Nerve growth factor increases choline acetyltransferase activity in developing basal forebrain neurons

Nerve growth factor (NGF) is a neuronotrophic protein. Its effects on developing peripheral sensory and sympathetic neurons have been extensively characterized, but it is not clear whether NGF plays a role during the development of central nervous system neurons. To address this point, we examined the effect of NGF on the activity of neurotransmitter enzymes in several brain regions. Intracerebroventricular injections of highly purified mouse NGF had a marked effect on the activity of choline acetyltransferase (ChAT), a selective marker of cholinergic neurons. NGF elicited prominent increases in ChAT activity in the basal forebrain of neonatal rats, including the septum and a region which contains neurons of the nucleus basalis and substantia innominata. NGF also increased ChAT activity in the hippocampus and neocortex, terminal regions for the fibers of basal forebrain cholinergic neurons. In analogy with the response of developing peripheral neurons, the NGF effect was shown to be selective for basal forebrain cholinergic cells and to be dose-dependent. Furthermore, septal neurons closely resembled sympathetic neurons in the time course of their response to NGF. These observations suggest that endogenous NGF does play a role in the development of basal forebrain cholinergic neurons.     

Tamoxifen enhances choline acetyltransferase mRNA expression in rat basal forebrain cholinergic neurons

Novel estrogen-like molecules known as SERMs (selective estrogen receptor modulators) produce many of the beneficial estrogen-like actions without the detrimental side-effects. The SERM, tamoxifen, an estrogen-like molecule with both agonist and antagonist properties, is widely prescribed for the treatment of breast cancer. While the effects of tamoxifen are being evaluated in many peripheral tissues, its effects in the central nervous system (CNS) have been largely ignored. In the present study, we begin to evaluate the effects of tamoxifen in the rat basal forebrain, a region known to be highly responsive to estrogen. We compared the effects of short-term (24 h) tamoxifen treatment to that of estrogen on ChAT mRNA expression in cholinergic neurons. In addition, we examined the effect of tamoxifen in the presence and absence of estrogen. Our results indicate that tamoxifen enhances ChAT expression in a manner similar to that of estrogen in several basal forebrain regions. In contrast, tamoxifen exhibits antagonist properties with respect to estrogen-induction of progesterone receptor mRNA in the medial preoptic nucleus. These results indicate tamoxifen has estrogenic properties with respect to cholinergic neurons, suggesting a previously unidentified effect of this agent in the CNS.     

Phasic relationship between the activity of basal forebrain neurons and cortical EEG in urethane-anesthetized rat

Previous studies have shown that a large number of neurons in the basal forebrain have higher firing rates when the cortical electroencephalogram (EEG) is characterized by low-voltage fast activity compared to states characterized by slow waves. A smaller number of cells with increased discharge rates during slow waves have also been observed. This putative ascending effect is thought to be tonic, but no attempt has been made to analyze a closer temporal correlation between the activity of basal forebrain neurons and the cortical EEG. Recordings were made from single units in the basal forebrain concurrently with the cortical EEG in urethane-anesthetized rats. A total of 52 neurons consistently showed higher firing during low-voltage fast activity (F-cells), whereas 14 neurons were consistently more active during cortical slow waves (S-cells). In most of the F- (90%) and S-cells (86%) the change in firing rate occurred prior to the change in the EEG. The average delay was 300–400 ms. At a deep level of anesthesia, the EEG was characterized by an alternation of flat periods and large waves. Most F-cells became active near the start of the first large wave, which is known to correspond to the onset of depolarization of cortical pyramidal neurons. In contrast, most S-cells were less active during the large waves. These data show that the activity of basal forebrain neurons is phasically correlated with the EEG in addition to the tonic correlation that has been demonstrated previously. Both types of basal forebrain neurons change their firing rate prior to the change in cortical EEG, suggesting that the basal forebrain neurons may have a regulatory influence on the EEG.^© 1997 Elsevier Science B.V. All rights reserved.     

