Folkert Postema

DIVERGENT AXON COLLATERALS OF RAT LOCUS COERULEUS NEURONS - DEMONSTRATION BY A FLUORESCENT DOUBLE LABELING TECHNIQUE

Abstract The distribution of the noradrenaline-containing neurons of the rat locus coeruleus has been investigated with retrograde labeling techniques using two different fluorescent tracers. Injections were placed in the prefrontal cortex, the striatum, the thalamus, the hippocampus, the cerebellar cortex and the lumbar spinal cord. No evidence for locus coeruleus projections to the striatum was found. Injections in the cortex, thalamus and hippocampus revealed not only ipsilateral but also contralateral labeling of cells in the locus coeruleus. Following unilateral or bilateral homo- or heterotopic injections of the two tracers several cells of the locus coeruleus were double labeled. Combined injections of the two fluorophores in any of these forebrain areas and in the spinal cord also produced double labeled cells. The majority of double labeled cells was located in an area between the ventral and the dorsal parts of the locus coeruleus. These results indicate that individual neurons of the locus coeruleus have the possibility to influence adrenergic receptors at remote areas in the central nervous system.

Increases in Striatal and Hippocampal Impedance and Extracellular Levels of Amino Acids by Cardiac Arrest in Freely Moving Rats

Abstract: The time course of changes in the tissue impedance and the levels of extracellular transmitter and non‐transmitter amino acids was studied in the striatum and hippocampus of the unanesthetized rat after cardiac arrest. Electrodes were implanted for the continuous measurement of tissue impedance so that a measure of the volume of extracellular space was provided. Alternatively, bilateral dialysis probes were used for monitoring levels of extracellular amino acids in subsequent 30‐s samples using an automated precolumn derivatization technique for reversed‐phase HPLC analysis and fluorimetric detection. The impedance started to rise ∼1.2 min following cardiac arrest, increased rapidly during the first 5 min, and increased almost linearly thereafter. After 15 min, a decrease of ∼50% in the extracellular space was calculated. The impedance rose more steeply in the striatum than in the hippocampus. The extracellular levels of taurine, which increased >300% within 5 min after cardiac arrest, most closely resembled the time course of the change in impedance. Glutamate and aspartate levels did not increase until 5 min after circulatory arrest, and at 15 min they had risen to a level of 465 and 265% for the striatum and 298 and 140% for the hippocampus of the resting release, respectively. The release of γ‐aminobutyric acid (GABA) was multiphasic and did not resemble that of any of the other—putative—transmitter amino acids. Fifteen minutes after cardiac arrest, the levels of GABA were 617 and 774% of the resting release in the striatum and hippocampus, respectively. Glycine and ala‐nine efflux substantially increased (232 and 151% in striatum and 141 and 154% in hippocampus, respectively) 15 min postmortem, whereas the glutamine level was slightly increased and levels of asparagine, histidine, threonine, ethanolamine, serine, arginine, and tyrosine were inconsistently higher in the two brain regions. At this time, the extracellular levels of glutamate, GABA, and aspartate were only slightly lower, as expected from the tissue levels and from levels of the other amino acids, an observation indicating that all the amino acids may diffuse through postmortem brain tissue to a nearly similar extent. This study provides evidence that extracellular levels of taurine reflect changes in distribution of electrolytes (and in membrane potentials), that the postmortem release of transmitter amino acids is multiphasic with a delay of at least 1 min, that postmortem shrinkage in extracellular volume cannot account for the increase in the content of transmitter amino acids in the dialysate, and that the massive overflow of glutamate, aspartate, GABA, and taurine seen during ischemia is the result of both release and the failure of uptake. Possible implications of the present findings for excitotoxic damage of the brain are discussed.

Agomelatine reverses the decrease in hippocampal cell survival induced by chronic mild stress.

