Maturation and maintenance of cholinergic medial septum neurons require glucocorticoid receptor signaling

Glucocorticoids have been shown to influence trophic processes in the nervous system. In particular, they seem to be important for the development of cholinergic neurons in various brain regions. Here, we applied a genetic approach to investigate the role of the glucocorticoid receptor (GR) on the maturation and maintenance of cholinergic medial septal neurons between P15 and one year of age by using a mouse model carrying a CNS-specific conditional inactivation of the GR gene (GRNesCre). The number of choline acetyltransferase and p75NTR immuno-positive neurons in the medial septum (MS) was analyzed by stereology in controls versus mutants. In addition, cholinergic fiber density, acetylcholine release and cholinergic key enzyme activity of these neurons were determined in the hippocampus. We found that in GRNesCre animals the number of medial septal cholinergic neurons was significantly reduced during development. In addition, cholinergic cell number further decreased with aging in these mutants. The functional GR gene is therefore required for the proper maturation and maintenance of medial septal cholinergic neurons. However, the loss of cholinergic neurons in the medial septum is not accompanied by a loss of functional cholinergic parameters of these neurons in their target region, the hippocampus. This pinpoints to plasticity of the septo-hippocampal system, that seems to compensate for the septal cell loss by sprouting of the remaining neurons.     

Regional Variation in γ-Aminobutyric Acid Turnover: Effect of Castration on γ-Aminobutyric Acid Turnover in Microdissected Brain Regions of the Male Rat

Abstract: This study compared the turnover of GABA neurons in different brain areas of the male rat and examined the effect of castration on GABA turnover in regions of the brain associated with the control of gonadotropin secretion. To estimate GABA turnover, GABA was quantified by HPLC in microdissected brain regions 0,30,60,90, and 120 min after inhibition of GABA degradation by aminooxyacetic acid (100 mg/kg, i.p.). GABA accumulation was linear in all areas for 90 min ( p < 0.01), and GABA turnover was estimated as the slope of the line formed by increased GABA concentration versus time, determined by linear regression. There was considerable regional variation both in the initial steady-state concentrations of GABA and in the rates of GABA turnover. Of 10 discrete brain structures, GABA turnover was highest in the medial preoptic nucleus and lowest in the caudate nucleus. Turnover times in the terminal fields of known GABAergic projection neurons ranged sevenfold, from 2.6 h in the substantia nigra to 0.4 h in the lateral vestibular nucleus. The effect of castration on GABA turnover in 13 microdissected brain regions was investigated by measuring regional GABA concentrations before and 30 min after injection of aminooxyacetic acid in intact rats or 2 or 6 days postcastration. Following castration, steady-state GABA concentrations were increased, and GABA turnover decreased in the diagonal band of Broca, the medial preoptic area, and the median eminence. GABA turnover increased in the medial septal nucleus and was unaffected in the cortex, striatum, and hindbrain. These results are consistent with the hypothesis that testosterone negative-feedback control of luteinizing hormone-releasing hormone involves steroid-sensitive GABAergic neurons in the rostral and medial basal hypothalamus.     

Cholinergic interneuron characteristics and nicotinic properties in the striatum

The neostriatum (dorsal striatum) is composed of the caudate and putamen. The ventral striatum is the ventral conjunction of the caudate and putamen that merges into and includes the nucleus accumbens and striatal portions of the olfactory tubercle. About 2% of the striatal neurons are cholinergic. Most cholinergic neurons in the central nervous system make diffuse projections that sparsely innervate relatively broad areas. In the striatum, however, the cholinergic neurons are interneurons that provide very dense local innervation. The cholinergic interneurons provide an ongoing acetylcholine (ACh) signal by firing action potentials tonically at about 5 Hz. A high concentration of acetylcholinesterase in the striatum rapidly terminates the ACh signal, and thereby minimizes desensitization of nicotinic acetylcholine receptors. Among the many muscarinic and nicotinic striatal mechanisms, the ongoing nicotinic activity potently enhances dopamine release. This process is among those in the striatum that link the two extensive and dense local arbors of the cholinergic interneurons and dopaminergic afferent fibers. During a conditioned motor task, cholinergic interneurons respond with a pause in their tonic firing. It is reasonable to hypothesize that this pause in the cholinergic activity alters action potential dependent dopamine release. The correlated response of these two broad and dense neurotransmitter systems helps to coordinate the output of the striatum, and is likely to be an important process in sensorimotor planning and learning.     

