Effects of dihydroergotoxine on central cholinergic neuronal systems and discrimination learning test in aged rats.

We evaluated changes in the cholinergic neuronal system and learning ability with aging. Choline acetyltransferase (ChAT) activity, a presynaptic index of the cholinergic system, was decreased in the cerebral cortex, hippocampus, striatum, and hypothalamus in the brain of aged rats compared with young adults. Muscarinic cholinergic binding sites (receptors, MCR), a postsynaptic index of the cholinergic system, were markedly decreased in all areas of the brain. However, intraperitoneal injection of 1 mg/kg of dihydroergotoxine (DHET) for 14 days normalized both ChAT and MCR in the cerebral cortex and hippocampus. In the striatum, ChAT was normalized, but MCR did not recover. Aged rats showed marked learning impairment in a 30-day operant type brightness discrimination learning test. Daily DHET administration restored the discrimination ability in the aged rats to nearly the young adult level. DHET had no effects on central cholinergic indices or learning test results in young adult rats. These findings suggest that learning is impaired in aged rats due to impairment in the central cholinergic neuronal system, and that DHET normalizes the decreased function in this system, restoring the learning ability.     

Nerve growth factor and choline acetyltransferase activity levels in the rat brain following experimental impairment of cerebral glucose and energy metabolism.

Intracerebroventricular (ICV) injection of streptozotocin (STZ) has been reported to impair cerebral glucose utilization and energy metabolism (Nitsch and Hoyer: Neurosci Lett, 128:199-202, 1991) and also to prejudice passive avoidance learning in adult rats (Mayer et al.: Brain Res 532:95-100, 1990). It is well established that the forebrain cholinergic system, whose integrity is essential for learning and memory functions, depends on the target-derived retrograde messenger nerve growth factor (NGF). Therefore, we measured NGF and choline acetyltransferase (ChAT) activity levels in the forebrain cholinergic system in adult rats that had received a single injection of either STZ or artificial cerebrospinal fluid into the left ventricle 1 or 3 weeks prior to sacrifice. One week after ICV STZ treatment, NGF content was significantly decreased (-32%) in the septal region, where NGF-responsive cell bodies are located and NGF exerts its neurotrophic action after retrograde transport from NGF-producing targets. In contrast, NGF levels in the cortex and hippocampus, which are target regions for the basal forebrain cholinergic neurons, and in the brainstem and cerebellum were increased (+12% to +47%) within 3 weeks after ICV STZ treatment. The alterations in NGF levels were not related to changes in ChAT activity that decreased in the hippocampus by only 15%. This might be due to masking effects exerted by compensatory NGF-mediated stimulation of ChAT activity in remaining functional neurons. It is suggested that impaired behavior which has been observed after STZ-induced impairment of cerebral glucose and energy metabolism may be at least partially related to a diminished capacity of central NGF-responsive neurons to bind and/or transport NGF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hippocampal nerve growth factor levels are related to spatial learning ability in aged rats.

Brain nerve growth factor (NGF) was determined in two groups of aged rats: 'good' and 'poor' performers. The animals were selected out of a population of 40 aged rats (26-28 months old) trained in a spatial learning task. Animals performing well in the test had significantly higher NGF in the hippocampus when compared to 'poor' performers. No differences in the levels of NGF were found in the cortex, septum and cerebellum. The results implicate hippocampal NGF in cognitive functioning of aged rats, and suggests that the forebrain cholinergic neuronal atrophy which has been observed in cognitively impaired aged rats may be due to reduced availability of target-derived NGF.     

