Effects of nerve growth factor (NGF), fluoxetine, and amitriptyline on gene expression profiles in rat brain

Evidence suggests that nerve growth factor (NGF) may have antidepressant properties but the pharmacological mechanisms remain unknown. Previously, we found that NGF improved performance in the forced swim test in Flinders Sensitive Line rats, but did not appear to have similar biochemical actions with the antidepressant fluoxetine. Gene expression profiles for neurotransmitter receptors and regulator-related genes in the amygdala/hippocampus were determined in rats treated for 14 days with NGF, fluoxetine, amitriptyline, or saline. Gene expression was measured using an RT² profiler PCR Array System to determine the basis for this effect. Compared with saline, there were numerous genes with significantly altered mRNA levels in the amygdala/hippocampal region. Overlap was found between the mRNA levels of genes altered by NGF and the two antidepressant medications including genes related to the cholinergic and dopaminergic systems. However, decreased mRNA levels of Drd5, Sstr3, Htr3a, and Cckar genes in the amygdala/hippocampus were uniquely regulated by NGF. The results of this study are consistent with a previous conclusion that the antidepressant effects of NGF are mediated through non-traditional receptors for traditionally considered neurotransmitters and may suggest a particular utility of NGF in treating comorbid depression and addiction.     

NGF delays rather than prevents the cholinergic terminal damage and delayed neuronal death in the hippocampus after ischemia

Cerebral ischemia induces damage of cholinergic terminals in the hippocampus, which preceded the delayed neuronal death (DND) of the CA1 pyramidal cells. We investigated the effects of nerve growth factor (NGF) on the cholinergic terminal damage after ischemia. Continuous NGF infusion (0.5 μg/7 days) into the lateral ventricle before and after 5 min ischemia prevented a decrease in choline acetyltransferase (ChAT)-immunoreactivity and disturbance of acetylcholine (ACh) release on the 4th day after ischemia, but not on day 7, i.e., NGF infusion caused delay in the progress of the cholinergic terminal damage. These findings show that the cholinergic terminal damage may result from deficiency of endogenous NGF in an ischemic brain. In addition, we investigated whether NGF would prevent the DND after ischemia. NGF infusion also caused delay in the progress of the DND until day 14. Our results suggested that the neuroprotective effect of NGF on the DND may be secondarily yielded by maintenance of communication between cholinergic terminal and the target CA1 cell, and that prevention of cholinergic terminal damage may be useful for the treatment of cerebrovascular disease.     

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.     

Selective antibody-induced cholinergic cell and synapse loss produce sustained hippocampal and cortical hypometabolism with correlated cognitive deficits.

The physiological interrelationships between cognitive impairments, neurotransmitter loss, amyloid processing and energy metabolism changes in AD, cholinergic dementia and Down's syndrome are largely unknown to date. This report contains novel studies into the association between cognitive function and cerebral metabolism after long-term selective CNS cholinergic neuronal and synaptic loss in a rodent model. We measured local cerebral rates of glucose utilization ((14)C-2-deoxyglucose) throughout the brains of awake rats 4.5 months after bilateral intraventricular injections of a cholinotoxic antibody directed against the low-affinity NGF receptor (p75 NGF) associated with cholinergic neurons (192 IgG-saporin). Permanent cholinergic synapse loss was demonstrated by [(3)H]-vesamicol in vitro autoradiography defining presynaptic vesicular acetylcholine (ACh) transport sites. While other metabolic studies have defined acute and transient glucose use changes after relatively nonspecific lesions of anatomical regions containing cholinergic neurons, our results show sustained reductions in glucose utilization in brain regions impacted by cholinergic synapse loss, including frontal cortical and hippocampal regions, relative to glucose use levels in control rats. In the same animals, impaired cognitive spatial performance in a Morris water maze was correlated with reduced glucose use rates in the cortex and hippocampus at this time point, which is consistent with increased postmortem cortical and hippocampal amyloid precursor protein (APP) levels (45, 46). These results are consistent with the view of cholinergic influence over metabolism, APP processing, and cognition in the cortex and hippocampus.     