The dimethylheptyl derivative of (-)-delta 8-tetrahydrocannabinol reduces the turnover rate of gamma-aminobutyric acid in the septum and nucleus accumbens

Accumulating evidence suggests that the cannabinoids exert their action to reduce the turnover rate of acetylcholine in the hippocampus by an action in the septum via inhibitory gamma-butyric acid (GABA) containing interneurons. In the studies presented here administration of the potent dimethylheptyl derivative of (-)-delta-tetrahydrocannabinol, which has previously been shown to reduce the turnover rate of acetylcholine in the hippocampus, reduces the turnover rate of GABA in the septum. A simple model in which cannabinoids transsynaptically activate inhibitory GABAergic septal neurons impinging on cholinergic septal neurons does not explain the data. A more complex model suggesting that inhibitory GABAergic septal interneurons innervate other inhibitory GABAergic septal interneurons has been hypothesized.     

Identification of a region from the human cholinergic gene locus that targets expression of the vesicular acetylcholine transporter to a subset of neurons in the medial habenular nucleus in transgenic mice

We use a transgenic mouse model system to elucidate the regulatory regions within the human cholinergic gene locus responsible for vesicular acetylcholine transporter gene expression in vivo. In this report we characterized two transgenes for their ability to confer cholinergic-specific expression of the encoded vesicular acetylcholine transporter. An 11.2 kb transgene (named hV11.2) that spanned from about 5 kb upstream of the start of vesicular acetylcholine transporter translation down to the first choline acetyltransferase coding exon gave expression in the somatomotor neurons and a subpopulation of cholinergic neurons in the medial habenular nucleus. The second transgene (named hV6.7), a 5-prime truncated version of hV11.2 that was devoid of 4.5 kb of gene-regulatory sequences completely lacked vesicular acetylcholine transporter expression in vivo. Our data indicate that vesicular acetylcholine transporter expression in somatomotor neurons and in the medial habenular nucleus is uniquely specified within the cholinergic gene locus, and separable from cholinergic expression elsewhere. The identification of these two subdivisions of the cholinergic nervous system suggests that other cholinergic neurons in the CNS and PNS are similarly regulated by additional discrete domains within the cholinergic gene locus.     

Modulation of cerebral acetylcholine metabolism by the dorsal diencephalic conduction system in the rat

We investigated the effects of interruption of the impulse flow in the habenulopeduncular pathways by local infusion of tetrodotoxin on the acetylcholine and choline content in selected dopamine rich regions in the forebrain and midbrain in rats. The tetrodotoxin infusion caused a marked increase in acetylcholine content in the medial frontal cortex, striatum and ventral tegmental area+interpeduncular nucleus, but not in the limbic area or the substantia nigra, whereas choline content was reduced only in both the striatum and ventral tegmental area+interpeduncular nucleus. There was an increase in 3,4-dihydroxyphenylacetic acid content in the striatum after the manipulation. These findings suggest that the dorsal diencephalic conduction system may be involved in the integration of the activity of cholinergic neurons in the forebrain and midbrain regions and striatal dopanine neurons may play a role in the modulation of cholinergic neurons.     

Organization of central cholinergic neurons revealed by combined in situ hybridization histochemistry and choline-O-acetyltransferase immunocytochemistry.