Long-term reversal of cholinergic neuronal decline in aged non-human primates by lentiviral NGF gene delivery

Spontaneous atrophy of basal forebrain cholinergic neurons occurs with aging in the non-human primate brain. Short-term reversal of this atrophy has been reported following ex vivo nerve growth factor (NGF) gene delivery, but long-term effects of in vivo NGF gene delivery in the aged primate brain have not to date been examined. We tested the hypothesis that long-term lentiviral NGF intraparenchymal gene delivery would reverse age-related cholinergic decline, without induction of adverse effects previously observed following sustained intracerebroventricular growth factor protein exposure. Three aged rhesus monkeys underwent intraparenchymal lentiviral NGF gene delivery to the cholinergic basal forebrain. 1 year later, cholinergic neuronal numbers were quantified stereologically and compared to findings in four controls, non-treated aged monkeys and four young adult monkeys. Safety was assessed on several variables related to growth factor exposure. We now report that lentiviral gene delivery of NGF to the aged primate basal forebrain sustains gene expression for at least 1 year, and significantly restores cholinergic neuronal markers to levels of young monkeys. Aging resulted in a significant 17% reduction (p < 0.05) in the number of neurons labeled for the cholinergic marker p75 among basal forebrain neurons. Lentiviral NGF gene delivery induced significant (p < 0.05) and nearly complete recovery of p75-labeled neuronal numbers in aged subjects to levels observed in young monkeys. Similarly, the size of cholinergic neurons in aged monkeys was significantly reduced by 16% compared to young subjects (p < 0.05), and lentiviral NGF delivery to aged subjects induced complete recovery of neuronal size. Intraparenchymal NGF gene delivery over a one-year period did not result in systemic leakage of NGF, activation of inflammatory markers in the brain, pain, weight loss, Schwann cell migration, or formation of anti-NGF antibodies. These findings indicate that extended trophic support to neurons in the non-human primate brain reverses age-related neuronal atrophy. These findings also support the safety and feasibility of lentiviral NGF gene transfer for potential testing in human clinical trials to protect degenerating cholinergic neurons in Alzheimer's disease.     

Effects of aging on nicotinic and muscarinic autoreceptor function in the rat brain: relationship to presynaptic cholinergic markers and binding sites

The main objective of the present work was to determine whether the regulation of ACh release by nicotinic and muscarinic autoreceptors is compromised in the aged rat brain. For this, the effects of the nicotinic agonist N-methylcarbamylcholine (MCC) and the muscarinic-M2 antagonist AF-DX 116 on ACh release from brain slices of young (3-month-old), adult (9-month-old), and aged (27-month-old) rats were tested. The ability of MCC to enhance spontaneous ACh release in hippocampal, cerebral cortical, and cerebellar slices was only modestly altered with age. In contrast, the sensitivity of muscarinic autoreceptors in the aged hippocampus and cerebral cortex, but not the striatum, to blockade by the muscarinic-M2 antagonist AF-DX 116 was severely attenuated. To assess whether the age-related changes in cholinergic autoreceptor function may be due to deficits in presynaptic cholinergic markers, we tested whether choline acetyltransferase (ChAT) activity, basal and evoked ACh release, and nicotinic and muscarinic binding sites are altered in the aged rats. ChAT activity in forebrain regions was decreased in the aged compared to the young and mature adult rats. Furthermore, the potassium-evoked, but not the spontaneous, release of ACh was markedly depressed in striatal, hippocampal, and cortical slices of aged rats. The densities of nicotinic and muscarinic-M2 binding sites, assessed using 3H-MCC and 3H-AF-DX 116 as selective ligands, respectively, were markedly reduced in homogenates of the striatum, hippocampus, cerebral cortex, and thalamus of aged rats. In contrast, muscarinic-M1 sites, selectively labeled with 3H-pirenzepine, were not affected. Therefore, it appears that age-related decrements in ChAT activity and in muscarinic-M2, but not nicotinic, binding sites in the rat brain are reflected in a decreased function of muscarinic-M2 autoreceptors. However, the positive correlation between loss of ChAT activity, decreased muscarinic-M2 binding sites, and impaired muscarinic autoreceptor function is clearly tissue dependent.     

