Effect of 6-hydroxydopamine on daily variations of 5-HT synthesis in the hypothalamus of the rat

In the cortical gray matter of human brains, which were obtained by permissible resection at the time of surgery, cyclic 3′, 5′-AMP was found in a range of concentrations between 42 and 143 pmole/mg protein. The subcortical white matter and various types of brain tumor contained much lower concentrations of the nucleotide. The level of the cyclic nucleotide in incubated slices of gray matter was elevated 20–50-fold by norepinephrine (0.5–1.0 mM) or veratridine (0.05 mM), 4–7-fold by histamine (1.0mM) or adenosine (0.2mM) and 1.5–2-fold by serotonin. With respect to the stimulatory effect on the cyclic AMP level in human brain slices, norepinephrine was inferior to epinephrine or isoproterenol, and was completely antagonized with a β-adrenergic blocking agent, propranolol, but only slightly by α-blocking agents, such as phentolamine and dibenamine.The particulate fraction of human brain homogenate was capable of synthesizing cyclic AMP from ATP. This enzymic activity was 0.19–0.24 nmole/min/mg protein. This activity was stimulated 2–3-fold by the addition of 10mM NaF but not by any of the biogenic amines tested. The activity of nucleotide phosphodiesterase of human brain was almost equal to that of rat brain: only a single K_m for the substrate cyclic AMP was observed around 0.9 × 10^?4M.     

Ontogenetic profile of ecto-5′-nucleotidase in rat brain synaptic plasma membranes

Ecto-5′-nucleotidase (CD73; EC 3.1.3.5, e-5NT) is regarded as the key enzyme in the extracellular formation of adenosine, which acts as a neuromodulator and important trophic and homeostatic factor in the brain. In the present study, we have investigated e-5NT activity, kinetic properties concerning AMP hydrolysis and the enzyme protein abundance in the purified synaptic plasma membrane (SPM) preparations isolated from whole female rat brain at different ages. We observed pronounced increase in AMP hydrolyzing activity in SPM during maturation, with greatest increment between juvenile (15-day-old) and pre-pubertal (30-day-old) rats. Immunodetection of e-5NT protein in the SPM displayed the reverse pattern of expression, with the maximum relative abundance at juvenile and minimum relative abundance in the adult stage. Negative correlation between the enzyme activity and the enzyme protein abundance in the SPM indicates that e-5NT has additional roles in the synaptic compartment during postnatal brain development, other than those related to AMP hydrolysis. Determination of kinetic parameters, K_m and V_max, suggested that the increase in the enzyme activity with maturation was entirely due to the increase in the enzyme catalytic efficiency (V_max/K_m). Finally, double immunofluorescence staining against e-5NT and presynaptic membrane marker syntaxin provided first direct evidence for the existence of this ecto-enzyme in the presynaptic compartment. The results of the study suggest that e-5NT may be a part of general scheme of brain development and synapse maturation and provide rationale for the previously reported inconsistencies between enzyme immunohistochemical and biochemical studies concerning localization of e-5NT in the brain.     

Proinflammatory cytokine regulation of cyclic AMP‐phosphodiesterase 4 signaling in microglia in vitro and following CNS injury

Cyclic AMP suppresses immune cell activation and inflammation. The positive feedback loop of proinflammatory cytokine production and immune activation implies that cytokines may not only be regulated by cyclic AMP but also conversely regulate cyclic AMP. This study examined the effects of tumor necrosis factor (TNF)‐α and interleukin (IL)‐1β on cyclic AMP‐phosphodiesterase (PDE) signaling in microglia in vitro and after spinal cord injury (SCI) or traumatic brain injury (TBI). TNF‐α or IL‐1β stimulation produced a profound reduction (>90%) of cyclic AMP within EOC2 microglia from 30 min that then recovered after IL‐1β but remained suppressed with TNF‐α through 24 h. Cyclic AMP was also reduced in TNF‐α‐stimulated primary microglia, albeit to a lesser extent. Accompanying TNF‐α‐induced cyclic AMP reductions, but not IL‐1β, was increased cyclic AMP‐PDE activity. The role of PDE4 activity in cyclic AMP reductions was confirmed by using Rolipram. Examination of pde4 mRNA revealed an immediate, persistent increase in pde4b with TNF‐α; IL‐1β increased all pde4 mRNAs. Immunoblotting for PDE4 showed that both cytokines increased PDE4A1, but only TNF‐α increased PDE4B2. Immunocytochemistry revealed PDE4B nuclear translocation with TNF‐α but not IL‐1β. Acutely after SCI/TBI, where cyclic AMP levels are reduced, PDE4B was localized to activated OX‐42⁺ microglia; PDE4B was absent in OX‐42⁺ cells in uninjured spinal cord/cortex or inactive microglia. Immunoblotting showed PDE4B2 up‐regulation from 24 h to 1 wk post‐SCI, the peak of microglia activation. These studies show that TNF‐α and IL‐1β differentially affect cyclic AMP‐PDE signaling in microglia. Targeting PDE4B2 may be a putative therapeutic direction for reducing microglia activation in CNS injury and neurodegenerative diseases. © 2012 Wiley Periodicals, Inc.     

