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.     

Configuration of the magnocellular nuclei in the basal forebrain of the human adult

With the use of gapless series of thick sections (800 microns) stained for lipofuscin pigment as a stable intraneuronal characteristic, it was found in the human brain that the three magnocellular nuclei in the basal forebrain-the medial septal nucleus, the nucleus of the diagonal band (vertical and horizontal limb) and the basal nucleus of Meynert located within the substantia innominata-were tightly connected with each other. A band-like anteromedial subnucleus and a semilunar posterolateral subnucleus could be delineated in the basal nucleus and were found to be embedded in a zone of low cell density, the substantia innominata, which medially blends into the horizontal limb of the diagonal band nucleus. A three-dimensional reconstruction was made to demonstrate the complex shape of the magnocellular basal forebrain nuclei.     

Levels of nerve growth factor and its mRNA in the central nervous system of the rat correlate with cholinergic innervation.

The levels of nerve growth factor (NGF) and its mRNA in the rat central nervous system were determined by two-site enzyme immunoassay and quantitative Northern blots, respectively. Relatively high NGF levels (0.4-1.4 ng NGF/g wet weight) were found both in the regions innervated by the magnocellular cholinergic neurons of the basal forebrain (hippocampus, olfactory bulb, neocortex) and in the regions containing the cell bodies of these neurons (septum, nucleus of the diagonal band of Broca, nucleus basalis of Meynert). Comparatively low, but significant NGF levels (0.07-0.21 ng NGF/g wet weight) were found in various other brain regions. mRNANGF was found in the hippocampus and cortex but not in the septum. This suggests that magnocellular cholinergic neurons of the basal forebrain are supplied with NGF via retrograde axonal transport from their fields of innervation. These results, taken together with those of previous studies showing that these neurons are responsive to NGF, support the concept that NGF acts as trophic factor for magnocellular cholinergic neurons.     

Estimation of the total number of cholinergic neurons containing estrogen receptor-alpha in the rat basal forebrain.

This study was undertaken to estimate the total number of cholinergic cells and the percentage of cholinergic cells that contain estrogen receptor-alpha (ER alpha) in the rat basal forebrain. Double immunostaining for choline acetyltransferase (ChAT) and ER alpha was carried out on 50-microm-thick free-floating sections. Because routine mounting method causes considerable flattening of the sections, we embedded immunostained sections in Durcupan, an epoxy resin known to cause virtually no shrinkage. When this procedure was used the section thickness was well preserved, individual cells could be clearly identified, and subcellular localization of ER alpha immunoreactivity was easy to verify. Cell counting in these sections revealed that the rat basal forebrain contains 26,390 +/- 1097 (mean +/- SEM) cholinergic neurons. This comprises 9674 +/- 504 in the medial septum-vertical diagonal band of Broca, 9403 +/- 484 in the horizontal diagonal band of Broca, and 7312 +/- 281 in the nucleus basalis. In these nuclei, 60%, 46%, and 14% of the cholinergic neurons were co-localized with ER alpha, respectively. We believe that our results are an improvement on existing data because of the better distinction of individual neurons that the Durcupan embedding method brings.     

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.     

Differential co-expression of AMPA receptor subunits in substance P receptor-containing neurons of basal forebrain regions of C57/BL mice

We are interested in cellular co-expression patterns of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptor subunits 1-4 (GluR1-4) in substance P receptor (SPR)-containing neurons of the basal forebrain, which may act as a morphological basis for interaction between neurokinins and glutamate-driven neuronal signaling and excitotoxicity. Immunohistochemistry and laser scanning confocal microscopy in adult C57/BL mice revealed that distribution of SPR-positive neurons overlapped with that of GluR1-4-containing ones in most basal forebrain regions, i.e. the medial septal nucleus, nucleus of diagonal band of Broca, magnocellular preoptic nucleus and substantia innominata. Neurons showing both SPR and GluR1-4-immunoreactivities were found in above cholinergic neurons-rich containing basal forebrain regions. Semi-quantification analysis indicated that about 57-95% of SPR-positive neurons displayed GluR1-4-immunoreactivity. The percentages of AMPA receptor subunits co-localizing in SPR-positive neurons were GluR4 (48%), GluR1 (47%), GluR2 (26%) and GluR3 (20%), respectively. However, the neurons co-expressing SPR and GluR1-4 were hardly detected in the basal nucleus of Meynert of the basal forebrain. The co-localization of SPR and AMPA receptors has provided a molecular basis for functional interaction between neurokinins and AMPA receptors-mediated signaling in basal forebrain neurons. This study has also implied that glutamate-driven neuronal transmission and excitotoxicity can be modulated by neurokinin peptides in most basal forebrain regions but not in the basal nucleus of Meynert, suggesting that neurokinins or SP may play certain roles in determining neuronal functional properties or excitotoxic susceptibility in the various basal forebrain regions of mammals.     