The antidepressant agomelatine is a MT(1)/MT(2) receptor agonist and 5-HT(2C) antagonist. Its antidepressant activity is proposed to result from the synergy between these sets of receptors. Agomelatine-induced changes in the brain have been reported under basal conditions. Yet, little is known about its effects in the brain exposed to chronic stress as a risk factor for major depressive disorder. Recently, we described agomelatine-induced changes on neuronal activity and adult neurogenesis in the hippocampus of rats subjected to chronic footshock stress. In order to better characterize the actions of agomelatine in the stress-compromised brain, here we investigated its effects on hippocampal neurogenesis in the chronic mild stress (CMS) model. Adult male rats were subjected to various mild stressors for 5 weeks, and treated with agomelatine during the last 3 weeks of the stress period. The sucrose preference test was performed weekly to measure anhedonia, and the marble burying test was carried out at the end of the experiment to assess anxiety-like behavior. In our model, the CMS paradigm did not change sucrose preference; however, it increased marble burying behavior, indicating enhanced anxiety. Interestingly, this stress model differentially affected distinct stages of the neurogenesis process. Whereas CMS did not influence the rate of hippocampal cell proliferation, it significantly decreased the newborn cell survival and doublecortin expression in the dentate gyrus. Importantly, treatment with agomelatine completely normalized stress-affected cell survival and partly reversed reduced doublecortin expression. Taken together, these data show that agomelatine has beneficial effects on hippocampal neurogenesis in the CMS paradigm.

Cyclic AMP in the rat cerebral cortex after stimulation of the locus coeruleus: decrease by antidepressant drugs.

The study concerned the effect of repeated treatment with antidepressant drugs on the elevation of cyclic AMP levels in the rat cerebral cortex following electrical stimulation of the locus coeruleus. Some of the tricyclic and tetracyclic antidepressant drugs inhibited the cyclic AMP response. Desmethylimipramine was the most potent (effective when given 5 mg/kg/day for 2 weeks). Imipramine and nomifensine (daily dose 10 mg/kg for 2 weeks) produced slight decreases, while iprindol and clomipramine were ineffective. After 6 weeks of treatment (daily 10 mg/kg) iprindol, clomipramine and mianserin were without effect. The cyclic AMP response was suppressed by higher doses of the latter two drugs (2 weeks, 20 mg/kg/day). These results indicate that tricyclic and tetracyclic antidepressant drugs are able to decrease cerebral noradrenergic neurotransmission of locus coeruleus neurons, as far as this is mediated by cyclic AMP. It is not clear, however, whether such modification is related to the therapeutic action of antidepressant drugs.

Chronic but not acute foot-shock stress leads to temporary suppression of cell proliferation in rat hippocampus.

Stressful experiences, especially when prolonged and severe are associated with psychopathology and impaired neuronal plasticity. Among other effects on the brain, stress has been shown to negatively regulate hippocampal neurogenesis, and this effect is considered to be exerted via glucocorticoids. Here, we sought to determine the temporal dynamics of changes in hippocampal neurogenesis after acute and chronic exposure to foot-shock stress. Rats subjected to a foot-shock procedure showed strong activation of the hypothalamic-pituitary-adrenal (HPA) axis, even after exposure to daily stress for 3 weeks. Despite a robust release of corticosterone, acute foot-shock stress did not affect the rate of hippocampal cell proliferation. In contrast, exposure to foot-shock stress daily for 3 weeks led to reduced cell proliferation 2 hours after the stress procedure. Interestingly, this stress-induced effect did not persist and was no longer detected 24 hours later. Also, while chronic foot-shock stress had no impact on survival of hippocampal cells that were born before the stress procedure, it led to a decreased number of doublecortin-positive granule neurons that were born during the chronic stress period. Thus, whereas a strong activation of the HPA axis during acute foot-shock stress is not sufficient to reduce hippocampal cell proliferation, repeated exposure to stressful stimuli for prolonged period of time ultimately results in dysregulated neurogenesis. In sum, this study supports the notion that chronic stress may lead to cumulative changes in the brain that are not seen after acute stress. Such changes may indicate compromised brain plasticity and increased vulnerability to neuropathology.