Biochemical properties of Na+/K+-ATPase in axonal growth cone particles isolated from fetal rat brain

Axonal growth cones (AGC) isolated from fetal rat brain have an important specific activity of N⁺/K⁺-ATPase. Kinetic assays of the enzyme in AGC showed that K_m values for ATP or K⁺ are similar to those reported for the adult brain enzyme. For Na⁺ the affinity (K_m) was lower. V_max for the three substrates was several times lower in AGC as compared to the adult value. We also observed two apparent inhibition constants of Na⁺/K⁺-ATPase by ouabain, one of low affinity, possibly corresponding to the α1 isoform and another of high affinity which is different to that described for the α2 isoform of the enzyme. These results support an important role for the sodium pump in the mantainance of volume and cationic balance in neuronal differentiating structures. The functional differences observed also suggest that the enzymatic complex of Na⁺/K⁺-ATPase in AGC is in a transitional state towards the adult configuration.     

Postnatal development of two nicotinic cholinergic receptors in seven mouse brain regions

The developmental profiles for binding of alpha-[125I]bungarotoxin and L-[3H]nicotine to putative nicotinic cholinergic receptors were determined in seven mouse brain regions. The overall pattern of development of alpha-bungarotoxin binding was similar in all of the regions. Neonatal binding values tended to be greater than those observed in adult brain regions. Maximal binding occurred within 10 days of birth and adult binding values were reached by 20 days of age. The patterns of development of nicotine binding in each of the seven brain regions differed according to region. Gross similarities in developmental profiles for nicotine binding were found among the more caudal and among the more rostral regions. In hindbrain and cerebellum, maximal nicotine binding was found at birth (5 days of age in cerebellum); binding declined approximately 4-fold by 20 days and remained relatively constant thereafter. In midbrain and hypothalamus, a less extensive decrease in nicotine binding occurred from birth to adulthood (midbrain, 25%; hypothalamus, 50%). Nicotine binding in hippocampus and cortex remained unchanged between birth and adulthood. The developmental pattern for nicotine binding in striatum differed from that found in the other brain regions. At 5 days of age, binding was about 65% of adult binding, which was reached at 30 days of age. In most of the brain regions the developmental profile for alpha-bungarotoxin binding was different from that of nicotine. This difference was especially notable in striatum, where adult nicotine binding was higher than neonatal nicotine binding, whereas adult alpha-bungarotoxin binding was lower than neonatal alpha-bungarotoxin binding.     

Diameter and internodal length of axons of spinal interneurones excited by group I afferents in the cat

Examination of various biochemical characteristics of the GABAnergic nervous system in the rat brain was made between 15 days of gestation and adulthood. At birth, the concentration of GABA in whole brain and most regions is approximately 50% of adult levels, whereas the medulla-pons has achieved adult levels by this time. Compared to GABA levels, there is a marked lag in the development of the activity of glutamic acid decar?ylase in all areas studied; however, the activity of the enzyme increases in a linear fashion from birth to adulthood. The development of the uptake of GABA into particulate fractions prepared from whole brain and regions differs markedly from that of GABA and glutamic acid decar?ylase, with uptake near adult levels by birth, peaking considerably above that of the adult between one to two weeks after birth and then declining toward adult activity by 4 weeks after birth. Examination of the kinetics of GABA uptake into resuspended P₂ fractions demonstrates that the developmental changes in the uptake reflects differences in the V_max whereas theK_t remains constant. Studies on the development of the apparent postsynaptic receptor for GABA reveals that in all regions binding is approximately 25% of adult up to 8 days after birth, at which time it increases dramatically, approximating adult levels by 4 weeks after birth. The rise in the density of the apparent postsynaptic GABA receptor after 8 days postpartum correlates best with the increase in the activity of glutamic acid decar?ylase, a presynaptic marker.     