Degenerative Changes in Forebrain Cholinergic Nuclei Correlate with Cognitive Impairments in Aged Rats

Degenerative changes in the forebrain cholinergic nuclei have been studied morphometrically in behaviourally characterized aged female Sprague-Dawley rats. In all regions analysed (medial septum, diagonal band of Broca, nucleus basalis, and striatum) the acetylcholinesterase-positive neurons were reduced in both size and number in the aged (24-months-old) rats as compared to the young (3-months-old) controls. The overall reduction in cell size amounted to between 20 and 30% and the overall reduction in cell number to between 27 and 45%. Impairment in learning and/or memory performance in the aged rats, as assessed in the Morris' water-maze task, was significantly correlated with both cholinergic cell size and cell number in the medial septum, and with cholinergic cell number in the diagonal band of Broca and in the striatum. In the nucleus basalis there was a trend in the same direction but it did not reach significance. In contrast to these degenerative changes in the cell body regions, no significant differences in cortical or hippocampal choline acetyltransferase activity were detected biochemically between the young and the aged rats, and the enzyme activity levels did not correlate with the degree of behavioural impairment in the aged rats. The present results provide evidence that all major forebrain cholinergic cell groups undergo degenerative changes with age in the rat, and that the most severe changes are found in those rats which display the most profound spatial learning impairments. Despite the severe changes at the cell body level, however, the choline acetyltransferase activity in the cortical projection areas are affected only to a minor degree, perhaps as a result of functional compensatory changes at the terminal level.     

Nerve growth factor does not influence the expression of β amyloid precursor protein mRNA in rat brain: in vivo and in vitro studies

We investigated the effect of NGF on amyloid precursor protein (APP) mRNA levels in the rat septal/nucleus basalis system. Total APP mRNA and APP 695 mRNA were determined in basal forebrain primary cell cultures exposed acutely and chronically to NGF (150–300 ng/ml) and, in vivo, in the septal area and striatum of rat pups after multiple intracerebroventricular injections of NGF. The trophic factor was able to affect cholinergic neurons in both paradigms, as evidenced by the significant increase of choline acetyltransferase (ChAT) activity induced by NGF in cell cultures (+80%) and in the striatum (+240%) of rat pups. In spite of this effect, no significant change of APP mRNA expression was observed in neuronal cultures and brain tissues. These data indicate that the neurotrophic effect of NGF on forebrain cholinergic neurons is not always associated with an alteration of APP expression.     

Nerve growth factor binding in aged rat central nervous system: effect of acetyl-L-carnitine

The nerve growth factor protein (NGF) has been demonstrated to affect neuronal development and maintenance of the differentiated state in certain neurons of the peripheral and central nervous system (CNS) of mammals. In the CNS, NGF has sparing effects on cholinergic neurons of the rodent basal forebrain (BF) following lesions where it selectively induces choline acetyltransferase (ChAT). NGF also induces ChAT in the areas to which BF provides afferents. In aged rats, there is a reduction in the NGF-binding capacity of sympathetic ganglia. Here, we wish to report that there is a decrease in the NGF-binding capacity of the hippocampus and basal forebrain of aged (26-month-old) rats as compared to 4-month-old controls but no change in NGF binding in cerebellum. In all instances, equilibrium binding dissociation constants did not differ significantly. Treatment of rats with acetyl-L-carnitine, reported to improve cognitive performance of aged rats, ameliorates these age-related deficits.     

Thyroid hormone regulation of NGF, NT-3 and BDNF RNA in the adult rat brain.

The effects of peripherally administered thyroid hormone (TH; 500 micrograms/kg; i.p.; q.d.) on the relative abundances of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) RNA were determined by rtPCR in the cortex and hippocampus of young adult rats. Corresponding changes in choline acetyltransferase (ChAT) activity were measured since NGF and BDNF have been shown to enhance the expression of this marker enzyme of central cholinergic pathways. Abundance levels of NGF and NT-3, relative to cyclophilin (cycl), were increased significantly (+50%, P < 0.05) in the hippocampus following TH treatment. Despite enhanced abundance of NGF in the hippocampus, ChAT activity was unchanged, whereas ChAT activity was modestly increased by 28% in the cortex without corresponding changes in NGF, NT-3 or BDNF. These results demonstrate that TH administration is capable of inducing the accumulation of NT-3, in addition to NGF but that the induction levels of RNA cannot be directly correlated with responsivity of the cholinergic system as measured by ChAT activity.     

