Glucocorticoid receptor-immunoreactivity in corticotrophin-releasing factor afferents to the locus coeruleus

Choline acetyltransferase (ChAT) is a specific phenotypic marker of cholinergic neurons. Previous reports showed that different upstream regions of the ChAT gene are necessary for cell type-specific expression of reporter genes in cholinergic cell lines. The identity of the mouse ChAT promoter region controlling the establishment, maintenance, and plasticity of the cholinergic phenotype in vivo is not known. We characterized a promoter region of the mouse ChAT gene in transgenic mice, using beta-galactosidase (LacZ) as a reporter gene. A 3,402-bp segment from the 5'-untranslated region of the mouse ChAT gene (from -3,356 to +46, +1 being the translation initiation site) was sufficient to direct the expression of LacZ to selected neurons of the nervous system; however, it did not provide complete cholinergic specificity. A larger fragment (6,417 bp, from -6,371 to +46) of this region contains the requisite regulatory elements that restrict expression of the LacZ reporter gene only in cholinergic neurons of transgenic mice. This 6.4-kb DNA fragment encompasses 633 bp of the 5'-flanking region of the mouse vesicular acetylcholine transporter (VAChT), the entire open reading frame of the VAChT gene, contained within the first intron of the ChAT gene, and sequences upstream of the start coding sequences of the ChAT gene. This promoter will allow targeting of specific gene products to cholinergic neurons to evaluate the mechanisms of diseases characterized by dysfunction of cholinergic neurons and will be valuable in design strategies to correct those disorders.     

Reduction of vesicular acetylcholine transporter mRNA in the rat septum following lead exposure

We have previously observed that maternal exposure to lead (Pb) results in a reduction of levels of mRNA coding for cholineacetyltransferase (ChAT) in the septum of developing rat without affecting the dams. Here we report that Pb similarly affects the expression of vesicular acetylcholine transporter (VAChT) mRNA in the rat septum. In close agreement with the time course of ChAT mRNA expression, septal VAChT mRNA levels increased from 30% at postnatal day 7 to 78% and 100% of adult levels at days 14 and 21, respectively. Maternal exposure to 0.2% lead acetate in drinking water from gestational day 16 resulted in an approximately 30% reduction of VAChT in 7 and 21-day-old rat pups without affecting VAChT mRNA levels in the dams. These results indicate a developmental stage-dependent interference by Pb with ChAT/VAChT gene expression in the rat septum.     

Fluctuations in relative levels of choline acetyltransferase mRNA in different regions of the rat basal forebrain across the estrous cycle: effects of estrogen and progesterone

Quantitative in situ hybridization techniques were used to compare relative cellular levels of choline acetyltransferase (ChAT) mRNA in different regions of the female rat basal forebrain at different stages of the estrous cycle and at different time points after the administration of physiological levels of estrogen and progesterone. Significant fluctuations in relative levels of ChAT mRNA were detected during the course of the estrous cycle. In the medial septum (MS) and striatum, the highest levels of ChAT mRNA were detected on diestrus 1. Fluctuations in the nucleus basalis magnocellularis (NBM) were highly variable, with the highest levels detected on diestrus 2. In ovariectomized animals, significant increases in ChAT mRNA were detected in the MS, NBM, and striatum within 1-3 d after a single administration of estradiol. In addition, the effects of estradiol on ChAT mRNA expression in the NBM and striatum were significantly enhanced by the subsequent administration of progesterone. The magnitude and timing of the effects of steroid replacement were consistent with the magnitude and time course of the fluctuations detected during the course of the estrous cycle. These data demonstrate that estrogen and progesterone can increase basal forebrain levels of ChAT mRNA significantly in specific regions of the rat basal forebrain, that the magnitude and time course of the effects vary between different subpopulations of cholinergic neurons, and that the effects are associated with changes in the functioning of specific basal forebrain cholinergic neurons across the estrous cycle.     

Cellular localizations of AMPA glutamate receptors within the basal forebrain magnocellular complex of rat and monkey

