Beneficial effects of antioxidant-enriched diet for tyrosine hydroxylase-positive neurons in ventral mesencephalic tissue in oculo grafts.

Supplementation of antioxidants to the diet has been proved to be beneficial in aging and after brain injury. Furthermore, it has been postulated that the locus coeruleus promotes survival of dopamine neurons. Thus, this study was performed to elucidate the effects of a blueberry-enriched diet on fetal ventral mesencephalic tissue in the presence or absence of locus coeruleus utilizing the in oculo grafting method. Sprague-Dawley rats were given control diet or diet supplemented with 2% blueberries, and solid tissue pieces of fetal locus coeruleus and ventral mesencephalon were implanted as single and co-grafts. The results revealed that the presence of locus coeruleus tissue or the addition of blueberries enhanced the survival of ventral mesencephalic tyrosine hydroxylase (TH)-positive neurons, whereas no additive effects were observed for the two treatments. The density of TH-positive nerve fibers in ventral mesencephalic tissue was significantly elevated when it was attached to the locus coeruleus or by blueberry treatment, whereas the innervation of dopamine-beta-hydroxylase-positive nerve fibers was not altered. The presence of locus coeruleus tissue or bluberry supplementation reduced the number of Iba-1-positive microglia in the ventral mesencephalic portion of single and co-grafts, respectively, whereas almost no OX6 immunoreactivity was found. Furthermore, neither the attachment of ventral mesencephalic tissue nor the addition of blueberries improved the survival of TH-positive neurons in the locus coerulean grafts. To conclude, locus coeruleus and blueberries are beneficial for the survival of fetal ventral mesencephalic tissue, findings that could be useful when grafting tissue in Parkinson's disease.

Regulation of trophic factor expression by innervating target regions in intraocular double transplants.

Trophic factors have been found to play a significant role both in long-term survival processes and in more rapid and dynamic processes in the brain and spinal cord. However, little is known regarding the regulation of expression of growth factors, and how these proteins interact on a cell-to-cell basis. We have studied protein levels of one growth factor known to affect the noradrenergic innervation of the hippocampal formation, namely brain-derived neurotrophic factor (BDNF). The purpose of the present study was to determine if appropriate innervation or contact between the LC noradrenergic neurons and their target, the hippocampus, affects expression of this growth factor in either brain region. Fetal brain stem tissue, containing the LC, and hippocampal formation were dissected from embryonic day 17 rat fetuses and transplanted together or alone into the anterior chamber of the eye of adult Fisher 344 rats. The tissue was grown together for 6 weeks, after which the animals were sacrificed and ELISAs for BDNF were undertaken. Transplantation to the anterior chamber of the eye increased the expression of BDNF in the hippocampal but not the brain stem tissue, compared with levels observed in fetal and adult rats in vivo. In addition, double grafting with hippocampal tissue more than tripled BDNF levels in brain stem grafts and doubled BDNF levels in the hippocampal portion of double grafts compared with hippocampal single grafts. Triple grafts containing basal forebrain, hippocampus, and brain stem LC tissue increased brain stem and hippocampal BDNF levels even further. Colchicine treatment of LC-hippocampal double grafts gave rise to a significant decrease in hippocampal BDNF levels to levels seen in single hippocampal grafts, while only a partial reduction of BDNF levels was seen in the brain stem portion of the same double grafts treated with colchicine. The findings suggest that an appropriate hippocampal innervation or contact with its target tissues is essential for regulation of BDNF expression in the brain stem, and that retrograde transport of BDNF can occur between double grafted fetal tissues in oculo.

Glial cell line-derived neurotrophic factor is essential for neuronal survival in the locus coeruleus-hippocampal noradrenergic pathway.

It has been shown that the noradrenergic (NE) locus coeruleus (LC)-hippocampal pathway plays an important role in learning and memory processing, and that the development of this transmitter pathway is influenced by neurotrophic factors. Although some of these factors have been discovered, the regulatory mechanisms for this developmental event have not been fully elucidated. Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor influencing LC-NE neurons. We have utilized a GDNF knockout animal model to explore its function on the LC-NE transmitter system during development, particularly with respect to target innervation. By transplanting various combinations of brainstem (including LC) and hippocampal tissues from wildtype or GDNF knockout fetuses into the brains of adult wildtype mice, we demonstrate that normal postnatal development of brainstem LC-NE neurons is disrupted as a result of the GDNF null mutation. Tyrosine hydroxylase immunohistochemistry revealed that brainstem grafts had markedly reduced number and size of LC neurons in transplants from knockout fetuses. NE fiber innervation into the hippocampal co-transplant from an adjacent brainstem graft was also influenced by the presence of GDNF, with a significantly more robust innervation observed in transplants from wildtype fetuses. The most successful LC/hippocampal co-grafts were generated from fetuses expressing the wildtype GDNF background, whereas the most severely affected transplants were derived from double transplants from null-mutated fetuses. Our data suggest that development of the NE LC-hippocampal pathway is dependent on the presence of GDNF, most likely through a target-derived neurotrophic function.

