A requirement of serotonergic p38α mitogen-activated protein kinase for peripheral immune system activation of CNS serotonin uptake and serotonin-linked behaviors.

Alterations in central serotonin (5-hydroxytryptamine, 5-HT) neurotransmission and peripheral immune activation have been linked to multiple neuropsychiatric disorders, including depression, schizophrenia and autism. The antidepressant-sensitive 5-HT transporter (SERT, SLC6A4), a critical determinant of synaptic 5-HT inactivation, can be regulated by pro-inflammatory cytokine signaling. Systemic innate immune system activation via intraperitoneal lipopolysaccharide (LPS) injection rapidly elevates brain SERT activity and 5-HT clearance. Moreover, the pro-inflammatory cytokine interleukin (IL)-1ß rapidly stimulates SERT activity in raphe nerve terminal preparations ex vivo, effects that are attenuated by pharmacological p38 MAPK inhibition. To establish a role of serotonergic p38α MAPK signaling in LPS/IL-1ß-induced SERT regulation and attendant behavioral responses, we pursued studies in mice that afford conditional elimination of p38α MAPK in 5-HT neurons (p38α(5HT-)). We found p38α(5HT-) and control (p38α(5HT+)) littermates to be indistinguishable in viability and growth and to express equivalent levels of SERT protein and synaptosomal 5-HT transport activity. Consistent with pharmacological studies, however, IL-1ß fails to increase SERT activity in midbrain synaptosomes prepared from p38α(5HT-) animals. Moreover, although LPS elevated plasma corticosterone and central/peripheral pro-inflammatory cytokines in p38α(5HT-) animals, elevations in midbrain SERT activity were absent nor were changes in depressive and anxiety-like behaviors observed. Our studies support an obligate role of p38α MAPK signaling in 5-HT neurons for the translation of immune activation to SERT regulation and 5-HT-modulated behaviors.

Molecular genetics of mouse serotonin neurons across the lifespan.

New molecular genetics approaches have been developed over the past several years to study brain serotonin (5-HT) neuron development and the roles of 5-HT neurons in behavior and physiology. These approaches were enabled by manipulation of the gene encoding the Pet-1 ETS transcription factor whose expression in the brain is restricted to developing and adult 5-HT neurons. Targeting of the Pet-1 gene led to the development of a mouse line with a severe and stable deficiency of embryonic 5-HT-synthesizing neurons. The Pet-1 transcription regulatory region has been used to create several new 5-HT neuron-type transgenic tools that have greatly increased the experimental accessibility of the small number of brain 5-HT neurons. Permanent and specific marking of 5-HT neurons with Pet-1-based transgenic tools have now been used for flow cytometry, whole cell electrophysiological recordings, progenitor fate mapping, and live time lapse imaging of these neurons. Additional tools provide multiple strategies for conditional temporal targeting of gene expression in 5-HT neurons at different stages of life. Pet-1-based approaches have led to advances in understanding the role of 5-HT neurons in respiration, thermoregulation, emotional behaviors, maternal behavior, and the mechanism of antipsychotic drug actions. In addition, these approaches have begun to reveal the molecular basis of 5-HT neuron heterogeneity and the transcriptional mechanisms that direct 5-HT neuron-type identity, maturation, and maintenance.

Contribution of 5-HT to locomotion - the paradox of Pet-1(-/-) mice.

Serotonin (5-HT) plays a critical role in locomotor pattern generation by modulating the rhythm and the coordinations. Pet-1, a transcription factor selectively expressed in the raphe nuclei, controls the differentiation of 5-HT neurons. Surprisingly, inactivation of Pet-1 (Pet-1(-/-) mice) that causes a 70% reduction in the number of 5-HT-positive neurons in the raphe does not impair locomotion in adult mice. The goal of the present study was to investigate the operation of the locomotor central pattern generator (CPG) in neonatal Pet-1(-/-) mice. We first confirmed, by means of immunohistochemistry, that there is a marked reduction of 5-HT innervation in the lumbar spinal cord of Pet-1(-/-) mice. Fictive locomotion was induced in the in vitro neonatal mouse spinal cord preparation by bath application of N-methyl-d,l-Aspartate (NMA) alone or together with dopamine and 5-HT. A locomotor pattern characterized by left-right and flexor-extensor alternations was observed in both conditions. Increasing the concentration of 5-HT from 0.5 to 5 µm impaired the pattern in Pet-1(-/-) mice. We tested the role of endogenous 5-HT in the NMA-induced fictive locomotion. Application of 5-HT(2) or 5-HT(7) receptor antagonists affected the NMA-induced fictive locomotion in both heterozygous and homozygous mice although the effects were weaker in the latter strain. This may be, at least partly, explained by the reduced expression of 5-HT(2A) R as observed by means of immunohistochemistry. These results suggest that compensatory mechanisms take place in Pet-1(-/-) mice that make locomotion less dependent upon 5-HT.