Changes in cortical EEG and cholinergic function in response to NGF in rats with nucleus basalis lesions

We examined whether recovery of cholinergic function in response to nerve growth factor (NGF) results in restoration of electrocortical activity. Rats received unilateral lesions of the nucleus basalis and were infused intracerebroventricularly (i.c.v.) over 3 weeks with NGF or vehicle. Cortical electrical activity was assessed at postoperative days 4, 7, 14, and 21 from 8 epidural electrodes. On day 21, choline acetyl transferase (ChAT) activity was measured in cortical tissue underlying each electrode site. Lesions resulted in increases in slow-wave (δ) power and decreases in high-frequency (β₂) power in the lesioned, as well as the non-lesioned hemisphere. Changes correlated topographically and in magnitude with losses of ChAT activity and suggested that regional electrocortical function was affected by cholinergic activity originating in the ipsilateral, as well as the contralateral hemisphere. NGF attenuated changes in cholinergic and electrocortical function bilaterally, though in the lesioned hemisphere, function did not return to control levels. Likewise, intact animals receiving NGF showed increases in β₂-power, as well as modest increases in ChAT activity. Changes in brain electrical activity in response to NGF occurred within 4–7 days without significant changes during the 2 weeks thereafter. Our results suggest that outcomes of future animal and human trials using unilateral i.c.v. infusions of NGF need to consider the reciprocal influences of hemispheric cholinergic function, as well as possible effects of NGF on intact brain.     

Comparison of nerve growth factor's effects on development of septum, striatum, and nucleus basalis cholinergic neurons in vitro

In the central nervous system, nerve growth factor (NGF) affects basal forebrain cholinergic neurons during early development and in the adult mammalian brain. These neurons are located in medial septum, diagonal band of Broca, and nucleus basalis of Meynert. While the effects of NGF on the development of septal cholinergic neurons are well documented, only little is known about the influence of NGF on development of cholinergic neurons in the nucleus basalis. In addition to the basal forebrain cholinergic neurons, there are cholinergic interneurons in the corpus striatum, which form an anatomically and functionally distinct population of cholinergic neurons. These striatal interneurons have been reported to respond to NGF during early development; however, it is not known whether the effects of NGF on their development are similar to those on septal cholinergic neurons. We prepared cultures of dissociated cells from fetal rat septum, striatum, and nucleus basalis and investigated the development of cholinergic neurons localized in these three different areas in the presence or absence of NGF. We now report that, first, cholinergic neurons of striatum and nucleus basalis develop a more extensive fiber network and contain more acetylcholinesterase (AChE) per neuron than do cholinergic neurons of septum. The amount of choline acetyltransferase (ChAT) per cholinergic neuron is approximately the same in all three culture types when grown in the absence of NGF. Second, NGF treatment increases and anti-NGF treatment decreases the number of AChE-positive neurons in cultures of low plating density, suggesting that NGF is able to promote survival of cholinergic neurons of all three areas studied. Third, NGF increases the total length of fibers and the number of branching points of cholinergic neurons in septal cultures but not in cultures of striatum and nucleus basalis. Fourth, NGF treatment increases AChE activity in septal but not in nucleus basalis or striatal cultures, suggesting that AChE activity reflects the extent of the fiber network of cholinergic neurons of all areas. Fifth, NGF treatment produces severalfold elevations in ChAT activity in septal cultures and more modest increases in cultures of nucleus basalis and striatum, suggesting that NGF is able to stimulate ChAT activity also in the absence of a stimulatory effect on survival and fiber growth. Our results demonstrate that, during early development, NGF is able to affect survival and differentiation of all three populations of forebrain cholinergic neurons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Spatial memory deficits in aged rats: contributions of the cholinergic system assessed by ChAT

Activity of choline acetylase (ChAT) was measured in basal forebrain cholinergic nuclei and in projection sites of these cells in the hippocampus and cortex of young rats and of aged rats who showed impaired performance on the radial arm maze. Decreased ChAT activity was found in the vertical diagonal band nucleus, the dentate gyrus and striatum of aged rats.     