Effects of estrogens on cholinergic neurons in the rat basal nucleus

Estrogen replacement in postmenopausal women may help prevent or delay development of Alzheimer's disease. Because loss of basal forebrain cholinergic neurons with reductions in choline acetyltransferase (ChAT) concentration are associated with Alzheimer's disease, we investigated the effect of estradiol (E(2)) and J 861, a non-feminizing estrogen, on cholinergic neurons in the basal forebrain. Ovariectomized rats received E(2), J 861 or vehicle, and basal forebrain sections through the substantia innominata, medial septum, and nucleus of the diagonal band were immunostained for ChAT. ChAT-immunoreactive cells in the basal forebrain were significantly reduced in the ovariectomized rats compared to intact rats, but those ovariectomized rats receiving estrogen replacement with E(2) and J 861 had near normal levels of ChAT-positive neurons. While retrograde tracing experiments with fluorogold injected into the prefrontal cortex showed no significant differences in the number of fluorogold-labeled cells among the groups, ChAT-immunoreactive cells and double-labeled cells were significantly lower in OVX rats than in intact and E(2) rats. Some substantia innominata cells in the J 861 rats were ChAT/estrogen receptor alpha-positive. These results suggest that E(2) and J 861 have positive effects on cholinergic neurons that project from the basal nucleus to the forebrain cortex.     

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.     

Expression of neuronal-NOS in developing basal forebrain cholinergic neurons: Regulation by NGF

Nerve growth factor (NGF) acts through the receptor tyrosine kinase trkA to serve as a trophic factor for cholinergic neurons in the medial septal nucleus and vertical limb of the diagonal band. We have previously shown that the neuronal isoform of nitric oxide synthase (NOS) is selectively expressed in a large fraction of trkA-expressing cholinergic neurons in these brain regions in the adult rat, and that NGF induces the expression of neuronal-NOS in these cells. Herein, we show that: 1) neuronal-NOS is also localized to these neurons in the developing septum; 2) the expression of neuronal-NOS is regulated in the developing medial septal nucleus and vertical limb of the diagonal band; 3) neuronal-NOS regulation parallels that for other markers of basal forebrain cholinergic neuron differentiation, such as cholineacetyltransferase; and 4) NGF infusion in the postnatal period induces robust increases in neuronal-NOS mRNA and in NOS activity in the basal forebrain. Taken together with earlier findings, our results suggest that neuronal-NOS has a role in the differentiation and mature function of septal cholinergic neurons. Through enhancing neuronal-NOS synthesis, endogenous NGF is likely to regulate NO functions in vivo. Special issue dedicated to Dr. Hans Thoenen.     

Alpha-adrenergic receptor (α2A ) is colocalized in basal forebrain cholinergic neurons: A light and electron microscopic double immunolabeling study

A variety of data suggest that noradrenaline and acetylcholine may interact in the basal forebrain, however no morphological studies have addressed whether indeed cholinergic neurons express adrenergic receptors. We have investigated the presence of alpha-adrenergic receptor subtype α_2A -AR in cholinergic neurons of the basal forebrain. Cholinergic neurons were identified with an antibody against choline acetyltransferase and the receptor with a polyclonal antibody raised against a 47 amino acid fragment of the third intracellular loop of the α_2A -AR. For double labeling at the light microscopic level the Ni-DAB/DAB technique was used, and for electron microscopy an immunoperoxidase/immunogold method was applied. We detected the α_2A -AR protein in cholinergic as well as in non-cholinergic neurons. Almost half of all cholinergic neurons contained this adrenergic receptor. Double-labeled neurons were distributed throughout the rostro-caudal extent of the basal forebrain cholinergic continuum, including the medial septum, vertical and horizontal diagonal band nuclei, pallidal regions, substantia innominata and the internal capsule. Non-cholinergic neurons that expressed the α_2A -AR outnumbered cholinergic/α_2A -AR neurons by several factors. Electron microscopy confirmed the presence of α_2A -AR in cholinergic neurons in the medial septum, vertical and horizontal diagonal band nuclei. Gold particles (10 nm) indicative of α_2A -AR were diffusely distributed in the cytoplasm and accumulated in cytoplasmic areas near the Golgi complex and cysterns of the endoplasmic reticulum and were associated with the cellular membranes at synaptic and non-synaptic locations. Since many of the α_2A -AR+/non-cholinergic neurons we detected are likely to be GABAergic cells, our data support the hypothesis that noradrenaline may act via basal forebrain cholinergic and non-cholinergic neurons to influence cortical activity.     

