Substance P responsiveness of smooth muscle cells is regulated by the integrin ligand, thrombospondin.

The extracellular factors that determine a cell's responsiveness to neurotransmitters are of particular relevance for pharmacologically diverse cell types such as neurons and smooth muscle. We previously demonstrated that matrix-associated factors are capable of dramatically and specifically suppressing the responsiveness of smooth muscle to the neuropeptide, substance P. We now demonstrate that this influence of extracellular matrix on the pharmacological phenotype of smooth muscle cells can be blocked specifically by an Arg-Gly-Asp (RGD)-containing antagonist of integrins. Of a battery of integrin ligands tested, only thrombospondin mimicked the effect of the extracellular matrix on substance P responsiveness. This effect of thrombospondin was dose dependent, RGD sensitive, and blocked by an antibody directed against the RGD-containing region of thrombospondin. Because the mRNA for thrombospondin is present in the cells of the chicken amnion, this extracellular factor may normally suppress substance P responsiveness in amniotic smooth muscle. The results suggest a role for matrix-associated integrin ligands in the regulation of cellular responses to specific neurotransmitters and hormones and in the development and maintenance of tissue-specific pharmacological properties.

Protein kinase A-independent modulation of ion channels in the brain by cyclic AMP.

Ion channels underlying the electrical activity of neurons can be regulated by neurotransmitters via two basic mechanisms: ligand binding and covalent modification. Whereas neurotransmitters often act by binding directly to ion channels, the intracellular messenger cyclic AMP is thought usually to act indirectly, by activating protein kinase A, which in turn can phosphorylate channel proteins. Here we show that cyclic AMP, and transmitters acting via cyclic AMP, can act in a protein kinase A-independent manner in the brain. In hippocampal pyramidal cells, cyclic AMP and norepinephrine were found to cause a depolarization by enhancing the hyperpolarization-activated mixed cation current, IQ (also called Ih). This effect persisted even after protein kinase A activity was blocked, thus strongly suggesting a kinase-independent action of cyclic AMP. The modulation of this current by ascending monoaminergic fibers from the brainstem is likely to be a widespread mechanism, participating in the state control of the brain during arousal and attention.

Aromatic-L-amino-acid decarboxylase, a pyridoxal phosphate-dependent enzyme, is a beta-cell autoantigen.

Different autoantigens are thought to be involved in the pathogenesis of insulin-dependent diabetes mellitus, and they may account for the variation in the clinical presentation of the disease. Sera from patients with autoimmune polyendocrine syndrome type I contain autoantibodies against the beta-cell proteins glutamate decarboxylase and an unrelated 51-kDa antigen. By screening of an expression library derived from rat insulinoma cells, we have identified the 51-kDa protein as aromatic-L-amino-acid decarboxylase (EC 4.1.1.28). In addition to the previously published full-length cDNA, forms coding for a truncated and an alternatively spliced version were identified. Aromatic L-amino acid decarboxylase catalyzes the decarboxylation of L-5-hydroxytryptophan to serotonin and that of L-3,4-dihydroxyphenylalanine to dopamine. Interestingly, pyridoxal phosphate is the cofactor of both aromatic L-amino acid decarboxylase and glutamate decarboxylase. The biological significance of the neurotransmitters produced by the two enzymes in the beta cells remains largely unknown.

The vesicular monoamine transporter 2 is present in small synaptic vesicles and preferentially localizes to large dense core vesicles in rat solitary tract nuclei.

In central neurons, monamine neurotransmitters are taken up and stored within two distinct classes of regulated secretory vesicles: small synaptic vesicles and large dense core vesicles (DCVs). Biochemical and pharmacological evidence has shown that this uptake is mediated by specific vesicular monamine transporters (VMATs). Recent molecular cloning techniques have identified the vesicular monoamine transporter (VMAT2) that is expressed in brain. This transporter determines the sites of intracellular storage of monoamines and has been implicated in both the modulation of normal monoaminergic neurotransmission and the pathogenesis of related neuropsychiatric disease. We used an antiserum against VMAT2 to examine its ultrastructural distribution in rat solitary tract nuclei, a region that contains a dense and heterogeneous population of monoaminergic neurons. We find that both immunoperoxidase and immunogold labeling for VMAT2 localize to DCVs and small synaptic vesicles in axon terminals, the trans-Golgi network of neuronal perikarya, tubulovesicles of smooth endoplasmic reticulum, and potential sites of vesicular membrane recycling. In axon terminals, immunogold labeling for VMAT2 was preferentially associated with DCVs at sites distant from typical synaptic junctions. The results provide direct evidence that a single VMAT is expressed in two morphologically distinct types of regulated secretory vesicles in central monoaminergic neurons.

