Subsets Of Cholinergic Neurons Research Articles

A role for the Erk MAPK pathway in modulating SAX-7/L1CAM-dependent locomotion in Caenorhabditis elegans.

L1CAMs are immunoglobulin cell adhesion molecules that function in nervous system development and function. Besides being associated with autism and schizophrenia spectrum disorders, impaired L1CAM function also underlies the X-linked L1 syndrome, which encompasses a group of neurological conditions, including spastic paraplegia and congenital hydrocephalus. Studies on vertebrate and invertebrate L1CAMs established conserved roles that include axon guidance, dendrite morphogenesis, synapse development, and maintenance of neural architecture. We previously identified a genetic interaction between the Caenorhabditis elegans L1CAM encoded by the sax-7 gene and RAB-3, a GTPase that functions in synaptic neurotransmission; rab-3; sax-7 mutant animals exhibit synthetic locomotion abnormalities and neuronal dysfunction. Here, we show that this synergism also occurs when loss of SAX-7 is combined with mutants of other genes encoding key players of the synaptic vesicle (SV) cycle. In contrast, sax-7 does not interact with genes that function in synaptogenesis. These findings suggest a postdevelopmental role for sax-7 in the regulation of synaptic activity. To assess this possibility, we conducted electrophysiological recordings and ultrastructural analyses at neuromuscular junctions; these analyses did not reveal obvious synaptic abnormalities. Lastly, based on a forward genetic screen for suppressors of the rab-3; sax-7 synthetic phenotypes, we determined that mutants in the ERK Mitogen-activated Protein Kinase (MAPK) pathway can suppress the rab-3; sax-7 locomotion defects. Moreover, we established that Erk signaling acts in a subset of cholinergic neurons in the head to promote coordinated locomotion. In combination, these results suggest a modulatory role for Erk MAPK in L1CAM-dependent locomotion in C. elegans.

VAChT-Cre. Fast and VAChT-Cre.Slow: postnatal expression of Cre recombinase in somatomotor neurons with different onset.

The cholinergic gene locus (CGL) consists of the genes encoding the choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT). To establish a cholinergic-specific Cre-expressing mouse, we constructed a transgene expression vector (VAChT-Cre) with 11.3 kb human CGL in which a Cre-IRES-EGFP unit was inserted in the VAChT open reading frame. The activity of Cre, whose expression was driven by the VAChT promoter, was examined by crossing a reporter mouse (CAG-CAT-Z) in which expression of LacZ is activated upon Cre-mediated recombination. Transgenic lines with the VAChT-Cre construct displayed the restricted Cre expression in a subset of cholinergic neurons in the somatomotor nuclei and medial habenular nucleus, but absent in visceromotor and other central and peripheral cholinergic neurons. Cre expression was first observed at postnatal day 7 and later detected in approximately 40-60% of somatomotor neurons. Based on the onset of Cre expression, we generated two mouse lines (two alleles; VAChT-Cre. Fast and VAChT-Cre.Slow) in which Cre expression reaches maximal levels fast and slow, respectively. The use of VAChT-Cre mice should allow us to deliver Cre to a subset of postnatal motor neurons, thereby bypassing lethality and facilitating analysis of gene function in adult motor neurons.

Abnormal chemosensory jump 6 is a positive transcriptional regulator of the cholinergic gene locus in Drosophila olfactory neurons.

Cholinergic neurons acquire their neurotransmitter phenotype, in part, by expressing the cholinergic gene locus. Previous studies have indicated that the 5' flanking DNA of the locus contains both positive and negative regulatory elements important for expression in different subsets of cholinergic neurons in Drosophila and other animals. Approximately 300 bases of proximal 5' flanking DNA control expression in Drosophila CNS neurons essential for viability, whereas more distal regulatory elements are important for expression in PNS sensory neurons. In this study we identify the POU domain transcription factor abnormal chemosensory jump 6 (Acj6) as a necessary positive transcriptional regulator for cholinergic locus expression in primary olfactory neurons. Choline acetyltransferase enzyme activity, protein levels, mRNA, and a fluorescent cholinergic reporter gene are all decreased in olfactory neurons of acj6 mutants. Decreased cholinergic expression was observed in both adults and larvae. The presence of a specific Acj6 binding site has been identified in the cholinergic locus 5' flanking DNA, suggesting that Acj6 may play a direct role in specifying the cholinergic neurotransmitter phenotype of most olfactory neurons. Transgenic expression of two different isoforms of Acj6 restricted to olfactory neurons indicates that additional trans factors may be required for cholinergic locus expression. Transgenic expression in all cholinergic neurons, however, results in lethality when a POU IV box element is absent but is essentially benign when present, indicating the importance of this motif in specifying different functional roles for Acj6.

