Delayed NGF infusion fails to reverse axotomy-induced degeneration of basal forebrain cholinergic neurons in adult p75(LNTR)-deficient mice

The p75 low-affinity neurotrophin receptor (p75^LNTR) appears to have various functions that include enhancing nerve growth factor (NGF)-mediated survival by increasing TrkA (high-affinity NGF receptor) efficiency, and mediating apoptosis by acting as a ligand-regulated pro-apoptotic receptor. Here, we investigated the role of p75^LNTR for adult cholinergic basal forebrain neurons by comparing neuronal responses to injury in control and p75^LNTR-deficient mice. In both types of mice, 70% of the cholinergic neurons in the ipsilateral medial septum had lost their markers choline acetyltransferase and tyrosine kinase A by 28 days following unilateral transection of the dorsal septohippocampal pathway (fimbria fornix). A 7-day delayed infusion of NGF that started 28 days after the injury resulted in reversal of choline acetyltransferase expression and cell atrophy in control, but not in p75^LNTR-deficient, mice. This lack of response to delayed NGF treatment in p75^LNTR-deficient mice was most likely not due to cell death, as all of the septohippocampal neurons, labeled with Fluorogold before the lesion, were present at 28 days post-lesion, similar to control mice. p75^LNTR-deficient cholinergic neurons can respond to NGF as they were protected by NGF infusions that started immediately after the injury. These observations, the fact that lesioned p75^LNTR-deficient neurons atrophy faster, and that non-lesioned neurons hypertrophy in response to NGF in control but not in p75^LNTR-deficient mice, suggest that p75^LNTR is needed for tyrosine kinase A and NGF signaling efficiency.

In conclusion, during adulthood p75^LNTR appears to play a beneficial role in the response of cholinergic neurons to injury, consistent with the proposed role of p75^LNTR in the enhancement of TrkA signaling and the transport of neurotrophins by these neurons.     

Cholinergic medial septum neurons do not degenerate in aged 129/Sv control or p75(NGFR)-/-mice

Cholinergic medial septum neurons express TrkA and p75 nerve growth factor receptor (p75(NGFR)) and interactions between TrkA and p75(NGFR) are necessary for high-affinity binding and signaling of nerve growth factor (NGF) through TrkA. In adult p75(NGFR)-deficient (-/-) mice, retrograde transport of NGF and other neurotrophins by these neurons is greatly reduced, however, these neurons maintain their cholinergic phenotype and size. Reduced transport of NGF has been proposed to play a role in Alzheimer's disease. Here, we investigated whether chronic and long-term absence of p75(NGFR) (and possibly reduced NGF transport and TrkA binding) would affect the cholinergic septohippocampal system during aging in mice. In young (6-8 months), middle aged (12-18 months), and aged (19-23 months) 129/Sv control mice the total number of choline acetyltransferase-positive medial septum neurons and the mean diameter and cross sectional area of the cholinergic cell bodies were similar. The cholinergic hippocampal innervation, as measured by the density of acetylcholinesterase-positive fibers in the outer molecular layer of the dentate gyrus was also similar across all ages. These parameters also did not change during aging in p75(NGFR) -/- mice and the number and size of the choline acetyltransferase-positive neurons and the cholinergic innervation density were largely similar as in control mice at all ages. These results suggest that p75(NGFR) does not play a major role in the maintenance of the number or morphology of the cholinergic basal forebrain neurons during aging of these mice. Alternatively, p75(NGFR) -/- mice may have developed compensatory mechanisms in response to the absence of p75(NGFR).     