Cerebral hemodynamics measured with simultaneous PET and near-infrared spectroscopy in humans

Evidence that Ca²⁺-activated K⁺ (K_Ca) channels play a role in cell volume changes and K⁺ homeostasis led to a prediction that astrocytes would have K_Ca channels near blood vessels in order to maintain K⁺ homeostasis. Consistent with this thinking the present study demonstrates that rSlo K_Ca channels are in glial cells of the adult rat central nervous system (CNS) and highly localized to specializations of astrocytes associated with the brain vasculature. Using confocal and thin-section electron microscopic immunolabeling methods the distribution of rSlo was examined in adult rat brain. Strong rSlo immunolabeling was present around the vasculature of most brain regions. Examination of dye-filled hippocampal astrocytes revealed rSlo immunolabeling polarized in astrocytic endfeet. Ultrastructural analysis confirmed that the rSlo staining was concentrated in astrocytic endfeet ensheathing capillaries as well as abutting the pia mater. Immunostaining within the endfeet was predominantly distributed at the plasma membrane directly adjacent to either the vascular basal lamina or the pial surface. The distribution of the aquaporin-4 (AQP-4) water channel was also examined using dye-filled hippocampal astrocytes. In confirmation of earlier reports, intense AQP-4 immunolabeling was generally observed at the perimeter of blood vessels, and coincided with perivascular endfeet and rSlo labeling. We propose that rSlo K_Ca channels, with their sensitivity to membrane depolarization and intracellular calcium, play a role in the K⁺ modulation of cerebral blood flow. Additional knowledge of the molecular and cellular machinery present at perivascular endfeet may provide insight into the structural and functional molecular elements responsible for the neuronal activity-dependent regulation of cerebral blood flow.     

Altered responsiveness of adenosine 3′,5′-monophosphate-generating systems in brain slices from adult rats after neonatal treatment with 6-hydroxydopamine

Sprague-Dawley rat pups were administered 6-hydroxydopamine (100 mg/kg) subcutaneously on each of the first 4 days after birth. At 4–5 mo of age the rats and untreated littermates were killed, and the accumulation of [¹⁴C]cyclic AMP elicited by various agents in [¹⁴C]adenine-labeled brain slices was investigated. The accumulation of cyclic AMP elicited by norepinephrine was significantly greater in slices from neocortex and midbrain of 6-hydroxydopamine-treated rats than in corresponding slices from littermate controls. Accumulations in slices from medulla-pons and cerebellum were not significantly altered by the neonatal treatment with 6-hydroxydopamine. Thus, an enhanced response to norepinephrine pertains both in a region, the neocortex, where norepinephrine-levels were permanently reduced by neonatal treatment with 6-hydroxydopamine and in a region, the midbrain, where adult levels of norepinephrine were nearly doubled by the neonatal treatment with 6-hydroxydopamine. Enhanced responses of cyclic AMP-generating systems in neocortical slices from 6-hydroxydopamine-treated rats also pertained with isoproterenol, prostaglandin E₁ and an adenosine-norepinephrine combination, but not with adenosine alone. Depletion of norepinephrine throughout the brain by administration of either reserpine or intraventricular 6-hydroxydopamine to adult rats enhanced responses to norepinephrine in slices from neocortex, but not midbrain. Reserpine had no effect on responses in either neocortex or midbrain of rats treated neonatally with 6-hydroxydopamine.     

