Compensation in behavioral disorders in rats receiving 5,7-DHT neonatally and by transplantation of embryonic raphe nucleus tissue

In 62 male Wistar rats the influence was studied of the transplanted embryonal tissue of raphe nuclei (NR) on the mechanisms of compensation of disturbances of exploratory activity, sensory attention, learning and emotional reactivity induced by neonatal injection of 5,7-DHT. In histochemical studies by Falk-Hillarp method the presence of yellow fluorescence confirmed the specificity of transplanted 5-HT neurones. It is found that NR transplantation causes in animals after 3 months recovery of orienting reaction to sensory stimuli, reduces rats reactivity in the open field, restores the ability to discrimination of emotionally positive influence, disturbed by neonatal injection of 5,7-DHT. The obtained data show the possibility of compensation of behaviour disturbances caused by chronic deprivation of 5-HT system activity by transplantation in the neocortex parenchyma of the embryonal tissue, containing serotoninergic neurones.     

Amygdaloid kindling in the rat after a lesion of the locus coeruleus induced by 6-hydroxydopamine

Amygdaloid kindling was studied after locus coeruleus lesion with 6-hydroxydopamine in the rat. Specific lesions, controlled with immunocytochemistry using an anti-norepinephrine antibody, facilitated the amygdaloid kindling. These results indicate that the noradrenergic system plays an inhibitory role in the development of amygdaloid kindling.     

The dynamic behavioral changes in rats after destruction of the locus coeruleus]

Destruction of locus coeruleus (LC) was conducted in adult rats (male rats of the Wistar line, 250-300 g) electrolytically or by means of 6-hydroxydopamine (6-OHDA). Behaviour of the intact animals sham-operated and of rats with destructed LC was evaluated before and every week after the operation during 5 weeks. Statistically significant alterations in the behaviour were revealed only in the initially low-active rats. These animals were characterized firstly, by low motor and exploratory activity in all tests (open field and automated devices), disturbance of the reactivity to novelty--only in one week after the operation with subsequent compensation of shifts; secondly, by low level of investigatory activity in the open field and absence of habituation to the repetitive tests--from the 21-st day after the operation.     

Quantitative evaluation of the cytoarchitectonic reorganization of the locus coeruleus in the brain stem under the influence of 6-hydroxydopamine

It has been demonstrated the reorganization of structure of locus coeruleus in the animals treated intracisternally with 300 micrograms of 6-hydroxydopamine (6-OHDA) which selectively causes the lesion in catecholaminergic system using the method of quantitative estimation of the degree of uniformity and isotropy of structures. After administration of specific neurotoxin in early stages chromatolytic changes were observed in neurons which were not yet destructive but contractive for long. After injection of 6-OHDA in 36 days in the cytoarchitectonic structure were observed marked changes in uniformity and isotropy of neuron distribution within some groups in locus coeruleus versus controls.     

Arousal effects of orexin-A correlate with GLU release from the locus coeruleus in rats

Although orexin was found to promote food intake, recent reports proposed its involvement in the regulation of vigilance. To study the mechanism of how orexin affects arousal, we analyzed glutamate (GLU) release from the locus coeruleus (LC) in rats after systemic injection of orexin-A. Baseline levels of orexin-A in the LC were significantly higher during the dark period than the light period. Intravenous administration of orexin-A increased GLU levels as well as orexin in the LC, simultaneously promoting wakefulness. These results suggest that increases in GLU release may reflect the arousal-inducing effects of orexin.     

Role of the hypothalamus in originating disorders of exploratory behavior and learning in rats with excised nuclei of the locus coeruleus

Open-field behaviour and emotionally differently reinforced learning were studied in male Wistar rats with bilaterally ablated Locus coeruleus. Histochemical analysis of the hypothalamic structures was carried out. Decrease of investigating activity and attention was found as well as disturbances of learning with emotionally-negative (painful) reinforcement. By means of histochemical methods, fluorescence characteristic for catecholamines was found to decrease sharply in paraventricular and supraoptic hypothalamic nuclei, eminentia medialis and the posterior lobe of the hypophysis.     