NADPH oxidase is required for the sensory plasticity of the carotid body by chronic intermittent hypoxia.

Respiratory motoneuron response to hypoxia is reflex in nature and carotid body sensory receptor constitutes the afferent limb of this reflex. Recent studies showed that repetitive exposures to hypoxia evokes long term facilitation of sensory nerve discharge (sLTF) of the carotid body in rodents exposed to chronic intermittent hypoxia (CIH). Although studies with anti-oxidants suggested the involvement of reactive oxygen species (ROS)-mediated signaling in eliciting sLTF, the source of and the mechanisms associated with ROS generation have not yet been investigated. We tested the hypothesis that ROS generated by NADPH oxidase (NOX) mediate CIH-evoked sLTF. Experiments were performed on ex vivo carotid bodies from rats and mice exposed either to 10 d of CIH or normoxia. Acute repetitive hypoxia evoked a approximately 12-fold increase in NOX activity in CIH but not in control carotid bodies, and this effect was associated with upregulation of NOX2 mRNA and protein, which was primarily localized to glomus cells of the carotid body. sLTF was prevented by NOX inhibitors and was absent in mice deficient in NOX2. NOX activation by CIH required 5-HT release and activation of 5-HT(2) receptors coupled to PKC signaling. Studies with ROS scavengers revealed that H(2)O(2) generated from O(2).(-) contributes to sLTF. Priming with H(2)O(2) elicited sLTF of carotid bodies from normoxic control rats and mice, similar to that seen in CIH-treated animals. These observations reveal a novel role for NOX-induced ROS signaling in mediating sensory plasticity of the carotid body.

Nicotinic acetylcholine receptor subunit proteins alpha7 and beta4 decrease in the superior cervical ganglion after axotomy.

Synaptic transmission in the superior cervical ganglion (SCG) is mediated by nicotinic acetylcholine receptors (nAChR). After transection of the postganglionic nerves of the SCG in the adult rat, the transcript levels of four of the five nAChR subunits present in the ganglion, alpha3, alpha5, alpha7, and beta4, decrease dramatically. In the present study, the effect of axotomy on nAChR subunit expression was examined at the protein level, focusing on the alpha7 and beta4 subunits. Immunohistochemistry with monoclonal antibody mAb306 (for the alpha7 subunit) and polyclonal antibody 4886 (for the beta4 subunit) showed that immunoreactivities for both alpha7 and beta4 subunits were concentrated in neurons in the intact ganglion. Results from double staining with antibodies to these subunits and to tyrosine hydroxylase, the enzyme that catalyzes the rate-limiting step in the biosynthesis of the sympathetic neurotransmitter norepinephrine, demonstrated that most neurons in the SCG express both the alpha7 and beta4 subunits. Three days after axotomy, the number of immunolabeled neurons and the intensity of the immunostaining per labeled neuron were decreased for both subunits. Decreases in subunit levels were also observed by Western blot analysis. Observing changes in these subunits over time after surgery revealed that, while the protein level of the alpha7 subunit recovered substantially within 2 weeks after the lesion, that of the beta4 subunit stayed low. These data demonstrate that decreases in nicotinic receptor subunits are among the changes in proteins that occur in axotomized sympathetic neurons, and suggest that these decreases may contribute to the depression in ganglionic synaptic transmission observed in axotomized ganglia.

Regulation of transcription in the neuronal nicotinic receptor subunit gene cluster by a neuron-selective enhancer and ETS domain factors.