The Emerging Role of Phosphodiesterases in Movement Disorders

Cyclic nucleotide phosphodiesterase (PDE) enzymes catalyze the hydrolysis and inactivation of the cyclic nucleotides cyclic adenosine monophosphate and cyclic guanosine monophosphate, which act as intracellular second messengers for many signal transduction pathways in the central nervous system. Several classes of PDE enzymes with specific tissue distributions and cyclic nucleotide selectivity are highly expressed in brain regions involved in cognitive and motor functions, which are known to be implicated in neurodegenerative diseases, such as Parkinson's disease and Huntington's disease. The indication that PDEs are intimately involved in the pathophysiology of different movement disorders further stems from recent discoveries that mutations in genes encoding different PDEs, including PDE2A, PDE8B, and PDE10A, are responsible for rare forms of monogenic parkinsonism and chorea. We here aim to provide a translational overview of the preclinical and clinical data on PDEs, the role of which is emerging in the field of movement disorders, offering a novel venue for a better understanding of their pathophysiology. Modulating cyclic nucleotide signaling, by either acting on their synthesis or on their degradation, represents a promising area for development of novel therapeutic approaches. The study of PDE mutations linked to monogenic movement disorders offers the opportunity of better understanding the role of PDEs in disease pathogenesis, a necessary step to successfully benefit the treatment of both hyperkinetic and hypokinetic movement disorders. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society     

The development of tetrahydrobiopterin and guanosine-5′-triphosphate cyclohydrolase: differential patterns in rat brain and pineal gland

The developmental patterns of appearance of 5,6,7,8-tetrahydrobiopterin (BH₄) and the first enzyme in BH₄ biosynthesis, guanosine-5′-triphosphate cyclohydrolase (GTPcyc), were examined in rat brain and pineal gland. A parallel relationship between BH₄ content and GTPcyc activity was evident in both tissues during development. In brain, the maximal content of BH₄ and activity of GTPcyc was observed 2 days prior to, and 10 days after, birth. In contrast, both pineal BH₄ content and GTPcyc activity became maximal postnatally. The influence of neural input on the developmental appearance of pineal BH₄ was examined in rats that had been superior cervical ganglionectomized shortly after birth. It was found that this procedure did not alter the developmental appearance of BH₄.     

mRNA expression patterns of the cGMP-hydrolyzing phosphodiesterases types 2, 5, and 9 during development of the rat brain

Recent evidence indicates that cGMP plays an important role in neural development and neurotransmission. Since cGMP levels depend critically on the activities of phosphodiesterase (PDE) enzymes, mRNA expression patterns were examined for several key cGMP-hydrolyzing PDEs (type 2 [PDE2], 5 [PDE5], and 9 [PDE9]) in rat brain at defined developmental stages. Riboprobes were used for nonradioactive in situ hybridization on sections derived from embryonic animals at 15 days gestation (E15) and several postnatal stages (P0, P5, P10, P21) until adulthood (3 months). At all stages PDE9 mRNA was present throughout the whole central nervous system, with highest levels observed in cerebellar Purkinje cells, whereas PDE2 and PDE5 mRNA expression was more restricted. Like PDE9, PDE5 mRNA was abundant in cerebellar Purkinje cells, although it was observed only on and after postnatal day 10 in these cells. In other brain regions, PDE5 mRNA expression was minimal, detected in olfactory bulb, cortical layers, and in hippocampus. PDE2 mRNA was distributed more widely, with highest levels in medial habenula, and abundant expression in olfactory bulb, olfactory tubercle, cortex, amygdala, striatum, and hippocampus. Double immunostaining of PDE2, PDE5, or PDE9 mRNAs with the neuronal marker NeuN and the glial cell marker glial fibrillary acidic protein revealed that these mRNAs were predominantly expressed in neuronal cell bodies. Our data indicate that three cGMP-hydrolyzing PDE families have distinct expression patterns, although specific cell types coexpress mRNAs for all three enzymes. Thus, it appears that differential expression of PDE isoforms may provide a mechanism to match cGMP hydrolysis to the functional demands of individual brain regions.     

Increased concentration and accelerated synaptosomal uptake of noradrenaline in the central nervous tissues of neonatally hemispherectomized adult rats

Neonatally hemispherectomized adult rat brain was analyzed. The content of noradrenaline (NA) had increased significantly in the brain stem (+ 116%) and the cerebellum (+ 100%) by the 3rd month after operation. The K_m for the high affinity uptake of NA by the brain stem synaptosomes stayed unchanged but V_max was almost doubled by the operation. Fluorescence histochemical findings also suggest sprouting of noradrenergic axons in specific brain regions.     