Prefrontal cortical projections to the cholinergic neurons in the basal forebrain

The prefrontal cortex (PFC) projections to the basal forebrain cholinergic cell groups in the medial septum (MS), vertical and horizontal limbs of the diagonal band of Broca (VDB and HDB), and the magnocellular basal nucleus (MBN) in the rat were investigated by anterograde transport of Phaseolus vulgaris leuco-agglutinin (PHA-L) combined with acetylcholinesterase (AChE) histochemistry or choline acetyltransferase (ChAT) immunocytochemistry. The experiments revealed rich PHA-L-labeled projections to discrete parts of the basal forebrain cholinergic system (BFChS) essentially originating from all prefrontal areas investigated. The PFC afferents to the BFChS display a topographic organization, such that medial prefrontal areas project to the MS, VDB, and the medial part of the HDB, whereas the orbital and agranular insular areas predominantly innervate the HDB and MBN, respectively. Since the recurrent BFChS projection to the prefrontal cortex is arranged according to a similar topography, the relationship between the BFChS and the prefrontal cortex is characterized by reciprocal connections. Furthermore, tracer injections in the PFC resulted in anterograde labeling of numerous "en passant" and terminal boutons apposing perikarya and proximal dendrites of neurons in the basal forebrain, which were stained for the cholinergic marker enzymes. These results indicate that prefrontal cortical afferents make direct synaptic contacts upon the cholinergic neurons in the basal forebrain, although further analysis at the electron microscopic level will be needed to provide conclusive evidence.     

Choline acetyltransferase expression does not identify early pathogenic events in fetal SMA spinal cord

We investigated the expression of choline acetyltransferase, a specific marker for cholinergic neurons, in control and spinal muscular atrophy fetuses and newborns. By immunoblot we observed at 12 and 15 weeks a similar pattern of choline acetyltransferase expression in spinal muscular atrophy with respect to controls, although at 22 weeks this expression was reduced, probably due to a smaller number of motor neurons in the spinal muscular atrophy spinal cord. By immunohistochemistry, the counting of positive and negative motor neurons for choline acetyltransferase immunostaining in control and spinal muscular atrophy fetuses showed a similar proportion at all stages analyzed. The choline acetyltransferase-negative motor neurons were of similar appearance in both groups. After birth, chromatolytic motor neurons were detected in spinal muscular atrophy, all of which were choline acetyltransferase-negative. Our results in spinal muscular atrophy fetuses indicate that choline acetyltransferase immunostaining does not identify early events in neuronal pathogenesis and suggest that the spinal muscular atrophy surviving motor neurons may not be dysfunctional during this period. Furthermore, spinal muscular atrophy choline acetyltransferase-negative motor neurons showed detectable pathological changes only after birth, indicating that choline acetyltransferase is a late marker for motor neuron degeneration and not a primary contributing factor in this process.     

Choline acetyltransferase immunopositive neurons in the lateral septum

A small cluster of choline acetyltransferase (ChAT)-positive neurons was identified immunohistochemically in rat and monkey brain in the ventral portion of the lateral septal nucleus. These multipolar neurons were smaller and much less intensely staining than the medial septal nucleus ChAT-immunopositive group.     

Choline acetyltransferase and acetylcholinesterase containing projections from the basal forebrain to the amygdaloid complex of the rat

The origin of the cholinergic innervation to the amygdaloid complex was investigated with the use of acetylcholinesterase (AChE) histochemistry and choline acetyltransferase (ChAT) assay of microdissected nuclei. Visualization of AChE-positive neurones in the ventral forebrain was facilitated by pretreatment of rats with 1.5 mg/kg di-isopropyl phosphofluoridate (DFP). The AChE-positive neurones in the ventral forebrain are distributed in a continuous system from the septum through the lateral preoptic area to the entopeduncular nucleus caudally. Knife cuts or kainic acid injections (1.5 microgram/l microliter) placed in the lateral preoptic area resulted in substantial depletion of the AChE and ChAT content of the amygdala nuclei. Kainic acid injections (1.5 microgram/l microliter) in the diagonal band area or cuts through the stria terminalis dorsally did not significantly modify the AChE staining or ChAT content of the amygdala (although diagonal band injections partially depleted the hippocampus of ChAT). Knife cuts severing both the so-called ventral pathway and the stria terminalis did not produce significantly greater ChAT depletion in the amygdala than those produced by the knife cuts or kainic acid injections in the lateral preoptic area. Parasagittal knife cuts undercutting the lateral pyriform cortex also failed to modify the AChE or ChAT content of the amygdala, but they depleted the undercut cortex of both ChAT and AChE; AChE-positive material accumulated ventrally and medially to the knife cut. It is suggested that the major source of the cholinergic innervation of the amygdala is the magnocellular AChE-positive neurones in the lateral preoptic area and adjacent regions of the ventral forebrain.     

Heptyl-physostigmine enhances basal forebrain control of cortical cerebral blood flow.