Digoxigenin-labeled riboprobes and in situ hybridization of choline-O-acetyltransferase mRNA, both alone and in combination with immunohistochemical procedures for the synthetic enzyme of acetylcholine, were used to map the topography of putative cholinergic neurons in the rat central nervous system. Only the anti-sense riboprobe yielded specific labeling, which was absent in brain sections processed with sense riboprobe. Telencephalic neurons demonstrating the mRNA for choline-O-acetyltransferase and choline-O-acetyltransferase-like immunoreactivity were found in the caudate-putamen nucleus, nucleus accumbens, olfactory tubercule, Islands of Calleja complex, medial septal nucleus, vertical and horizontal limbs of the diagonal band, substantia innominata, nucleus basalis, and nucleus of the ansa lenticularis, as well as occasionally in the amygdala. Neurons in the cerebral cortex, hippocampus, and primary olfactory structures did not demonstrate hybridization signal, even though some cells in those areas were observed to exhibit choline-O-acetyltransferase-like immunopositivity. Thalamic cells were devoid of hybrido- and immunoreactivity, with the exception of several neurons located primarily in the ventral two-thirds of the medial habenula. A few cell bodies labeled with riboprobe and co-localizing choline-O-acetyltransferase-like immunopositivity were found in the lateral hypothalamus, caudal extension of the internal capsule, and zona incerta. Neurons in the pedunculopontine and laterodorsal tegmental nuclei evinced moderate hybridization signal, whereas cells of the parabigeminal nucleus were very weakly reactive. In contrast, motor neurons of the cranial nerve nuclei demonstrated high levels of choline-O-acetyltransferase mRNA and choline-O-acetyltransferase-like immunoreactivity. Putative cholinergic somata in the ventral horns and intermediolateral cell columns of the spinal cord and around the central canal were also labeled with riboprobe. It is concluded that hybridocytochemistry with digoxigenin-labeled riboprobes confirms the existence of cholinergic neurons in most of the neural regions believed to contain them on the basis of acetylcholinesterase pharmacohistochemistry and choline-O-acetyltransferase immunocytochemistry, with the prominent exceptions of the cerebral cortex, hippocampus, olfactory bulb, anterior olfactory nucleus, and caudal raphe nuclei, which apparently do not possess neurons expressing detectable levels of the mRNA for the synthetic enzyme of acetylcholine.     

大鼠基底前脑NGF-R及CHAT免疫反应阳性神经元的分布:免疫双标法

本文用免疫组化双标法观察了神经生长因子受体(NGF-R)及胆碱乙酰转移酶(ChAT)免疫反应阳性神经元在成鼠基底前脑内的分布,结果发现嗅结节、隔内侧核、斜角带核、腹侧苍白球及基底大细胞核均有NGF-R及ChAT免疫反应阳性神经元.免疫组化双标染色发现,大部分免疫反应阳性神经元的NGF-R与ChAT共存,部分神经元呈单纯NGF-R或ChAT阳性,但这种NGF-R和ChAT的共存情况在不同区域不完全相同.在隔内侧核和斜角带核,大多数的NGF-R阳性神经元和ChAT阳性神经元共存,但在腹侧仓白球和基底大细胞核,两者共存的神经元较前两区为少.此外ChAT阳性神经元在尾壳核中分布较均匀,而NGF-R阳性神经元较少见.研究结果表明,大多数胆碱能神经元有NGF-R,提示NGF对胆碱能神经元的保护和激活作用,部分可能是通过直接与NGF受体的结合而发生作用.     

Effect of specific serotonergic lesions on cholinergic neurons in the hippocampus,cortex and striatum

Local injection of 5, 7-dihydroxytryptamine into the median raphe nucleus of rats pretreated with desipramine decreases the serotonin content of the hippocampus and cortex. The turnover of acetylcholine, as measured by the rate of decline of acetylcholine content after hemicholinium-3, the rate of decline of acetylcholine content after hemicholinium-3, is not affected in the hippocampus or the striatum, but is increased in the cortex by such treatment. Local injection of 5, 7-dihydroxytryptamine into the dorsal raphe nucleus of desipramine-treated rats decreases the serotonin content of the hippocampus, cortex, and striatum. The turnover of acetylcholine is increased in the hippocampus and cortex, but not affected in the striatum. Thus, serotonergic neurons from the median raphe nucleus appear to tonically inhibit cholinergic neurons in the cortex, and serotonergic neurons from the dorsal raphe nucleus appear to tonically inhibit cholinergic neurons in the hippocampus and cortex. These serotonergic neurons do not appear to act tonically on striatal cholinergic neurons.     

Whole-Brain Mapping of Inputs to Projection Neurons and Cholinergic Interneurons in the Dorsal Striatum