Neurosecretory vesicles can be hybrids of synaptic vesicles and secretory granules.

We have investigated the relationship of the so-called small dense core vesicle (SDCV), the major catecholamine-containing neurosecretory vesicle of sympathetic neurons, to synaptic vesicles containing classic neurotransmitters and secretory granules containing neuropeptides. SDCVs contain membrane proteins characteristic of synaptic vesicles such as synaptophysin and synaptoporin. However, SDCVs also contain membrane proteins characteristic of certain secretory granules like the vesicular monoamine transporter and the membrane-bound form of dopamine beta-hydroxylase. In neurites of sympathetic neurons, synaptophysin and dopamine beta-hydroxylase are found in distinct vesicles, consistent with their transport from the trans-Golgi network to the site of SDCV formation in constitutive secretory vesicles and secretory granules, respectively. Hence, SDCVs constitute a distinct type of neurosecretory vesicle that is a hybrid of the synaptic vesicle and the secretory granule membranes and that originates from the contribution of both the constitutive and the regulated pathway of protein secretion.

Regulatory region in choline acetyltransferase gene directs developmental and tissue-specific expression in transgenic mice.

Acetylcholine, one of the main neurotransmitters in the nervous system, is synthesized by the enzyme choline acetyltransferase (ChAT; acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6). The molecular mechanisms controlling the establishment, maintenance, and plasticity of the cholinergic phenotype in vivo are largely unknown. A previous report showed that a 3800-bp, but not a 1450-bp, 5' flanking segment from the rat ChAT gene promoter directed cell type-specific expression of a reporter gene in cholinergic cells in vitro. Now we have characterized a distal regulatory region of the ChAT gene that confers cholinergic specificity on a heterologous downstream promoter in a cholinergic cell line and in transgenic mice. A 2342-bp segment from the 5' flanking region of the ChAT gene behaved as an enhancer in cholinergic cells but as a repressor in noncholinergic cells in an orientation-independent manner. Combined with a heterologous basal promoter, this fragment targeted transgene expression to several cholinergic regions of the central nervous system of transgenic mice, including basal forebrain, cortex, pons, and spinal cord. In eight independent transgenic lines, the pattern of transgene expression paralleled qualitatively and quantitatively that displayed by endogenous ChAT mRNA in various regions of the rat central nervous system. In the lumbar enlargement of the spinal cord, 85-90% of the transgene expression was targeted to the ventral part of the cord, where cholinergic alpha-motor neurons are located. Transgene expression in the spinal cord was developmentally regulated and responded to nerve injury in a similar way as the endogenous ChAT gene, indicating that the 2342-bp regulatory sequence contains elements controlling the plasticity of the cholinergic phenotype in developing and injured neurons.

Avaliação da inervação entérica e análise proteômica do intestino delgado de ratos expostos à dose aguda ou crônica de fluoreto