Expression of neurotransmitter receptors encoded by the nicotinic acetylcholine receptor (nAchR) subunit gene cluster depends on coexpression of the beta4, alpha3, and alpha5 subunits in certain kinds of neurons. One way in which coexpression might be achieved is through the regulation of promoters in the cluster by neuron-selective enhancers. The beta43' enhancer is located between the beta4 and alpha3 promoters and it directs cell type-specific expression in cell lines. It is not known, however, whether beta43' is active in neurons. Therefore, we assayed beta43' in dissociated rat sympathetic ganglia cultures, which contain nAchR-positive neurons as well as nAchR-negative non-neuronal cells. Reporters controlled by the alpha3 promoter and beta43' were expressed in a neuron-selective manner; greater than 90% and up to 100% of luciferase expression was detected in neurons. Neuron selectivity was maintained when beta43' was placed next to ubiquitously active viral promoters. In contrast, replacing beta43' with the SV40 enhancer eliminated neuron selectivity. The enhancer is composed of at least two separate but functionally interdependent elements, each of which interacts with a different type of ETS domain factor. These findings support a model in which beta43' controls neuronal expression of one or more genes in the cluster through interactions with a combination of ETS factors.

Transcriptional control of the neuronal nicotinic acetylcholine receptor gene cluster by the beta43' enhancer, Sp1, SCIP and ETS transcription factors.

Receptors assembled from the products of a neuronal beta4alpha3alpha5 NAChR gene cluster depend on these genes being coordinately regulated in particular populations of neurons. Little is known, however, about the transcriptional mechanisms that are likely to underlie their co-expression in correct neuronal cell types. We have identified several regulatory elements and transcription factors that influence transcription of the alpha3 and beta4 genes. The promoters of these genes appear to contain a common cis element that binds Sp1 transcription factors. They can be activated by the POU-domain factor SCIP and activation does not require SCIP binding sites. Between these two promoters is a cell type specific enhancer called beta43'. This enhancer has little activity in non-neuronal cells and is preferentially active in particular populations of central neurons. The clustered genes are potential targets of ETS factors as the ETS domain factor, Pet-1 can activate beta43'-dependent transcription. The neuron-selective properties of beta43' and its location suggest that it is a component of the cis regulatory information required to control expression of the beta4 and alpha3 genes in specific populations of neurons.

The ETS domain factor Pet-1 is an early and precise marker of central serotonin neurons and interacts with a conserved element in serotonergic genes.

Serotonin (5-HT) plays a crucial neuromodulatory role in numerous physiological and behavioral functions, and dysfunction of the serotonergic system has been implicated in several psychiatric disorders. Despite the widespread importance of the central serotonergic neurotransmitter system, little is known about the molecular mechanisms controlling the development of 5-HT neurons. We previously identified an ETS domain transcription factor, Pet-1, that is expressed in a small number of tissues, including the brain. Here, we show that expression of Pet-1 RNA in the brain is restricted to, and marks, the entire rostrocaudal extent of rat serotonergic hindbrain raphe nuclei. Remarkably, Pet-1 RNA colocalizes with tryptophan hydroxylase-positive neurons in raphe nuclei but not with their nonserotonergic neuron or non-neuronal neighbors. Pet-1 RNA is limited to two domains in the developing hindbrain, which precedes the appearance of 5-HT in each domain by approximately a half day. Conserved Pet-1 binding sites are present in or near the promoter regions of the human and mouse 5-HT1a receptor, serotonin transporter, tryptophan hydroxylase, and aromatic L-amino acid decarboxylase genes whose expression is characteristic of the serotonergic neuron phenotype. These sites are capable of supporting transcriptional activation through interactions with the Pet-1 ETS domain and can function as enhancers. Together, our findings establish Pet-1 as an early and precise marker of 5-HT neurons and suggest that it functions specifically in the differentiation and maintenance of these neurons.

Pet-1, a novel ETS domain factor that can activate neuronal nAchR gene transcription.