CEREBRAL CATION SHIFTS IN HYPOXIC ISCHEMIC BRAIN-DAMAGE ARE PREVENTED BY THE SODIUM-CHANNEL BLOCKER TETRODOTOXIN

We investigated the effect of the sodium channel blocker, tetrodotoxin, in two animal models of brain pathology. In the first, an acute model, we recorded the interstitial brain potential in the striatum of rats after cardiac arrest. The time of deflection of this potential, an indication of changes in cerebral cation concentrations, was determined in control rats, and in rats pretreated with intrastriatal tetrodotoxin. In control rats a deflection of the brain potential was noted 2 min after cardiac arrest; tetrodotoxin pretreatment delayed this deflection to about 5 min. The second, a survival model, was based on the Levine preparation in rats. A combination of ischemia and hypoxia produced unilateral, cerebral infarcts, which were characterized by a decrease of brain [K+], and by increases of [Ca2+] and [Na+] and thus of the Na+:K+ ratio. Data on the cation shifts, determined by chemical assay methods, were complemented by those of more conventional methods of assessment of brain damage, such as the determination of survival, of Evans blue staining, and of brain water content. Cation shifts could be prevented locally by tetrodotoxin. In conclusion, the drug can, at least partially, prevent the detrimental effects of an ischemic insult. In addition, our results showed that protective effects observed in the acute model may sometimes offer an indication of the effects to be expected in the survival model. Furthermore, the effect of tetrodotoxin on the brain potentials in the acute model showed that its protective action in the survival model may be brought about by delaying cell depolarization and by shortening the actual duration of the depolarized state. We conclude that Na+ influx and, consequently, neurotransmission may play a crucial role in the development of cerebral damage.

Contribution of the locus coeruleus to the adrenergic innervation of the rat Spinal cord: A biochemical study

The possible existence and magnitude of a noradrenergic innervation from the locus coeruleus (LC) to the spinal cord was investigated in the rat with various techniques. Horseradish peroxidase, injected into the lumbar spinal cord produced heavy labelling of presumably noradrenaline (NA)-containing neurons in the ventral region of the LC, while cells in the dorsal region of the LC were only lightly labelled. The effects of electrothermic destruction and electrical stimulation of the LC on levels of NA in various parts of the spinal cord, the cerebral cortex and the hippocampus were studied. Fourteen days after unilateral destruction of the LC there were decreases in NA levels of about 85% in the cerebral cortex and hippocampus and of about 15% in the cervical and thoracic segments of the spinal cord (ipsilateral versus contralateral). Fourteen days after bilateral lesioning of the LC significant decreases (about 25%) in NA levels were observed in all spinal cord segments. Unilateral stimulation in or near the LC induced decreases of NA levels in all areas of the central nervous system investigated. In this experiment the levels of NA in the spinal cord were significantly lowered in the ipsilateral cervical (16%), thoracic (12%) and lumbar/sacral (15%) segments of the spinal cord. These findings together indicate that a small part (no more than 30%) of the NA levels in the rat spinal cord are dependent upon the integrity and activity of NA-containing neurons of the predominantly ipsilaterally localized LC.

The Novel Antidepressant Agomelatine Normalizes Hippocampal Neuronal Activity and Promotes Neurogenesis in Chronically Stressed Rats