Decreased level of nerve growth factor (NGF) and its messenger RNA in the aged rat brain

Trophic factors such as nerve growth factor (NGF) are thought to support survival, differentiation and maintenance of neurons. Recent results indicate that NGF produced in cortical and hippocampal areas is required for the function of cholinergic neurons in the basal forebrain. With the use of enzyme immunoassay and RNA blot hybridization we studied the NGF protein and NGF mRNA, respectively, in regions of the brain innervated by basal forebrain cholinergic neurons in adult and aged rats. Levels of NGF protein were decreased by 40% in hippocampus of aged (28 months) Fischer 344 rats compared with adults (6 months), whereas no alterations were observed in cerebral cortex. Moreover, a reduction by 50% in the NGF mRNA was found in samples of the aged forebrain (cerebral cortex, hippocampus, basal forebrain and hypothalamus) compared to the adult. NGF deficiencies may thus account for the loss of cholinergic neurons in the basal forebrain generally found to accompany normal aging and resulting in altered cognitive functions.     

Exogenous NGF restores endogenous NGF distribution in the brain of the cognitively impaired aged rat

Alzheimer's disease and normal aging may impair retrograde transport of nerve growth factor (NGF) from cortical areas to basal forebrain cholinergic neurons. We demonstrate a relationship between performance in a spatial reference memory task and NGF distribution in the aged rat brain. In addition, exogenous NGF restored endogenous NGF distribution in cognitively impaired aged rats. These data suggest that NGF administration restores utilization of endogenous growth factor in the brain of cognitively impaired aged rats.     

Altered NGF response but not release in the aged septo-hippocampal cholinergic system

An important aspect of aging and Alzheimer's disease (AD) pathology includes the degeneration of basal forebrain cholinergic neurons (BFCNs), possibly due to disrupted nerve growth factor (NGF) signaling. Previous studies on disrupted NGF signaling have focused on changes in retrograde transport. This study focuses on two other possible mechanisms for loss of trophic support: diminished release of NGF from hippocampal neurons or diminished TrkA receptor response of BFCNs to NGF. We measured NGF levels in the effluent of hippocampal slices from young and aged rats in response to potassium chloride and glutamate. We found that release of NGF was not altered in aged hippocampal slices compared to slices from young controls. To measure the in situ response of the BFCNs to NGF, we injected NGF intraparenchymally into the right hippocampus of young and aged rats. Injections of cytochrome C served as controls. Fifteen minutes post-administration, a dramatic increase in TrkA immunoreactivity was found in the cell bodies of medial septal neurons. We found that this rapid response was blunted in aged rats compared to young adult controls. To determine whether retrograde transport was necessary for this rapid response, we injected colchicine prior to NGF injection. The NGF-induced upregulation was not blocked by colchicine, suggesting that this acute response was not dependent on classical retrograde transport. Since cholinergic degeneration coupled with altered levels of NGF and TrkA receptors are also seen in human aging and AD, the loss of acute responsivity to NGF in the BFCNs may also play a role in these processes.     

The cholinergic system, nerve growth factor and the cytoskeleton

Cholinergic neurons of the basal forebrain provide the major cholinergic innervation to the cortex and hippocampus, and play a key role in memory and attentional processes. Dysfunction of basal forebrain cholinergic neurons (BFCN) is a cardinal feature of Alzheimer's disease (AD) and correlates with cognitive decline. Survival of BFCN neurons depends upon binding of nerve growth factor (NGF), which is synthesized and secreted by cells in the cortex and hippocampus, with high-affinity (TrkA) and low-affinity (p75^NTR) neurotrophin receptors produced within BFCN neurons. NGF released from target cells activates TrkA on axon terminals and triggers activation of PI3K/Akt, MEK/ERK, and PLCγ (phospholipase C) signaling pathways. The signal then travels retrogradely along axon to cell body to promote neuronal survival. However, the nature of the retrograde signal remains mysterious. p75^NTR receptors could mediate a fundamentally different signaling pathway leading to apoptic cell death. Dysfunction of NGF and its receptors has been suggested to underlie the selective degeneration of the BFCN in end stage Alzheimer disease. In this regard, NGF, the founding member of the neurotrophin family, has generated great interest as a potential target for the treatment of AD. This review focuses on NGF-cholinergic dependency, NGF/receptor binding, signal transduction, retrograde transport, regulation of specific cellular endpoints, and the potential involvement of cytoskeleton dysfunction in defected NGF signaling.     