不同年龄大鼠斜角带核水平支神经元内TrkA和ChAT的表达——免疫组织化学研究

目的 了解大鼠基底前脑斜角带核水平支神经元内酪氨酸激酶Ａ（ｔｙｒｏｓｉｎｅ ｋｉｎａｓｅ,Ａ,ＴｒｋＡ）、胆碱乙酰化转移酶（ｃｈｏｌｉｎｅ ａｃｅｔｙｌｔｒａｎｓｆｅｒａｓｅ,ＣｈＡＴ）样阳性神经元的生后发育规律及两乾的相互关系。方法 用免疫组织化学方法结合图像分析仪检测大鼠基底前脑斜角带核水平支ＴｒｋＡ、ＣｈＡＴ样阳性神经元的数量、面积和灰度值。结果 ＴｒｋＡ、ＣｈＡＴ分布于基底前脑神经元。生后１ｄ可见ＴｒｋＡ表达,但生后５ｄ才出现ＣｈＡＴ表达。生后２０ｄＴｒｋＡ、ＣｈＡＴ表达至高峰;生后３０ｄ下调,成年时维持相对较高水平。老年鼠ＴｒｋＡ、ＣｈＡＴ样阳性神经元出现萎缩、数量也分别减少３９．７％、３３．３％;胞体平均面积分别减少１５．７％、１２．８％;平均灰度值分别减少２９．９％、９．９％。同时,不同年龄大鼠Ｔ     

Selective disarrangement of the rostral telencephalic cholinergic system in heterozygous reeler mice

Reelin (RELN) is a key molecule for the regulation of neuronal migration in the developing CNS. The reeler mice, which have spontaneous autosomal recessive mutation in the RELN gene, reveal multiple defects in brain development. Morphological, neurochemical and behavioral alterations have been detected in heterozygous reeler (HR) mice, suggesting that not only the presence, but also the level of RELN influences brain development. Several studies implicate an involvement of RELN in the pathophysiology of neuropsychiatric disorders in which an alteration of the cholinergic cortical pathways is implicated as well. Thus, we decided to investigate whether the basal forebrain (BF) cholinergic system is altered in HR mice by examining cholinergic markers at the level of both cell body and nerve terminals. In septal and rostral, but not caudal, basal forebrain region, HR mice exhibited a significant reduction in the number of choline acetyltransferase (ChAT) immunoreactive (ir) cell bodies compared with control mice. Instead, an increase in ChAT ir neurons was detected in lateral striatum. This suggests that an alteration in ChAT ir cell migration which leads to a redistribution of cholinergic neurons in subcortical forebrain regions occurs in HR mice. The reduction of ChAT ir neurons in the BF was paralleled by an alteration of cortical cholinergic nerve terminals. In particular, the HR mice presented a marked reduction of acetylcholinesterase (AChE) staining accompanied by a small reduction of cortical thickness in the rostral dorsomedial cortex, while the density of AChE staining was not altered in the lateral and ventral cortices. Present results show that the cholinergic basalo-cortical system is markedly, though selectively, impaired in HR mice. Rostral sub-regions of the BF and rostro-medial cortical areas show significant decreases of cholinergic neurons and innervation, respectively.     

Amelioration of cholinergic neurons dysfunction in aged rats depends on the continuous supply of NGF

The present study was designed to examine whether NGF-induced improvement in morphology of senile basal forebrain cholinergic neurons persist after discontinuation of NGF treatment. Trophic effect of continuous intraventricular infusion of NGF was tested in the 4- and 28 months old male Wistar rats immediately after cessation of NGF and 3 or 6 weeks after termination of treatment. Immunohistochemical procedure for ChAT, TrkA, and p75(NTR) receptor has been applied to identify cholinergic cells in the basal forebrain structures. Using the quantitative image analyzer, morphometric and densitometric parameters of cholinergic cells were measured. In untreated 28-month-old rats a reduction in the number, size and intensity of staining of cholinergic neurons was observed in all basal forebrain structures. NGF significantly improved morphological parameters of ChAT- and TrkA-positive cells in aged rats. In 28-month-old rats tested within 3 and 6 weeks after discontinuation of infusion a renewed progressive deterioration of cholinergic phenotype of basal forebrain neurons was observed when compared with the NGF-treated immediately tested group. The parallel staining for p75(NTR) revealed normal morphology of the basal forebrain neurons, despite of the age of rats or the NGF treatment. Analysis of Nissl stained sections also showed that 28-month-old rats did not display significant losses of neurons in the basal forebrain when compared with the young animals. These findings demonstrate that senile impairment of cholinergic neurons is induced by a loss of cholinergic phenotype rather than an acute degeneration of cell bodies. NGF may be beneficial in enhancing cholinergic neurochemical parameters, but the protective effects seem to be dependent on the continuous supply of NGF.     

Neurotrophic effect of brain-derived neurotrophic factor on basal forebrain cholinergic neurons in culture from postnatal rats.

