Beth J. Hoffman

Localization and Dynamic Regulation of Biogenic Amine Transporters in the Mammalian Central Nervous System

The monoamines, serotonin, dopamine, norepinephrine, epinephrine and histamine, play a critical role in the function of the hypothalamic-pituitary-adrenal axis and in the integration of information in sensory, limbic, and motor systems. The primary mechanism for termination of monoaminergic neurotransmission is through reuptake of released neurotransmitter by Na+, CI-dependent plasma membrane transporters. A second family of transporters packages monoamines into synaptic and secretory vesicles by exchange of protons. Identification of those cells which express these two families of neurotransmitter transporters is an initial step in understanding what adaptive strategies cells expressing monoamine transporters use to establish the appropriate level of transport activity and thus attain the appropriate efficiency of monoamine storage and clearance. The most recent advances in this field have yielded several surprises about their function, cellular and subcellular localization, and regulation, suggesting that these molecules are not static and most likely are the most important determinants of extracellular levels of monoamines. Here, information on the localization of mRNAs for these transporters in rodent and human brain is summarized along with immunohistochemical information at the light and electron microscopic levels. Regulation of transporters at the mRNA level by manipulation in rodents and differences in transporter site densities by tomographic techniques as an index of regulation in human disease and addictive states are also reviewed. These studies have highlighted the presence of monoamine neurotransmitter transporters in neurons but not in glia in situ. The norepinephrine transporter is present in all cells which are both tyrosine hydroxylase (TH)- and dopamine beta-hydroxylase-positive but not in those cells which are TH- and phenyl-N-methyltransferase-positive, suggesting that epinephrine cells may have their own, unique transporter. In most dopaminergic cells, dopamine transporter mRNA completely overlaps with TH mRNA-positive neurons. However, there are areas in which there is a lack of one to one correspondence. The serotonin transporter (5-HTT) mRNA is found in all raphe nuclei and in the hypothalamic dorsomedial nucleus where the 5-HTT mRNA is dramatically reduced following immobilization stress. The vesicular monoamine transporter 2 (VMAT2) is present in all monoaminergic neurons including epinephrine- and histamine-synthesizing cells. Immunohistochemistry demonstrates that the plasma membrane transporters are present along axons, soma, and dendrites. Subcellular localization of DAT by electron microscopy suggests that these transporters are not at the synaptic density but are confined to perisynaptic areas, implying that dopamine diffuses away from the synapse and that contribution of diffusion to dopamine signalling may vary between brain regions. Interestingly, the presence of VMAT2 in vesicles underlying dendrites, axons, and soma suggests that monoamines may be released at these cellular domains. An understanding of the regulation of transporter function may have important therapeutic consequences for neuroendocrine function in stress and psychiatric disorders.

Substance P receptor expression in intestinal epithelium in Clostridium difficile toxin A enteritis in rats

We previously reported that the inflammatory effects of Clostridium difficile toxin A on rat intestine can be significantly inhibited with a specific neurokinin-1 receptor (NK-1R) antagonist. In this study we investigated the localization and expression of NK-1R mRNA and protein in rat intestine by in situ hybridization, Northern blot analysis, and immunohistochemistry, respectively, after exposure to toxin A. Northern blot analysis showed increased mucosal levels of NK-1R mRNA starting 30 min after toxin A administration. In situ hybridization showed that toxin A increased NK-1R mRNA expression in intestinal epithelial cells after 30, 120, and 180 min. In rats pretreated with the NK-1R antagonist CP-96345 the increase in NK-1R mRNA levels after exposure to toxin A was inhibited, indicating that NK-1R upregulation is substance P (SP) dependent. One hour after exposure to toxin A many of the intestinal epithelial cells showed staining for NK-1R compared with controls. Specific 125I-SP binding to purified epithelial cell membranes obtained from ileum exposed to toxin A for 15 min was increased twofold over control and persisted for 4 h. This report provides evidence that NK-1R expression is increased in the intestinal epithelium shortly after exposure to toxin A and may be important in toxin A-induced inflammation.