Existence of [3H]serotonin binding sites in the rat spinal cord: A developmental study

[³H]5-HT specific binding sites have been characterized in the rat spinal cord. Experimental conditions allowed us to study a single class of sites possibly related to the postsynaptic receptor for 5-HT. Binding constants (K_D and B_max) are described for adult and developing animals. No substantial changes in affinity were observed but the number of receptors increased from birth up to day 20 postnatally, stabilizing thereafter. The developmental pattern of a presynaptic marker, tryptophan-5-hydroxylase, was similar to that of binding sites.     

Aquaporin-4 expression contributes to decreases in brain water content during mouse postnatal development

The water channel protein aquaporin-4 (AQP4) is implicated to facilitate water efflux from the brain parenchyma into the blood and CSF, playing a critical role in maintaining brain water homeostasis. Nevertheless, its contribution to decreases in brain water content during postnatal development remains unknown. A quantitative Western blot analysis was performed to investigate developmental expression of AQP4 in the whole mouse brain and showed that AQP4 expression level in 1 week-old brain was only 21.3% of that in the adult brain, but significantly increased to 67.4% of the adult level by 2 weeks after birth. Statistical analysis demonstrated that increased AQP4 expression partially relates to decreased brain water content in postnatal mice (r² = 0.92 and P = 0.002). Moreover, AQP4 null mice had greater brain water content than littermate controls from 2 weeks up to adult age. Consistently, mature pattern of AQP4 localization at the brain–blood and brain–CSF interfaces were completed at approximately at 2 weeks after birth. In addition, AQP4 expression in the brain stem and hypothalamus was earlier than that in the cerebral cortex and cerebellum, suggesting a brain regional variation in developmental expression of AQP4. These results characterize the developmental feature of AQP4 expression in the postnatal brain and provide direct evidence for a role of AQP4 in postnatal brain water uptake.     

Effects of Dibutyryl Cyclic AMP on Na+, K+-ATPase Activity and Intracellular Na+ and K+ in Primary Cultures of Astrocytes from DBA and C57 Mice

Summary: Effects of chronic treatment of dibutyryl cyclic AMP (db-cyclic AMP) on Na⁺, K⁺-ATPase activity in cell homogenates and intracellular N a f and K+ contents [(Na⁺)_i and (K⁺)_i] were studied in primary cultures of astrocytes derived from cerebral cortex of neonatal audiogenic seizure-susceptible DBA and audiogenic seizure-resistant C57 mice. Na⁺, K⁺-ATPase activity in cell homogenates was greater and (Na⁺)_i was less in DBA astrocytes than in C57 astrocytes. There was no difference in (K⁺)_i between astrocytes from DBA and C57 mice. Addition of db-cyclic AMP to the medium from day 14 to day 21 in culture (final concentration 0.25 mM) increased Na⁺, K⁺-ATPase activity in cell homogenates and decreased (Na⁺)_i, but had no significant effect on (K⁺)_i in astrocytes from either DBA or C57 mice. Chronic treatment with db-cyclic AMP altered cell growth. Protein and DNA content of cultured astrocytes from both DBA and C57 mice was decreased. DNA was more affected than protein. Modifying K⁺ and Na⁺ concentration in medium altered Na⁺, K⁺-ATPase activity in cell homogenates as well as (Na⁺)_i and (K⁺)_i in cultured astrocytes of both DBA and C57 mice. Changes in (Na⁺)_i and (K⁺)_i at different K⁺ concentrations in medium paralleled those in Na⁺, K⁺-ATPase activity in cell homogenates. Results indicate that the ability to transport Na⁺ across the cell membrane and the response of Na⁺, K⁺-ATPase to db-cyclic AMP and to the changes in K + in medium of cultured astrocytes from audiogenic seizure-susceptible DBA mice are sufficient.     