We examined the effect of brain-derived neurotrophic factor (BDNF) on cholinergic neurons in culture from postnatal rat basal forebrain by assay of choline acetyltransferase (ChAT) activity and cytochemical staining for acetylcholinesterase (AChE). BDNF was found to increase the ChAT activities but failed to promote the survival of AChE-positive neurons in cultures from neonatal (P3) rats, suggesting that its main role is cholinergic differentiation. In contrast, an enhancement of the survival of AChE-positive neurons and of ChAT activity was observed in cultures from P15-16 rats, suggesting that BDNF's main action is the maintenance of cholinergic neurons. Our results indicate a similarity between BDNF and nerve growth factor effects on the responses of cholinergic neurons of postnatal rat basal forebrain in culture.     

In vivo MRI identifies cholinergic circuitry deficits in a Down syndrome model

In vivo quantitative magnetic resonance imaging (MRI) was employed to detect brain pathology and map its distribution within control, disomic mice (2N) and in Ts65Dn and Ts1Cje trisomy mice with features of human Down syndrome (DS). In Ts65Dn, but not Ts1Cje mice, transverse proton spin–spin (T₂) relaxation time was selectively reduced in the medial septal nucleus (MSN) and in brain regions that receive cholinergic innervation from the MSN, including the hippocampus, cingulate cortex, and retrosplenial cortex. Basal forebrain cholinergic neurons (BFCNs) in the MSN, identified by choline acetyltransferase (ChAT) and nerve growth factor receptors p75^NTR and TrkA immunolabeling were reduced in Ts65Dn brains and in situ acetylcholinesterase (AChE) activity was depleted distally along projecting cholinergic fibers, and selectively on pre- and postsynaptic profiles in these target areas. T₂ effects were negligible in Ts1Cje mice that are diploid for App and lack BFCN neuropathology, consistent with the suspected relationship of this pathology to increased App dosage. These results establish the utility of quantitative MRI in vivo for identifying Alzheimer's disease-relevant cholinergic changes in animal models of DS and characterizing the selective vulnerability of cholinergic neuron subpopulations.     

Short-term consequences of N-methyl-D-aspartate excitotoxicity in rat magnocellular nucleus basalis: effects on in vivo labelling of cholinergic neurons.

Cholinergic neurons of the basal forebrain form one of the neuron populations that are susceptible to excitotoxic injury. Whereas neuropharmacological studies have aimed at rescuing cholinergic neurons from acute excitotoxic attacks, the short-term temporal profile of excitotoxic damage to cholinergic nerve cells remains largely elusive. The effects of N-methyl-D-aspartate (NMDA) infusion on cytochemical markers of cholinergic neurons in rat magnocellular nucleus basalis were therefore determined 4, 24 and 48 h post-lesion. Additionally, the influence of excitotoxic damage on the efficacy of in vivo labelling of cholinergic neurons with carbocyanine 3-192IgG was investigated. Carbocyanine 3-192IgG was unilaterally injected in the lateral ventricle. Twenty-four hours later, NMDA (60 nM/microl) was infused in the right magnocellular nucleus basalis, while control lesions were performed contralaterally. Triple immunofluorescence labelling for carbocyanine 3-192IgG, NMDA receptor 2A and B subunits and choline-acetyltransferase (ChAT) was employed to determine temporal changes in NMDA receptor immunoreactivity on cholinergic neurons. The extent of neuronal degeneration was studied by staining with Fluoro-Jade. Moreover, changes in the numbers of ChAT or p75 low-affinity neurotrophin receptor immunoreactive neurons, and the degree of their co-labelling with carbocyanine 3-192IgG were determined in basal forebrain nuclei. The effects of NMDA-induced lesions on cortical projections of cholinergic nucleus basalis neurons were studied by acetylcholinesterase (AChE) histochemistry. Characteristic signs of cellular damage, as indicated by decreased immunoreactivity for NMDA receptors, ChAT and p75 low-affinity neurotrophin receptors, were already detected at the shortest post-lesion interval investigated. Fluoro-Jade at 4 h post-lesion only labelled the core of the excitotoxic lesion. Longer survival led to enhanced Fluoro-Jade staining, and to the decline of ChAT immunoreactivity reaching a maximum 24 h post-surgery. Significant loss of p75 low-affinity neurotrophin receptor immunoreactivity and of cortical AChE-positive projections only became apparent 48 h post-lesion. Carbocyanine 3-192IgG labelling in the ipsilateral basal forebrain exceeded that of the contralateral hemisphere at all time points investigated and progressively declined in the damaged magnocellular nucleus basalis up to 48 h after NMDA infusion.The present study indicates that excitotoxic lesion-induced alteration of cholinergic neuronal markers is a rapid and gradual process reaching its maximum 24 h post-surgery. Furthermore, in vivo labelling of cholinergic neurons may be applied to indicate neuronal survival under pathological conditions, and enable to follow their degeneration process under a variety of experimental conditions.     

