Cholinergic neurons contain Calbindin-D28k in the monkey medial septal nucleus and nucleus of the diagonal band: an immunocytochemical study

The distribution patterns of choline acetyltransferase (CAT), as a marker for cholinergic neurons, and Calbindin-D_28k (CaBP) immunoreactivities in the forebrain basal ganglia of the Japanese monkeyMacaca fuscata were compared. Similar distribution patterns of CAT and CaBP immunoreactivities were found in the medial septal nucleus (MS) and the nucleus of the diagonal band of Broca (DBB). Double-labeling fluorescence immunocytochemistry revealed that most, but not all, cholinergic neurons were CaBP-immunoreactive in the MS and DBB. The results suggest that CaBP may play a role in the septohippocampal cholinergic neuron system of the monkey.     

Basal Forebrain Cholinergic Neurons Selectively Drive Coordinated Motor Learning in Mice

Motor control requires precise temporal and spatial encoding across distinct motor centers that is refined through the repetition of learning. The recruitment of motor regions requires modulatory input to shape circuit activity. Here, we identify a role for the baso-cortical cholinergic pathway in the acquisition of a coordinated motor skill in mice. Targeted depletion of basal forebrain cholinergic neurons results in significant impairments in training on the rotarod task of coordinated movement. Cholinergic neuromodulation is required during training sessions as chemogenetic inactivation of cholinergic neurons also impairs task acquisition. Rotarod learning is known to drive refinement of corticostriatal neurons arising in both medial prefrontal cortex (mPFC) and motor cortex, and we have found that cholinergic input to both motor regions is required for task acquisition. Critically, the effects of cholinergic neuromodulation are restricted to the acquisition stage, as depletion of basal forebrain cholinergic neurons after learning does not affect task execution. Our results indicate a critical role for cholinergic neuromodulation of distant cortical motor centers during coordinated motor learning.SIGNIFICANCE STATEMENT Acetylcholine release from basal forebrain cholinergic neuron terminals rapidly modulates neuronal excitability, circuit dynamics, and cortical coding; all processes required for processing complex sensory information, cognition, and attention. We found that depletion or transient silencing of cholinergic inputs to anatomically isolated motor areas, medial prefrontal cortex (mPFC) and motor cortex, selectively led to significant impairments on coordinated motor learning; disrupting this baso-cortical network after acquisition elicited no effect on task execution. Our results indicate a pivotal role for cholinergic neuromodulation of distant cortical motor centers during coordinated motor learning. These findings support the concept that cognitive components (such as attention) are indispensable in the adjustment of motor output and training-induced improvements in motor performance.     

弥漫性脑损伤诱发的前脑胆碱能神经元变化

目的 本实验通过实验性研究,以了解弥漫性脑损伤（ＤＢＩ）是否引起胆碱能神经元的显性减少。方法 采用Ｍａｒｍａｒｏｕ打击装置建立ＤＢＩ动物模型,行胆碱酯酶组织化学染色以显示基底前胆碱能神经元。结果 ①基底前脑胆碱能神经元在重伤组、轻伤组、对照组有显差异（Ｐ〈０．０１）;②损伤后二周组神经元减少与一周组比较有显差异（Ｐ〈０．０１或Ｐ〈０．０５）。结论 ①在本实验所观察到的ＤＢＩ后基底前脑胆碱能神     

Age-Related Vulnerability of Developing Cholinergic Basal Forebrain Neurons Following Excitotoxic Lesions of the Hippocampus