This study sought to determine the effect of heptylphysostigmine (H-PHY), a reversible cholinesterase (ChE) inhibitor with greater lipophilicity and longer duration of action than physostigmine, on resting and basal forebrain (BF)-elicited increases in cortical cerebral blood flow (CBF). Laser-doppler flowmetry (LDF) was used to monitor changes in frontal cortical microvascular perfusion in urethane anesthetized rats. Responses were measured before, early after, and 1 hr following H-PHY, 3 mg/kg, i.m. Electrical stimulation (100 microA) of the BF elicited up to 220% increases in CBF at 50 Hz, an effect that was graded with frequency. At 15 min following H-PHY (3 mg/kg) resting cortical CBF was unchanged, whereas BF-elicited increases were potentiated 47% at 50 Hz. At 1 hour, resting cortical CBF remained unchanged, and the BF-elicited responses were remarkably potentiated by 354% at 10 Hz and 67% at 50 Hz. Acetylcholinesterase (AChE) activity measured in the tissue directly beneath the LDF probe was decreased by 84% at a time when these CBF responses were enhanced. These data suggest that H-PHY substantially enhances the regulation of cortical CBF by the BF, an effect that may be linked to inhibition of cortical AChE activity. This enhancement of cortical CBF may contribute to the efficacy of H-PHY as a treatment for Alzheimer's disease.     

Neonatal lesions of the basal forebrain cholinergic neurons result in abnormal cortical development

The effect of electrolytic lesions of the neonatal forebrain on the morphogenesis of the mouse neocortex has been examined. Balb/C mice were lesioned unilaterally within 24 h of birth. The development of cortical cytoarchitecture was assessed in Nissl-stained sections, and the levels of presynaptic markers for cholinergic, noradrenergic and serotonergic afferents were measured in the fronto-parietal cortex ipsilateral and contralateral to the lesion at various postnatal ages and in adulthood. The basal forebrain (nBM) lesion resulted in a transient but severe reduction of cortical cholinergic markers and in abnormal cortical cytoarchitecture. Cytoarchitectural abnormalities were expressed as delay in the emergence of differentiated cell populations and affected sequentially more superficial layers with maturation following lesion. Furthermore, the location and extent of these morphologic abnormalities appeared to correlate with the degree of cholinergic denervation. Cortical monoamines were also temporarily reduced as a result of the lesion; however, pharmacologic lesions of the monoaminergic projections alone did not result in the abnormal cortical cytoarchitecture. Thus, the basal forebrain cholinergic projection appears to serve a role in regulating cortical differentiation.     

Choline acetyltransferase activity in mouse cerebellar cultures

The finding of the acetylcholine synthetic enzyme, choline acetyltransferase, has been reported in mouse cerebellar cultures, and it has been used as an index of neuronal survival and maturation. These results are curious in light of immunocytochemical studies which show that this enzyme is localized within mossy fiber terminals in glomerular structures of the cerebellar cortex. Since most mossy fibers are of extracerebellar origin, a significant population of mossy fiber terminals would not be expected to be present in cerebellar cultures. The origin of this acetylcholine synthetic activity has been examined in mouse cerebellar cultures. Two groups of explants, one with and the other without incorporated dorsal pontine tissue, were cultivated. Only cultures that included pons showed well developed glomerular structures with mossy fiber rosettes. Homogenates of the cultures were assayed for their ability to synthesize acetylcholine, and the synthesis was shown to be due to choline acetyltransferase by use of the specific inhibitor, (naphthylvinyl)pyridine. Cultures lacking dorsal pontine tissue had only low levels of enzyme activity, whereas those which included pons had 20–60 times greater synthetic activity. These results indicate that the choline acetyltransferase activity arises from pontine tissue in cerebellar cultures and are consistent with mossy fibers being the source of this enzyme.     

Age-related variations in plasticity of rat basal forebrain cholinergic neurons after cortical lesions

The enzymatic activity of choline acetyltransferase (ChAT) in the nucleus basalis (NBM) of young rats (30 days old at the time of the operation) drops by 50% thirty days after cortical damage. This is followed by a spontaneous recovery of the enzymatic activity at 120 days after the lesion. In the present study, similar changes were observed in rats which were lesioned at maturity (4 months old). However, a different response was noted when surgery was performed on aged rats (2 years old at the time of operation). In these aged rats the drop in enzymatic activity in the NBM at 30 days post-lesion was as marked as in the young and mature animals, and no recovery was observed, even at 120 days. These results are discussed in the context of age-related neurodegenerative disease with cholinergic involvement.     

Choline acetyltransferase activity in murine thymus.