Haloperidol alters rat CNS cholinergic system: enzymatic and morphological analyses

Chemical and morphological changes in cholinergic marker enzymes, acetylcholinesterase (AChE), and choline acetyltransferase (ChAT) of striatum, hippocampus, and cerebral cortex were studied following haloperidol treatment of rats. After short-term (7-21 days) haloperidol treatment, the levels of both enzymes (AChE and ChAT) were increased in striatum and hippocampus (greater than 25%), but not in cortex. After long-term (+40 days) haloperidol treatment, the level of AChE activity returned to control levels in all brain areas, whereas the levels of striatal and hippocampal ChAT decreased by 26% and 29%, respectively. No change in levels of both enzymes was detected after acute treatment (single dose) of haloperidol or chronic treatment with either clozapine or imipramine. Morphological analysis of cholinergic neurons and their processes using monoclonal antibody to ChAT showed two types of changes following 40 days of haloperidol treatment. First, parallel to the observed decrease in the levels of ChAT activity there was a visual decrease in the immunoreactivity in neurons as well as in their processes in striatum and hippocampus. Second, there was an apparent reduction in the size and number of stained neurons and their processes. No changes were seen in immunoreactivity after an acute treatment with haloperidol. These results indicate that the chronic haloperidol treatment in rats causes changes in central cholinergic systems that may be relevant to the pathophysiology of schizophrenia and its treatment.     

Effect of nerve growth factor on the lesioned septohippocampal cholinergic system of aged rats

Nerve growth factor (NGF) was injected intraventricularly into aged (24 months) rats with unilateral lesions of the lateral fimbria. The activity of choline acetyltransferase (ChAT) was determined in the septum and hippocampus from the normal unlesioned rats, lesioned and cytochrome c-treated rats (controls), and lesioned and NGF-treated rats at different times after the lesion. NGF-injection for 15 days after the lesion resulted in an increase of the ChAT activity in both the contralateral hippocampus and the entire septum, to about 130% of that in the normal animals, but resulted in a slight increase in the ipsilateral lesioned hippocampus, when compared to the activity in the ipsilateral side of the cytochrome c-treated controls. NGF-injection for 30 days after the lesion resulted in a 48% increase of the ChAT activity in the ipsilateral hippocampus as compared to cytochrome c-treated controls, but failed to result in a significant increase in the contralateral hippocampus. These findings indicate that atrophic cholinergic neurons in aged animals are similarly responsive to NGF treatment, like these in the young animals. Moreover, these findings suggest that the responses of basal forebrain cholinergic neurons to NGF treatment varies with time after the lesion and imply that the NGF administration can promote the collateral sprouting from spared cholinergic fibers after the lesion in the aged forebrain.     

Electroacupuncture in rats normalizes the diabetes‐induced alterations in the septo‐hippocampal cholinergic system