大鼠Meynert基底核TrkA和ChAT的表达

本实验用免疫组织化学方法研究了 Trk A在大鼠 Meynert基底核的分布及 Trk A、Ch AT免疫反应神经元的生后发育及两者表达的相互关系。用图像分析仪检测 Meynert基底核 Trk A-、Ch AT-IR神经元的数量、截面积和灰度值。结果表明 :　Tr-k A、Ch AT分布于 Meynert基底核神经元。生后 1d可见 Trk A表达 ,但未见 Ch AT表达 ;生后 5 d方出现 Ch AT表达 ;生后 2 0 dTrk A、Ch AT表达达高峰 ;生后 3 0 d下降 ,到成年时维持相对较高水平。老龄鼠 Trk A-、Ch AT-IR神经元萎缩 ,数量分别减少 41.3 8%和 5 1.61% ,胞体平均截面积分别减少 3 9.4%和 3 0 .4% ,平均灰度值分别减少 11.8%和 9.9%。大鼠 Trk A-、Ch AT-IR神经元截面积呈正相关。大鼠 Meynert基底核神经元 Trk A表达早于 Ch AT表达 ,从生后 5 d开始 ,Trk A、Ch AT表达有相似的时间模式。 Trk A可能参与 Meynert基底核胆碱能神经元的发育、分化、成熟和老化。老龄鼠 Trk A、Ch AT表达下降可能是老年动物和老年性痴呆病人基底前脑胆碱能神经元变性的易感性增加的原因之一     

Expression of dopamine D2 receptor and choline acetyltransferase mRNA in the dopamine deafferented rat caudate-putamen

Summary In situ hybridization was used to study dopamine D2 receptor (D2R) and choline acetyltransferase (ChAT) mRNA expression in neurons of the rat forebrain, both on control animals and after a unilateral 6-hydroxydopamine (6-OHDA) lesion of midbrain dopamine neurons. D2R mRNA expressing neurons were seen in regions which are known to be heavily innervated by midbrain dopamine fibers such as caudate-putamen, nucleus accumbens and olfactory tubercle. ChAT mRNA expressing neurons were seen in caudate-putamen, nucleus accumbens and septal regions including vertical limb of the diagonal band. In caudate-putamen, approximately 55% of the medium sized neurons, which is the predominating neuronal cell-size in this region, were specifically labeled with the D2R probe. In addition, approximately 95% of the large size neurons in caudate-putamen were specifically labeled with both the D2R and ChAT probes, suggesting that most cholinergic neurons in the caudate-putamen express D2R mRNA. After a unilateral lesion of midbrain dopamine neurons, no change in the level of either D2R or ChAT mRNA were seen in the large size intrinsic cholinergic neurons in caudate-putamen. Similarily, no evidence was obtained for altered levels of D2R mRNA in medium size neurons in medial caudate-putamen, or nucleus accumbens. However, an increase in the number of medium size neurons expressing D2R mRNA was observed in the lateral part of the dopamine deafferented caudateputamen. Thus, it appears that midbrain dopamine deafferentation causes an increase in D2R mRNA expression in a subpopulation of medium size neurons in the lateral caudate-putamen.     