The cellular distributions of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors within the rodent and nonhuman primate basal forebrain magnocellular complex (BFMC) were demonstrated immunocytochemically using anti-peptide antibodies that recognize glutamate receptor (GluR) subunit proteins (i.e., GluR1, GluR4, and a conserved region of GluR2, GluR3, and GluR4c). In both species, many large GluR1-positive neuronal perikarya and aspiny dendrites are present within the medial septal nucleus, the nucleus of the diagonal band of Broca, and the nucleus basalis of Meynert. In this population of neurons in rat and monkey, GluR2/3/4c and GluR4 immunoreactivities are less abundant than GluR1 immunoreactivity. In rat, GluR1 does not colocalize with ChAT, but, within many neurons, GluR1 does colocalize with GABA, glutamic acid decarboxylase (GAD), and parvalbumin immunoreactivities. GluR1- and GABA/GAD-positive neurons intermingle extensively with ChAT-positive neurons. In monkey, however, most GluR1-immunoreactive neurons express ChAT and calbindin-D28 immunoreactivities. The results reveal that noncholinergic GABAergic neurons, within the BFMC of rat, express AMPA receptors, whereas cholinergic neurons in the BFMC of monkey express AMPA receptors. Thus, the cellular localizations of the AMPA subtype of GluR are different within the BFMC of rat and monkey, suggesting that excitatory synaptic regulation of distinct subsets of BFMC neurons may differ among species. We conclude that, in the rodent, BFMC GABAergic neurons receive glutamatergic inputs, whereas cholinergic neurons either do not receive glutamatergic synapses or utilize GluR subtypes other than AMPA receptors. In contrast, in primate, basal forebrain cholinergic neurons are innervated directly by glutamatergic afferents and utilize AMPA receptors.     

Few cholinergic neurons in the rat basal forebrain coexpress galanin messenger RNA

The concept that galanin (GAL) is cosecreted with acetylcholine (ACh) into the ventral hippocampus is a major component of the current model delineating GAL regulation of the cholinergic memory pathways in the rat. Although GAL-immunoreactivity coexists in 50-70% of cholinergic neurons in the basal forebrain (BF) of colchicine-treated rats, the actual coexistence of these neurotransmitters in the basal state may be lower, because colchicine treatment was recently shown to both induce GAL gene expression and inhibit choline acetyltransferase (ChAT) gene expression in this brain region. We have used single and double in situ hybridization histochemistry to examine the distribution and coexistence of GAL and ChAT mRNAs in the BF of male and female rats. Compared with other forebrain regions, few GAL mRNA-expressing neurons are present within the cholinergic fields of the BF. The greatest number of GAL mRNA-expressing cells in this region are located within the nucleus of the horizontal limb of the diagonal band; but, even in this region, they represent only a small percentage (<20%) of ChAT mRNA-expressing cells. Our results indicate that few cholinergic neurons in the rat BF coexpress GAL mRNA and suggest that, in the basal state, GAL is not widely cosecreted with ACh into hippocampal memory centers.     

Obsessive-compulsive disorder in schizophrenia: epidemiologic and biologic overlap

AF64A (ethylcholine mustard aziridinium ion) was stereotaxically administered bilaterally (1 nmol/side) into rat lateral cerebral ventricles. Choline acetyltransferase (ChAT) activity and ChAT mRNA levels were measured at predetermined time points in the septo-hippocampal pathway and striatum, both well identified as rich in cholinergic neurons. AF64A caused a rapid but transient increase in ChAT mRNA (167%, P < 0.05) and ChAT activity (164%, P < 0.01) in the septum. By day 7 post treatment, there was a significant decrease in ChAT mRNA (42.5% of control, P < 0.05) in the septum although the ChAT activity still stayed high. This decreased ChAT mRNA level in the septum lasted for at least four weeks, and was paralleled by a long-lasting decrease in ChAT activity in the hippocampus. In the striatum, on the other hand, there were no observed changes in either ChAT activity or ChAT mRNA. These data suggest that the long term effect of AF64A on the septo-hippocampal cholinergic pathway may, at least in part, be due to an action of AF64A on gene expression in the cholinergic neuron. The difference in the response to AF64A between the septo-hippocampal and striatal cholinergic systems might be due to their difference in neuron types.     

Role of Leukotriene-degrading enzymes in pulmonary response to antigen infusion in sensitized guinea pigs in vivo

Although the expression of nerve growth factor (NGF) in the rat striatum is the highest at 2 postnatal weeks (P2w), the action of NGF at that age has not been studied in detail. We examined the effects of several neurotrophic factors, including NGF, on striatal cholinergic neurons cultured from P2w rats. We also examined the effects of a cyclic AMP (cAMP) analog and high K(+)-evoked depolarization. NGF specifically promoted the survival of choline acetyltransferase (ChAT)-positive neurons, and consequently increased the ChAT activity per well, whereas it did not induce the ChAT activity per cholinergic neuron. NGF-responsiveness was the highest in striatal cultures from P2w rats, but it was almost lost in cultures from P4w rats. Brain-derived neurotrophic factor (BDNF), neurotrophin-4/5 (NT-4/5), and a cAMP analog had survival-promoting effects on striatal total neurons including cholinergic neurons. On the other hand, high K+ hardly promoted the survival of striatal cholinergic neurons in cultures from P2w rats, although it increased the viable number of total striatal neurons. High K+ did not increase the ChAT activity in any tested cultures from postnatal 3- to 28-day-old rats. These results demonstrated that NGF prevented the death of striatal cholinergic neurons in cultures from P2w rats, but not from P4w rats, and that high K+ could not rescue these deaths. We propose that cholinergic neurons in the striatum are programmed to die at P2w, and that this programmed cell death can be restored by neurotrophins, but not by depolarization.     

