single graft to the dorsal caudateputamen and were screened for rotational behavior following 5 mg/kg methamphetamine. The grafts restored dopamine concentrations in the caudateputamen from initially less than 0.5% to an average of 13.6% of normal in rats with behavioral compensation. The ratio of 3,4‐dihydroxyphenylacetic acid to dopamine, which is a measure of the rate of transmitter turnover, were equivalent in transplanted and normal control rats. Moreover, measurements of DOPA accumulation for a 30‐min period after DOPA decarboxylase inhibition indicated similar fractional dopamine turnover rates in normal and transplantreinnervated tissues. Correlations between rotational behavior and dopamine concentrations showed that reinnervation to only 3% of normal was sufficient to counterbalance the motor asymmetry. Measurements of glucose utilization by [14C]deoxyglucose autoradiography indicated equivalent metabolic rates for the grafted tissue and the intact substantia nigra. 6‐Hydroxydopamine denervation of the caudate‐putamen had no significant effect on neuronal metabolism in that region, nor did subsequent reinnervation from a graft. Grafts, however, were associated with a 16% reduction of glucose uptake in the ipsilateral globus pallidus, indicating a significant transsynaptic influence of the nigral transplants on neuronal metabolism in the host brain. Overall the results indicate that behaviorally functional neuronal grafts spontaneously metabolize dopamine and utilize glucose at rates characteristic of the intact nigrostriatal system. This provides further evidence that ectopic intracortical nigral trans‐plants can reinstate dopaminergic neurotransmission in regions of the host brain initially denervated by the 6‐hydroxydopamine lesion.

Regeneration of the septohippocampal pathways in adult rats is promoted by utilizing embryonic hippocampal implants as bridges

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.

Intracerebral grafting of dissociated CNS tissue suspensions: a new approach for neuronal transplantation to deep brain sites

Suspensions of central nervous tissue, prepared by dissociation of selected embryonic brain regions, may be viably transplanted by intraparenchymal injection into a variety of sites in the depth of adult rat host brains. Such grafted neurons can mediate considerable reinnervation of a previously denervated brain region, and they can replace neurons intrinsic to a particular target, such as the caudate-putamen, previously damaged by the neurotoxin kainic acid. The present technique should open entirely new possibilities for experiments on neuronal reconstruction following brain lesions.

Evidence for regenerative axon sprouting of central catecholamine neurons in the rat mesencephalon following electrolytic lesions

Abstract By means of histochemical fluorescence techniques, evidence has been obtained for regenerative sprouting of axons from catecholamine neurons in the rat mesencephalon 1–7 weeks after electrolytic destruction of the substantia nigra and part of the ventromedial midbrain tegmentum. Three days after the lesion, accumulations of catecholamine occurred in coarse, beaded and distorted fibers on the borders of the lesion within the ascending catecholamine fiber tracts. Such catecholamine accumulations in proximal portions of severed axons or axon collaterals are well known; these accumulations showed a progressive decrease after approximately 1 week. In the present study an additional phenomenon was discovered between 7 and 19 days after lesion, namely the appearance of numerous, densely packed, fine, varicose, fluorescent fibers, probably identical with sprouts from catecholamine neurons. These fibers had an abnormal distribution outlining the lesion border and the electrode tracks, sometimes coursing through the lesion and even invading the walls of blood vessels penetrating, or situated close to, the lesion. These numerous newly appearing fluorescent fibers, including those invading the blood vessel walls, were equally prominent in animals with their intracranial arteries sympathectomized by bilateral removal of the superior cervical ganglia. Hence, these fibers were of central origin. The fluorescence of the newly formed fibers was characteristic for catecholamines, and the presence of noradrenaline could be established by means of microspectrofluorometric analysis in fibers invading sympathectomized vascular walls. Although the catecholamine fluorescence in the abnormal fibers on the border of the lesion had decreased at 7 weeks, it persisted in the new fibers invading the vessel walls, which may thus constitute a terminal area of distribution for the regenerating central catecholamine neurons. The phenomenon of regenerative sprouting in the central nervous system has previously been demonstrated, but this is the first direct evidence of regeneration of central monoamine fibers.