GABAergic circuits mediate the reinforcement-related signals of striatal cholinergic interneurons

Neostriatal cholinergic interneurons are believed to be important for reinforcement-mediated learning and response selection by signaling the occurrence and motivational value of behaviorally relevant stimuli through precisely timed multiphasic population responses. An important problem is to understand how these signals regulate the functioning of the neostriatum. Here we describe the synaptic organization of a previously unknown circuit that involves direct nicotinic excitation of several classes of GABAergic interneurons, including neuroptide Y-expressing neurogilaform neurons, and enables cholinergic interneurons to exert rapid inhibitory control of the activity of projection neurons. We also found that, in vivo, the dominant effect of an optogenetically reproduced pause-excitation population response of cholinergic interneurons was powerful and rapid inhibition of the firing of projection neurons that is coincident with synchronous cholinergic activation. These results reveal a previously unknown circuit mechanism that transmits reinforcement-related information of ChAT interneurons in the mouse neostriatal network.     

The p75 neurotrophin receptor and neuronal apoptosis

Although evidence continues to accumulate for the apoptosis-inducing role of the p75 neurotrophin receptor, several outstanding questions remain. One of these concerns the signal transduction pathway of p75, which continues to be elusive. The evidence for the roles of ceramide, c-jun kinase and NF-kappaB is discussed: none of these are able to account satisfactorily for p75 death signalling. Negative modulation of Trk signalling by p75 could account for part of the pro-apoptotic effect, but is unlikely to be a major component. Although recent evidence indicates that the juxtamembrane region is critical for causing cell death, p75 has a well-conserved death domain. This may be important for functions other than killing. In glial cells and some neurons that express p75 but not TrkA, p75 causes cell death in response to nerve growth factor (NGF) binding. In sensory neurons and PC12 cells, p75 appears to signal constitutively. In cholinergic forebrain neurons, p75 expression leads to atrophy and downregulation of cholinergic markers, rather than cell death. The major challenges in p75 research are to define its signalling pathways, and particularly the intracellular proteins with which it interacts. Another major challenge is to develop a model that reconciles the different facets of p75, such as its ability in some situations to assist TrkA to rescue NGF-dependent neurons, but to stimulate apoptosis in others.     

Immunohistochemical staining of cholinergic neurons in the human brain using a polyclonal antibody to human choline acetyltransferase

Antibodies against human placental choline acetyltransferase (ChAT) were used to immunohistochemically stain cholinergic neurons in the neostriatum and nucleus basalis of Meynert in human brain. Cells in both regions were intensely stained as were nerve fibers. Comparable cells were stained in these same brain regions in the rat. This anti-human ChAT antibody will enable the further detailed characterization of cholinergic neurons in the human brain in both health and disease.     

Targeted silencing of TrkA expression in rat forebrain neurons via the p75 receptor

Basal forebrain neurons express the neurotrophin receptors, p75NTR and tyrosine kinase receptor A (TrkA). We tested the hypothesis that impairment of memory in rats could be achieved by RNA interference (RNAi) -induced silencing of TrkA specifically within these neurons. A novel fusogenic, karyophilic immunoporter (fkAb(p75)-ipr) was constructed from the antibody, MC192 (monoclonal antibody to the rat neurotrophin receptor p75NTR, Ab(p75)), poly-l-lysine together with the hemagglutinin 2 and VP1 nuclear localization peptides of influenza and SV40 virus, respectively. Plasmid DNA constructs containing short hairpin sequences inhibitory to tyrosine kinase receptor A expression (TrkAi) and the gene encoding cGFP (green fluorescent protein from coral fish) was produced. These TrkAi plasmids were mixed with the immunoporter, forming the immunogene, TrkAi-fkAb(p75). A control TrkAsc complexed with fkAb(p75) (TrkAsc-fkAb(p75)) immunogene was constructed from a scrambled sequence (TrkAsc) and fkAb(p75)-ipr. Rats were infused using an osmotic mini-pump into the third ventricle with either TrkAi-fkAb(p75) or TrkAsc-fkAb(p75). Naive rats were also included as additional controls. After 7 days, examination of gene expression on forebrain sections of some rats revealed cGFP expression in TrkA neurons. Fifteen to 19 days after infusion, rats were tested in a Morris water maze apparatus. Animals that received TrkAi-fkAb(p75) showed significantly impaired spatial memory learning ability compared with naive or TrkAsc-fkAb(p75)-treated rats. Western blot and immunofluorescence analysis showed that TrkA protein levels and numbers of TrkA positive neurons were reduced by 60% and 55% respectively in TrkAi-fkAb(p75)-infused rats compared with infused controls or naive animals. We conclude that p75-receptor-mediated RNAi-induced silencing of genes offers a novel and powerful way to study the function of specific endogenous genes within distinct neuronal subpopulations of the brain.     