Choline acetyltransferase in cochlear root neurons

Cochlear root neurons are a distinct group of cells located in the auditory nerve root in small rodents. Their transmitter is still unknown. Some of our preparations showed immunoreactivity of somata of cochlear root neurons with both polyclonal and monoclonal antibodies against choline acetyltransferase (ChAT) which, despite their very weak histochemical reaction for acetylcholinesterase (AChE), suggested that cochlear root neurons might be cholinergic. To test this, we used a radiometric assay to measure ChAT activities of rat auditory nerve root samples containing cochlear root neurons and of adjacent samples not containing them. There was no significant difference between the low mean ChAT activities of these two groups of samples. Thus, cochlear root neurons are not likely to be cholinergic.     

Technical note: a blind test of mandibular ramus flexure as a morphologic indicator of sexual dimorphism in the human skeleton

Following axotomy most medial septal neurons in the adult rat brain have dramatically reduced numbers of choline acetyltransferase (ChAT) positive neurons. Since leukemia inhibitory factor (LIF) promotes cholinergic expression in several neuronal populations, the aim of this study was to determine if LIF would continue to support cholinergic expression in axotomized medial septal neurons. Mini-osmotic pumps were used to infuse saline or LIF into the lateral cerebral ventricle. Counts of ChAT and low-affinity nerve growth factor (p75NGFR) immunostained neurons indicated that LIF-treated animals retained ChAT expression in > 90% of axotomized neurons whereas in saline-infused animals this was < 30%. Also, LIF was equally effective in maintaining p75NGFR expression levels in axotomized medial septal neurons.     

EYA4, a novel vertebrate gene related to Drosophila eyes absent

NMDA receptors are composed of proteins from two families: NMDAR1 and NMDAR2. We used quantitative double-label in situ hybridization to examine in rat brain the expression of NMDAR1, NMDAR2A, NMDAR2B, and NMDAR2C mRNA in six neurochemically defined populations of striatal neurons: preproenkephalin (ENK) and preprotachykinin (SP) expressing projection neurons, and somatostatin (SOM), glutamic acid decarboxylase 67 (GAD67), parvalbumin (PARV), and choline acetyltransferase (ChAT) expressing interneurons. NMDAR1 was expressed by all striatal neurons: strongly in ENK, SP, PARV and ChAT neurons, and less intensely in SOM and GAD67 positive cells. NMDAR2A mRNA was present at moderate levels in all striatal neurons except those containing ChAT. Labeling for NMDAR2B was strong in projection neurons and ChAT interneurons, and only moderate in SOM, GAD67 and PARV interneurons. NMDAR2C was scarce in striatal neurons, but a low level signal was detected in GAD67 positive cells. NMDAR2C expression was also observed in small cells not labeled by any of the markers, most likely glia. These data suggest that all striatal neurons have NMDA receptors, but different populations have different subunit compositions which may affect function as well as selective vulnerability.     

Acidic fibroblast growth factor mRNA is expressed by basal forebrain and striatal cholinergic neurons

Evidence for the importance of the basal forebrain cholinergic system in the maintenance of cognitive function has stimulated efforts to identify trophic mechanisms that protect this cell population from atrophy and dysfunction associated with aging and disease. Acidic fibroblast growth factor (aFGF) has been reported to support cholinergic neuronal survival and has been localized in basal forebrain with the use of immunohistochemical techniques. Although these data indicate that aFGF is present in regions containing cholinergic cell bodies, the actual site of synthesis of this factor has yet to be determined. In the present study, in situ hybridization techniques were used to evaluate the distribution and possible colocalization of mRNAs for aFGF and the cholinergic neuron marker choline acetyltransferase (ChAT) in basal forebrain and striatum. In single-labeling preparations, aFGF mRNA-containing neurons were found to be codistributed with ChAT mRNA+ cells throughout all fields of basal forebrain, including the medial septum/diagonal band complex and striatum. By using a double-labeling (colormetric and isotopic) technique, high levels of colocalization (over 85%) of aFGF and ChAT mRNAs were observed in the medial septum, the diagonal bands of Broca, the magnocellular preoptic area, and the nucleus basalis of Meynert. The degree of colocalization was lower in the striatum, with 64% of the cholinergic cells in the caudate and 33% in the ventral striatum and olfactory tubercle labeled by the aFGF cRNA. These data demonstrate substantial regionally specific patterns of colocalization and support the hypothesis that, via an autocrine mechanism, aFGF provides local trophic support for cholinergic neurons in the basal forebrain and the striatum.     