猫基底前脑胆碱能皮质投射神经元区的P物质样免疫反应神经元

应用免疫组织化学和邻位切片法,研究了猫基底前脑胆碱能皮质投射神经元区的Ｐ物质样免疫反应神经元的分布特征,及其与胆碱乙酰化酶样免疫反应神经元的分布关系,从内侧隔核至Ｍｅｙｎｅｒｔ基底核,２种神经元分布范围相近,且形态,大小相似。但在邻位切片,未见分别呈此二免疫反应的同一神经元的对应剖面。Ｐ物质样免疫反应神经元在内侧隔核和斜角带核数量较多,但在基底核明显减少,在Ｃｈ４间质部及腹侧苍白球连合下部仅为偶见     

Telencephalic sources of the afferents of the main olfactory bulb in the frog Rana temporaria

Following horseradish peroxidase iontophoretic application into the main olfactory bulb (MOB) retrograde neuronal labeling was examined in the telencephalon in the frog. Labeled neurons, the sources of the MOB afferents are found in the mitral cell layer of the contralateral MOB, pallial and some subpallial areas. Very heavy labeling is observed in the pars ventralis of the lateral pallium, and to a lesser extent in the medial pallium, pars dorsalis of the lateral pallium and in the dorsal pallium. In subpallium labeled neurons are found in the eminentia postolfactoria, the rostral part of the medial septal nucleus, and in the nucleus of the ventro-medial telencephalic wall, which is probably homologous to the nucleus of the diagonal band (Broca) of mammals. No labelled neurons were found in the caudal portion of the MOB granular layer, usually referred to as the anterior olfactory nucleus. The arrangement of the MOB centrifugal innervation in amphibians is discussed in comparison with that in mammals.     

Neuronal activity in the nucleus basalis magnocellularis during performance of an instrumental task

Spike activity of 95 neurons in the rabbit basal forebrain forebrain magnocellular nucleus was recorded during spontaneous behavior and instrumental conditioned performance. Almost half of the neurons (48.4%) displayed a significant (p > 0.05) negative correlation between their spontaneous discharge rate and the power of the delta rhythm in the EEG of the frontal cortex; most of these cells can be classified as projection cholinergic neurons. During instrumental conditioned performance, neurons of this subgroup responded with excitation to the conditioned stimulus, whereas presumably noncholinergic nonprojection neurons responded to the conditioned stimulus with inhibition. Excitatory response of cells in the basal forebrain magnocellular nucleus was significantly more intense as compared to trials without the conditioned reaction. On the whole, our data testify that the basal forebrain magnocellular nucleus maintains the level of arousal and attention required for the instrumental conditioned performance.     

Age-related changes of hypocretin in basal forebrain of guinea pig

Hypocretin-1 (hcrt-1) and hypocretin-2 (hcrt-2) have been implicated in a wide variety of functions including sleep and wakefulness as well as related behaviors. Many of these functions of the hypocretins involve the activation of cholinergic neurons in the basal forebrain (BF). These neurons have been shown to exhibit age-related changes in a variety of species. In the present experiment, in adult and aged guinea pigs, we compared hypocretin immunoreactivity in regions of the BF that include the medial septal nucleus (MS), the vertical and horizontal limbs of the diagonal band of Broca (VDB and HDB) and the magocellular preoptic nucleus (MCPO). In adult guinea pigs (3–5 months of age), all of the preceding BF regions contained dense hypocretin fibers with varicosities. On the contrary, in old guinea pigs (27–28 months), although the MS exhibited a similar intensity of hypocretin immunoreactivity compared with the adult guinea pig, there was a significant decrease in the intensity of immunoreactivity of hypocretinergic fibers in the VDB, HDB and MCPO. These data indicate that the hypocretinergic innervation of specific nuclei of the BF is compromised during the aging process. We suggest that the reduction in hypocretinergic innervation of the BF nuclei may contribute to the age-related changes in the states of sleep and wakefulness as well as deficits in related systems that occur in old age.     

NGF induction of NGF receptor gene expression and cholinergic neuronal hypertrophy within the basal forebrain of the adult rat

Chronic infusion of nerve growth factor (NGF) into the forebrain of the adult rat produced increases in NGF receptor (NGF-R) mRNA hybridization, NGF-R immunoreactivity, choline acetyltransferase (ChAT) mRNA hybridization, and neuronal hypertrophy, when compared with vehicle infusion or noninfused rat brain. In situ hybridization showed NGF induction of NGF-R gene expression, documented by increases in the number of NGF-R mRNA-positive cells within the medial septum, diagonal band, and nucleus basalis magnocellularis. NGF also produced hypertrophy of ChAT mRNA-positive neurons. These results suggest that NGF produces cholinergic neuronal hypertrophy through induction of NGF-R gene expression within the basal forebrain.     