Ca activated and pH sensitive cyclic AMP phosphodiesterase in the nervous system of the molluscPleurobranchaea

Regulation of cyclic AMP through its synthesis is known to be important in modulating the activity of molluscan neurons; however, no data exists regarding the regulation of cyclic AMP degradation.We find that cyclic AMP phosphodiesterase (PDE) activity in homogenates of the nervous system of the molluscPleurobranchea is significantly stimulated by calcium ion. Ca²⁺ stimulation is suppressed by the calmodulin antagonist trifluoperazine (TFP), indicating resemblance to the Ca²⁺-calmodulin PDEs of mammalian neurons. Ca²⁺ also accentuates the pH sensitivity of PDE. The qualities of Ca²⁺ and pH sensitivity of PDE are fitted into a model for cAMP regulation of neuronal activity in an identified feeding command neuron; the postulated role of PDE is consistent with effects of cAMP, TFP, and pH on the neuron's activity.     

Biochemical characteristics of γ-glutamyl transpeptidase in capillaries from entorhinohippocampal complex of quinolinate-lesioned rat brain

Quinolinic acid (QUIN) is an endogenous excitotoxic agonist of theN-methyl-d-aspartate (NMDA) type of glutamate receptor, which causes slowly progressing degeneration of vulnerable neurons in some brain regions. Using changes in the activity of membrane-bound γ-glutamyl transpeptidase (GGT) as a marker of cell damage, we found a significant decrease of this enzyme activity, which was preferentially located in the ipsilateral hippocampal formation and entorhinal cortex, 4 d after the unilateral intracerebroventricular (icv) injection of 0.5 μmol QUIN. The dose of QUIN divided into two half-doses injected bilaterally led to a symmetrical decline of GGT activity in hippocampal areas. The lesion was characterized by a suppression of GGT activity in hippocampal and entorhinal capillaries, corresponding to 60 and 81% of their initial value, respectively, but no significant changes were ascertained in synaptosomal membranes. The changes in the activity of capillary GGT were associated with the decrease of apparent maximal velocityV _max ^app, whereas apparent Michaelis constantK _m ^app (0.69–0.79 mM) remained unaffected. In the nonlesioned brain, concanavalin A (Con A) affinity chromatography revealed five glycoforms of synaptosomal GGT in contrast to only one found in hippocampal and entorhinal capillaries. The results document that neither the saccharide moiety of GGT nor the value of enzymeK _m ^app is significantly affected by the QUIN-induced lesion of the rat brain. However, the suppression of GGT activity, which is accompanied by a decrease in the value ofV _max ^app in brain microvessels, may suggest dysfunction of the blood-brain barrier (BBB) in the QUIN-injuried rat brain.     

Kinetic similarity of enzymes in human blood serum and cerebrospinal fluid: aspartate aminotransferase and lactate dehydrogenase

Michaelis constants (K_m) for l-aspartate and 2-oxoglutarate were calculated for aspartate aminotransferase of normal human blood serum and cerebrospinal fluid (CSF), and human brain supernatant and mitochondria. K_m for pyruvate of lactate dehydrogenase from the same sources was also measured at a saturating concentration of NADH. The kinetic constants for enzymes from serum and CSF were similar, and differed significantly from those of brain enzymes, suggesting that CSF enzymes are derived from the blood stream by passage across the blood-CSF barrier.     

N-phenylisopropyladenosine stimulates in normal and inhibits in adrenalectomized rats the low KM cyclic AMP phosphodiesterase in the brain

In crude rat brain membranes, apparent adenylate cyclase activity tested in the absence of phosphodiesterase inhibitors was dose-dependently inhibited by the adenosine ‘R-site’ agonist N⁶-phenylisopropyladenosine (N⁶-PIA). In membranes from adrenalectomized rats, however, N⁶-PIA induced, under the same conditions, an activation. However, in the presence of the phosphodiesterase inhibitor Ro-20-1724, brain adenylate cyclase responsiveness to N⁶-PIA resulted in a dose-dependent inhibition in both sham-operated and adrenalectomized rats. Thus, the low K_M cyclic AMP phosphodiesterase activity present in these brain membranes was investigated. Although this activity (tested in the presence of GTP) was unaltered by adrenalectomy, the dose-response curve of this enzyme to low concentrations of N⁶-PIA showed an activation in sham-operated and an inhibition in adrenalectomized rats, two effects which were suppressed by sodium (80 mM). These results showing that N⁶-PIA modulates both adenylate cyclase and cyclic AMP phosphodiesterase in the brain, provide an additional argument for a potential role of adenosine in the regulation of cyclic AMP metabolism in normal as well as in pathological brain.