Diabetes induces early sufferance in the cholinergic septo‐hippocampal system, characterized by deficits in learning and memory, reduced hippocampal plasticity and abnormal pro‐nerve growth factor (proNGF) release from hippocampal cells, all linked to dysfunctions in the muscarinic cholinergic modulation of hippocampal physiology. These alterations are associated with dysregulation of several cholinergic markers, such as the NGF receptor system and the acetylcholine biosynthetic enzyme choline‐acetyl transferase (ChAT), in the medial septum and its target, the hippocampus. Controlled and repeated sensory stimulation by electroacupuncture has been proven effective in counteracting the consequences of diabetes on cholinergic system physiology in the brain. Here, we used a well‐established Type 1 diabetes model, obtained by injecting young adult male rats with streptozotocin, to induce sufferance in the septo‐hippocampal system. We then evaluated the effects of a 3‐week treatment with low‐frequency electroacupuncture on: (a) the expression and protein distribution of proNGF in the hippocampus, (b) the tissue distribution and content of NGF receptors in the medial septum, (c) the neuronal cholinergic and glial phenotype in the septo‐hippocampal circuitry. Twice‐a‐week treatment with low‐frequency electroacupuncture normalized, in both hippocampus and medial septum, the ratio between the neurotrophic NGF and its neurotoxic counterpart, the precursor proNGF. Electroacupuncture regulated the balance between the two major proNGF variants (proNGF‐A and proNGF‐B) at both gene expression and protein synthesis levels. In addition, electroacupuncture recovered to basal level the pro‐neurotrophic NGF receptor tropomyosin receptor kinase‐A content, down‐regulated in medial septum cholinergic neurons by diabetes. Electroacupuncture also regulated ChAT content in medial septum neurons and its anterograde transport toward the hippocampus. Our data indicate that repeated sensory stimulation can positively affect brain circuits involved in learning and memory, reverting early impairment induced by diabetes development. Electroacupuncture could exert its effects on the septo‐hippocampal cholinergic neurotransmission in diabetic rats, not only by rescuing the hippocampal muscarinic responsivity, as previously described, but also normalizing acetylcholine biosynthesis and NGF metabolism in the hippocampus.     

Protective Role of Nerve Growth Factor against Excitatory Amino Acid Injury during Neostriatal Cholinergic Neurons Postnatal Development

The interaction between excitatory amino acids (EAAs) and nerve growth factor (NGF) levels were studied on neostriatal cholinergic neurons during postnatal development. Striatal choline acetyltransferase (ChAT) activity and NGF levels were determined 7 days following EAA injection in 7-, 15-, 21-, 30-, and 50-day-old rats. ChAT activity was decreased 7 days after kainate (KA), quinolinate (QUIN), or quisqualate (QUIS) lesion. The reduction was most pronounced in 30-day-old rats. KA injection produced the greatest decrease in ChAT activity. Conversely, KA did not change NGF levels. QUIN and QUIS increased NGF protein and these effects were maximal with lesions in 21-day-old rats. In order to further characterize the effect of EAAs on NGF levels and ChAT activity, the time-course of the lesion was studied. We used 30-day-old rats as the maximal sensitivity of cholinergic neurons to EAAs was observed at this age. ChAT activity decreased 2 days following QUIN or QUIS injection and 1 day after KA. The EAA agonists also changed NGF levels. QUIN induced an increase in NGF levels 1 day after lesion. This effect was maintained to the last time point examined. In contrast, KA and QUIS induced transient increases in NGF levels that were only detected 2 and 4 days after injection, respectively. To study whether NGF is able to regulate EAA excitotoxicity on striatal cholinergic neurons, we studied ChAT activity 7 days after simultaneous injection of NGF plus QUIN, KA, or QUIS. Intrastriatal injection of exogenous NGF was able to block the decrease in ChAT activity observed following EAA injection alone. In conclusion, our results show that striatal cholinergic neurons have different vulnerabilities to excitotoxicity induced by EAAs during development. ChAT activity was decreased and NGF was increased by EAAs. However, those EAAs (QUIN and QUIS) that increased NGF had less effect on ChAT activity than KA which had little effect on NGF levels, suggesting that an increase in endogenous NGF levels by these agents may decrease their toxicity. This was confirmed by our finding that exogenous NGF protects cholinergic neurons against excitotoxic lesion. The combined results suggest that sensitivity to EAAs and the regulation of NGF may be crucial to the development of striatal cholinergic neurons.