The dorsal striatum integrates inputs from multiple brain areas to coordinate voluntary movements, associative plasticity, and reinforcement learning. Its projection neurons consist of the GABAergic medium spiny neurons (MSNs) that express dopamine receptor type 1 (D1) or dopamine receptor type 2 (D2). Cholinergic interneurons account for a small portion of striatal neuron populations, but they play important roles in striatal functions by synapsing onto the MSNs and other local interneurons. By combining the modified rabies virus with specific Cre- mouse lines, a recent study mapped the monosynaptic input patterns to MSNs. Because only a small number of extrastriatal neurons were labeled in the prior study, it is important to reexamine the input patterns of MSNs with higher labeling efficiency. Additionally, the whole-brain innervation pattern of cholinergic interneurons remains unknown. Using the rabies virus-based transsynaptic tracing method in this study, we comprehensively charted the brain areas that provide direct inputs to D1-MSNs, D2-MSNs, and cholinergic interneurons in the dorsal striatum. We found that both types of projection neurons and the cholinergic interneurons receive extensive inputs from discrete brain areas in the cortex, thalamus, amygdala, and other subcortical areas, several of which were not reported in the previous study. The MSNs and cholinergic interneurons share largely common inputs from areas outside the striatum. However, innervations within the dorsal striatum represent a significantly larger proportion of total inputs for cholinergic interneurons than for the MSNs. The comprehensive maps of direct inputs to striatal MSNs and cholinergic interneurons shall assist future functional dissection of the striatal circuits.     

A light and electron microscopic study of the CB1 cannabinoid receptor in monkey basal forebrain

The distribution of the CB1 cannabinoid receptor was studied in the monkey basal forebrain by immunocytochemistry and electron microscopy, using an antibody to the CB1 brain cannabinoid receptor. Large numbers of labelled neurons were observed in the medial septum, nucleus of the diagonal band, and the nucleus basalis of Meynert. The labelled neurons had dimensions similar to those of cholinergic neurons and were larger than those of GABAergic neurons. Double immunolabelling with an antibody to the synthetic enzyme for acetylcholine, choline acetyl transferase (ChAT) showed that CB1-positive neurons were also positive for ChAT, whilst electron microscopy confirmed that CB1-labelled neurons contained lipofuscin granules and dense clusters of rough endoplasmic reticulum, characteristic of cholinergic neurons. The dense labelling of cholinergic neurons for CB1 is interesting from the standpoint of neuroprotection. The CB1 receptor has been shown to couple in an inhibitory manner to voltage dependent calcium channels, and the dense labelling of CB1 in cholinergic neurons would therefore suggest that CB1 receptors could be important in limiting calcium influx through voltage dependent calcium channels in these neurons. This could serve to limit intracellular calcium concentrations, and consequent calcium mediated injury, in these neurons.     

Pallial origin of basal forebrain cholinergic neurons in the nucleus basalis of Meynert and horizontal limb of the diagonal band nucleus

The majority of the cortical cholinergic innervation implicated in attention and memory originates in the nucleus basalis of Meynert and in the horizontal limb of the diagonal band nucleus of the basal prosencephalon. Functional alterations in this system give rise to neuropsychiatric disorders as well as to the cognitive alterations described in Parkinson and Alzheimer's diseases. Despite the functional importance of these basal forebrain cholinergic neurons very little is known about their origin and development. Previous studies suggest that they originate in the medial ganglionic eminence of the telencephalic subpallium; however, our results identified Tbr1-expressing, reelin-positive neurons migrating from the ventral pallium to the subpallium that differentiate into cholinergic neurons in the basal forebrain nuclei projecting to the cortex. Experiments with Tbr1 knockout mice, which lack ventropallial structures, confirmed the pallial origin of cholinergic neurons in Meynert and horizontal diagonal band nuclei. Also, we demonstrate that Fgf8 signaling in the telencephalic midline attracts these neurons from the pallium to follow a tangential migratory route towards the basal forebrain.     

Thyroid Hormone Causes Sexually Distinct Neurochemical and Morphological Alterations in Rat Septal-Diagonal Band Neurons

Sex differences were investigated in cholinergic neurons of the septal-diagonal band region of adult rats subjected to neonatal treatment with 3,3',5-triiodo-L-thyronine (T3). Neonatal hyperthyroidism resulted in a 44% increase in specific activity of choline acetyltransferase (ChAT; EC 2.3.1.6) in adult male rat septal-diagonal band region, whereas no change in ChAT activity could be detected in either dorsal or ventral hippocampus. An increase in muscarinic cholinergic receptors, as measured by [3H]quinuclidinyl benzilate [( 3H]QNB) binding, was discovered in both septum-diagonal band and dorsal hippocampus of the T3-treated male rats. Immunohistochemistry in the septal-diagonal band region indicated a more intense staining in the neonatally T3-treated adult male rats than in controls, with larger and more abundant ChAT-positive and nerve growth factor receptor (NGF-R)-positive varicosities. ChAT immunocytochemistry showed a substantial decrease in cell body area in the medial septum and in the vertical limb of the diagonal band of T3-treated male rats, while cell density increased twofold. Female littermates subjected to the same treatment showed no changes in any of the biochemical or immunohistochemical cholinergic markers. Only in the medial septum was morphology significantly altered in the female T3-treated rats in that ChAT-positive cell body area increased. These results indicate a marked sexual variation in the septal-diagonal band region with respect to the sensitivity of postnatally developing cholinergic neurons to the actions of excess thyroid hormone.     