Release properties and functional integration of noradrenergic-rich tissue grafted to the denervated spinal cord of the adult rat.

Noradrenaline- (NA-) containing grafts of central (embryonic locus coeruleus, LC) or peripheral (juvenile adrenal medullary, AM, autologous superior cervical ganglionic, SCG) tissue were implanted unilaterally into rat lumbar spinal cord previously depleted of its NA content by 6-hydroxydopamine (6-OHDA) intraventricularly. A microdialysis probe was implanted in the spinal cord 3-4 months after transplantation, and extracellular levels of noradrenaline were monitored in freely moving animals during basal conditions and following administration of pharmacological or behavioural stimuli. Age-matched normal and lesioned animals both served as controls. Morphometric analyses were carried out on horizontal spinal sections processed for dopamine-beta-hydroxylase (DBH) immunocitochemistry, in order to assess lesion- or graft-induced changes in the density of spinal noradrenergic innervation, relative to the normal patterns. In lesioned animals, the entire spinal cord was virtually devoid of DBH-positive fibers, resulting in a dramatic 88% reduction in baseline NA, compared with that in controls, which did not change in response to the various stimuli. LC and SCG grafts reinstated approximately 80% and 50% of normal innervation density, respectively, but they differed strikingly in their release ability. Thus, LC grafts restored baseline NA levels up to 60% of those in controls, and responded with significantly increased NA release to KCl-induced depolarization, neuronal uptake blockade and handling. In contrast, very low NA levels and only poor and inconsistent responses to the various stimuli were observed in the SCG-grafted animals. In AM-grafted animals, spinal extracellular NA levels were restored up to 45% of those in controls, probably as a result of nonsynaptic, endocrine-like release, as grafted AM cells retained the chromaffine phenotype, showed no detectable fibre outgrowth and did not respond to any of the pharmacological or behavioural challenges. Thus, both a regulated, impulse-dependent, and a diffuse, paracrine-like, NA outflow may play roles in the recovery of lesion-induced sensory and/or motor impairments previously reported with these types of grafts following transplantation into the severed spinal cord.

The hyaluronan receptor RHAMM in noradrenergic fibers contributes to axon growth capacity of locus coeruleus neurons in an intraocular transplant model.

The hyaluronan receptor for hyaluronic acid-mediated motility (RHAMM) plays a role in cell migration and motility in many systems. Recent observations on the involvement of RHAMM in neurite motility in vitro suggest that it might also be important in axon outgrowth in situ. This was addressed directly by investigating both RHAMM expression in the rat CNS and the ability of anti-RHAMM reagents to interfere with tissue growth and axon outgrowth in intraocular brainstem transplants. By western blotting, anti-RHAMM antibody detected a RHAMM isoform of 75,000 mol. wt in both whole brain homogenate and synaptosome preparations, and a 65,000 mol. wt isoform in synaptosomes. Immunofluorescence of adult brain sections revealed RHAMM-like immunoreactivity in varicose fibers that were also positive for the noradrenergic marker dopamine-beta-hydroxylase. Not all noradrenergic fibers contained RHAMM, nor was RHAMM detected in other monoaminergic fiber types. Lesions of noradrenergic fiber systems with beta-halobenzylamine-N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) eliminated RHAMM-positive fibers, but noradrenergic axons that sprouted extensively after this treatment were strongly RHAMM-positive. To assess RHAMM's role in fiber outgrowth, fetal brainstem tissue containing noradrenergic neurons was grafted into the anterior chamber of the eye. Treatment of grafts with anti-RHAMM antibody caused significant inhibition of tissue growth and axon outgrowth, as did a peptide corresponding to a hyaluronan binding domain of RHAMM. These agents had no such effects on transplants containing serotonergic and dopaminergic neurons. These results suggest that RHAMM, an extracellular matrix receptor previously shown to contribute to migratory and contact behavior of cells, may also be important in the growth and/or regenerative capacity of central noradrenergic fibers originating from the locus coeruleus.