Previous studies have demonstrated that depleting the hippocampus of endogenous neurotrophins via excitotoxic lesions fails to alter the viability of adult cholinergic septal/diagonal band neurons. Since cholinergic basal forebrain neurons may be more vulnerable during development, we investigated whether excitotoxic lesions produced in neonatal animals alter the viability of these cells. Postnatal Day 7, 10, 14, and 28 rats pups received unilateral intrahippocampal injections of ibotenic acid and were sacrificed 4 weeks later. At 7, 10, and 14 days of age, significant reductions in the number of choline acetyltransferase (ChAT)- and p75 nerve growth factor receptor (NGFr)-immunoreactive neurons were observed within the medial septum ipsilateral to the hippocampal lesion. In contrast, rats receiving similar lesions on Day 28 failed to display a significant reduction in ChAT-immunoreactive medial septal neurons. The magnitude of ChAT-immunoreactive neuronal loss within the medial septum and the age at which the lesion was made were inversely correlated (r² = 0.887), indicating that cholinergic septal neurons become less vulnerable to target removal as the cells develop. Similar results were observed in the vertical limb of the diagonal band although a small but significant loss of ChAT-immunoreactive neurons was seen in this structure ipsilateral to the hippocampal lesion when lesions were performed on Postnatal Day 28. At all age groups, many remaining cholinergic septal/diagonal band neurons appeared dystrophic with stunted fiber outgrowth. The present study demonstrates that unlike adult rats, removal of hippocampal target neurons during development alters the viability and morphology of cholinergic neurons of the medial septum and diagonal band. This suggests that target neurons which synthesize endogenous neurotrophins are needed for normal development of cholinergic basal forebrain neurons, but may not be required for the normal maintenance of the adult cell.     

Cyto- and Chemoarchitecture of Basal Forebrain Cholinergic Neurons in the Common Marmoset (Callithrix Jacchus)

The cyto- and chemoarchitecture of basal forebrain cholinergic neurons (BFCN) was investigated in the lower primate, the common marmoset (Callithrix jacchus). A large population of magnocellular, hyperchromic, and choline acetyltransferase (ChAT)-positive neurons was detected in the marmoset basal forebrain. The distribution of these neurons was similar to those in higher primates. Thus, ChAT-positive neurons were observed in the medial septum (Ch2), the vertical (Ch2) and horizontal (Ch3) limbs of the diagonal band of Broca, and the nucleus basalis of Meynert (Ch4). The Ch4 complex was relatively well differentiated and displayed distinct sectors. We detected anterior (Ch4a, with a medial and a lateral subdivision), intermediate (Ch4i, with a dorsal and a ventral subdivision), and posterior (Ch4p) sectors in the marmoset Ch4. The Ch4i was relatively small while the Ch4p was large. Similar to the rodent, the marmoset Ch1 extended quite a distance posteriorly, and the Ch4p displayed a major interstitial component distributed within the globus pallidus, its medullary laminae, and the internal capsule. Virtually all of the marmoset BFCN displayed acetylcholinesterase activity, and low affinity (p75^NTR) and high affinity (Trk) neurotrophin receptor immunoreactivity. A majority contained immunoreactivity for calbindin-D_28K and calretinin. Many of the Ch4 neurons also displayed tyrosine hydroxylase immunoreactivity. The BFCN lacked galanin immunoreactivity, but were innervated by galanin-positive fibers. None of the marmoset BFCN were NADPH-d-positive. Thus, the BFCN display major anatomical and biochemical differences in the marmoset when compared with higher primates. The marmoset BFCN also display many characteristics common to other primates. This fact, combined with the relatively short life span of the marmoset, indicates that this species may be ideal for studies of age-related changes in the BFCN.     

BDNF Is Needed for Postnatal Maturation of Basal Forebrain and Neostriatum Cholinergic Neurons In Vivo

Neurotrophins regulate survival, neurite outgrowth, and phenotypic maturation of developing neurons. Brain-derived neurotrophic factor (BDNF) can promote the survival of developing cholinergic forebrain neurons in vitro and reduce their degeneration following injury in adult rats. We investigated the role of endogenous BDNF during postnatal development of these cholinergic neurons by analyzing homozygous BDNF-deficient (−/−) mice and their littermates (+/+, +/−). At P6, the number of choline acetyltransferase- (ChAT) positive neurons in the medial septum was 23% lower in BDNF−/− mice, although their brain and body weight was normal. At P15, control (+/+) littermates had 45% more and 45% larger ChAT-positive neurons and a much denser cholinergic hippocampal innervation than at P6, indicative of maturation of the septohippocampal system. In BDNF−/− mice, the number, size, and ChAT-immunostaining intensity of the cholinergic neurons remained the same between P6 and P15 (few mice survive longer). BDNF−/− mice had about three times more TUNEL-labeled (a marker of apoptosis) cells in the medial septum at P6, consistent with (but not proof of) the possibility that the cholinergic neurons were dying. The cholinergic hippocampal innervation in BDNF−/− mice expanded to a lesser extent than in controls and had reduced levels of acetylcholinesterase staining at P15. The developmental deficits were largely similar in the neostriatum of BDNF−/− mice. These findings suggest that BDNF is critical for postnatal development and maturation of cholinergic forebrain neurons.     