Acetylcholine synthesis by choline acetyltransferase of a peripheral type as demonstrated in adult rat dorsal root ganglion

pChAT is a splice variant of a peripheral type encoded alternatively by the gene for choline acetyltransferase of the common type (cChAT), the enzyme responsible for acetylcholine synthesis. Immunohistochemistry using pChAT antiserum has successfully visualized many known peripheral cholinergic cells, whereas most cChAT antibodies failed to do so. As, however, accumulating evidence indicates that pChAT expression also occurs in various non-cholinergic neurons, we examined possible acetylcholine production by pChAT in rat dorsal root ganglion as a model. The present study indicated that the ganglion neurons possessed pChAT, but never cChAT, mRNA and protein. Our detailed analysis further showed that, despite low enzyme activities of both choline acetyltransferase and acetylcholinesterase, the level of acetylcholine in the ganglion was as high as to that in various brain regions receiving cholinergic innervation. By using immunoprecipitation methods, we here provide evidence that pChAT definitely has enzyme activity enough to supply physiological concentrations of acetylcholine in the ganglion. We propose that pChAT contributes both to acetylcholine neurotransmission in physiologically identified cholinergic cells and to functions yet unknown in non-cholinergic neurons. Thus pChAT provides a new window on the role of neuronal acetylcholine.     

Neurotransmitter Release from the Medial Hyperstriatum Ventrale of the Chick Forebrain Accompanying Filial Imprinting Behavior,Measured by In Vivo Microdialysis

The imprinting behavior of chicks was quantified as a preference score (correct response ratio) achieved in a running wheel apparatus. A total of 249 chicks were exposed to an imprinting stimulus and tested for stimulus-approaching behavior. The chicks were then classified as good learners (imprinted), poor learners (non-imprinted) and a gray-zone group, those were 46%, 31% and 23% of the total chicks respectively. Using the classified chicks, the acetylcholine (ACh) and glutamate releases from the medial hyperstriatum ventrale (MHV) of the chick forebrains were determined by in vivo microdialysis. The non-imprinted chicks were used as yoked controls. Increases of ACh and glutamate released were observed in the imprinted chicks during exposure to the imprinting stimulus, whereas there were no changes in the release of these neurotransmitters in the non-imprinted chicks during the imprinting exposure. These results might be indicated that cholinergic and glutamatergic synapses which are newly formed as functioning synapses with imprinting stimulus in the MHV are involved in the performance of imprinting behavior.     

Molecular cloning and functional identification of mouse vesicular glutamate transporter 3 and its expression in subsets of novel excitatory neurons

We have cloned and functionally characterized a third isoform of a vesicular glutamate transporter (VGLUT3) expressed on synaptic vesicles that identifies a distinct glutamatergic system in the brain that is partly and selectively promiscuous with cholinergic and serotoninergic transmission. Transport activity was specific for glutamate, was H(+)-dependent, was stimulated by Cl(-) ion, and was inhibited by Rose Bengal and trypan blue. Northern analysis revealed higher mRNA levels in early postnatal development than in adult brain. Restricted patterns of mRNA expression were observed in presumed interneurons in cortex and hippocampus, and projection systems were observed in the lateral and ventrolateral hypothalamic nuclei, limbic system, and brainstem. Double in situ hybridization histochemistry for vesicular acetylcholine transporter identified VGLUT3 neurons in the striatum as cholinergic interneurons, whereas VGLUT3 mRNA and protein were absent from all other cholinergic cell groups. In the brainstem VGLUT3 mRNA was concentrated in mesopontine raphé nuclei. VGLUT3 immunoreactivity was present throughout the brain in a diffuse system of thick and thin beaded varicose fibers much less abundant than, and strictly separated from, VGLUT1 or VGLUT2 synapses. Co-existence of VGLUT3 in VMAT2-positive and tyrosine hydroxylase -negative varicosities only in the cerebral cortex and hippocampus and in subsets of tryptophan hydroxylase-positive cell bodies and processes in differentiating primary raphé neurons in vitro indicates selective and target-specific expression of the glutamatergic/serotoninergic synaptic phenotype.     