Serotonin 5-HT2C Receptor Stimulates Cyclic GMP Formation in Choroid Plexus†

Abstract: The serotonin 5‐HT2C receptor (formerly designated the 5‐HT1C receptor) of the choroid plexus triggers phosphoinositide turnover. In the present study, we demonstrate that receptor activation also triggers the formation of cyclic GMP (cGMP). Application of 1 µM 5‐HT to porcine choroid plexus tissue slices resulted in stimulation of cGMP formation to a maximum of five‐fold basal level, with an EC50 of 11 nM. This response was not inhibited by muscarinic or β‐adrenergic receptor antagonists. Serotonin receptor antagonists inhibited cGMP formation with apparent Ki values of 1.3 (mianserin), 200 (ketanserin), and 5,500 (spiperone) nM, respectively. Neither serotonin‐stimulated cGMP formation nor PI turnover was inhibited by pertussis toxin pretreatment. Preliminary biochemical studies suggested that serotonin‐stimulated cGMP formation was calcium, phospholipase A2, and lipoxygenase dependent, as incubation in low calcium buffers or inclusion of the phospholipase A2 or lipoxygenase inhibitors p‐bromophenacyl bromide or BW 755c resulted in significant reduction of cGMP formation. The present results suggest that in addition to triggering phosphoinositide turnover, choroid plexus serotonin 5‐HT2C receptors trigger cGMP formation in a calcium‐sensitive manner.

ONTOGENY OF VESICULAR MONOAMINE TRANSPORTER MRNAS VMAT1 AND VMAT2. I. THE DEVELOPING RAT CENTRAL NERVOUS SYSTEM

We used in situ hybridization histochemistry to study the expression of the mRNA of the two vesicular monoamine transporters (VMAT1 and VMAT2) during embryonic and postnatal development of the central nervous system (CNS) in the rat. In the adult rat, VMAT2 mRNA is present exclusively in monoaminergic cell groups of the CNS and VMAT1 mRNA was reported to be present in the adrenal medulla and certain intestinal epithelial cells. In contrast to the above, the expression of VMAT1 mRNA has previously never been detected in the central nervous system. This study shows the first evidence that both transporter molecules are expressed in CNS during ontogenesis. We here demonstrate four main expression patterns detected during development: 1. VMAT2 mRNA expression in monoaminergic neurons of the brainstem beginning as early as embryonic day E13. 2. Expression of VMAT2 mRNA in all major sensory relay nuclei of central nervous system. 3. Co-expression of VMAT1 and VMAT2 mRNA in most limbic structures, basal ganglia, as well as in some hypothalamic nuclei. 4. Exclusive expression of VMAT1 mRNA in the neocortical subventricular zone, in the amygdala at early (E15-18) and late (P1-P28) timepoints, the granular cell layer of cerebellum, and in several brainstem motor nuclei. Based on their distribution during development we suggest that monoamines, released in a controlled fashion, might affect wiring of sensory and also motor circuits. VMAT1 mRNA expression may reflect a specific effect of monoamines in glial differentiation and cerebellar granule cell migration and/or differentiation.

Dopamine Produced by the Stomach May Act as a Paracrine/ Autocrine Hormone in the Rat

Dopamine (DA) has been suggested to be a protective factor in the gastrointestinal tract but neither a source of DA nor its exact targets of action have been identified. In this study, we demonstrate high levels of DA (and DOPA) which persist after chemical sympathectomy in the gastric juice of rats. Immunostaining and in situ hybridization histochemistry reveal the presence of tyrosine hydroxylase (TH), DA transporter and vesicular monamine transporters in the acid-producing parietal cells. Like DA, TH enzyme activity remains after chemical sympathectomy. We also demonstrate active reuptake and storage of DA that indicates a regulated release of this neurohormone from parietal cells. DA D1b receptor mRNA is the most abundant DA receptor subtype in gastric and duodenal epithelium. Therefore, we suggest that selective DA D1b receptor agonists may be useful adjuncts in the treatment of duodenal and gastric ulcers. Gastric epithelia possess the hallmarks of functional DA neuroendocrine cells, suggesting that DA has an important role in self-protective mechanisms of the gastrointestinal tract. These findings should allow elucidation of DA role in normal and disease states in the stomach and duodenum.

ONTOGENY OF VESICULAR MONOAMINE TRANSPORTER MRNAS VMAT1 AND VMAT2. II. EXPRESSION IN NEURAL CREST DERIVATIVES AND THEIR TARGET SITES IN THE RAT

We used in situ hybridization histochemistry to study the expression of the two vesicular monoamine transporters (VMAT1 and VMAT2) during embryonic development in the rat. In the adult rat VMAT2 is present exclusively in neuronal tissues and VMAT1 is present in the adrenal medulla and in certain intestinal endocrine cells. We found that both transporter molecules are more widely expressed during development. We demonstrate a complete overlap of the two VMAT mRNAs in the sympathetic nervous system between E13 and E21 days. In addition, VMAT2 (and to some extent VMAT1) mRNA is expressed in ganglionic cells of the parasympathetic nervous system and in cranial ganglia (trigeminal, vestibular and spiral ganglia) between E12 and E21. The sensory neurons of the dorsal root ganglia, which are also neural crest derivatives, express VMAT2 mRNA (E11-E21), exclusively. Both VMAT mRNAs are found in the developing GI system, but in different cells. VMAT1 mRNA was detected in organs of the endocrine system (pituitary gland, adrenal gland, testis, seminal vesicle), some connective tissue cells, and the thymus. We observed expression of both VMAT mRNAs in two separate cell groups in the placenta (E8-E10). Based on their distribution during development we suggest that monoamines, released in a controlled fashion, might affect migration and differentiation of neural crest derivatives.