Abnormal Chemosensory Jump 6 Is a Positive Transcriptional Regulator of the Cholinergic Gene Locus inDrosophilaOlfactory Neurons

Cholinergic neurons acquire their neurotransmitter phenotype, in part, by expressing the cholinergic gene locus. Previous studies have indicated that the 5′ flanking DNA of the locus contains both positive and negative regulatory elements important for expression in different subsets of cholinergic neurons in <i>Drosophila</i> and other animals. Approximately 300 bases of proximal 5′ flanking DNA control expression in <i>Drosophila</i> CNS neurons essential for viability, whereas more distal regulatory elements are important for expression in PNS sensory neurons. In this study we identify the POU domain transcription factor abnormal chemosensory jump 6 (Acj6) as a necessary positive transcriptional regulator for cholinergic locus expression in primary olfactory neurons. Choline acetyltransferase enzyme activity, protein levels, mRNA, and a fluorescent cholinergic reporter gene are all decreased in olfactory neurons of<i>acj6</i> mutants. Decreased cholinergic expression was observed in both adults and larvae. The presence of a specific Acj6 binding site has been identified in the cholinergic locus 5′ flanking DNA, suggesting that Acj6 may play a direct role in specifying the cholinergic neurotransmitter phenotype of most olfactory neurons. Transgenic expression of two different isoforms of Acj6 restricted to olfactory neurons indicates that additional <i>trans</i>factors may be required for cholinergic locus expression. Transgenic expression in all cholinergic neurons, however, results in lethality when a POU IV box element is absent but is essentially benign when present, indicating the importance of this motif in specifying different functional roles for Acj6.

Nitric-oxide-induced cGMP synthesis in cholinergic neurons in the rat brain.

Nitric oxide (NO)-mediated cGMP synthesis is localized throughout the rat brain in close proximity to the NO-synthase-containing structures. However, characterization of the cGMP synthesizing structures in terms of co-localization with the classical neurotransmitter systems has not yet been reported. Here we present evidence, using double immunostaining for cGMP and the vesicular acetylcholine transporter, that virtually all of the cholinergic fibers in the cerebral cortex and the majority of the cholinergic fibers in the basal ganglia accumulate cGMP in response to a NO donor. In these areas, only few cGMP-containing fibers were observed not to be part of the cholinergic system. Co-localization between cGMP and the vesicular acetylcholine transporter was only observed to a minor degree in the ventral forebrain, the hippocampus, the reticular thalamic nucleus, and the nucleus ambiguus. No association of cGMP synthesis with the cholinergic system was observed to a similar extent in other brain areas. These results, in combination with literature data on the distribution of cholinergic receptors in the rat brain, suggest that NO has an anterograde and/or retrograde signaling function on subsets of cholinergic neurons.

Galanin regulates the postnatal survival of a subset of basal forebrain cholinergic neurons.

The neuropeptide galanin colocalizes with choline acetyltransferase, the synthetic enzyme for acetylcholine, in a subset of cholinergic neurons in the basal forebrain of rodents. Chronic intracerebroventricular infusion of nerve growth factor induces a 3- to 4-fold increase in galanin gene expression in these neurons. Here we report the loss of a third of cholinergic neurons in the medial septum and vertical limb diagonal band of the basal forebrain of adult mice carrying a targeted loss-of-function mutation in the galanin gene. These deficits are associated with a 2-fold increase in the number of apoptotic cells in the forebrain at postnatal day seven. This loss is associated with marked age-dependent deficits in stimulated acetylcholine release, performance in the Morris water maze, and induction of long-term potentiation in the CA1 region of the hippocampus. These data provide unexpected evidence that galanin plays a trophic role to regulate the development and function of a subset of septohippocampal cholinergic neurons.