[The differentiation of locus coeruleus neurons after their allotransplantation into the preliminarily denervated hippocampus of white rats]. / Differentsirovka neironov golubovatogo mesta posle ikh allotransplantatsii v predvaritel'no denervirovannyi gippokamp belykh krys.

The suspension of embryonic locus coeruleus (LC) was transplanted into outbred albino rat hippocampus after its preliminary 6-hydroxy-dopamine-induced denervation. Immunohistochemical and morphometric analysis revealed that 3 months after the transplantation, embryonic noradrenergic LC cells which have completed their histogenesis in recipient hippocampus, appear as differentiated multipolar and fusiform cells, typical to LC. Intrahippocampal allotransplants of rat embryonic LC were also demonstrated to normalize the level of orientation activity in an open area, that was significantly reduced after administration of 6-hydroxy-dopamine to the animals.

Specific innervation of the rat thalamus by grafted noradrenergic locus coeruleus neurons.

Growth and distribution of noradrenaline (NA) fibres from the implant into the thalamus of host rats were examined at 5-13 months after the implantation by immunohistochemistry using NA or tyrosine hydroxylase antisera. Cell suspension dissociated from the locus coeruleus (LC) region of 14-day-old rat fetuses was implanted into the center of the unilateral thalamus in adult rats from which the noradrenergic afferents to the thalamus had been eliminated with 6-hydroxydopamine treatment. A dense network of varicose NA-immunoreactive (NA-IR) fibres extended laterally into the posterior thalamic nuclear group and the ventral posterolateral thalamic nucleus from the implant in a pattern similar to that the intrinsic noradrenergic fibres form in the normal thalamus, i.e. laterally rich and medially poor NA fibres. Electron microscopic observations revealed that varicosities of NA-IR fibres formed symmetrical as well as asymmetrical axodendritic synapses and axo-axonic synapses with the host neurons as seen in the normal thalamus. labelled dendrite-like fibres of graft origin penetrated deep into the host brain and received afferents from non-labelled axon terminals. Varicosities of NA-IR fibres in the LC implanted animal formed axo-dendritic synapses at the higher ratio than those in the normal animal did. These results show that implanted fetal noradrenergic neurons innervate target regions of the thalamus specifically as the noradrenergic fibres in the normal thalamus do and maintain the innervation for a long time in the noradrenergically denervated rats.

Transplantation of embryonic noradrenergic neurons in two models of adult rat spinal cord injury: ultrastructural immunocytochemical study.

The synaptic connections established by grafted noradrenergic (NA) neurons into the lesioned adult rat spinal cord were analysed using immunocytochemistry at the electron microscopic level. An embryonic cell suspension of the locus coeruleus region from E-13 rat embryos was transplanted into the spinal cord following either: (1) spinal cord transection or (2), partial selective denervation by 6-hydroxy dopamine (6-OH DA). One month after grafting, the NA-neurons established, in the two models, an innervation pattern similar to that found in the intact spinal cord. In both models, the transplanted NA-immunoreactive neurons formed extensive synaptic contacts with dendrites, spines and perikarya. The proportion of axodendritic and axospinous contacts was inverse in the two models. The first model thus reproduced more closely the normal synaptic pattern prefering dendritic targets, which could correspond to a better integration of the graft. In the second model, a partially NA-denervated spinal cord, there existed a competition between residual intrinsic and grafted neuron-derived fibres, which presumably affects synaptogenesis. In conclusion, the present study illustrate the complexity of cell interations conducting to the formation of a specific circuitry. Recognition phenomenon are likely modulated by space constraints, which ultimately shape-up the geometry of synaptic contacts.

Intraspinal noradrenergic-rich implants reverse the increase of alpha 1 adrenoceptors densities caused by complete spinal cord transection or selective chemical denervation: a quantitative autoradiographic study.

This study examined, in the adult rat, whether the intraspinal transplantation of a cell suspension of embryonic day (ED)13 rat locus coeruleus primordia was able to normalize the lesion-induced increase of spinal alpha 1-adrenoceptors. Two experimental models of spinal denervation were studied. The first model consisted of a complete spinal cord transection (thoracic vertebrae level T8-T9) and 1 week later, the cell suspension was transplanted below the section; the second one was obtained by a selective chemical lesion of the noradrenergic (NA) system and one month later, the cell suspension was implanted at the same level as in transected rats. Five weeks after grafting, all animals were sacrificed and spinal cord tissue sections were processed for immunohistochemical detection of noradrenaline or for quantification of alpha 1-adrenoceptors binding sites densities using [3H]prazosin as a ligand. After 6-OHDA lesion, as well as caudally to the transection, a significant increase by 21% (P < 0.01) to 68% (P < 0.001) of alpha 1-adrenoceptors densities was detected. The implantation of embryonic NA neurons into the denervated spinal cord led to a reversal of the lesion-induced increase of spinal alpha 1-adrenoceptors, five weeks later. Moreover, this reversal seems to be more effective after mechanical than after chemical denervation.