NGF content in the cerebral cortex of non-dementedpatients with amyloid-plaques and in symptomaticALZHEIMERS disease

There is increasing evidence that in Alzheimers disease nerve growth factor (NGF)protein and NGF mRNA content in post-mortem cortex is not decreased, but may evenbe elevated although the NGF-sensitive cholinergic basal forebrain neurons are preferentiallyaffected. However, only little is known about the early pathophysiological events leading toAlzheimers disease. We therefore measured the post-mortem NGF concentrations intemporal and frontal cortex of Alzheimers disease patients, non-demented controls withoutAlzheimers disease-related pathology, as well as non-demented patients with βA4plaques who might be classified as preclinical cases. In the Alzheimers disease group we found upto 43% increase in NGF concentrations in the frontal and temporal cortex as compared to the twoother groups. In a subgroup analysis of the non-demented patients with plaques, NGFconcentrations were lower in the frontal cortex when βA4 plaques were present (46% ofthe control temporal area) than in patients without evidence of frontal plaques (81% of the controltemporal area). This NGF decrease was paralleled to a similar decrease of cholineacetyltransferase activity, which is regulated by NGF in the cholinergic basal forebrain. Thesefindings support the hypothesis of lower cortical NGF content at the onset of plaque formationand of elevated NGF levels in the clinically manifest and neuropathologically advanced stage ofthe disease.     

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.     

No nerve growth factor response to treatment with memantine in adult rats

Summary. Nerve growth factor (NGF) is the most widely examined neurotrophin in the experimental models of Alzheimers disease (AD) and has been shown to prevent the retrograde degeneration of cholinergic neurons. In this study we examined NGF and cholineacetyltransferase (ChAT) changes in several rat brain regions after excitotoxic lesion of the entorhinal cortex with quinolinic acid and tested the effect of memantine on spatial learning in the radial maze after lesion. We observed a significant increase (+26%, p=0.02) of NGF concentrations in the hippocampus of the lesioned rats when compared to sham-lesioned rats. Chronic treatment with memantine showed no significant effect on the NGF increase in the hippocampus (p=0.72). The ChAT activity was significantly increased in the lesioned rats when compared to controls (+16%, p<0.05) and did not depend on treatment with memantine. In spite of this, memantine improved performance of the radial maze. This indicates that memory improving effects of memantine observed in experimental animals and in clinical studies are probably not related to changes in brain NGF content, whereas the observed NGF increase in the denervated hippocampus is probably trauma-related reflecting impaired retrograde transport of hippocampal NGF.Present address: Solvay Pharmaceuticals BV, Weesp, NiederlandePresent address: Institute of Pharmacology PAN, Cracow, Poland     

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.     

Effects of tetrahydro-9-aminoacridine on cortical and hippocampal neurons in the rat: an in vivo and in vitro study

The effects of tetrahydro-9-aminoacridine (THA), an anticholinesterase drug, have been studied in the rat both in vivo (cerebral cortex) and in vitro (CA1 field of the hippocampus) and compared with those of physostigmine. In the cerebral cortex THA potentiated the excitatory effect of acetylcholine in most neurons, including cortical neurons recorded from chronic unanesthetized animals. In vitro, THA (but not physostigmine) had a depolarizing, atropine- and tetrodotoxin-insensitive effect. This effect is associated with an increase in membrane resistance which suggests a direct effect of THA on hippocampal neurons. In addition THA blocked the slow inhibitory postsynaptic potential. At the same concentration THA potentiated the slow cholinergic excitatory postsynaptic potential produced by electrical stimulation of the cholinergic afferents. Its potency was, however, about 10 times lower than that of physostigmine. These results show that THA: (1) is an anticholinesterase much less potent than physostigmine; but (2) has also direct effects on central neurons, not observed with physostigmine and unrelated to its anticholinesterase activity.     