Impairment of Basal Forebrain Cholinergic Neurons Associated with Aging and Long-Term Loss of Ovarian Function

Recent studies suggest that women are at greater risk for Alzheimer's disease than men and that estrogen replacement can help to reduce the risk and severity of Alzheimer's-related dementia in postmenopausal women. We have hypothesized that the increased risk for Alzheimer's-related dementia is due, in part, to the loss of ovarian function in postmenopausal women and to the effects that decreased levels of ovarian hormones have on basal forebrain cholinergic function. In the present study, the effects of aging and ovariectomy on cholinergic neurons in the rat basal forebrain were examined to determine (1) whether aging differentially affects cholinergic neurons in the basal forebrain of males vs females, and (2) whether long-term loss of ovarian function produces deficits in basal forebrain cholinergic function beyond those associated with aging and sex. In part I of the study, gonadally intact male and female rats were sacrificed at 13, 19, and 25 months of age and the effects of aging on cholinergic neurons in the medial septum (MS) and nucleus basalis magnocellularis (NBM) were compared. In part II of the study, female rats were ovariectomized at 13 months of age and then sacrificed 3 and 6 months later along with gonadally intact, age-matched controls. Adjacent sections through the MS and NBM were processed for either immunocytochemical detection of choline acetyltransferase (ChAT) and p75NTR-like immunoreactivity or forin situhybridization detection and quantification of ChAT and trkA mRNA. Results from part I revealed no significant effects of age on the relative size or density of cholinergic neurons in the MS and NBM of gonadally intact animals. Likewise, no significant effects on the relative numbers of cholinergic neurons expressing p75NTR protein were detected. However, a significant decrease in trkA mRNA was detected in the MS of gonadally intact females, but not males, between 13 and 25 months of age. No significant effects of aging on ChAT mRNA were detected. Results from part II revealed significant decreases in both ChAT and trkA mRNA in the MS and NBM of female rats sacrificed 6 months, but not 3 months, following ovariectomy relative to age-matched, gonadally intact controls. Short-term estrogen replacement initiated 6 months following ovariectomy and administered for 3 days prior to sacrifice partially restored ChAT mRNA levels in the MS and trkA mRNA levels in the NBM. These findings suggest that ovarian hormones play a role in maintaining normal levels of ChAT and trkA expression in the MS and NBM. The fact that ChAT mRNA was decreased in the MS and NBM at 6 months following ovariectomy suggests that long-term loss of ovarian function produces a decrease in the functional status of basal forebrain cholinergic neurons projecting to the hippocampus and cortex. In addition, we hypothesize that the decreases in trkA mRNA detected both in the MS as a function of aging, and in the MS and NBM in response to ovariectomy, reflect decreases in the production of high affinity nerve growth factor (NGF) receptors, and a decrease in the responsiveness of the cholinergic neurons to endogenous NGF. This, in turn, may increase the susceptibility of the cholinergic neurons to the effects of aging and disease and thereby contribute to basal forebrain cholinergic decline. We conclude that long-term loss of ovarian function combined with aging has a negative impact on basal forebrain cholinergic neurons. These effects may contribute to the risk and severity of cognitive decline associated with aging and Alzheimer's disease in postmenopausal women.     