We report a cDNA clone prepared from adrenal chromaffin-derived PC12 cell RNA that encodes a novel ETS-domain factor, Pet-1. The deduced primary structure of Pet-1 is composed of 340 amino acids and the encoded polypeptide has a predicted molecular mass of 35.4 kD. The pattern of Pet-1 gene expression in the neonatal rat is highly restricted and suggests that Pet-1 functions primarily in the nervous system. Adrenal gland expresses the highest level of Pet-1 among the tissues examined. In situ hybridization indicates that Pet-1 is expressed in the adrenal medulla but not the adrenal cortex. Slightly weaker Pet-1 hybridization is detected in brain and low levels are detectable in intestine and eye. Pet-1 can bind specifically to a PEA3 ETS DNA-binding motif and can modulate transcription of synthetic promoter constructs in a sequence-specific manner. We recently identified a neural cell-type specific enhancer, beta43', within the 3'-untranslated exon of the neuronal nicotinic acetylcholine receptor (nAchR) beta4 subunit gene. Similar to Pet-1, the beta4 gene is also expressed in PC12 cells. The presence of putative ETS-domain binding sites in the beta43' enhancer led us to hypothesize that members of the ets gene family activate neuronal nAchR genes. Cotransfection assays show that Pet-1 can activate reporter gene transcription in a beta43' enhancer-dependent and cell type-dependent manner. Our results lead us to hypothesize that Pet-1 acts as a transcriptional regulator of downstream target genes involved in cholinergic neurotransmission.

Differential regulation of levels of nicotinic receptor subunit transcripts in adult sympathetic neurons after axotomy.

Axotomy of adult peripheral neurons produces decreases in the levels of transcripts for a number of proteins involved in synaptic transmission. For example, tyrosine hydroxylase and neuropeptide Y mRNA decrease in axotomized sympathetic neurons in the superior cervical ganglion (SCG). In the present study, the effects of axotomy on the expression of nicotinic receptor subunit transcripts were examined in the SCG and the results were compared to those produced by deafferentation and explantation. Normally, neurons in the SCG express five different nicotinic subunits: alpha3, alpha5, alpha7, beta2, and beta4. Forty-eight hours after axotomy in vivo or explantation, dramatic decreases in these transcripts were seen, except for beta2, which increased. In contrast, deafferentation of the SCG had negligible effects on any of these transcripts. Both leukemia inhibitory factor (LIF) and nerve growth factor (NGF) have been shown to play a role in the decrease in neuropeptide Y mRNA expression after axotomy. In the cases of these nicotinic receptor transcripts, however, similar decreases were seen in wild-type and LIF knockout animals. Furthermore, administration of an antiserum to NGF in intact animals produced no changes in transcript levels. On the other hand, providing exogenous NGF to axotomized SCG in vivo or in explant cultures partially prevented the decreases in the transcripts for alpha3, alpha5, alpha7, and beta4. These data indicate that axotomy produces dramatic decreases in the expression of several nicotinic receptor subunit transcripts, and that the molecular signals underlying these changes differ from those previously shown to mediate the decrease in neuropeptide Y expression.

beta43': An enhancer displaying neural-restricted activity is located in the 3'-untranslated exon of the rat nicotinic acetylcholine receptor beta4 gene.

Members of a neuronal nicotinic acetylcholine receptor subunit gene cluster ordered beta4, alpha3, alpha5 in the vertebrate genome are expressed in highly restricted patterns in the PNS and CNS. Nothing is known, however, about the regulatory elements that control transcription of these genes in selected neuronal cell populations. We report here a novel enhancer, designated beta43', that is positioned in the beta4 3'-untranslated exon. It is composed of two nearly identical 37 bp direct repeats that are separated by 6 bp. Multimerization of the enhancer upstream of the alpha3 minimal promoter results in synergistic activation. Analysis in different cell types, including three neural lines and primary keratinocytes, shows that beta43' is preferentially active in the neural line PC12, which expresses all members of the cluster. Mobility shift assays reveal a cell-type-specific complex, which forms with the first repeat of the enhancer and PC12 extracts. Complexes co-migrating with the PC12 cell complex are not detected with extracts from other lines, which suggests that PC12 cells contain a differentially expressed factor that may be important for the restricted activity of beta43'. The cell-type-specific activity of the beta43' enhancer suggests that it is important for regulating restricted expression patterns of one or more clustered neuronal acetylcholine receptor genes. Its location within the beta4 gene may be a selective pressure for maintaining tight linkage of clustered neuronal nAchR genes.

Elements between the protein-coding regions of the adjacent beta 4 and alpha 3 acetylcholine receptor genes direct neuron-specific expression in the central nervous system.