Neurotrophin effects on survival and expression of cholinergic properties in cultured rat septal neurons under normal and stress conditions

These studies tested the hypothesis that survival-promoting effects of neurotrophins on basal forebrain cholinergic neurons are enhanced under stress. Septal neurons from embryonic day 14-15 rats exposed for 10-14 d to neurotrophin [nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), or neurotrophin-4 (NT-4), each at 100 ng/ml] showed a two- to threefold increase in choline acetyltransferase (ChAT) activity, with little evidence of synergistic interactions. Neurotrophins produced no significant increase in the survival of total or acetylcholinesterase (AChE)-positive neurons at moderate plating density (1200-1600 cells/mm2). However, with very low plating densities (2-28 cells/mm2) BDNF, NT-3, and NT-4 (but not NGF) increased total neuronal survival, and BDNF increased survival of AChE-positive neurons. NGF and BDNF enhanced ChAT activity and survival of cholinergic neurons after a 24 hr hypoglycemic stress, even when added 1 hr after stress onset. All four tested neurotrophins increased total neuronal survival after hypoglycemic stress. These results suggest that neurotrophins are important for preservation of central cholinergic function under stress conditions, with different neurotrophins protecting against different stresses. The stress-associated survival-promoting effects of neurotrophins were not limited to the cholinergic subpopulation.     

Graft-derived cholinergic reinnervation of the hippocampus prevents a lasting increase of hippocampal noradrenaline concentration induced by septohippocampal damage in rats

Long-Evans female rats sustained aspirative lesions of the septohippocampal pathways and, 2 weeks later, received into the dorsal hippocampus grafts prepared from the septal area (rich in cholinergic neurons; Group Sep) or from the mesencephalic raphe (poor in cholinergic neurons; Group Rap) of rat fetuses. Lesion-only (Group Les) and virtually intact (Group Sham) rats served as controls. Between 9.5 and 10.5 months after grafting surgery, we found the lesions to decrease choline acetyltransferase activity (ChAT), high affinity synaptosomal uptake of [3H]choline (HACU) and serotonin concentration ([5-HT]), as well as to increase the noradrenaline concentration ([NA]) in the dorsal hippocampus. Raphe grafts increased [5-HT] to 456% of normal, but had only weak or no effects on the other lesion-induced modifications in brain neurochemistry. Septal grafts dramatically increased ChAT activity and HACU, enhanced [5-HT], and reduced [NA] to near-normal levels. We also found a significant negative correlation between HACU and [NA] in rats with lesions, whether grafted or not. These data show that grafts providing the denervated hippocampus with a new cholinergic innervation might be able to exert inhibitory effects on the lesion-induced increase of [NA]. Since such an increase is indicative of sympathetic sprouting, the finding of reduced [NA] in rats with graft-derived cholinergic reinnervation of the hippocampus is in line with the hypothesis that hippocampal cholinergic denervation plays a crucial role in the induction of sympathetic sprouting. However, our data do not allow to distinguish whether grafts rich in cholinergic neurons inhibited the sympathetic sprouting itself, or rather reduced the NA content of sprouted fibers.     

The specification of sympathetic neurotransmitter phenotype depends on gp130 cytokine receptor signaling

Sympathetic ganglia are composed of noradrenergic and cholinergic neurons. The differentiation of cholinergic sympathetic neurons is characterized by the expression of choline acetyltransferase (ChAT) and vasoactive intestinal peptide (VIP), induced in vitro by a subfamily of cytokines, including LIF, CNTF, GPA, OSM and cardiotrophin-1 (CT-1). To interfere with the function of these neuropoietic cytokines in vivo, antisense RNA for gp130, the common signal-transducing receptor subunit for neuropoietic cytokines, was expressed in chick sympathetic neurons, using retroviral vectors. A strong reduction in the number of VIP-expressing cells, but not of cells expressing ChAT or the adrenergic marker tyrosine hydroxylase (TH), was observed. These results reveal a physiological role of neuropoietic cytokines for the control of VIP expression during the development of cholinergic sympathetic neurons.