Loss of Developing Cholinergic Basal Forebrain Neurons Following Excitotoxic Lesions of the Hippocampus: Rescue by Neurotrophins

Previous studies have demonstrated that the viability of developing cholinergic basal forebrain neurons is dependent upon the integrity of neurotrophin-secreting target cells. In the present study, we examined whether infusions of nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF) could prevent the loss of cholinergic septal/diagonal band neurons following excitotoxic lesions of their target neurons within the hippocampus. Postnatal Day 10 rat pups received unilateral intrahippocampal injections of ibotenic acid. Rats then received intracerebroventricular (icv) injections of nerve growth factor (30 μg/injection), brain-derived neurotrophic factor (60 μg/injection), or saline immediately following the lesion and continuing every third day for 27 days. Both saline- and BDNF-treated rats displayed a significant loss of septal/diagonal band neurons expressing the protein and mRNA for choline acetyltransferase (ChAT) and p75 low-affinity nerve growth factor receptor ipsilateral to the lesion. The magnitude of this loss was significantly attenuated in BDNF-treated rats. Many remaining neurons were atrophic with stunted dendritic processes. In contrast, NGF treatment completely rescued these cells and prevented the shrinkage of remaining cholinergic septal neurons. In addition, both NGF and BDNF induced a sprouting of cholinergic processes within the residual hippocampal remnant ipsilateral to the infusions. The present study demonstrates that icv injections of NGF, and to a lesser extent BDNF, prevent the loss of developing basal forebrain neurons which occurs following removal of normal target cells. Diffusion studies revealed relatively poor penetration of BDNF into brain parenchyma. Thus, it remains to be determined whether the failure of BDNF to provide optimal trophic support for these cells is biological or due to restricted bioavailability of this trophic factor.     

Selective Immunolesion of the Basal Forebrain Cholinergic Neurons: Effects on Hippocampal Activity During Sleep and Wakefulness in the Rat

Intracerebroventricular injection of the toxin 192 IgG-saporin (4μg) kills the cholinergic neurons of the basal forebrain bearing the low affinity NGF receptor (NGFr). The effect of this cholinergic denervation on the hippocampal and cortical electrical activity (EEG) was studied during sleep and wakefulness. EEG was recorded under freely-moving conditions in lesioned (n=10) and control (n=6) rats (8–16 days post-injection). In lesioned rats, active (AW) and quiet (QW) wakefulness episode durations were similar to those of controls whereas the REM sleep duration was reduced, 8 days post-lesion (P<0.01). Bouts of REM sleep were more numerous but shorter. The hippocampal theta activity was still present in lesioned-rats during AW (type 1 theta), QW (type 2 theta) and REM sleep. The frequency was unchanged but the amplitude of the three types of theta was significantly reduced (P<0.01). Type 2 theta occurred with shorter and less regular bouts (P<0.05). Abnormal slow waves (2–4 Hz) were observed during wakefulness. Histology showed a dramatic loss of NGFr-positive neurons in the basal forebrain and a decline in hippocampal and cortical acetylcholinesterase activity. These results suggest that the cholinergic septohippocampal input is not the primary pacemaker for the hippocampal theta rhythm.     

Low-Affinity Neurotrophin Receptor p75 Promotes the Transduction of Targeted Lentiviral Vectors to Cholinergic Neurons of Rat Basal Forebrain

Basal forebrain cholinergic neurons (BFCNs) are one of the most affected neuronal types in Alzheimer’s disease (AD), with their extensive loss documented at late stages of the pathology. While discriminatory provision of neuroprotective agents and trophic factors to these cells is thought to be of substantial therapeutic potential, the intricate topography and structure of the forebrain cholinergic system imposes a major challenge. To overcome this, we took advantage of the physiological enrichment of BFCNs with a low-affinity p75 neurotrophin receptor (p75^NTR) for their targeting by lentiviral vectors within the intact brain of adult rat. Herein, a method is described that affords selective and effective transduction of BFCNs with a green fluorescence protein (GFP) reporter, which combines streptavidin–biotin technology with anti-p75^NTR antibody-coated lentiviral vectors. Specific GFP expression in cholinergic neurons was attained in the medial septum and nuclei of the diagonal band Broca after a single intraventricular administration of such targeted vectors. Bioelectrical activity of GFP-labeled neurons was proven to be unchanged. Thus, proof of principle is obtained for the utility of the low-affinity p75^NTR for targeted transduction of vectors to BFCNs in vivo.     