A possible mechanism of participation of dopaminergic cells and striatal cholinergic interneurons in the conditioned selection of motor activity

A possible mechanism of participation of cholinergic striatal interneurons and dopaminergic cells in conditioned selection of a certain types of motor activity is proposed. This selection is triggered by simultaneous increase in the activity of dopaminergic cells and a pause in the activity of cholinergic interneurons in response to a conditioned stimulus. This pause is promoted by activation of striatal inhibitory interneurons and action of dopamine at D2 receptors on cholinergic cells. Opposite changes in dopamine and acetylcholine concentration synergistically modulate the efficacy of corticostriatal inputs, modulation rules for the "strong" and "weak" corticostriatal inputs are opposite. Subsequent reorganization of neuronal firing in the loop cortex--basal ganglia--thalamus--cortex results in amplification of activity of the group of cortical neurons that strongly activate striatal cells, and simultaneous suppression of activity of another group of cortical neurons that weakly activate striatal cells. These changes can underlie a conditioned selection of motor activity performed with involvement of the motor cortex. As follows from the proposed model, if the time delay between conditioned and unconditioned stimuli does not exceed the latency of responses of dopaminergic and cholinergic cells (about 100 ms), conditioned selection of motor activity and learning is problematic.     

Repeated exposure to cocaine alters the modulation of mesocorticolimbic glutamate transmission by medial prefrontal cortex Group II metabotropic glutamate receptors

Repeated cocaine exposure enhances glutamatergic output from the medial prefrontal cortex to subcortical brain regions. Loss of inhibitory control of cortical pyramidal neurons may partly account for this augmented cortical glutamate output. Recent research indicated that repeated cocaine exposure reduced the ability of cortical Group II metabotropic glutamate receptors to modulate behavioral and neurochemical responses to cocaine. Thus, experiments described below examined whether repeated cocaine exposure alters metabotropic glutamate receptor regulation of mesocorticolimbic glutamatergic transmission using in vivo microdialysis. Infusion of the Group II metabotropic glutamate receptor antagonist LY341495 into the medial prefrontal cortex enhanced glutamate release in this region, the nucleus accumbens and the ventral tegmental area in sensitized animals, compared to controls, following short-term withdrawal but not after long-term withdrawal. Additional studies demonstrated that vesicular (K(+)-evoked) and non-vesicular (cystine-evoked) glutamate release in the medial prefrontal cortex was enhanced in sensitized animals, compared to controls, that resulted in part from a reduction in Group II metabotropic glutamate receptor modulation of these pools of glutamate. In summary, these findings indicate that the expression of sensitization to cocaine is correlated with an altered modulation of mesocorticolimbic glutamatergic transmission via reduction of Group II metabotropic glutamate receptor function.     

The distribution of cholinergic neurons in the human central nervous system

Choline acetyltransferase (ChAT), the enzyme responsible for the biosynthesis of acetylcholine, is presently the most specific marker for identifying cholinergic neurons in the central and peripheral nervous systems. The present article reviews immunohistochemical and in situ hybridization studies on the distribution of neurons expressing ChAT in the human central nervous system. Neurons with both immunoreactivity and in situ hybridization signals of ChAT are observed in the basal forebrain (diagonal band of Broca and nucleus basalis of Meynert), striatum (caudate nucleus, putamen and nucleus accumbens), cerebral cortex, mesopontine tegmental nuclei (pedunculopontine tegmental nucleus, laterodorsal tegmental nucleus and parabigeminal nucleus), cranial motor nuclei and spinal motor neurons. The cerebral cortex displays regional and laminal differences in the distribution of neurons with ChAT. The medial septal nucleus and medial habenular nucleus contain immunoreactive neurons for ChAT, which are devoid of ChAT mRNA signals. This is probably because there is a small number of cholinergic neurons with a low level of ChAT gene expression in these nuclei of human. Possible connections and speculated functions of these neurons are briefly summarized.