Murine thymus has been demonstrated to contain both cholinergic receptors and acetylcholinesterase activity. In the present study we have investigated the presence of the enzyme choline acetyltransferase in this organ, which is responsible for the synthesis of acetylcholine. Results reported here demonstrate that (1) an appreciable amount of the enzyme is already present in the thymus on the day of birth; (2) its expression is developmentally regulated; and (3) thymic atrophy, induced in young (2-week-old) and adult (6-week-old) mice by i.p. injection of hydrocortisone for 2 days, is accompanied by significant reduction of choline acetyltransferase activity only in young mice. Altogether these results demonstrate the presence in the murine thymus of functionally relevant markers of the cholinergic system that might interface the interactions between the nervous and immune systems.     

Choline acetyltransferase activity in human muscular diseases

Zusammenfassung Die Cholinacetyltransferase-Aktivität wurde in Muskeln von Patienten mit Neuromyopathien gemessen. Die Muskelbiopsien wurden bei Patienten mit Myasthenia gravis, mit verschiedenen neurogenen Atrophien der Muskeln und mit Muskeldystrophie vom geschlechtsgebundenen Typus entnommen. Die CAT-Aktivität ist bei den Muskeldystrophien sehr nennenswert und signifikant vermindert (–48%), während keine statistisch signifikante Verminderung bei der Myasthenie nachweisbar ist. Bei den neurogenen Atrophien ist die Verminderung der CAT-Aktivität parallel zum Ausmaß der Erkrankung. Es wird auf die möglichen Zusammenhänge zwischen der CAT-Aktivitätsverminderung im Muskel und dem neurogenen Prozeß hingewiesen.     

Development of cholinergic markers in mouse forebrain. I. Choline acetyltransferase enzyme activity and acetylcholinesterase histochemistry

Measurements of choline acetyltransferase (ChAT) activity were made during the development of the neocortical cholinergic innervation, and correlated with the development of the acetylcholinesterase (AChE) staining pattern in mouse cerebral cortex and several other areas of the forebrain between the time of initial onset and maturity ChAT activity can first be measured on postnatal day 6 (P6). The enzyme reaches 40% of adult activity by P18 and adult values by 7 weeks postnatal. The onset of AChE staining varies for different regions of the forebrain and for various areas within the cerebral cortex. The earliest appearance of AChE is seen in several basal forebrain nuclei including the striatum, the ventromedial region of the globus pallidus and the hypothalamus on embryonic day 18 (E18). In neocortex and olfactory cortex, AChE-stained axons are seen in the white matter before birth, but do not enter cingulate cortex and hippocampus until P2. By P2. almost all areas of the basal forebrain and diencephalon have acquired some AChE staining pattern. The adult distribution of AChE staining is reached by 3 weeks postnatal in all areas of the forebrain. Adult cerebral cortex shows a characteristic pattern of alternating AChE dense and AChE sparse bands which vary in depth depending on the cortical area. The cortical banding pattern develops in an 'inside-out' fashion, starting in layer VI and gradually entering more superficial layers. In parallel with the AChE pattern of development in cortex, transient AChE staining can be observed in some thalamic nuclei and in some forebrain fiber systems. In the neostriatum patches of intense AChE staining first develop along the ventrolateral border, then spread throughout the whole nucleus and finally coalesce to a uniform high density over the entire neostriatum. We discuss the close spatial and temporal correspondence between AChE pattern development and reported data on synapse formation, and speculate on the role of the cortical cholinergic system in development.     

Choline acetyltransferase activity in skeletal muscles after denervation

The activity of choline acetyltransferase (ChAc) in rat hind-limb muscles started to decrease rapidly between 10 and 24 hr after transection of the mixed nerve supply. The time course of changes in the tibialis anterior, extensor digitorum longus, and soleus muscles was very similar. Semilogarithmic plots of ChAc changes vs time revealed that the changes in enzyme activity proceed in two steps, both of which have an exponential time course. A steep decline occurs during the first 72–96 hr after denervation; this is followed by a slow decline. During the period of steep decline the activity of ChAc in the muscles decreased by 79–92%. After 7 mo, the mean ChAc activity in denervated tibialis anterior was 3.4 and in extensor digitorum longus 3.3% of that in contralateral control muscles. It is suggested that the ChAc disappearing from the muscles during the period of steep decline is the enzyme present in the motor nerve terminals and intramuscular branches of the motor nerve, whereas the enzyme remaining in the muscle after 4 days of denervation constitutes a different pool (or different pools) of ChAc. It appears likely that this pool of ChAc is also present in the muscle before denervation. No secondary increase of ChAc at later stages of denervation was observed. The level of nerve transection affected the time course of the steep ChAc decline; a 50% decrease of ChAc was attained 9 hr earlier when the nerve was cut near the muscle than when it was cut 34 mm away from the muscle.     

