Retrograde axonal transport of beta-adrenoreceptors in rat brain: effect of reserpine

Free amino acids were studied in the olfactory bulb of the rabbit during basal conditions and veratridine-induced depolarization, in vitro with a tissue slice preparation and in vivo with a perfusion-dialysis technique. In vivo, basal extracellular concentrations of GABA, beta-alanine and aspartate were low, while glutamine showed the highest level. The basal steady-state concentration ratio between the total tissue pool of free amino acids and amino acids in the extracellular fluid was high for GABA, aspartate and glutamate, while low for glutamine and other 'non-transmitter' amino acids. Veratridine induced a marked TTX-sensitive release of GABA (40-50 times the control) both in vivo and in vitro. In vivo, the GABA release showed a peak during the first minutes of veratridine perfusion. The TTX-sensitive release of aspartate and glutamate, on the other hand, was approximately 5 times higher in vitro than in vivo. Furthermore, a prolonged response to veratridine was seen for glutamate and aspartate in vivo consisting of an early peak, followed by a sustained release. Taurine showed a time-delayed veratridine response, both in vivo and in vitro, whereas glutamine displayed a slow, TTX-sensitive decrease. No effect of veratridine was seen on beta-alanine or carnosine-threonine levels.     

Reserpine and the role of axonal transport in the independent regulation of pre- and postsynaptic β-adrenoreceptors

The response of pre- and postsynaptic β-adrenoreceptors to depletion of brain norepinephrine (NE) with reserpine in the rat was characterized by studying the anterograde and retrograde axonal transport of presynaptic receptors and the receptor binding changes induced in postsynaptic frontal cortex cells. Anterograde transport was shown to occur by the linear accumulation of [³H]dihydroalprenolol ([³H]DHA) binding sites (by in vitro binding assay) proximal to a 6-hydroxydopamine (6-OHDA) lesion placed in the ascending pathway of the locus coeruleus and was blocked by more proximal lesions in the pathway. Retrograde transport was demonstrated by the accumulation of [¹²⁵I]iodocyanopindolol binding distal to similar lesions. Autoradiograms from sections of 6-OHDA injected brains were produced with [³H]DHA binding in the presence of theβ₂-agonist, zinterol, and suggested that the antegrade accumulation of binding sites was primarily of theβ₁-subtype. A single injection of reserpine (5 mg/kg, i.p.) produced a long lasting (6–8 weeks), biphasic decrease in cortical NE levels with nadirs and 4 and 28 days (10% and 45% of control, respectively). Frontal cortex binding of [³H]DHA increased to a maximum at 7–14 days and again at 28 days post-reserpine (230% and 167% of control, respectively). These increases were not prevented by the destruction of presynaptic noradrenergic nerve terminals with intraventricular administration of 6-OHDA 1 day prior to sacrifice and therefore appeared to take place solely in postsynaptic cells. Presynaptic, anterograde axonal transport of β-receptors was completely blocked from 4–14 days post-reserpine, increased to 323% of control at 21 days, was blocked again at 6 weeks and returned to control by 8 weeks. Retrograde transport of β-receptors followed a similar pattern suggesting that the presynaptic alterations in β-receptors in noradrenergic neurons of the locus coeruleus take place independently from those in postsynaptic cortical β-receptors as a response to NE depletion by reserpine.     

Modulation of rat brain α- and β-adrenergic receptor populations by lesions of the dorsal noradrenergic bundle

Bilateral lesion of the ascending noradrenergic fibers in the dorsal bundle of adult Wistar rats with 4 μg 6-hydroxydopamine caused extensive depletion of norepinephrine in all forebrain areas, but led to a 54% increase in norepinephrine levels in the cerebellum. β-Adrenergic receptor binding of [³Hdihydroalprenolol was significantly increased in all forebrain areas depleted of norepinephrine except hypothalamus. The increase in [³Hdihydroalprenolol binding was due to 62% and 34% increases in the number of β-receptor sites in the frontal cerebral cortex and hippocampus respectively. Binding of [³HWB-4101 toα₁-adrenergic receptors after dorsal bundle lesion was augmented generally to a lesser extent than β-receptor binding, with significantly increased numbers of sites only in the frontal cortex (74%), thalamus (20%) and septum. Bothα₁-andβ-receptor binding sites were reduced in number by 25–28% in the cerebellum of dorsal bundle-lesioned rats, whereas intraventricular administration of 6-hydroxydopamine to adult rats, which depletes norepinephrine in the cerebellum by 96%, increased cerebellarα₁-andβ-receptor binding by 33–40%. Binding of [³Hclonidine to forebrainα₂-adrenergic receptors was significantly elevated in the frontal cortex, but reduced in the amygdala and septum, after dorsal bundle lesion.     