Discharge Properties of Juxtacellularly Labeled and Immunohistochemically Identified Cholinergic Basal Forebrain Neurons Recorded in Association with the Electroencephalogram in Anesthetized Rats

Multiple lines of evidence indicate that cholinergic basal forebrain neurons play an important role in the regulation of cortical activity and state. However, the discharge properties of cholinergic cells in relation to the electroencephalogram (EEG) are not yet known. In the present study, cells were recorded in the basal forebrain in association with cortical EEG activity in urethane-anesthetized rats, and their discharge was examined during EEG irregular slow activity and during stimulation-induced cortical activation, characterized by rhythmic slow (theta) and high-frequency (gamma) activities. Recorded cells were labeled with Neurobiotin (Nb), using the juxtacellular technique and identified as cholinergic by immunohistochemical staining for choline acetyltransferase (ChAT). Nb-positive/ChAT-positive neurons were distinctive and significantly different from Nb-positive/ChAT-negative neurons, which were heterogeneous in their discharge properties. All Nb(+)/ChAT(+) cells increased their discharge rate with stimulation, and most shifted from an irregular tonic discharge during EEG slow irregular activity to a rhythmic burst discharge during rhythmic slow activity. The stimulation-induced rhythmic discharge was cross-correlated with the EEG rhythmic slow activity. In some units the rhythmic discharge matched the rhythmic slow activity of the retrosplenial cortex; in others, it matched that of the prefrontal cortex, which occurred at a slower frequency, suggesting that subsets of cholinergic neurons may influence their cortical target areas rhythmically at particular frequencies. Cholinergic basal forebrain neurons thus may evoke and enhance cortical activation via both an increase in rate and a change in pattern to rhythmic bursting that would stimulate rhythmic slow (theta-like) activity in cortical fields during active waking and paradoxical sleep states.

Nitric oxide synthase in ventral forebrain grafts and in early ventral forebrain development

Embryonic ventral forebrain (VFB) grafts to cortex contain neurons that synthesize acetylcholine and partially ameliorate behavioral deficits caused by excitotoxic damage to the nucleus basalis magnocelullaris in rats. An additional neurotransmitter, nitric oxide (NO), is synthesized by a subset of cholinergic neurons in rat ventral forebrain. If this neurotransmitter is expressed also by grafted cholinergic neurons (which include the embryonic medial septum and diagonal band), its functional contribution should be considered. Six to twelve months after transplantation of embryonic VFB tissue rats were sacrificed. Brain tissue was processed either for in situ hybridization of nNOS and neuropeptide Y (NPY) or for immunohistochemistry of choline acetyltransferase (ChAT) and neuronal nitric oxide synthase (nNOS). Quantification of messenger ribonucleic acid (mRNA) for nNOS was performed with radioactively labeled probes (silver grains were counted) and a preliminary comparison was made of graft sections to sections of the ventral forebrain of developing rats. Plots of silver grain counts against cell size revealed similar patterns in the grafts and in the ventral forebrain of developing rats. The rates of expression of mRNA for nNOS in the grafts were intermediate between those of the ventral forebrain of postnatal day 19 and those of postnatal day 12. Double immunohistochemical labeling revealed that 45.87+8.26% of cells expressing ChAT also expressed nNOS in the grafts, significantly higher than 33.16+3.9% which was the rate of co-expression observed in the adult ventral forebrain. This study suggests that possible contribution of NO to graft-associated modulation of behavior should be examined. © 1997 Elsevier Science B.V. All rights reserved.

Immunocytochemical study of choline acetyltransferase in Drosophila melanogaster: an analysis of cis-regulatory regions controlling expression in the brain of cDNA-transformed flies.

We have analyzed the cis-regulatory regions in the 5' flanking DNA of the Drosophila melanogaster choline acetyltransferase (ChAT; E.C. 2.3.1.6) gene by using germline transformants. These transformants are carrying wild-type ChAT cDNA fused to different lengths of 5' flanking sequence of the ChAT gene. Appropriate genetic crosses were used to introduce the transgene into animals with a presumptive null genetic background for endogenous ChAT. Expression of ChAT protein could thus be attributed exclusively to the transgene. Using a monoclonal antibody against Drosophila ChAT, we have investigated the spatial distribution of transgenic ChAT and compared it to the normal distribution of ChAT protein in wild-type animals. The brains of 7.4 kb cDNA transformants showed a ChAT expression pattern similar to that of wild-type animals in the first- and second-order sensory neuropil but reduced expression in other highly ordered neuropil. Several lines that were transformed with 1.2 kb or 0.8 kb of 5' flanking DNA demonstrated relatively normal expression in sensory neuropil. In addition, these lines also showed ectopic expression in higher order neuropil. In the optic lobe, the expression pattern directed by 7.4 kb of 5' flanking DNA was very similar to that of wild-type ChAT expression. In contrast, 1.2 kb or 0.8 kb transformants showed reduced levels of expression and a more limited pattern of distribution in the optic lobe. Our results suggest that the 5' flanking DNA of the ChAT gene can be divided into several separable positive and negative regulatory regions, which define various subsets of cholinergic neurons in the nervous system.