O trato gastrointestinal (TGI) é a principal rota de exposição ao fluoreto (F) e o seu mais importante sítio de absorção. Acredita-se que a toxicidade do F comprometa a fisiologia do intestino, devido à relevante sintomatologia gastrointestinal relatada em consequência da exposição excessiva ao F. A função intestinal é controlada por uma complexa rede neuronal interligada e incorporada à parede deste órgão, denominada Sistema Nervoso Entérico (SNE). Embora os efeitos tóxicos do F sobre o Sistema Nervoso Central sejam descritos na literatura, não há estudos relacionados à sua toxicidade sobre o SNE. Neste estudo realizado em ratos, foi avaliado o efeito da exposição aguda ou crônica ao F, sobre a população geral de neurônios entéricos e sobre as subpopulações que expressam os principais neurotransmissores entéricos: Acetilcolina (ACh), Óxido Nítrico (NO), Peptídeo Vasoativo Intestinal (VIP), Peptídeo Relacionado ao Gene da Calcitonina (CGRP) e Substância P (SP). Os animais foram divididos em 5 grupos: 3 destinados à exposição crônica (0 ppm, 10 ppm ou 50 ppm de F na água de beber) e 2 à exposição aguda (0 ou 25 mgF/Kg por gavagem gástrica). Foram coletados os 3 segmentos do intestino delgado (duodeno, jejuno e íleo) e processados para a detecção da HuC/D, ChAT, nNOS, VIP, CGRP e SP, através de técnicas de imunofluorescência, no plexo mioentérico. Foram obtidas imagens para a realização da análise quantitativa dos neurônios da população geral (HuC/D) e nitrérgicos (imunorreativos à nNOS); e morfométrica dos neurônios imunorreativos à HuC/D ou nNOS; e das varicosidades imunorreativas à ChAT, VIP, CGRP ou SP. Amostras dos 3 segmentos intestinais foram preparadas e coradas em Hematoxilina e Eosina para análise histológica da morfologia básica. O segmento intestinal considerado mais afetado na análise morfométrica da população geral de neurônios, o duodeno, foi selecionado para a realização da análise proteômica, com o objetivo de oferecer o seu perfil proteico e determinar diferenças na expressão proteica em decorrência da exposição crônica ou aguda ao F. A análise da concentração de F no plasma sanguíneo foi realizada para a confirmação da exposição. Na análise quantitativa, o grupo de 50 ppm F, apresentou uma diminuição significativa na densidade da população geral de neurônios do jejuno e do íleo e na densidade dos neurônios imunorreativos à nNOS no duodeno e no jejuno. Quanto à análise morfométrica, a população geral e as subpopulações neuronais entéricas avaliadas apresentaram alterações morfológicas significativas, tanto após a exposição crônica quanto a aguda. Para a análise proteômica do duodeno, verificou-se que da associação de seus genes a um termo, e assim classificadas de acordo com diferentes processos biológicos. No caso do grupo da dose aguda, o processo biológico com a maior porcentagem de genes associados foi a geração de metabólitos precursores e energia (27% das proteínas); enquanto para os grupos de 10 e 50 ppm F foram o processo metabólico da piridina (41%) e a polimerização proteica (33%), respectivamente.

Countermanding in rats as a practical model for investigation of adaptive control of behaviour, lifespan changes in behavioural control and neurotransmitter function

The countermanding paradigm was designed to investigate the ability to cancel a prepotent response when a stop signal is presented and allows estimation of the stop signal response time (SSRT), an otherwise unobservable behaviour. Humans exhibit adaptive control of behaviour in the countermanding task, proactively lengthening response time (RT) in expectation of stopping and reactively lengthening RT following stop trials or errors. Human performance changes throughout the lifespan, with longer RT, SSRT and greater emphasis on post-error slowing reported for older compared to younger adults. Inhibition in the task has generally been improved by drugs that increase extracellular norepinephrine. The current thesis examined a novel choice response countermanding task in rats to explore whether rodent countermanding performance is a suitable model for the study of adaptive control of behaviour, lifespan changes in behavioural control and the role of neurotransmitters in these behaviours. Rats reactively adjusted RT in the countermanding task, shortening RT after consecutive correct go trials and lengthening RT following non-cancelled, but not cancelled stop trials, in sessions with a 10 s, but not a 1 s post-error timeout interval. Rats proactively lengthened RT in countermanding task sessions compared to go trial-only sessions. Together, these findings suggest that rats strategically lengthened RT in the countermanding task to improve accuracy and avoid longer, unrewarded timeout intervals. Next, rats exhibited longer RT and relatively conserved post-error slowing, but no significant change in SSRT when tested at 12, compared to 7 months of age, suggesting that rats exhibit changes in countermanding task performance with aging similar to those observed in humans. Finally, acute administration of yohimbine (1.25, 2.5 mg/kg) and d-amphetamine (0.25, 0.5 mg/kg), which putatively increase extracellular norepinephrine and dopamine respectively, resulted in RT shortening, baseline-dependent effects on SSRT, and attenuated adaptive RT adjustments in rats in the case of d-amphetamine. These findings suggest that dopamine and norepinephrine encouraged motivated, reward-seeking behaviour and supported inhibitory control in an inverted-U-like fashion. Taken together, these observations validate the rat countermanding task for further study of the neural correlates and neurotransmitters mediating adaptive control of behaviour and lifespan changes in behavioural control.