Fibrin glue used as an adhesive agent in CNS tissues.

One of the limitations of many bridging experiments in neural transplantation is that the CNS tissues cannot be sutured. Fibrin glue is a two-component system derived from whole blood which, when mixed, reproduces the final stage of blood coagulation and solidifies. Many experimental studies of humans and animals show that fibrin glue repair of peripheral nerves is almost equivalent to microsurgical sutures. In this study, we attempted to extend its use to CNS tissues and transplants. Two techniques were tried: (1) Bilateral parietal knife cuts were performed by stereotaxic technique in six rats. Fibrin glue was applied in the right-side cortical lesion. Immunohistochemistry using antisera to tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP), laminin and neurofilament (NF) was essentially similar between the control and treatment groups. The immunoreactivity of each marker revealed no significant differences between the two groups on days 1, 7 and 30. There was no difference in terms of gliosis or microvascular proliferation. (2) Embryonic day 16 fetal locus coeruleus was grafted together with E16 cortex to the anterior chamber of sympathectomized eyes. In the six eyes of the glue treatment group, the parietal cortical piece and the locus coeruleus piece were joined together before grafting by immersing them in the solution of fibrin glue. In the eight eyes of the control group, pieces of parietal cortex and locus coeruleus were introduced individually and approximated by gently pressing the cornea. The sizes of double grafts showed no significant difference between group during six weeks postgrafting. The immunohistochemical pictures using antisera against TH, GFAP and laminin were similar in both groups. Catecholaminergic fibers from the grafted locus coeruleus were found bridging over into the parietal cortical piece in both the control and treatment groups. There was no significant difference in TH-positive nerve fiber density between tissue glue-joined and control double intraocular grafts. In conclusion, fibrin glue can be used as an adhesive agent in CNS tissues without hampering the outgrowth of neurites or causing adverse tissue reactions in fetal or adult nervous tissues.

Seizure development and noradrenaline release in kindling epilepsy after noradrenergic reinnervation of the subcortically deafferented hippocampus by superior cervical ganglion or fetal locus coeruleus grafts.

Solid pieces of fetal locus coeruleus (LC) or superior cervical ganglion (SCG) were placed into a fimbria-fornix lesion cavity in 6-hydroxydopamine-treated, noradrenaline (NA)-denervated rats. Six to 8 months later, all animals were subjected to electrical kindling stimulations in the hippocampus until they had reached the fully kindled state. Nongrafted lesioned animals showed markedly increased kindling rate which was partly attenuated by LC but not SCG grafts. In both LC- and SCG-grafted animals, dopamine beta-hydroxylase immunocytochemistry demonstrated a high density of graft-derived noradrenergic fibers in the dorsal hippocampus, whereas reinnervation of the ventral hippocampus was much more sparse. Subregional distribution of these fibers within the hippocampus was different in the two grafted groups. Both grafts partly restored basal extracellular NA levels in the hippocampus and reacted to generalized seizures by a significant (two- to threefold) increase of NA release, as measured by intracerebral microdialysis. Our data indicate (i) that seizure activity can regulate transmitter release from noradrenergic neurons in both LC and SCG grafts, (ii) that only fetal LC grafts retard seizure development in kindling, and (iii) that the inability of SCG implants to influence kindling epileptogenesis could be due to a lack of synaptic contacts between the graft-derived ganglionic fibers and host hippocampal neurons.

Transmitter release from transplants of fetal ventral mesencephalon or locus coeruleus in the rat frontal cortex and nucleus accumbens: effects of pharmacological and behaviorally activating stimuli.