The expression patterns of three clustered neuronal nicotinic acetylcholine receptor (nAchR) subunit genes ordered beta 4, alpha 3, and alpha 5 overlap extensively in the peripheral nervous system (PNS) but only partially in the central nervous system (CNS). We have begun to investigate cell type-specific cis elements regulating these genes by analyzing in both cell culture and transgenic mice, a 2.8-kb fragment (-2732/+47) containing the alpha 3 promoter region, the beta 4/alpha 3 intergenic region, and a portion of the beta 4 3'-untranslated exon. The -2732/+47 fragment is preferentially active in PC12 cells relative to nonneural cell lines. Deletion analysis revealed a cell type-specific positive transcriptional element positioned in the beta 4 3'-untranslated exon. The positive element is likely to be an enhancer and not a second alpha 3 promoter, because no alpha 3 exons are present in this region. Having shown in cell culture that cell-type specific cis elements are positioned between the beta 4 and alpha 3 coding regions, we investigated the activity of -2732/+47 in vivo. Transgenic mice were generated, which carry the lacZ gene fused downstream of -2732/+47. Expression of the lacZ transgene is restricted to neurons of the CNS; no expression was detected in the PNS or in nonneural tissues. LacZ-positive cells were detected virtually exclusively in a subset of CNS nuclei that transcribe the endogenous alpha 3 gene. Some overlap was seen with the beta 4 gene, but nearly none with the alpha 5 gene. Our results demonstrate that cis elements positioned between the alpha 3 and beta 4 coding regions are important for establishing part of the restricted CNS patterns of beta 4, alpha 3, and alpha 5 gene transcription.

The POU domain of SCIP/Tst-1/Oct-6 is sufficient for activation of an acetylcholine receptor promoter.

In the PC12 neuroendocrine line, the neuronal nicotinic acetylcholine receptor alpha3 gene promoter is activated by SCIP/Tst-1/Oct-6, a POU domain transcription factor proposed to be important for regulating the development of specific neural cell populations. In this study, we have investigated the SCIP polypeptide domains involved in alpha3 promoter activation. The characteristics of activation by a chimeric effector in which the GAL4 DNA binding domain was substituted for the SCIP POU domain were dramatically different from those of wild-type SCIP. At low effector masses, the chimeric polypeptide weakly activated alpha3 in a GAL4 binding-site-dependent manner but then squelched transcription at higher masses. In contrast, wild-type SCIP activation was not modulated by the presence of multimerized SCIP binding sites, and squelching was not observed. Analysis of wild-type SCIP truncations revealed that deletion of the previously characterized SCIP amino-terminal activation domain did not destroy activity of the factor. Surprisingly, a truncation expressing nothing more than the POU domain was nearly as active as wild-type SCIP. Moreover, cotransfection of a GAL4-VP16 effector with an effector expressing just the SCIP POU domain resulted in synergistic activation of the promoter. Synergistic activation did not depend on an Sp1 motif that is the only functional alpha3 cis element outside the transcription start site region. Our results show that the DNA binding domain of a POU factor is capable of transcriptional activation probably through protein-protein interactions with components of the basal transcription complex.

Transcriptional analysis of acetylcholine receptor alpha 3 gene promoter motifs that bind Sp1 and AP2.

In this study, we performed an analysis of the neuronal nicotinic acetylcholine receptor alpha 3 subunit gene promoter region, -238/+47, to identify cis and trans elements that are important for basal activity in PC12 cells. Sequence analyses of the alpha 3 promoter and footprint assays revealed an Sp1 binding site between -79 and -57 (termed the alpha 3 GA motif) and an AP2 binding site between -30 and -7. Using mobility shift analysis, we found that PC12 cell extracts contain proteins that specifically bind to the alpha 3 GA motif and are immunologically related to Sp1. Mutation of the alpha 3 GA motif, which prevented binding of Sp1, resulted in a 75% decrease in promoter activity. Mutation of the AP2 site resulted in only a minor loss of promoter activity, which is consistent with the lack of AP2 binding activity in PC12 extracts. In Drosophila Schneider line 2 (S2) cell cotransfection assays, Sp1 activated the alpha 3 promoter in a GA motif-dependent manner. Furthermore, multimerization of the GA motif upstream of the beta-globin TATA box conferred Sp1 responsiveness. Our results indicate that Sp1 can activate transcription through direct interaction with the alpha 3 GA motif and that this motif plays a major role in alpha 3 promoter basal activity in PC12 cells.