Inference of binding affinity from neuronal receptors in humans

Tese de mestrado, Bioinformática e Biologia Computacional (Bioinformática), Universidade de Lisboa, Faculdade de Ciências, 2016

Differential involvement of N-type calcium channels in transmitter release from vasoconstrictor and vasodilator neurons

1 The effects of calcium channel blockers on co-transmission from different populations of autonomic vasomotor neurons were studied on isolated segments of uterine artery and vena cava from guinea-pigs. 2 Sympathetic, noradrenergic contractions of the uterine artery (produced by 200 pulses at 1 or 10 Hz; 600 pulses at 20 Hz) were abolished by the N-type calcium channel blocker omega-conotoxin (CTX) GVIA at 1-10 nM. 3 Biphasic sympathetic contractions of the vena cava (600 pulses at 20 Hz) mediated by noradrenaline and neuropeptide Y were abolished by 10 nM CTX GVIA. 4 Neurogenic relaxations of the uterine artery (200 pulses at 10 Hz) mediated by neuronal nitric oxide and neuropeptides were reduced < 50% by CTX GVIA 10-100 nM. 5 Capsaicin (3 muM) did not affect the CTX GVIA-sensitive or CTX GVIA-resistant neurogenic relaxations of the uterine artery. 6 The novel N-type blocker CTX CVID (100-300 nM), P/Q-type blockers agatoxin IVA (10-100 nM) or CTX CVIB (100 nM), the L-type blocker nifedipine (10 muM) or the 'R-type' blocker SNX-482 (100 nM), all failed to reduce CTX GVIA-resistant relaxations. The T-type channel blocker NiCl2 (100-300 muM) reduced but did not abolish the remaining neurogenic dilations. 7 Release of different neurotransmitters from the same autonomic vasomotor axon depends on similar subtypes of calcium channels. N-type channels are responsible for transmitter release from vasoconstrictor neurons innervating a muscular artery and capacitance vein, but only partly mediate release of nitric oxide and neuropeptides from pelvic vasodilator neurons.

Adenosine triphosphate acts as both a competitive antagonist and a positive allosteric modulator at recombinant N-methyl-D-aspartate receptors

ATP and glutamate are fast excitatory neurotransmitters in the central nervous system acting primarily on ionotropic P2X and glutamate [N-methyl-D-aspartate (NMDA) and non-NMDA] receptors, respectively. Both neurotransmitters regulate synaptic plasticity and long-term potentiation in hippocampal neurons. NMDA receptors are responsible primarily for the modulatory action of glutamate, but the mechanism underlying the modulatory effect of ATP remains uncertain. In the present study, the effect of ATP on recombinant NR1a + 2A, NR1a + 2B, and NR1a + 2C NMDA receptors expressed in Xenopus laevis oocytes was investigated. ATP inhibited NR1a + 2A and NR1a + 2B receptor currents evoked by low concentrations of glutamate but potentiated currents evoked by saturating glutamate concentrations. In contrast, ATP potentiated NR1a + 2C receptor currents evoked by nonsaturating glutamate concentrations. ATP shifted the glutamate concentration-response curve to the right, indicating a competitive interaction at the agonist binding site. ATP inhibition and potentiation of glutamate-evoked currents was voltage-independent, indicating that ATP acts outside the membrane electric field. Other nucleotides, including ADP, GTP, CTP, and UTP, inhibited glutamate-evoked currents with different potencies, revealing that the inhibition is dependent on both the phosphate chain and nucleotide ring structure. At high concentrations, glutamate outcompetes ATP at the agonist binding site, revealing a potentiation of the current. This effect must be caused by ATP binding at a separate site, where it acts as a positive allosteric modulator of channel gating. A simple model of the NMDA receptor, with ATP acting both as a competitive antagonist at the glutamate binding site and as a positive allosteric modulator at a separate site, reproduced the main features of the data.