The present study was performed in order to establish whether dopamine (DA) release from behaviorally functional intracerebral DA transplants is dependent on changes in neuronal impulse flow, and is under control of the host brain. Rats were subjected to combined intraventricular and ventral tegmental injections of 6-hydroxydopamine (6-OHDA) in order to obtain a severe bilateral lesion of the ascending mesocorticolimbic DA projections. Cell suspension grafts of fetal ventral mesencephalic neurons were thereafter implanted into the medial frontal cortex (MFC) and the nucleus accumbens (NAc). Since the neurotoxin injections removed also the ascending noradrenergic systems, fetal locus coeruleus neurons were added to the graft suspension in one group of animals. Age-matched lesion-only and normal animals served as controls. The lesion-induced alterations in spontaneous, amphetamine- and apomorphine-induced locomotor activity and in a skilled paw reaching task were evaluated before transplantation, and at 3 and 6 months post-grafting. Microdialysis probes were finally implanted in the MFC and NAc in order to monitor extracellular DA and noradrenaline (NA) levels (i) during administration of pharmacological agents which augment or depress catecholamine release in the intact brain; (ii) during exposure of the rats to stressful manipulations (handling and immobilization) or appetitive stimuli (eating) known to enhance cortical and limbic DA or NA release in intact animals. The lesion-induced reduction in amphetamine-induced locomotor activity was reversed in all grafted animals, which also showed a higher than normal spontaneous overnight activity. Daytime spontaneous locomotor activity (which was reduced in the lesion-only rats) as well as apomorphine-induced hyperactivity was reversed by the grafts of DA neurons only. By contrast, the lesion-induced impairment in skilled forelimb use was not alleviated by the grafts. The grafted DA neurons restored normal steady-state DA overflow in the NAc, whereas they enhanced cortical DA overflow to significantly higher than normal levels. Restoration of both cortical and striatal NA overflow was observed in the group that received mixed DA and NA grafts, whereas animals that received DA grafts only did not differ from the lesioned controls. The changes in extracellular DA and NA levels measured in the grafted MFC and NAc under potassium depolarization (100 mM KCl), inhibition of terminal catecholamine reuptake (10 microM nomifensine), and sodium channel blockade (1 microM TTX) indicated that graft-derived DA or NA release had normal neuronal properties, and was dependent on an intact axonal impulse flow.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of intraventricular locus coeruleus transplants on seizure severity in genetically epilepsy-prone rats following depletion of brain norepinephrine.

Audiogenic seizures (AGS) in genetically epilepsy-prone rats (GEPR) of the moderate-seizure substrain (GEPR-3s) were investigated to determine whether norepinephrine (NE) depletion induced by 6-hydroxydopamine (6-OHDA) microinfusion into the locus coeruleus (LC) could alter the efficacy of intraventricular NE tissue grafts in promoting reductions in seizure severity in AGS. GEPR-3s were stereotaxically infused with 6-OHDA (4 micrograms/side/rat), or vehicle into the region of the LC. Following 6-OHDA treatment all animals were subjected to 3 AGS tests. GEPR-3s seizure severities were increased in 39.5% of the animals after microinfusion of 6-OHDA into the region of the LC. Following the third AGS test, each rat was stereotaxically implanted with 17 gestational day rat fetal tissue obtained from the dorsal pons and containing the primordia of the LC or with tissue obtained from the neocortex or were sham-grafted. Subsequent to grafting, rats were subjected to 3 additional AGS tests. 53% (10/19) of 6-OHDA treated GEPRs showed a significant reduction in seizure severity following transplantation of fetal LC tissue. In contrast, only 20% (1/5) of GEPRs infused with saline rather than 6-OHDA showed a reduction of seizure severity following fetal LC transplantation. NE content in the cortex and pons/medulla was decreased by 78% and 46% respectively following 6-OHDA microinfusion into the LC. Prominent grafts with numerous TH positive neurons and neurites were present within the third ventricle of grafted animals, while cortex grafts contained no TH immunostained structures. These findings suggest that the efficacy of fetal LC tissue to promote reductions in seizure severity in GEPRs is increased following depletion of central NE by microinfusion of 6-OHDA.

Plastic changes in Ara C-treated and "transplanted" coeruleocerebellar cultures.

Locus coeruleus axons project to cerebellar cortex in coeruleocerebellar cultures, where they make functional contacts, and also appear as fine fibers in the outgrowth zones. The predominant catecholamine of locus coeruleus neurons in culture is dopamine. When coeruleocerebellar cultures are exposed to cytosine arabinoside to destroy cerebellar granule cells and functionally compromise glia, there is a resultant increase of Purkinje cell survival and a sprouting of Purkinje cell recurrent axon collaterals, plus an increase of catecholaminergic axons accompanied by a doubling of tissue dopamine content. If such reorganized cultures are transplanted with granule cells and glia, a second round of plastic changes ensues in which the Purkinje cell population and the recurrent axon collaterals are reduced to control levels, but catecholaminergic axons and dopamine content remain increased. The maintenance of catecholaminergic axons does not appear to depend on the persistence of target neurons.