The developmental increase in ACh current densities on rat sympathetic neurons correlates with changes in nicotinic ACh receptor alpha-subunit gene expression and occurs independent of innervation.

Determining factors that control the expression of neurotransmitter receptors and the mechanisms by which these factors operate is essential to understand how synapses form during development and how receptor numbers change in the adult. In this study, we have investigated one such factor, the influence of innervation, on the developmental expression of nicotinic ACh receptors (nAChRs) on neonatal rat sympathetic neurons, both in terms of ACh current densities, and in terms of mRNA levels for the transcripts that encode these receptors. To date, nine genes have been cloned that encode neuronal nAChRs subunits in mammals. We demonstrate that mRNA encoding five nAChR subunits, alpha 3, alpha 5, alpha 7, beta 2, and beta 4, are present in neonatal rat sympathetic neurons. We show that mRNA levels for alpha 3 and alpha 7 subunits increase by more than threefold over the first 2 postnatal weeks, a period when most synapses are forming on the neurons; however, we observed no significant change in mRNA levels for alpha 5, beta 2, or beta 4. Using whole-cell voltage-clamp techniques, we show that the increase in alpha-subunit mRNA correlates with increases in ACh current densities, which double over the same period. To investigate the role of innervation, we cut the preganglionic nerve at birth and measured subunit mRNA levels and ACh current densities in denervated neurons 1-2 weeks later. Our results indicate that the preganglionic nerve differentially affects the mRNA level for the five nAChR transcripts, yet it has little influence on the developmental increase in ACh current densities.

Characterization of an acetylcholine receptor alpha 3 gene promoter and its activation by the POU domain factor SCIP/Tst-1.

Genes encoding neuronal nicotinic acetylcholine receptors exhibit restricted patterns of expression in the nervous system. We are interested in elucidating the molecular mechanisms responsible for establishing these patterns of expression. This paper presents the characterization of regulatory elements upstream of the neuronal nicotinic acetylcholine receptor alpha 3 gene. We have identified a GC-rich multistart site promoter adjacent to the alpha 3 coding region. Similar alpha 3 start sites were identified in PC12 cells and sympathetic ganglion neurons, suggesting similar control mechanisms in the clonal line and peripheral neurons. The start site region lacks TATA-like sequences but does contain initiator-like sequences. We show, in transient transfection assays, that the POU domain transcription factor, SCIP/Tst-1, specifically activates alpha 3 in a neural context. Other POU domain factors tested only weakly activated or repressed alpha 3. Unexpectedly, we found that alpha 3 basal activity and SCIP/Tst-1 activation of alpha 3 is not dependent on the SCIP/Tst-1 binding sites found upstream of the gene. In addition, mutations in the SCIP/Tst-1 coding region that prevent the factor from binding to DNA with high affinity do not obliterate alpha 3 activation. These results lead us to propose that alpha 3 activation by SCIP/Tst-1 is achieved via protein-protein interactions between SCIP/Tst-1 and a specific complement of transcription factors that act directly on the promoter.

The expression of nicotinic acetylcholine receptors by PC12 cells treated with NGF.

The expression of neuronal nicotinic ACh receptors (nAChRs) and the subunits that compose these receptors by PC12 cells exposed to NGF has been studied. The analysis of total RNA reveals that the neuronal nAChR subunits alpha 3, alpha S, beta 2, beta 3, and beta 4, but not alpha 2 and alpha 4, are expressed in our PC12 cells. Within 48 hr of adding NGF to cultures, the RNA corresponding to alpha 3, alpha 5, beta 3, and beta 4 is decreased, but the beta 2 RNA increases for up to 6 d after NGF treatment. To determine the influence of NGF treatment on subunit protein expression, subunit-specific antisera were prepared. Immunocytochemistry detected antigen for alpha 3, alpha 5, beta 2, beta 3, and beta 4 (but not alpha 2 and alpha 4) in both NGF-treated and nontreated PC12 cells. The expression of nAChR subunit proteins, as measured by direct binding of antibodies to PC12 cells, does not change subsequent to 6 d of treatment with NGF. Whole-cell recording of PC12 cells shows that both the individual cell current density and response to the agonist cytisine were not altered after 5-7 d in NGF. However, the number of cells exhibiting detectable ACh-induced currents doubled. These results indicate that NGF increases the number of PC12 cells expressing ACh-sensitive nAChR currents but the activation is not the result of altering the amounts of individual nAChR subunit proteins. These data, taken together with the decrease in most nAChR subunit RNAs (except beta 2), suggest that NGF regulation of nAChRs may be through a posttranscriptional mechanism.