Effects of Cholinergic Deafferentation and NGF on Brain Electrical Coherence

Rats received unilateral lesions of the nucleus basalis and were infused intracerebroventricularly (i.c.v.) over 3 weeks with nerve growth factor (NGF) or vehicle. Electrocortical coherence was assessed at postoperative days 4, 7, 14, and 21 from all possible pairs of eight epidural electrodes in the delta (1–4 Hz), theta (4–8 Hz), alpha (8–12 Hz), beta-1 (12–20 Hz), and beta-2 (20–28 Hz) bands. On day 21 choline acetyltransferase (ChAT) activity was measured in cortical tissue underlying each electrode site. Lesions resulted in losses of interhemispheric, as well as bilateral intrahemispheric coherence in the theta band

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, with no significant differences seen in other bands. Changes were accentuated during immobility compared with walking and exploratory behavior. Intrahemispheric changes were greatest within the lesioned hemisphere in long connections between electrode pairings connecting frontal to posterior brain regions. Nerve growth factor (NGF) attenuated losses in ChAT

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and intrahemispheric coherence , whereas interhemispheric coherence showed no significant response. Intact animals receiving NGF showed increases in intrahemispheric coherence, as well as modest increases in ChAT. Increases in coherence in response to NGF occurred within 4–7 days following brain lesions, with no significant change during the 2 weeks thereafter. Our results suggest that coherence is sensitive to cholinergic deafferentation, particularly of long corticocortical connections. NGF differentially restores coherence within hemispheres, as opposed to between hemispheres. Our study suggests that brain function in Alzheimer’s disease related to damage of transcallosal fiber tracts may not be responsive to cholinergic treatments. Future studies may wish to evaluate the cognitive relevance of NGF’s effects on intact brain.     

Altered NGF protein levels in different brain areas after immunolesion

Nerve growth factor (NGF) provides critical trophic support to the cholinergic basal forebrain neurons that express high levels of the low-affinity NGF receptor (p75^NGFR) in the adult rat brain. Intraventricular injection of 192 IgG-saporin, made by coupling the monoclonal antibody to p75^NGFR 192 IgG to the cytotoxin saporin, selectively destroys the p75^NGFR-bearing neurons in the basal forebrain and was used here to examine the effects of selective cholinergic lesions on brain NGF protein levels. We showed that 192 IgG-saporin produced significant long-lasting elevation of NGF protein levels in the hippocampus, cortex, and olfactory bulb, with profound reductions of ChAT activities representing complete cholinergic deafferentations of these areas. NGF level was maintained in the basal forebrain, even though there was almost complete loss of p75^NGFR-immunoreactive cells and significant decrease of ChAT activity. In addition, a mild glial response was observed in the basal forebrain, and most of the activated astroglia expressed NGF-like immunoreactivity there. The increases in NGF protein levels in the target areas of the basal forebrain were most likely due to loss of cholinergic basal forebrain neurons and retrograde transport of NGF from these areas. Glial-derived NGF is partially responsible for the maintained level of NGF in the basal forebrain after the loss of cholinergic neurons. The accumulation of NGF protein in the target areas may have some effects on synaptic rearrangement in denervated tissues. © 1996 Wiley-Liss, Inc.     