Nicotine increases the expression of neurotrophin receptor tyrosine kinase receptor A in basal forebrain cholinergic neurons

In this study, we investigated whether the potential positive effects of nicotine in Alzheimer's disease (AD) may involve neurotrophic factors, such as nerve growth factor (NGF), closely associated with basal forebrain (BF) cholinergic function and survival. To this aim, we studied the effects of prolonged nicotine treatment on neurotrophin receptors expression and on NGF protein levels in the rat BF cholinergic circuitry. Both in vivo and in vitro experiments were conducted. We found that s.c. nicotine infusion (1.2 mg free base/kg/d delivered by mini-pumps for 7 days) induced in vivo an increase in tyrosine kinase receptor A (TrkA)—but not TrkB, TrkC or low affinity neurotrophin receptor p75 (p75)—expression in BF cholinergic neurons targeting the cerebral cortex. Nicotine did not produce statistically significant long-lasting effects on NGF levels in the cerebral cortex, or in the BF. In vitro experiments performed on primary BF neuronal cultures, showed that 72 h exposure to nicotine increased both TrkA expression, and NGF release in culture medium. Neutralization experiments with an anti-NGF antibody showed that NGF presence was not necessary for nicotine-induced increase of TrkA levels in cultured cholinergic neurons, suggesting that nicotine may act through NGF-independent mechanisms. This study shows that nicotine, independently of its action on NGF levels, may contribute to the restoration of the trophic support to BF cholinergic neurons by increasing TrkA levels.     

Co-expression of the P75 neurotrophin receptor and neurotrophin receptor-interacting melanoma antigen homolog in the mature rat brain

The p75 neurotrophin receptor (p75(NTR)) is involved in the regulation of neuronal survival and phenotype, but its signal transduction mechanisms are poorly understood. Recent evidence has implicated the cytoplasmic protein NRAGE (neurotrophin receptor-interacting MAGE (from Melanoma AntiGEn) homolog) in p75(NTR) signaling. To gain further insight into the role of NRAGE, we investigated the co-expression of NRAGE and p75(NTR) in mature rat brain. In all areas examined, NRAGE appeared to be confined to neurons. In the basal forebrain cholinergic complex, NRAGE immunoreactivity was evident in all p75(NTR)-positive neurons. There were many more NRAGE-positive than p75(NTR)-positive neurons in these regions, however. NRAGE was also expressed in areas of the basal forebrain that did not express p75(NTR), including the lateral septal nucleus and the nucleus accumbens. A finding in marked contrast to previous studies was the presence of p75(NTR) immunoreactivity in neuronal cell bodies in the hippocampus. Hippocampal p75(NTR) immunoreactivity was apparent in rats 6 months and older, and was localized to the dentate gyrus and stratum oriens. All p75(NTR)-positive neurons in the dentate gyrus and hippocampal formation were positive for NRAGE. The majority of granular cells of the dentate gyrus and pyramidal cells in the hippocampal formation were positive for NRAGE and negative for p75(NTR). NRAGE was also present in some neuronal populations that express p75(NTR) after injury, including striatal cholinergic interneurons, and motor neurons. A region of marked disparity was the cerebral cortex, in which NRAGE immunoreactivity was widespread whereas p75(NTR) was absent. The results are consistent with an important role for NRAGE in p75(NTR) signaling, as all cells that expressed p75(NTR) also expressed NRAGE. The wider distribution of NRAGE expression suggests that NRAGE may also participate in other signaling processes.     

P75 nerve growth factor receptor is important for retrograde transport of neurotrophins in adult cholinergic basal forebrain neurons