AMPA-induced Lesions of the Basal Forebrain Differentially Affect Cholinergic and Non-cholinergic Neurons: Lesion Assessment Using Quantitative In Situ Hybridization Histochemistry

The direct and transynaptic effects of lesions of the basal forebrain induced by α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and ibotenic acid were investigated using quantitative in situ hybridization histochemistry. Probes complementary to the sequences of choline acetyltransferase mRNA, glutamate decarboxylase mRNA and preproenkephalin mRNA were used to assess direct lesion effects within the basal forebrain and probes for postsynaptic M-1 and M-3 muscarinic receptors were used to assess long-term changes in neocortical muscarinic receptor mRNA expression following cholinergic deafferentation. AMPA-induced basal forebrain lesions destroyed significantly more neurons that expressed choline acetyltransferase mRNA than ibotenic acid-induced lesions (90 versus 60%), but significantly fewer neurons which expressed either glutamate decarboxylase or preproenkephalin mRNA (61 versus 83% reduction in glutamate decarboxylase mRNA and 56 versus 79% reduction in preproenkephalin mRNA). AMPA-induced lesions did, however, destroy a significant proportion of the neurons which expressed glutamate decarboxylase and preproenkephalin mRNA (-60%). The neurons spared following AMPA-induced lesions were typically situated dorsolaterally within the dorsal pallidum, although neurons expressing glutamate decarboxylase or preproenkephalin mRNA were frequently observed within the areas of greatest cholinergic neuronal loss, i.e. the region of the nucleus basalis magnocellularis. These findings suggest that there is a population of non-cholinergic pallidal neurons which are insensitive to AMPA but not to ibotenic acid, reflecting a possibly heterogeneous distribution of NMDA and non-NMDA subtypes of glutamate receptors within the rat basal forebrain. AMPA-induced lesions of the basal forebrain were, however, without significant effect on the levels of expression of M-1 and M-3 muscarinic receptor mRNAs in the cerebral neocortex.     

NGF预防大鼠额叶皮质损伤后基底前脑胆碱能神经元变性

目的：为了观察神经生长因子（NGF）对大鼠额叶皮质损伤后基底前脑胆碱能神经元变性的预防效果。方法：24只SD大鼠,随机平均分为3组：（1）实验组（NGF）,（2）对照组（等容量的生理盐水）,（3）正常组（假手术）,用外科手术造成双侧额叶皮质局限性损伤,动物的学习,记忆机能及基底前脑含乙酰胆碱酯酶（AChE)活性神经元则分别用Y型迷宫和组织化学方法进行检测。结果：额叶皮质损伤4周后,动物的学习,记忆机能明显下降（P＜0．01）,基底前脑含AChE活性神经元明显减少（P＜0．01）,NGI治疗可有效地防止额叶皮质损伤大鼠学习,记忆障碍的发生及基底前脑含AChE活性神经元的减少（P＜0．05）。结论：NGF对双侧额叶皮质损伤大鼠的学习,记忆机能障碍及基底前脑胆碱能神经元变性有预防效果。     

Thalamic and Basal Forebrain Afferents Modulate the Development of Parvalbumin and Calbindin D28k Immunoreactivity in the Barrel Cortex of the Rat