Retrograde transport of dopamine β-hydroxylase antibodies in sympathetic neurons: Effects of drugs modifying noradrenergic transmission

Antibodies to dopamine beta-hydroxylase (anti-D beta H) were taken up by noradrenergic nerve terminals in the iris following attachment to D beta H, and were transported back to, and accumulated in, the superior cervical ganglion (SCG). Concurrent, or prior destruction of noradrenergic terminals with 6-hydroxydopamine, injected intraocularly, blocked the retrograde transport of anti-D beta H. However, recovery was rapid, reaching 50% of control values within 1 day. Such transport was characterized by a shorter time period before accumulation could be detected in the SCG and by a slower rate of accumulation. These results suggest that noradrenergic neurons recover their ability to turn over synaptic vesicles by exocytosis and transport these back to the ganglion early during the period of axonal regeneration when the axonal length is shorter than normal. The uptake and transport of anti-D beta H was regulated by alpha-adrenergic agents administered locally in the vicinity of noradrenergic nerve terminals. Thus intraocular injection of phentolamine resulted in an increased accumulation of anti-D beta H in the SCG, while amphetamine and the postsynaptic alpha-receptor antagonist, phenylephrine, decreased accumulation. Clonidine and desipramine, which have a predominant presynaptic action, failed to influence the transport of anti-D beta H. These results suggest that in vivo the uptake of anti-D beta H can be increased more by local postsynaptic reflex actions than by a mechanism depending on the inhibition of presynaptic alpha-receptors.     

Glial localization of adenylate-cyclase-coupled β-adrenoreceptors in rat forebrain slices

Fluorocitrate (FC), a selective inhibitor of glial cell respiration, was used to estimate the extent to which glial cells contain adenylate cyclase-coupled β-adrenoreceptors in rat brain slices. The drug blocked 75–95% of the elevation of cyclic AMP caused by the β-agonist, isoproterenol, in the 4 forebrain regions sampled (frontal and parietal cortex, caudate nucleus, olfactory tubercle). Intracellular recording of neurons in the treated slices confirmed that they were unaffected by FC. Treatment with the neurotoxin, kainic acid, eliminated all electrophysiological activity but did not affect the cAMP response. The results indicate that glial cells contain the preponderance of adenylate-cyclase-coupled β-adrenoreceptors in slices of the rat forebrain and may constitute an important target of the central noradrenergic system in vivo.     

Distribution of α-adrenergic, β-adrenergic and dopaminergic receptors in discrete hypothalamic areas of rat