Agomelatine is a novel antidepressant which acts as a melatonergic (MT1/MT2) receptor agonist and serotonergic (5‐HT2C) receptor antagonist. The antidepressant properties of agomelatine have been demonstrated in animal models as well as in clinical studies. Several preclinical studies reported agomelatine‐induced effects on brain plasticity, mainly under basal conditions in healthy animals. Yet, it is important to unravel agomelatine‐mediated changes in the brain affected by psychopathology or exposed to conditions that might predispose to mood disorders. Since stress is implicated in the etiology of depression, it is valid to investigate antidepressant‐induced effects in animals subjected to chronic stress. In this context, we sought to determine changes in the brain after agomelatine treatment in chronically stressed rats. Adult male rats were subjected to footshock stress and agomelatine treatment for 21 consecutive days. Rats exposed to footshock showed a robust increase in adrenocorticotropic hormone (ACTH) and corticosterone. Chronic agomelatine treatment did not markedly influence this HPA‐axis response. Whereas chronic exposure to daily footshock stress reduced c‐Fos expression in the hippocampal dentate gyrus, agomelatine treatment reversed this effect and normalized neuronal activity to basal levels. Moreover, chronic agomelatine administration was associated with enhanced hippocampal cell proliferation and survival in stressed but not in control rats. Furthermore, agomelatine reversed the stress‐induced decrease in doublecortin expression in the dentate gyrus. Taken together, these data show a beneficial action of agomelatine in the stress‐compromised brain, where it restores stress‐affected hippocampal neuronal activity and promotes adult hippocampal neurogenesis.

BILATERALLY DIVERGING AXON COLLATERALS AND CONTRALATERAL PROJECTIONS FROM RAT LOCUS COERULEUS NEURONS, DEMONSTRATED BY FLUORESCENT RETROGRADE DOUBLE LABELING AND NOREPINEPHRINE METABOLISM

Evans Blue (EB) and a mixture of 4′-6-diamidino-2-phenylindol 2 HCl and primuline (DAPI-Pr), fluorescing at different wave-lengths were injected into the rat hippocampus, frontal cortex or lateral part of the thalamus. After unilateral injection either of the two substances was retrogradely transported not only to ipsilateral but also to contralateral locus coeruleus (LC) neurons. Moreover after simultaneous injections of EB and DAPI-Pr respectively into the opposite brain structures of individual animals double-labeled neurons were observed in the bilateral LC. Unilateral electrical stimulation of the LC induced significant decreases of norepinephrine and increases of 3-methoxy-4-hydroxyphenylethyleneglycol in both the ipsi- and contralateral frontal cortex and whole forebrain, respectively. These ipsi- and contralateral alterations of the amine and its metabolite correlated highly significantly. These results indicate that several LC neurons have both contralateral and bilateral projections to the brain areas mentioned above.

Regional Calcium Accumulation and Cation Shifts in Rat Brain by Kainate

Abstract: Following local application of kainic acid, changes in the contents of Na+, K+, Ca2+, and Mg2+ of the striatum, cerebellum, and hippocampus of the rat were observed at various times after surgery. Within 1 h the levels of K+ decreased 20% whereas the levels of Na+ and Ca2+ increased at least 50% and 20%, respectively. These changes persisted for more than 8 weeks. Ca2+ levels rose further, to more than 10‐fold during 8 weeks. The Mg2+ levels were slightly and only transiently decreased. Unilateral injections of kainate into the striatum affected the contents of these cations not only in this area, but also in the overlying cerebral cortex, the olfactory tubercle, and the ipsilateral substantia nigra. The Ca2+ increases were less when rats were kept on a diet deficient in Ca2+ and vitamin D. 45Ca2+, intravenously administered, accumulated significantly more in the kainate‐lesioned striatum and substantia nigra than in the homotopic contralateral areas. Electron microscopic examination of the localization of Ca2+ with the oxalate‐pyroantimonate technique showed the appearance of extracellularly located deposits and the accumulation of Ca2+ in (possibly degenerating) myelinated axons in kainate‐lesioned striata. This study provides evidence that calcification of cerebral tissue is closely associated with neurodegenerative processes and shows that kainate may serve as a tool to elucidate the mechanism of brain calcification. The results are discussed in relation to idiopathic calcinosis (striopallidodentate calcinosis, Fahrs disease).