Differential involvement of rat medial prefrontal cortex dopamine receptors in modulation of hypothalamic-pituitary-adrenal axis responses to different stressors

Recent investigations have implicated the medial prefrontal cortex (mPFC) in modulation of subcortical pathways that contribute to the generation of behavioural, autonomic and endocrine responses to stress. However, little is known of the mechanisms involved. One of the key neurotransmitters involved in mPFC function is dopamine, and we therefore aimed, in this investigation, to examine the role of mPFC dopamine in response to stress in Wistar rats. In this regard, we infused dopamine antagonists SCH23390 or sulpiride into the mPFC via retrodialysis. We then examined changes in numbers of cells expressing the c-fos immediate-early gene protein product, Fos, in subcortical neuronal populations associated with regulation of hypothalamic-pituitary-adrenal (HPA) axis stress responses in response to either of two stressors; systemic injection of interleukin-1beta, or air puff. The D-1 antagonist, SCH23390, and the D-2 antagonist, sulpiride, both attenuated expression of Fos in the medial parvocellular hypothalamic paraventricular nucleus (mpPVN) corticotropin-releasing factor cells at the apex of the HPA axis, as well as in most extra-hypothalamic brain regions examined in response to interleukin-1beta. By contrast, SCH23390 failed to affect Fos expression in response to air puff in any brain region examined, while sulpiride resulted in an attenuation of the air puff-induced response in only the mpPVN and the bed nucleus of the stria terminalis. These results indicate that the mPFC differentially processes the response to different stressors and that the two types of dopamine receptor may have different roles.

Immunocytochemistry and metamorphic fate of the larval nervous system of Triphyllozoon mucronatum (Ectoprocta : Gymnolaemata : Cheilostomata)

The development of gymnolaemate Ectoprocta includes a larval stage of either the coronate or the cyphonautes type. Herein, we provide the first description of the larval neural anatomy of a coronate larva using immunocytochemical methods. We used antibodies against the neurotransmitters serotonin and FMRFamide and followed the fate of immunoreactive cells through metamorphosis. The larval serotonergic nervous system of Triphyllozoon mucronatum consists of an apical commissure, one pair of lateral axons, a coronate nerve net, an internal nerve mesh, and one pair of axons innervating the frontal organ. FMRFamide is only found in the larval commissure and in the lateral axons. The entire serotonergic and FMRFamidergic nervous system is lost during metamorphosis and the adult neural structures form independent of the larval ones. In the postlarval zooid, both neurotransmitters are detected in the cerebral commissure, in cell bodies located at the base of the lophophore, and in neurites connecting these somata to the cerebral commissure. These findings differ significantly from that observed in other lophotrochozoans, where certain larval neural features are either incorporated in the adult nervous system and/or have inductive functions during its ontogeny. The occurrence of a larval commissure and the lack of a serotonergic or FMRFamidergic apical organ in T. mucronatum are unique among lophotrochozoan larvae, which usually have a distinct apical organ containing serotonergic cells. Our data show that the larval neuroanatomy and the processes that underlie the reorganization of larval organ systems during metamorphosis may vary much more among lophotrochozoan taxa than previously thought.

Cholinergic and adrenergic innervation of lingual salt glands of the estuarine crocodile, Crocodylus porosus

Many marine reptiles and birds possess extrarenal salt glands that facilitate the excretion of excess sodium and chloride ions accumulated as a consequence of living in saline environments. Control of the secretory activity of avian salt glands is under neural control, but little information is available on the control of reptilian salt glands. Innervation of the lingual salt glands of the salt water crocodile, Crocodylus porosus, was examined in salt water-acclimated animals using histological methods. Extensive networks of both cholinergic and adrenergic nerve fibres were identified close to salt-secreting lobules and vasculature. The identification of both catecholamine-containing and cholinergic neurons in the salt gland epithelium and close to major blood vessels in the tissue suggests the action of the neurotransmitters on the salt-secreting epithelium itself and the rich vascular network of the lingual salt glands.