Catecholamine receptor binding sites were measured in discrete hypothalamic nuclei or regions as well as in certain extrahypothalamic areas of the adult male rat. For each assay, discrete areas were microdissected from frozen tissue sections and pooled from several animals. Specific high affinity binding sites were assessed at fixed ligand concentrations for [³H]p-aminoclonidine (PAC) and [³H](2-C 2′,6′-(CH₃O)₂ phenoxyethylamino)-methylbenzodioxan (WB-4101) for α-adrenergic receptor sites, for [³H]dihydroalprenolol (DHA) for β-adrenergic receptor sites, and for [³H]2-amino-6, 7-dihydroxy-1,2,3,4-tetrahydronaphtalene (ADTN) and [³H]spiroperidol in the presence of cinanserin for dopaminergic receptor sites.Regional variations in [³H]WB-4101 binding were relatively small in magnitude, with most hypothalamic and extrahypothalamic areas possessing between 60 and 90% of the binding in frontal cortex. [³H]PAC binding showed a wider range of binding density across brain areas than did [³H]WB-4101, but, in general, variations in [³H]PAC binding paralleled those in [³H]WB-4101 binding. In hypothalamus, binding was characterized as being predominantly to α₁-receptors in the of [³H]WB-4101 and to α₂-receptors in the case of [³H]PAC. The medial hypothalamic areas exhibited a somewhat higher density of these α-adrenergic sites than did the lateral hypothalamus (perifornical hypothalamus and medial forebrain bundle). Also, the ratio of [³H]PAC to [³H]WB-4101 binding differed in different hypothalamic areas, ranging from 1.5:1 to 4:1. The median eminence was exceptional in that it contained appreciable [³H]PAC but no significant [³H]WB-4101 binding sites at the radioligand concentrations tested. Binding of [³H]DHA to β-adrenergic receptors varied over approximately a 3-fold range in the different hypothalamic areas, with binding highest in the medial forebrain bundle and the medial preoptic area, and lowest in the periventricular, dorsomedial and posterior hypothalamic nuclei, the median eminence and the zona incerta. The ratio of β-adrenergic to α-adrenergic binding sites was generally lower in the medial than in the lateral hypothalamic areas and higher in the extrahypothalamic areas examined than in the hypothalamus. With regard to [³H]spiroperidol and [³H]ADTN binding to dopaminergic sites, the striatum, nucleus accumbens and olfactory tubercle showed a greater density of [³H]spiroperidol than of [³H]ADTN sites, in contrast to the hypothalamus where [³H]ADTN binding was more predominant. Within the hypothalamus, [³H]ADTN binding was relatively uniform, while [³H]spiroperidol binding was quite high in four hypothalamic areas (lateral perifornical area, medial forebrain bundle, paraventricular and dorsomedial nuclei), intermediate in the median eminence and arcuate nucleus, and low or not detectable in all other hypothalamic areas.     

Monoamines block kainate‐ and carbachol‐induced γ‐oscillations but augment stimulus‐induced γ‐oscillations in rat hippocampus in vitro

Monoamines are implicated in a cognitive processes in a variety of brain regions, including the hippocampal formation, where storage and retrieval of information are facilitated by synchronous network activities. We have investigated the effects of norepinephrine, serotonin, and dopamine on carbachol‐, kainate‐, and stimulus‐induced hippocampal γ‐oscillations employing combined extra‐ and intracellular recordings. Monoamines dose‐dependently and reversibly suppressed kainate‐ and carbachol‐induced γ‐oscillations while increasing the frequency. The effect of serotonin was mimicked by fenfluramine, which releases serotonin from presynaptic terminals. Forskolin also suppressed kainate‐ and carbachol‐induced γ‐oscillations. This effect was mimicked by 8‐Br‐cAMP and isoproterenol, an agonist of noradrenergic β‐receptor suggesting that the monoamines‐mediated suppression of these oscillations could involve intracellular cyclic adenosine 3′,5′‐cyclic monophosphate (AMP). By contrast, stimulus‐induced γ‐oscillations were dose‐dependently augmented in power and duration after monoamines application. Intracellular recordings from pyramidal cells revealed that monoamines prolonged the stimulus‐induced depolarization and membrane potential oscillations. Stimulus‐induced γ‐oscillations were also suppressed by isoproterenol, the D1 agonist SKF‐38393 forskolin, and 8‐Br‐cAMP. This suggests that the augmentation of stimulus‐induced γ‐oscillations by monoamines involves—at least in part—different classes of cells than in case of carbachol‐ and kainate‐induced γ‐oscillations. © 2009 Wiley‐Liss, Inc.     