Choline acetyltransferase activity in muscles of old rats

The total activity of choline acetyltransferase (ChAc) in the rat extensor digitorum longus (EDL) and soleus muscles increased by 50 and 55%, respectively, between 3 and 9 months of age. In rats 28 to 29 months old, the activity of ChAc in EDL and soleus diminished to 41 and 40%, respectively, of the activity observed in 9-month-old animals. Age changes of ChAc activity in the diaphragm were not significant. The number of muscle fibers in EDL and soleus muscles of rats 28 to 29 months old decreased by 44 and 38% respectively, in comparison with younger animals. Mean muscle fiber diameters did not change between 3 and 9 months of age and decreased by 24, 35 and 9% in the EDL, soleus and diaphragm, respectively, in the 28- to 29-month-old rats. The activity of ChAc expressed in relation to one muscle fiber was about the same in the EDL and soleus muscles. It increased between 3 and 9 months and decreased between 9 and 28 to 29 months of age. The observation that ChAc activity per muscle fiber was identical in the fast EDL and slow soleus muscle suggests that the physiological differences between the two muscles are not caused by a difference in the capacity of their motor nerves to synthesize ACh. In the diaphragm the activity of ChAc per muscle fiber apparently did not diminish in old age. The decrease in the total ChAc activity in the limb muscles of old animals seems due both to a decrease in the number of nerve terminals in the muscles and to a decrease in the amount of enzyme present in individual terminals. We suggest that the maintenance of ChAc activity in the motor nerve terminals in the diaphragm of old rats is due to the continuous activity of this muscle and its motor nerves.     

Estradiol increases choline acetyltransferase activity in specific basal forebrain nuclei and projection areas of female rats

Administration of estradiol to gonadectomized female, but not male rats, is associated with increased activity of choline acetyltransferase in the medial aspect of the horizontal diagonal band nucleus, the frontal cortex, and CA1 of the dorsal hippocampus. Four other basal forebrain cholinergic nuclei did not show changes in choline acetyltransferase activity after estradiol. These data have implications for possible benefits of estradiol administration to patients with senile dementia of the Alzheimer's type.     

Nitric oxide synthase is critical in mediating basal forebrain regulation of cortical cerebral circulation.

This study sought to determine whether the activity of nitric oxide synthase (NOS) is an important physiological link required to mediate increases in cortical cerebral blood flow (CBF) elicited by electrical microstimulation of the basal forebrain (BF). Changes in cortical CBF were assessed in urethane anesthetized rats using laser-Doppler flowmetry. Microstimulation of the BF elicited stimulus-locked increases in CBF that were dependent on frequency and current intensity (up to 280% of control at 50 Hz). Infusion of the potent NOS inhibitor NG-nitro-L-arginine (L-NNA) resulted in significant dose-related reductions in the BF-elicited response at 50 Hz (3.75-60 mg/kg, i.v.), significant elevation in resting mean arterial pressure (MAP) from 106 to 160 mmHg, and modest 21% reductions in resting CBF. The stereoisomer NG-nitro-D-arginine (D-NNA) was without any effect on CBF, although at higher concentrations MAP was elevated to levels comparable to those obtained with L-NNA. Infusion of arginase was also without effect on resting or BF-elicited CBF responses. In contrast, L-arginine (100-400 mg/kg, i.v.) significantly potentiated the BF-elicited response up to an additional 38%, without affecting resting CBF or MAP. This study suggests that NO, or a related nitroso precursor formed by NOS, has a critical role in mediating regulation of cortical CBF by BF neurons.     

Regulation of cerebral cortical blood flow by the basal forebrain cholinergic fibers and aging

This article reviews the study of neural vasodilator mechanisms of the cerebral cortex by basal forebrain cholinergic nerve fibers and their age-related function in rats. During the last decade, we have demonstrated a neural regulatory system of cerebral blood flow in rats involving intracerebral cholinergic vasodilator nerve fibers originating in the basal forebrain and projecting to the cerebral cortex. Activation of these cholinergic vasodilator fibers results in the release of acetylcholine (ACh) within the cortex, activation of both nicotinic and muscarinic ACh receptors, and vasodilatation without coupling to glucose metabolic rates. This cholinergic vasodilator system has been shown to decline with age in rats mainly due to age-related declines of nicotinic ACh receptor activity. However, muscarinic ACh receptor activity and the release of ACh into the extracellular space in the cortex are well maintained during aging. The present age-related decline of the intracerebral cholinergic vasodilator system found in rats seems to affect cognitive function during aging, although this cholinergic vasodilator system has not yet been demonstrated in humans.