Human fetal xenografts of brainstem tissue containing locus coeruleus neurons: functional and structural studies of intraocular grafts in athymic nude rats.

Fetal human brainstem tissue including the nucleus locus coeruleus was transplanted to the anterior eye chamber of athymic nude rats. Most transplants survived and grew in the anterior chamber of the eye. After 9-15 months, the host animals were anesthetized and electrophysiological or in vivo electrochemical recordings were performed. The brainstem transplants contained spontaneously active neurons with regular single-spike firing patterns. The neurons responded to ipsilateral light stimulation with an increase in firing rate and to the alpha 2-receptor agonist clonidine with significantly decreased firing rates. In vivo electrochemical studies demonstrated reproducible noradrenergic overflow after local application of potassium. Immunohistochemical evaluation of the brainstem transplants showed an abundance of tyrosine hydroxylase-positive neurons and neurites in all transplants and a dense network of neurofilament-, synapsin-, and glial fibrillary acidic protein-positive profiles throughout the grafts. Taken together, the present physiological and histochemical data indicate that it is possible to obtain transplants containing a specific monoaminergic population within the brainstem from human fetal fragments and to maintain these transplants in oculo in athymic nude rats for at least 15 months, during which time noradrenergic neurons develop.

Effects of neural transplantation on seizures in the immature genetically epilepsy-prone rat.

To study the hypothesis that neural transplantations can alter seizure susceptibility in a genetic animal model of epilepsy, 93 pubescent genetically epilepsy-prone rats with stage 9 seizures received either bilateral inferior colliculi (N = 21) or lateral ventricle (N = 42) transplants or sham transplants (N = 30). The grafts consisted of embryonic locus ceruleus, neocortical, or cerebellar tissue. Starting 2 days after the transplantation the rats were subjected to audiogenic stimulations every other day for 61 days. Latency to the running and tonic phase, seizure severity score, and duration of the tonic and clonic phase were compared in the neural transplant and sham-operated controls. Rats that received transplants had a longer latency to the tonic phase and a shorter duration of the clonic phase than the controls. At age 110 days the rats had electrodes implanted bilaterally into the angular bundle and were kindled. No difference in kindling rate was found between the rats that received neural grafts and the sham-operated controls. Cerebrospinal fluid concentration of norepinephrine was not altered by the transplants. This study demonstrates that the anticonvulsant effects of neural transplants, using the genetically epilepsy-prone model of epilepsy, are mild.

Electrophysiological effects of ethanol on hippocampal and cerebellar neurons cografted with locus coeruleus in oculo: role of the noradrenergic circuitry.

Nucleus locus coeruleus (LC) was sequentially transplanted with hippocampus or cerebellum from rat fetuses to the anterior eye chamber of adult rat hosts. Histological, electrophysiological and pharmacological studies indicate that the LC neurons survive and functionally innervate neurons in hippocampal and cerebellar cografts. Ethanol, when superfused over the double transplants in urethane-anesthetized hosts, caused excitations of hippocampal neuronal activity at doses between 1 and 30 mM, whereas applications above 30 mM depressed the activity of grafted hippocampal neurons. Similar results were observed in cerebellar Purkinje neurons cografted in oculo, except that cerebellar neurons were more sensitive to both the excitatory and the depressant effects of ethanol. The excitations caused by lower ethanol doses in double grafts were prevented by the cosuperfusion of 0.5 to 1.0 microM clonidine, a treatment which effectively removed the inhibitory influence of the LC neurons from the grafted neuronal circuit by depressing the LC neuronal activity. Ethanol-induced excitations were also not observed in single grafts of hippocampus, which lack a catecholamine innervation. Furthermore, in double grafts, when the noradrenergic inhibition was blocked postsynaptically with the alpha adrenergic antagonist phentolamine, ethanol-induced excitations were prevented, although ethanol did not alter the postsynaptic actions of norepinephrine. Our data suggest that the ethanol-induced excitations in the cerebellar and hippocampal grafts appear to be disinhibitions mediated by an ethanol-induced depression of the inhibitory noradrenergic input to these target tissues from LC cografts. Indeed, the doses of ethanol that induced neuronal excitations in hippocampal transplants also elicited marked depressions of LC neurons.