Early locus coeruleus lesions increase the density of β-adrenergic receptors in the main olfactory bulb of rats

Norepinephrine is supplied to both deep and superficial layers of the olfactory bulb through dense projections from the locus coeruleus.¹⁶ Beta-adrenergic receptors are located in nearly all bulb laminae, with high-density foci of β-1 and β-2-adrenoceptors present in the glomerular layer.²⁹ Early olfactory experiences that increase norepinephrine levels in the bulb also decrease the density of β-1- and β-2-adrenoceptors, as well as the number of high-density glomerular foci of β-2-receptors.³⁰ Changes in bulb norepinephrine levels, therefore, may affect the density of β-adrenoceptors in the bulb. In the current study, we test this hypothesis by performing unilateral lesions of the locus coeruleus with 6-hydroxydopamine on postnatal day 4, and examining the density of β-1- and β-2-adrenergic receptors in the main olfactory bulb of the rat using¹²⁵I-labeled iodopindolol receptor autoradiography on postnatal day 19. Locus coeruleus destruction resulted in a statistically significant increase in the density of adrenergic receptors in the ipsilateral bulb compared to the contralateral bulb. Both β-1- and β-2-adrenoceptor subtypes increased in density with this manipulation, although the number of glomerular layer high-density β-2 foci was not significantly different between the two bulbs. These results are consistent with the hypothesis that changes in olfactory bulb norepinephrine can regulate the density of β-adrenergic receptors in the bulb.     

Distribution of α2A-adrenergic receptor-like immunoreactivity in the rat central nervous system

In this study, we analyzed immunohistochemically the distribution of the A subtype of α₂-adrenergic receptor (α_2A-AR) in the rat central nervous system using light level immunohistochemistry. By using affinity-purified antisera, we found perikaryal labeling was diffuse and/or punctate; immunoreactive puncta were heterogeneous in size and number in a region-specific manner. Dense deposits of immunoreaction product were found associated with neuropil also, particularly in the lateral parabrachial nucleus, locus coeruleus, lateral septum, diagonal band, stratum lacunosum-moleculare of CA1, and various nuclei of the amygdala and extended amygdala. Prominently immunoreactive olfactory structures include the anterior olfactory nucleus and the granular layer of the olfactory bulb. The cortex was generally light to moderately labeled with greater immunoreactivity in the cingulate and insular cortices. α_2A-AR-like immunoreactivity was intense in the basal forebrain and continuous from the nucleus accumbens through the substantia innominata and fundus of the striatum. Most immunoreactivity in the diencephalon was restricted to the hypothalamus with light to moderate labeling in the thalamus. Generally light immunoreactivity was observed in midbrain structures. In the pons and medulla, both perikaryal and neuropil labeling were observed. Together with the accompanying paper describing the neural distribution of α_2C-AR-like immunoreactivity, our results provide an extensive immunohistochemical cartography of α₂-ARs in the adult rat central nervous system. © 1996 Wiley-Liss, Inc.     

Distribution of α2C-adrenergic receptor-like immunoreactivity in the rat central nervous system

The distribution of α_2C-adrenergic receptors (ARs) in rat brain and spinal cord was examined immunohistochemically by using an affinity purified polyclonal antibody. The antibody was directed against a recombinant fusion protein consisting of a 70-amino-acid polypeptide portion of the third intracellular loop of the α_2C-AR fused to glutathione-S-transferase. Selectivity and subtype specificity of the antibody were demonstrated by immunoprecipitation of [¹²⁵I]-photoaffinity-labeled α₂-AR and by immunohistochemical labeling of COS cells expressing the individual rat α₂-AR subtypes. In both cases the antibody recognized only the α_2C-AR subtype, and immunoreactivity was eliminated by preadsorption of the antibody with excess antigen. In rat brain, α_2C-AR-like immunoreactivity (α_2C-AR-LI) was found primarily in neuronal perikarya, with some labeling of proximal dendrites; analysis by confocal microscopy revealed the intracellular localization of some of the immunoreactivity. Areas of dense immunoreactivity include anterior olfactory nucleus, piriform cortex, septum, diagonal band, pallidum, preoptic areas, supraoptic nucleus, suprachiasmatic nucleus, paraventricular nucleus, amygdala, hippocampus (CA1 and dentate gyrus), substantia nigra, ventral tegmental area, raphe (pontine and medullary), motor trigeminal nucleus, facial nucleus, vestibular nucleus, dorsal motor nucleus of the vagus, and hypoglossal nucleus. Labeling was found in specific laminae throughout the cortex, and a sparse distribution of very darkly labeled cells was observed in the striatum. At all levels of the spinal cord there were small numbers of large, darkly labeled cells in layer IX and much smaller cells in layer X. In general, the pattern of α_2C-LI throughout the neuraxis is consistent with previously published reports of the distribution of receptor mRNA detected by hybridization histochemistry. © 1996 Wiley-Liss, Inc.     