In the adult barrel cortex of the rat the calcium-binding proteins calbindin D28k (CALB) and parvalbumin (PARV) are found in separate populations of GABAergic nonpyramidal neurons. In layers II to IV of the barrel cortex most PARV-immunoreactive neurons are likely to derive from a subpopulation of CALB-immunoreactive neurons whose CALB immunoreactivity ceases when they begin to express PARV between the second and third postnatal weeks. The aim of this study was to investigate the influence of subcortical afferents on the neurochemical differentiation of cortical PARV- and CALB-immunoreactive nonpyramidal neurons during development of the barrel cortex. We produced unilateral excitotoxic lesions with a single injection of ibotenic acid (0.5 μ, 0.05 M) in different subcortical nuclei in 7-to 8-day-old rats. Lesions involving the ventroposterior thalamic nuclei resulted in delayed development of PARV and CALB immunoreactivity in the barrel cortex. One week after ibotenic acid injections a transient decrease in the number of PARV-immunoreactive neurons in layer IV was observed, together with increased numbers of CALB-immunoreactive neurons in all cortical layers. The number of nonpyramidal neurons displaying coexistence of PARV and CALB in the lesioned hemisphere also increased compared with the numbers in the control hemisphere or control littermates. In contrast, lesions affecting the globus pallidus, zona incerta and reticular thalamic nucleus transiently increased the number of PARV-immunoreactive neurons in layers II and III, but had no effect on the number of CALB-positive cells. From 3 weeks onwards no differences were found between control and iesioned hemispheres after injections into either the ventroposterior thalamic nuclei or the magnocellular basal forebrain. These results suggest that CALB and PARV expression in nonpyramidal cortical neurons can be reversibly modulated in opposite directions by different cortical afferents during postnatal development.     

Selective Immunolesioning of the Basal Forebrain Cholinergic System Disrupts Short-term Memory in Rats

Selective depletion of nerve growth factor receptor-bearing neurons in the basal forebrain cholinergic nuclei by the immunotoxin 192 IgG-saporin offers a new and highly useful tool for the study of the role of the forebrain cholinergic system in cognitive functions. In the present study, we have tested the effects of 192 IgG—saporin in an operant delayed matching-to-position task which has previously been used to discriminate between delay-dependent learning impairments and delay-independent disturbances of non-mnemonic processes. Rats were first trained to criterion performance and then received intraventricular injections of 5 μg of 192 IgG—saporin 4 weeks prior to a second testing session. Rats with 192 IgG—saporin lesions displayed a significant delay-dependent decline in performance compared to normal controls, indicating a deficit in short-term memory. Administration of the muscarinic blocker scopolamine (0.5 mg/kg, i.p.) produced more pronounced impairment in the performance of the normal control rats across all delays, and induced further impairment also in animals with 192 IgG-saporin lesions. These effects were not observed following control injections of methyl scopolamine, suggesting that the impairment induced by scopolamine was due to the blockade of central muscarinic receptors. No improvement in performance was observed in either group following systemic treatment with the muscarinic cholinergic agonist arecoline (1.0 mg/kg). Biochemical and morphological analyses confirmed the selective and severe (>90–95%) depletion of cholinergic neurons throughout the septal-diagonal band area and the nucleus basalis region by the intraventricular 192 IgG—saporin treatment. Although the immunotoxin was observed to produce additional damage to the cerebellar Purkinje cells, no gross motor abnormalities were observed that could contribute to the effects on accuracy in the task used here. In conclusion, the results show that selective combined lesions of the basal forebrain cholinergic neurons in the septal—diagonal band area and nucleus basalis produce long-lasting impairments in short-term memory, thus providing further support for a role of this system in cognitive functions.     

Neuropathology of Mice Carrying Mutant APPswe and/or PS1M146L Transgenes: Alterations in the p75 Cholinergic Basal Forebrain Septohippocampal Pathway

Cholinergic basal forebrain (CBF) projection systems are defective in late Alzheimer's disease (AD). We examined the brains of 12-month-old singly and doubly transgenic mice overexpressing mutant amyloid precursor protein (APP_swe) and/or presenilin-1 (PS1_M146L) to investigate the effects of these AD-related genes on plaque and tangle pathology, astrocytic expression, and the CBF projection system. Two types of β-amyloid (Aβ)-immunoreactive (ir) plaques were observed: type 1 were darkly stained oval and elongated deposits of Aβ, and type 2 were diffuse plaques containing amyloid fibrils. APP_swe and PS1_M146L mouse brains contained some type 1 plaques, while the doubly transgenic (APP_swe/PS1_M146L) mice displayed a greater abundance of types 1 and 2 plaques. Sections immunostained for the p75 NGF receptor (p75^NTR) revealed circular patches scattered throughout the cortex and hippocampus of the APP_swe/PS1_M146L mice that contained Aβ, were innervated by p75^NTR-ir neurites, but displayed virtually no immunopositive neurons. Tau pathology was not seen in any transgenic genotype, although a massive glial response occurred in the APP_swe/PS1_M146L mice associated with amyloid plaques. Stereology revealed a significant increase in p75^NTR-ir medial septal neurons in the APP_swe and PS1_M146L singly transgenic mice compared to the APP_swe/PS1_M146L mice. No differences in size or optical density of p75^NTR-ir neurons were observed in these three mutants. p75^NTR-ir fibers in hippocampus and cortex were more pronounced in the APP_swe and PS1_M146L mice, while the APP_swe/PS1_M146L mice showed the least p75^NTR-ir fiber staining. These findings suggest a neurotrophic role for mutant APP and PS1 upon cholinergic hippocampal projection neurons at 12 months of age.     