Chronic 1,25-dihydroxyvitamin D3-mediated induction of nerve growth factor mRNA and protein in L929 fibroblasts and in adult rat brain

We have proposed that elevating levels of nerve growth factor (NGF) in the CNS is a rational strategy for treating certain neurodegenerative disorders. The present studies were conducted to determine: (1) if pharmacologically induced levels of NGF could be sustained for an extended time, and (2) if correlations exist between increases in NGF mRNA and NGF protein in L929 cells and in vivo. Short-term treatment of L929 cells with 1,25-dihydroxyvitamin D₃ resulted in a two-fold increase in both NGF mRNA and NGF protein. These increases were sustained for up to 48 h with continuous exposure to 1,25-dihydroxyvitamin D₃. In rats, 1,25-dihydroxyvitamin D₃ (2.5 nmol; i.c.v.) induced NGF mRNA transiently, with peak two-fold increased observed 4 h post-injection. In contrast to L929 cells, 1,25-dihydroxyvitamin D₃ did not elicit an increase in NGF protein after a single administration in vivo. However, consistent with long-term exposure in L929 cells, chronic 6 day infusion of 1,25-dihydroxyvitamin D₃ resulted in induction of both NGF mRNA and NGF protein in the brain. These results indicate that 1,25-dihydroxyvitamin D₃-mediated NGF induction in cultured L929 cells may predict of NGF induction in vivo, suggesting that L929 cells may have utility in studying underlying mechanisms of NGF induction by 1,25-dihydroxyvitamin D₃. On the basis of NGF's ability to increase cholinergic function in animal models of cholinergic degeneration, these results are supportive of a role for NGF inducers as potential drugs for neurodegenerative disorders.     

Cortico-hippocampal APP and NGF levels are dynamically altered by cholinergic muscarinic antagonist or M1 agonist treatment in normal mice

To determine whether altered cholinergic neurotransmission can modify the long-term secretion of amyloid precursor protein (APP), endogenous levels of APP and nerve growth factor (NGF), we administered a selective M1 muscarinic receptor agonist (RS86) or the muscarinic antagonist, atropine, for 7 days in vivo into young adult mice (C57BL/6j). The levels of NGF and total APP in the hippocampus, frontal cortex, striatum, parietal cortex and cerebrospinal fluid (CSF) were examined by ELISA and Western blot. We found that this repeated i.m. administration of M1 receptor agonist resulted in decreased total APP levels in the hippocampus, frontal cortex and parietal cortex, and increased secreted alpha-APPs levels in the CSF. M1 agonist treatment also resulted in decreased NGF levels in the hippocampus and CSF. These effects of the M1 muscarinic agonist could be blocked by atropine, which by itself elevated tissue levels of total APP. Interestingly, we found that the decrease of total APP in the hippocampus and striatum after M1 agonist treatment inversely correlated with the change in NGF levels. These data suggest that a sustained increased cholinergic, M1-mediated neurotransmission will enhance secretion of alpha-APPs in CSF and adaptively reduce the levels of total APP and NGF in the corticohippocampal regions of normal mice. The dynamic and adaptive regulation linking total APP and NGF levels in normal adult mice is relevant for understanding the pathophysiology of conditions with cholinergic and APP related pathologies, like Alzheimer's disease and Down's syndrome.     

Neurotrophic factor mRNA expression in dentate gyrus is increased following angular bundle transection

In the central nervous system, the highest levels of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) mRNA are found in the hippocampal formation. In the present study, we report that a unilateral transection of the angular bundle, which relays cortical information via the entorhinal cortex to the hippocampal formation, increases NGF and BDNF mRNA in the ipsilateral dentate gyrus. Within 4 hours following transection, the hybridization signal for NGF and BDNF mRNA increases in stratum granulosum 3- and 5-fold, respectively, compared to control levels. This lesion-induced increase of both mRNA returns to control levels within 24 hours and is maintained for at least 5 days. The induction is not prevented by pretreatment with AP-5, CNQX, or cholinergic denervation due to transection of the fimbria-fornix. Finally, the induction of neurotrophin mRNA is preceded by an increase in c-fos mRNA. These results provide evidence that transection of the cortical input to the hippocampal formation upregulates NGF and BNDF mRNA selectively in stratum granulosum. We suggest that the increased expression of NGF and BDNF mRNA may be an early step in the synaptic rearrangement of neurotrophin responsive cholinergic afferents observed following damage to the entorhinal cortex.