Alternative non‐coding exons support serotonin transporter mRNA expression in the brain and gut

A number of studies in recent years have linked polymorphisms within the serotonin transporter (5HTT) gene to affective disorders and anxiety traits. The human 5HTT mRNA is alternatively spliced, and the splice variants are equally expressed in the human placental cell line and dorsal raphe. In this study, using 5′ rapid amplification of cDNA ends, we show that the rat 5HTT mRNA is alternatively spliced, leading to three distinct mRNAs differing in the 5′ untranslated region. To determine whether the three alternatively spliced mRNA species that contain one of the following untranslated regions (i) exon 1A, 63 bp (ii) exon 1A + 1B, 125 bp or (iii) exon 1C, 101 bp, were expressed in a tissue‐specific manner, we used RT–PCR and exon‐specific oligonucleotide hybridization. Our results suggest two of the variants (1A + 1B and 1A) may utilize the same promoter; however, they are not equally expressed. While in the adult CNS and adrenal medulla, the shorter mRNA consisting of exon 1A was considerably more abundant, in the stomach and heart, the two variants were equally expressed. The third splice variant exon 1C is only expressed in the gut and to a lesser extent in the heart. The data from this study suggest the splice variant consisting of exon 1C may utilize a distinct promoter compared to the other two.

Gastrin-Producing Endocrine Cells: A Novel Source of Histamine in the Rat Stomach

Gastrin and histamine both potently stimulate secretion of acid into the gastric lumen. How these agents interact and how their release is controlled is poorly understood. Therefore, we decided to look for histamine in the antral portion of the rat stomach where the gastrin-producing G cells are located. We used immunocytochemical methods to visualize histamine, histidine decarboxylase (HDC, the enzyme that converts histidine to histamine), and the type 1 vesicular monoamine transporter (VMAT1, the protein responsible for moving histamine into vesicles for storage and release). We were surprised to find that histamine, HDC, and VMAT1 were all present in G cells. Our results suggest that G cells synthesize and secrete gastrin and histamine. Whether histamine acts in concert with gastrin to stimulate acid secretion, or functions as an autocrine inhibitor of gastrin release remains to be seen.

Ontogeny of vesicular monoamine transporter mRNAs VMAT1 and VMAT2

We used in situ hybridization histochemistry to study the expression of the two vesicular monoamine transporters (VMAT1 and VMAT2) during embryonic development in the rat. In the adult rat VMAT2 is present exclusively in neuronal tissues and VMAT1 is present in the adrenal medulla and in certain intestinal endocrine cells. We found that both transporter molecules are more widely expressed during development. We demonstrate a complete overlap of the two VMAT mRNAs in the sympathetic nervous system between E13 and E21 days. In addition, VMAT2 (and to some extent VMAT1) mRNA is expressed in ganglionic cells of the parasympathetic nervous system and in cranial ganglia (trigeminal, vestibular and spiral ganglia) between E12 and E21. The sensory neurons of the dorsal root ganglia, which are also neural crest derivatives, express VMAT2 mRNA (E11-E21), exclusively. Both VMAT mRNAs are found in the developing GI system, but in different cells. VMAT1 mRNA was detected in organs of the endocrine system (pituitary gland, adrenal gland, testis, seminal vesicle), some connective tissue cells, and the thymus. We observed expression of both VMAT mRNAs in two separate cell groups in the placenta (E8-E10). Based on their distribution during development we suggest that monoamines, released in a controlled fashion, might affect migration and differentiation of neural crest derivatives.

Regulation of norepinephrine transporter and tyrosine hydroxylase mRNAs after kainic acid-induced seizures.

Noradrenergic locus coeruleus (LC) efferents to the forebrain suppress seizures in several models of epilepsy. Using in situ hybridization, we demonstrate that tyrosine hydroxylase (TH) and norepinephrine transporter (NET) but not vesicular monoamine transporter 2 (VMAT2) mRNA levels are transiently elevated in LC neurons following kainic acid-induced status epilepticus. These increases of TH and NET mRNAs and presumably of the proteins themselves might enhance synthesis and reuptake of NE postictally.