Role of the locus coeruleus in the development of cerebrovascular disorders in acute myocardial ischemia

Experiments on rats were made to examine the total cerebral blood flow during locus coeruleus (LC) stimulation, acute myocardial ischemia and in the presence of acute myocardial ischemia after LC precoagulation . LC electric stimulation caused a decrease in the cerebral blood flow. The most profound cerebrovascular disorders were observed in animals with acute myocardial ischemia without LC precoagulation and were followed by cardiac arrhythmias. Cerebrovascular hemodynamic disorders occurring in acute myocardial ischemia were prevented by LC coagulation. It is suggested that the cerebrovascular disorders are consequent on the formation in the LC of the hyperactive determinant structure and play a role of a secondary pathogenetic factor in heart regulation disorders.     

Effect of intraventricular administration of noradrenaline and dopamine on the levels of corticosterone in rats and denervation hypersensitivity resulting from intraventricular administration of 6-hydroxydopamine.

The effects of intraventricular administration of noradrenaline (NA) on the resting levels, stress-induced rises and dexamethasone-induced decreases of plasma corticosterone (B) were studied in rats. The effect of pretreatment with intraventricular administration of 6-hydroxydopamine (6-OHDA) on the effects of NA or dopamine (DA), which was injected intraventricularly, was also examined. The results obtained were as follows: 1) Intraventricular administration of 1.0 μg of NA did not cause a decrease in concentrations of plasma B. 2) Ten μg of NA injected intraventricularly resulted in a rise of the levels of plasma B. 3) The stimulating action of centrally administered NA was more marked when the pre-injection concentrations of B were lower. 4) Pretreatment with intraventricular administration of 6-OHDA facilitated the action of intraventricularly administered NA in the regulation of pituitary-adrenocortical functions. The result suggests a development of denervation hypersensitivity caused by the pretreatment. 5) Intraventricular administration of NA did not block stress-induced rises of plasma B. 6) Intraventricular administration of NA counteracted dexamethasone-induced decrements of plasma B. 7) This counteraction was enhanced by pretreatment with intraventricular administration of 6-OHDA. This also suggests a development of denervation hypersensitivity resulting from intraventricular administration of 6-OHDA. 8) Intraventricular administration of 1.0 μg of DA caused no change in the concentrations of plasma B in either control or 6-OHDA treated animals.     

Intranuclear membranous inclusions in the CNS neurons of rats detectable after the administration of ascorbic acid and 6-hydroxydopamine

Intranuclear membranous inclusions were found in neurons of the rat's cerebral cortex and caudate nucleus 2-21 days following the intraperitoneal injection of ascorbic acid (0.2 and 2.0 g/kg) or the intracisternal infusion of 6-hydroxydopamine (300 micrograms). The intranuclear inclusions were mostly round, occasionally irregular in shape, consisting of one or several concentric membranes; in addition, they were electron-lucid with the diameter of 0.2-0.5 microns, sometimes up to 1 micron. Possible relationship between the formation of intranuclear membranous inclusions and the acceleration of intranuclear metabolism, particularly lipid peroxidation processes, are discussed.     

Increased neuronal activity in locus coeruleus from adult rats undernourished at perinatal age: its reversal by desipramine.

The spontaneous activity of locus coeruleus (LC) noradrenergic neurons was assessed by single unit recording in adult recovered rats undernourished at perinatal age as compared with wellnourished animals. Locus coeruleus activity, measured by the firing rate of noradrenergic neurons and the number of spontaneously active cells/track was significantly higher in deprived rats than in controls. In addition, dose-response curves for the inhibitory LC activity of clonidine showed a shift to the right in deprived animals indicating a subsensitivity of alpha2-adrenergic autoreceptors. This fact suggests an alteration in the negative feedback mechanism mediated by somatodentritic alpha2 autoreceptors that modulate the activity of LC neurons, and may account for the behavioral alterations attributed to early undernutrition. Repeated desipramine (DMI) administration to deprived rats reduced LC activity to values comparable to controls, which were not affected after a similar treatment. These data extend to previous reports on long-lasting or permanent plastic changes in the CNS induced by early undernutrition, which may be reverted by pharmacological manipulations. In addition, these results support the hypothesis that alterations induced by early undernutrition are in the same direction as and resemble those described for patients with panic disorders. Furthermore, together with behavioral alterations and selective anxiolytic effect of DMI and other drugs with antipanic effects described in early malnourished rats, the present data support the proposal that perinatally deprived rats may be a useful model for screening drugs with potential antipanic activity.     