Effects of chronic social defeat on expression of dopamine β‐hydroxylase in rat brains

It is documented that stress activates the locus coeruleus‐norepinephrine system. However, there are far few reports regarding effects of stress on the expression of dopamine β‐hydroxylase, a hallmark enzyme of the noradrenergic neuron. In the present study, adult Fischer 344 rats were subjected to chronic social defeat for 4 weeks. Dopamine β‐hydroxylase expressional levels in the locus coeruleus and its terminal regions were measured by in situ hybridization and western blotting. The results showed that immediately following chronic social defeat there are significantly increased mRNA and protein levels of dopamine β‐hydroxylase in the locus coeruleus, and dopamine β‐hydroxylase protein levels in the hippocampus, frontal cortex and amygdala, compared with those in the control. This chronic social defeat‐induced upregulation of dopamine β‐hydroxylase was completely abolished by adrenalectomy, and/or by treatment with corticosteroid receptor antagonists, mifepristone and spironolactone, either alone or in combination. Furthermore, treatment with desipramine, an antidepressant with specific inhibitory effects on norepinephrine transport, prevented an increased dopamine β‐hydroxylase expression by chronic social defeat in the locus coeruleus and its main terminal regions such as the hippocampus, frontal cortex and amygdala. However, treatment with fluoxetine, an antidepressant with specific inhibition for serotonin transport, only selectively blocked increased dopamine β‐hydroxylase protein levels in the hippocampus caused by CSD. The present findings indicate that chronic social defeat activates the locus coeruleus‐norepinephrine system by upregulating the expression of dopamine β‐hydroxylase, which may increase norepinephrine synthesis. This chronic social defeat induced upregulation of DBH expression was mediated through corticosterone and corticosteroid receptors, with possible interference from antidepressants. Synapse, 2013. © 2013 Wiley Periodicals, Inc.     

α-Tubulin is not detyrosylated during axonal transport

We have examined the question of whether α-tubulin is detyrosylated during axonal transport in retinal ganglion cell axons and axons of spinal motor neurons. The degree of tyrosylation of α-tubulin was estimated from immunocytochemistry and immunoblotting with two anti-α-tubulin monoclonals, one of which (YL1/2) recognizes only the tyrosylated form of α-tubulin. In the case of retinal ganglion cells, the axons were depleted of tyrosylated α-tubulin both in the retina and proximal region of the optic nerve. Distal regions of the axons, in the optic tract, gave a pattern of staining consistent with a reduction in the total level of α-tubulin at the expense of detyrosylated α-tubulin. Axons within the L5 ventral root, the sciatic nerve and tibial nerve were consistently unstained by YL1/2 indicating that these axons were depleted in tyrosylated α-tubulin in all 3 segments. The results indicate that α-tubulin destined for axonal microtubules is detyrosylated close to or in cell bodies and not progressively during its transport. Therefore the segregation of detyrosylated α-tubulin to axonal microtubules may occur at their site of assembly.     

Stress and β-adrenergic receptor binding in the rat's brain

The effect of acute and chronic electric shocks on β-adrenergic receptor binding in the rat's brain was investigated. β-adrenergic receptor subsensitivity in the corter was induced by chronic shocks, but not by acute shocks. This reduction appears to be due to a decreased number of receptors. It seems that stress, by increasing intrasynaptic norepinephrine levels resulting from an accelerated turnover rate, causes β-adrenergic receptor subsensitivity.     