The Distribution of Neurons Coexpressing Immunoreactivity to AMPA-sensitive Glutamate Receptor Subtypes (GluR1-4) and Nerve Growth Factor Receptor in the Rat Basal Forebrain

The regional distribution of neurons containing a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor (GluR1-4) subunit immunoreactivity, relative to the distribution of cholinergic neurons within the basal forebrain of rats, was assessed using single- and dual-antigen immunocytochemistry. Analysis of serial sections stained with antibodies to nerve growth factor receptor (NGFr) and antibodies against each of the AMPA receptor subunits, GluR1-4, revealed a regional codistribution between NGFr- and GluR1- and GluR4-immunoreactive neurons in the medial septum, diagonal band nuclei and nucleus basalis magnocellularis. Quantitative dual-labelling immunocytochemistry using NGFr in combination with each of the GluR antibodies revealed >65% colocalization between NGFr and GluR4 in each of the major cholinergic nuclei in the basal forebrain and 10–15% colocalization between NGFr, GluR1 and GluR2-3. The reticular nucleus of the thalamus, a structure known to be highly susceptible to AMPA-induced neurotoxicity, expressed GluR4 immunoreactivity exclusively. The observation that cholinergic neurons of the basal forebrain are also highly sensitive to AMPA and express the GluR4 subunit suggests that GluR4 may be important in AMPA receptor-mediated excitotoxicity.     

NGF Amplifies Expression of NGF Receptor Messenger RNA in Forebrain Cholinergic Neurons of Rats

In order to study the ligand-mediated regulation of NGF receptors in vivo, we assessed NGF receptor mRNA in the septal area of both neonatal and adult rats following intraventricular NGF administration. In neonatal rats NGF treatment, in comparison with cytochrome c, elicited a pronounced augmentation in the level of NGF receptor mRNA. A similar effect was also observed following continuous intraventricular NGF infusion in young adult rats. In addition, in this latter case, the increase in NGF receptor mRNA was associated with an increase in NGF receptor-related immunoreactivity, most likely associated with the cholinergic neurons, in the septal area. These results show that NGF itself may regulate expression of NGF receptor mRNA and corresponding protein levels in forebrain cholinergic neurons and suggest that NGF effects in the CNS may be mediated by an up-regulation of NGF receptors.     

Synergistic Trophic Actions on Rat Basal Forebrain Neurons Revealed by a Synthetic NGF/BDNF Chimaeric Molecule

Basal forebrain cholinergic neurons, which degenerate in Alzheimer's disease, respond to multiple trophic factors, including the neurotrophins, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). This dual responsiveness prompted us to investigate the effects of a synthetic chimaeric molecule, containing the active domains of both NGF and BDNF. The NGF/BDNF chimaeric factor exhibited synergistic actions, and was 100-fold more potent than wild-type BDNF in enhancing survival of cultured dissociated basal forebrain cholinergic neurons. This effect was apparently due to true BDNF/NGF synergy, since addition of the two wild-type trophins simultaneously reproduced the effect of the chimaera. Synergy was selective for neurons which respond to both factors; substantia nigra dopaminergic neurons, which respond to BDNF but not NGF, exhibited no potentiation. The chimaeric factor thus revealed a synergy that may normally occur in the brain, and constitutes a potentially novel therapeutic agent with greater potency than naturally occurring individual trophins.