Nicotine binding and nicotinic receptor subunit RNA after chronic nicotine treatment.

DBA mice were chronically treated with nicotine by continuous intravenous infusion of 4.0 mg/kg/hr for 10 d. Drug-treated mice were tolerant to the acute effects of nicotine on locomotor activity and body temperature. The effect of chronic treatment on the amount of L-3H-nicotine binding and RNA encoding for alpha 4, the most widely expressed nicotinic alpha-subunit, was measured in three brain regions. Chronic treatment increased L-3H-nicotine binding in cortex and midbrain but had no effect in cerebellum. In contrast, chronic treatment had no effect on the levels of mRNA encoding for alpha 4 in any of the three brain regions. Subsequently brains were sectioned and L-3H-nicotine binding was measured using quantitative autoradiographic methods. In addition, the relative amounts of mRNA for the major nicotinic receptor subunits (alpha 4 and beta 2), as well as for three additional minor subunits (alpha 2, alpha 3, and alpha 5), were determined by in situ hybridization histochemistry followed by quantitation of image intensity. Chronic nicotine treatment resulted in increases in the amount of L-3H-nicotine binding in many but not all brain areas measured. In contrast, chronic treatment had little effect on the intensity of the hybridization signal for the nicotinic subunit mRNA. The results suggest that chronic treatment with nicotine under conditions resulting in maximal steady-state increases in L-3H-nicotine binding has little effect on RNA levels encoding any of four nicotinic alpha-subunits and the beta 2-subunit.

The characterization and localization of the glutamate receptor subunit GluR1 in the rat brain.

The cloning of cDNAs that encode functional glutamate receptors makes it possible to produce antibodies that can be used as high-affinity probes for the localization and characterization of these receptors in the mammalian brain. We have made antibodies to different regions of the first cloned member of this family, GluR1, using bacterially overproduced antigen. On Western blots, these antisera detect glycoprotein(s) of 105 kDa present in crude membranes of the hippocampus and cerebellum. The 105-kDa band is associated with postsynaptic densities, and it is observed in cultured cells upon transfection with the GluR1 cDNA. Although glutamate receptors are thought to be the most prevalent excitatory ligand-gated ion channel in the mammalian brain, immunohistochemistry reveals that the receptors recognized by these antisera are localized predominantly in neurons of the cerebellum and some structures of the limbic system, including the hippocampus, the central nucleus of the amygdala, and portions of the septum. This pattern of expression is, in general, consistent with the distribution of GluR1 mRNA as determined by in situ hybridization histochemistry. Our results suggest that glutamate excitatory circuits recognized by these antisera are predominantly found in regions of the limbic system that are reciprocally interconnected.

The nicotinic receptor genes.

The causative factor(s) of Alzheimer's disease (AD) are presently unknown. However, it has been shown that the number as well as the fraction of high- to low-affinity nicotine binding sites is altered in patients suffering from this disease. This finding, along with the identification of seven genes which code for nicotinic receptors expressed in the mammalian brain, has led to the idea that one nicotinic receptor subtype may be specifically altered in AD. The present article reviews how, through a molecular genetic approach, a family of genes coding for nicotinic acetylcholine receptor subtypes was uncovered. Also discussed is the use of in situ hybridization to determine the distribution of expression of the mRNA encoding for each receptor subtype and the patch clamp technique to characterize their biophysical properties. Determination of the promoters of these genes, as well as the properties of the expressed receptor subtypes, may make it possible to design new specific nicotinic receptor subtype drugs that will treat not only the symptoms of AD but the progression of the disease process as well.