Homotopic transplantation of embryonic neocortical tissue into the brain of adult rats after its damage

Structural characteristics (survival, growth, connections) have been studied in the transplant of the cerebral cortex tissue in Wistar rat embryos (18-day-old), implanted into the brain of mature rats of the same line at various time after a partial lesion of the sensomotor cortex. In 3-5 months after transplantation the light microscopy methods demonstrate that spatial interconnections of the transplant and the injured brain of the recipient depend on time interval between the cerebral lesion and transplantation of the embryonal nervous tissue. Horseradish peroxidase (HP) is ionophoretically injected into the recipient's cerebral tissue away from the place of transplantation. In the transplant retrogradely labelled HP neurons are revealed. This demonstrates efferent connections of the implanted tissue with the host's brain. Presence of the anterogradely labelled nervous terminals in the transplant tissue demonstrates existence of afferent connections of the transplant with the recipient's tissue. Possible mechanisms of survival, growth and formation of connections of the transplant in the injured brain of the mature animal are discussed.     

Chromatographic analysis of the enkephalin immunoreactivity in the nucleus locus coeruleus of the cat after local colchicine administration

Cell bodies immunoreactive for methionine- and leucine-enkephalin are found in the area of the locus coeruleus (dorsolateral pons) of the cat after injection of colchicine in the ascending projections of the nucleus. Using radioimmunoassay procedures, it is shown that colchicine induces a significant increase in methionine- and leucine-enkephalin-immunoreactive material in this area of the brain. High pressure liquid chromatography analysis demonstrated that the immunoreactive materials were authentic methionine- and leucine-enkephalin. The methionine- and leucine-enkephalin patterns were identical in the colchicine injected and non-injected sides of the dorsolateral pons. It is suggested that, in this area of the brain, colchicine (i) does not significantly modify the processing of proenkephalin to form the pentapeptides methionine- and leucine-enkephalin, and (ii) does not induce the appearance of new substances reactive to the enkephalin antisera employed.     

Changes in the development of central noradrenaline neurones following neonatal administration of 6-hydroxydopamine

Abstract— The effects of the neurotoxic compound 6-hydroxydopamine on central noradrenaline (NA) neurones have been investigated in the adult rat after systemic administration of the drug at birth. This treatment produced a permanent and selective reduction in endogenous noradrenaline, [³H]noradrenaline uptake in vitro and the number of histochemically demonstrable noradrenaline nerve terminals in the forebrain, certainly related to neuroneal degeneration. The fluorescence morphology of the noradrenaline perikarya in the locus coeruleus was not notably affected. In the pons-medulla region, the 6-hydroxydopamine treatment led to an almost two-fold increase in endogenous noradrenaline with a similar increase in [³H]noradrenaline uptake and formation of ³H-catecholamines from [³H]tyrosine. Fluorescence histochemistry revealed an increased number of noradrenaline nerve terminals which in addition showed an increased fluorescence intensity. Subcellular distribution studies of endogenous noradrenaline in pons—medulla disclosed the highest relative noradrenaline increase in the microsomal fraction after 6-hydroxydopamine at birth. Sucrose gradient centrifugations disclosed that the pons-medulla synaptosomes from 6-OH-DA treated rats sedimented at a higher sucrose concentration than those from untreated controls. It is concluded that treatment of neonate rats with 6-hydroxydopamine produces a selective degeneration of noradrenaline nerve terminals in the forebrain, especially in the cerebral cortex, whereas in the pons-medulla this treatment leads to an increased intraneuronal noradrenaline concentration due to accumulation of noradrenaline in collateral systems not affected by 6-hydroxydopamine and probably also to an increased outgrowth of noradrenaline nerve terminals.     