The role of the p75 nerve growth factor receptor in the retrograde transport of neurotrophins in the adult CNS was investigated by comparing the transport of 125I-labeled neurotrophins by normal and p75 nerve growth factor receptor-deficient cholinergic septohippocampal neurons. In control mice, nerve growth factor was selectively transported from the hippocampal formation to the cholinergic neurons in the septum. Nerve growth factor labeling was found in three to four times as many septal cholinergic neuronal cell bodies than labeling for neurotrophin-3 or neurotrophin-4/5, and transported brain-derived neurotrophic factor was barely detectable. Cells were considered as labeled when the number of grains per cell exceeded five times background. In p75 nerve growth factor receptor-deficient mice, the number of cholinergic neurons labeled with each of the neurotrophins was reduced by 85-95%. Retrograde labeling of septohippocampal neurons with Fluorogold was not obviously reduced in p75 nerve growth factor receptor-deficient mice, suggesting that general transport mechanisms were not impaired. Despite the reduced neurotrophin transport, cholinergic neurons of p75 nerve growth factor receptor-deficient mice were larger than controls and had an apparently normal density of immunostaining for choline acetyltransferase. Since nerve growth factor is reportedly involved in size regulation and choline acetyltransferase expression, this raises the possibility that the retrograde transport itself is not essential for these events. Thus, p75 nerve growth factor receptor plays an important, although not exclusive, role in the transport of neurotrophins by cholinergic basal forebrain neurons, and retrograde transport of nerve growth factor may not be needed for regulating certain cellular processes.     

Distribution and co-localization of choline acetyltransferase and p75 neurotrophin receptors in the sheep basal forebrain: implications for the use of a specific cholinergic immunotoxin

The basal forebrain cholinergic system is involved in different forms of memory. To study its role in social memory in sheep, an immunotoxin, ME20.4 immunoglobulin G (IgG)-saporin, was developed that is specific to basal forebrain cholinergic neurons bearing the p75 neurotrophin receptor. The distribution of sheep cholinergic neurons was mapped with an antibody against choline acetyltransferase. To assess the localization of the p75 receptor on basal forebrain cholinergic neurons, the distribution of p75 receptor-immunoreactive neurons with ME20.4 IgG was examined, and a double-labeling study with antibodies against choline acetyltransferase and p75 receptor was undertaken. The loss of basal forebrain cholinergic neurons and acetylcholinesterase fibers in basal forebrain projection areas was assessed in ewes that had received intracerebroventricular injections of the immunotoxin (50, 100 or 150 microg) alone, as well as, in some of the ewes treated with the highest dose, with bilateral immunotoxin injections in the nucleus basalis (11 microg/side). Results indicated that choline acetyltransferase- and p75 receptor-immunoreactive cells had similar distributions in the medial septum, the vertical and horizontal limbs of the band of Broca, and the nucleus basalis. The double-labeling procedure revealed that 100% of the cholinergic neurons are also p75 receptor positive in the medial septum and in the vertical and horizontal limbs of the band of Broca, and 82% in the nucleus basalis. Moreover, 100% of the p75 receptor-immunoreactive cells of these four nuclei were cholinergic. Combined immunotoxin injections into ventricles and the nucleus basalis produced a near complete loss (80-95%) of basal forebrain cholinergic neurons and acetylcholinesterase-positive fibers in the hippocampus, olfactory bulb and entorhinal cortex. This study provides the first anatomical data concerning the basal forebrain cholinergic system in ungulates. The availability of a selective cholinergic immunotoxin effective in sheep provides a new tool to probe the involvement of basal forebrain cholinergic neurons in cognitive processes in this species.     

Development of the basal forebrain cholinergic system: phenotype expression prior to target innervation.

We measured choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities in the rat to determine the time course of development, maturity, and senescence of ChAT activity. Tissue was obtained from Sprague-Dawley rats ranging in age from embryonic day 14 through 23 months. Seven regions were examined, including the magnocellular preoptic/substantia innominata region, frontal cortex, medial septal region, hippocampus, diagnoal band, and medial and lateral striatum. ChAT and AChE activities were first detected as early as E18 in the medial septum, diagonal band and magnocellular preoptic area, all regions of cholinergic cell bodies. Enzyme activity subsequently developed in terminal fields of these cholinergic perikarya (hippocampus and frontal cortex) as well as in the striatum. For all regions, enzyme activity rose during the first four postnatal weeks. This increase in enzyme activity was transient and, in most instances, decreases were observed between postnatal days 30 and 60. Most dramatic were the decreases in enzyme activity in the magnocellular preoptic/substantia innominata and diagonal band regions. Age-related declines also occurred in the frontal cortex, hippocampus, magnocellular preoptic/substantia innominata region, and the striatum. Cholinergic systems undergo dynamic changes especially during development and adulthood.