Analysis of cis-regulatory elements in the 5' flanking region of the Drosophila melanogaster choline acetyltransferase gene

We have analyzed the cis-regulatory elements in the 5' flanking region of the Drosophila choline acetyltransferase gene (ChAT, E.C.2.3.1.6). DNA fragments were fused to the Escherichia coli lacZ reporter gene and introduced into the Drosophila germ line by P-element-mediated transformation. A 7.4 kb 5' flanking sequence directed beta-galactosidase expression in the adult optic lobes and other well-defined CNS structures with a pattern very similar to the distribution of endogenous ChAT protein. In contrast, the proximal 3.3 kb and 1.2 kb of 5' flanking DNA directed lacZ expression in only selected subsets of the structures seen with the 7.4 kb lacZ construct. Our results indicate that both qualitative and quantitative regulatory elements are present in the 5' flanking DNA and that these elements distinguish various subsets of cholinergic neurons. We have also fused the same 5' flanking DNA sequences to wild-type ChAT cDNA and used these constructs to transform Chatsl mutant flies. Not only the 7.4 kb cDNA construct, but also the 3.3 and 1.2 kb constructs, rescued Chatsl from temperature-dependent paralysis and adult lethality, indicating that the regulatory information in any of these genomic fragments can drive sufficient wild-type ChAT expression to overcome these mutant phenotypes.

Embryonic development of four different subsets of cholinergic neurons in rat cervical spinal cord

The developmental stage at which a neuron becomes committed to a neurotransmitter phenotype is an important time in its ontogenetic history. The present study examines when choline acetyltransferase (ChAT) is first detected within each of four different subsets of cholinergic neurons previously identified in the cervical enlargement of the spinal cord: namely, motor neurons, partition cells, central canal cluster cells, and dorsal horn neurons. By examining the temporal sequence of embryonic development of these cholinergic neurons, we can infer the relationships between ChAT expression and other important developmental events. ChAT was first detected reliably on embryonic day 13 (E13) by both biochemical and immunocytochemical methods, and it was localized predominantly within motor neurons. A second group of primitive-appearing ChAT-positive cells was detected adjacent to the ventricular zone on E14. These neurons seemed to disperse laterally into the intermediate zone by E15, and, on the basis of their location, were tentatively identified as partition cells. A third group of primitive ChAT-immunoreactive cells was detected on E16, both within and around the ventral half of the ventricular zone. By E17, some members of this "U"-shaped group appeared to have dispersed dorsally and laterally, probably giving rise to dorsal horn neurons as well as dorsal central canal cluster cells. Other members of this group remained near the ventral ventricular zone, most likely differentiating into ventral central canal cluster cells. Combined findings from the present study and a previous investigation of neurogenesis (Phelps et al.: J. Comp. Neurol. 273:459-472, '88), suggest that premitotic precursor cells have not yet acquired the cholinergic phenotype because ChAT is not detectable until after the onset of neuronal generation for each of the respective subsets of cholinergic neurons. However, ChAT is expressed in primitive bipolar neurons located within or adjacent to the germinal epithelium. Transitional stages of embryonic development suggest that these primitive ChAT-positive cells migrate to different locations within the intermediate zone to differentiate into the various subsets of mature cholinergic neurons. Therefore, it seems likely that spinal cholinergic neurons are committed to the cholinergic phenotype at pre- or early migratory stages of their development. Our results also hint that the subsets of cholinergic cells may follow different migration routes. For example, presumptive partition cells may use radial glial processes for guidance, whereas dorsal horn neurons may migrate along nerve fibers of the commissural pathway. Cell-cell interactions along such diverse migratory pathways could play a role in determining the different morphological, and presumably functional, phenotypes expressed by spinal cholinergic neurons.