Neuropeptide FF2 receptor distribution in the human brain. An immunohistochemical study

Human neuropeptide FF2 (hFF2) receptor has been postulated to mediate central autonomic regulation by virtue of its ability to bind with high affinity to many amidated neuropeptides. In the present immunohistochemical study, we identified hFF2 positive neurons in the forebrain and medulla oblongata of individuals, who died suddenly of mechanical trauma or hypothermia. Morphologically, these neurons demonstrated features identified with both projection neurons and interneurons. In the forebrain, the highest density of hFF2 expressing neurons was observed in the anterior amygdaloid area and dorsomedial hypothalamic nucleus, especially in its caudal part. A lesser density of hFF2 neurons was identified in the ventromedial hypothalamic nucleus, lateral and posterior hypothalamic areas whereas few cells were visualized in the paraventricular hypothalamic nucleus, perifornical nucleus, horizontal limb of the diagonal band, ventral division of the bed nucleus of the stria terminalis, nucleus basalis of Meynert and ventral tegmental area. In the medulla, significant numbers of hFF2 neurons were observed in the dorsal motor nucleus of vagus and to a lesser extent in the area of catecholaminergic cell groups, A1/C1. These data provide first immunohistochemical evidence of hFF2 localization in the human brain, which is consistent with that reported for tissue distribution of FF2 mRNA and FF2 binding sites within the brain of a variety of mammalian species. The distribution of hFF2 may help in identifying the role of amidated neuropeptides in the human brain within the context of central autonomic and neuroendocrine regulation.     

Ethylcholine mustard aziridinium ion lesions of the rat cortex result in retrograde degeneration of basal forebrain cholinergic neurons: Implications for animal models of neurodegenerative disease

Multiple injections of 2 nmols of cyclised ethylcholine mustard aziridinium ion (ECMA), a putative cholinergic neurotoxin, were made (unilaterally) into the cortical terminal field of cholinergic neurons projecting from the nucleus basalis of Meynert (NBM) in the rat basal forebrain. After 30 days, choline acetyltransferase enzymatic activity, a marker for cholinergic function, was significantly lowered in both ipsilateral cortex and NBM, and cholinergic cell bodies in the latter reduced in cross-sectional area, a spectrum of effects characteristic of retrograde degeneration of this pathway. These results are discussed in the context of neurodegenerative diseases affecting cholinergic function.     

Localization of nerve growth factor receptor mRNA in the rat basal forebrain within situ hybridization histochemistry

1. In situ hybridization histochemistry was used to localize nerve growth factor receptor (NGFR) mRNA in the adult rat basal forebrain. 2. In emulsion-dipped sections 35S-labeled RNA antisense probes produced a high density of silver grains over cells located in the medial septum, vertical and horizontal limbs of the diagonal band of Broca, and nucleus basalis. 3. This distribution of NGFR mRNA overlaps with the distribution of NGFR protein localized using immunocytochemical techniques. 4. No hybridization signal was detected when sections were hybridized with a 35S-labeled RNA sense (control) probe. 5. We suggest that NGFRs are synthesized in these basal forebrain nuclei and transported to terminal areas where NGF is thought to be bound and internalized, an initial step in the many actions of this neurotrophic factor.     

A light and electron microscopic study of the GABA transporter GAT-3 in the monkey basal ganglia and brainstem

The present study aimed to elucidate the distribution of the GABA transporter GAT-3 in the monkey basal ganglia and brainstem. Very dense GAT-3 immunoreactivity was observed in the medial septum, diagonal band, basal nucleus of Meynert, thalamus, globus pallidus, and substantia nigra. Moderate levels were observed in the subthalamic nucleus, periaqueductal grey, spinal trigeminal and vestibular nuclei. A general light level of staining was observed in the remainder of the brainstem regions, and very light staining was observed in the caudate nucleus and putamen. Electron microscopy showed that GAT-3 immunoreactivity was present in cell bodies with light cytoplasm and dense bundles of glial filaments, and features of astrocytes. Large numbers of astrocytic processes were also labeled in the neuropil. The cell bodies and processes of neurons were unlabeled. Further study is necessary to elucidate GAT-3 expression in neurological conditions, including hyperalgesia and Parkinson's disease.