Effects of systemic administration of 6-hydroxydopamine on the circumventricular organs in nonhuman primates

6-Hydroxydopamine (6-OH-DA) has been shown to produce degenerative changes in noradrenergic nerve terminals and preterminals in the CNS following intracisternal, intraventricular or direct injection into the brain parenchyma. Systemic injection of 6-OH-DA is known to result in degenerative changes in noradrenergic terminals in the peripheral nervous system. However, only a few studies have been carried out on the effects of systemic injections of 6-OH-DA on noradrenergic terminals in the CNS. In the present study cynomolgus and squirrel monkeys were injected intravenously on two successive days with total doses of 350 mg/kg and 150 mg/kg of 6-OH-DA, respectively, and sacrificed at 2 and 24 h following the second injection. Degenerative changes in the area postrema (AP) neurons in all injected animals were characterized by a generalized increase in electron density of cytoplasmic elements in axonal terminals and preterminals. Multilamellar bodies, clusters of clear and dense core vesicles, increased numbers of secondary lysosomes, and an increase in the number of glycogen increased markedly in injected animals, but no other glial alterations were observed. The number of mast cells in the AP was greater in injected than in noninjected animals, both in the perivascular spaces (PVS) and in parenchymal locations. Cell processes in the PVS were occasionally observed to contain electron dense bodies, and degenerative changes were seen in supraependymal processes in some injected animals.     

In vitro tolerance to inhibition by ethanol of N-methyl-D-aspartate-induced depolarization in locus coeruleus neurons of behaviorally ethanol-tolerant rats

Intracellular recordings were made in pontine slice preparations of the rat brain containing the locus coeruleus (LC). Ethanol at 100 mM, but not at 10 or 30 mM inhibited depolarizing responses to pressure-applied N-methyl-D-aspartate (NMDA) in LC neurons of ethanol-naive rats. Ethanol (100 mM) had a similar effect in LC neurons of ethanol-naive rats, of rats treated with ethanol for 14 days (3 g/kg daily, i.p.) and of rats treated with equicaloric amounts of saccharose (5 g/kg daily, i.p.). The blood concentration of ethanol was markedly decreased at 4 h, and was below the detection limit at 24 h after the last injection. Behavioral measurements in the open-field system demonstrated the development of tolerance in rats receiving ethanol for 14 days. Moreover, an anxiety-related reaction was shown to develop when the acute effect of the last ethanol injection vanished. Therefore, in subsequent in vitro experiments, ethanol (10 mM) was continuously present in the superfusion medium in order to mimic a steady blood concentration and to prevent a withdrawal-like situation. Under these conditions, ethanol (100 mM) still continued to inhibit the NMDA-induced depolarization in slices of untreated rats, but became ineffective in slices of ethanol-treated rats at 4 h after the last injection. By contrast, a supersensitivity to ethanol developed in brain slices at 24 h after the last ethanol injection. In conclusion, in vitro tolerance between systemically and locally applied ethanol at LC neurons could only be demonstrated when a low concentration of ethanol was added to the superfusion medium to simulate the blood concentration of this compound.     

Effects of systemic administration of 6-hydroxydopamine on the circumventricular organs in nonhuman primates

Summary The neurotoxin 6-hydroxydopamine (6-OH-DA) has been shown to produces degenerative changes in noradrenergic nerve terminals and preterminals in the CNS following intracisternal, intraventricular, and stereotaxic injection into the brain parenchyma. Systemic injections of this drug are also known to result in degenerative changes in noradrenergic terminals in the peripheral nervous system and in the circumventricular organs (CVO; areas of the CNS which lie outside the blood brain barrier). In the present study eight adult female cynomolgus monkeys were employed. The four experimental animals were injected on two successive days with 150 and 200 mg/kg 6-OH-DA, respectively. The four controls received only the diluent consisting of 0.1% ascorbic acid in normal saline. Two animals from each of the experimental and control groups were sacrificed at 2 h and 24 h after the second injection. Degenerative changes in the SFO neurons were characterized by a generalized increase in electron density of cytoplasmic elements in axonal terminals and preterminals. Multilamellar bodies, and increases in the number of dense core vesicles, dense bodies and secondary lysosomes were also observed after treatment with 6-OH-DA. The neurons showed clumping of mitochondria, which also appeared to be undergoing degenerative changes. The vacuoles in some supraependymal cells were greatly dilated as was the Golgi apparatus in the ependymal cells. The ependymal cell layer appeared to be intact, but there were areas immediately deep to this cell layer that contained large extracellular spaces. This increase in extracellular space was also commonly observed surrounding the perivascular spaces. These phenomena greatly contribute to the spongy appearance that the SFO takes on after 6-OH-DA administration.Supported by: NASA-Ames NSG-2139 and NIH RR00164-16