6-hydroxydopamine-induced blockade of hypothalamic obesity: Critical role of brain dopamine-norepinephrine interaction

1. Adult female rats received 3 weekly intracisternal injections of 6-hydroxydopamine or ascorbic acid vehicle following systemic pretreatment with desipramine or pargyline to deplete brain dopamine alone or in combination with norepinephrine. 2. Three weeks after the last injection, all rats received bilateral radiofrequency lesions of the medial hypothalamus to induce overeating and obesity. 3. Compared to normal controls, all lesioned groups over ate and became equally obese except the group receiving desipramine + 6-hydroxydopamine, which failed to show either response. 4. Terminal assays of striatal and neocortical monoamines revealed that hypothalamic obesity was blocked only when dopamine was selectively depleted, but not when equivalent dopamine depletion occurred with concurrent norepinephrine depletion. 5. The ability of central 6-hydroxydopamine to block hypothalamic obesity seems due to an imbalance created in the neural interactions of brain dopamine and norepinephrine systems.     

Glucocorticoid and β‐adrenergic regulation of hippocampal dendritic spines

Glucocorticoid hormones are particularly potent with respect to enhancing memory formation. Notably, this occurs in close synergy with arousal (i.e., when norepinephrine levels are enhanced). In the present study, we examined whether glucocorticoid and norepinephrine hormones regulate the number of spines in hippocampal primary neurons. We report that brief administration of corticosterone or the β‐adrenergic receptor agonist isoproterenol alone increases spine number. This effect becomes particularly prominent when corticosterone and isoproterenol are administered together. In parallel, corticosterone and isoproterenol alone increased the amplitude of miniature excitatory postsynaptic currents, an effect that is not amplified when both hormones are administered together. The effects of co‐application of corticosterone and isoproterenol on spines could be prevented by blocking the glucocorticoid receptor antagonist RU486. Taken together, both corticosterone and β‐adrenergic receptor activation increase spine number, and they exert additive effects on spine number for which activation of glucocorticoid receptors is permissive.     

Analysis of age dependent changes of Topoisomerase II α and β in rat brain

Eukaryotic Topoisomerase II (Topo II) is present in two isoforms alpha and beta. The alpha isoform is predominantly localized in proliferative tissue, while beta isoform is present in all tissues. In the present study we report the activity and protein levels of Topoisomerase II alpha and beta in rat brains of different age groups viz.: E11 (Embryo day 11), E18 (Embryo day 18), post-natal day 1, young (<10 days), adult (<6 months) and old (>2 years). Topoisomerase II beta isoform is found to be the predominant form in brain tissue but Topoisomerase II alpha is found in embryos up to post-natal day 1. The studies to examine the regional distribution of Topoisomerase II beta in brain showed highest activity in cerebellar region and that too only neuronal cell fraction. There was a significant age-dependent decline in this activity. Hence, Topoisomerase II beta may have some unknown function in cerebellum and the low levels of Topoisomerase II beta activity in ageing cerebellum may contribute to the genomic instability in cerebellar region of ageing brain.     

Selective interruption of axonal transport of neurofilament proteins in the visual system by β,β′-iminodipropionitrile (IDPN) intoxication

β,β′-iminodipropionitrile (IDPN) is an agent that produces a marked impairment in the transport of neurofilaments. Its effect on other slowly transported cytoskeletal components sucas tubulin and actin is variable. Previous studies have evaluated transport of neurofilaments after IDPN intoxication in a neurofilament-ricsystem (sciatic motor nerves) and in a system devoid of neurofilaments (axons of the dorsal motor nucleus of the vagus). In the former, IDPN impairs the transport of tubulin and actin but to a lesser degree than it does neurofilament proteins. In the latter, tubulin and actin transport were not impaired, and neurofilament proteins were not present. In this study we evaluated the transport of the cytoskeletal components in a system witan intermediate amount of neurofilaments (the visual system). In the visual system, there is a selective and marked (50%) impairment in the transport of neurofilaments witno impairment in transport of tubulin or microtubule-associated proteins (tau group). We conclude that these different patterns of impairment in transport reflect the differences in pre-intoxication neurofilament content of the nerves examined, the effect of IDPN on the transport of the other components of slow transport being secondary to the presence of stagnated neurofilaments. This model also suggests that transport of neurofilaments can be selectively impaired without producing an effect on other major slow transport components.