Changes In Choline Acetyltransferase Activity Research Articles

Arachidonic acid increases choline acetyltransferase activity in spinal cord neurons through a protein kinase C-mediated mechanism.

Arachidonic acid (AA) plays an important role as a signaling factor in the CNS. Therefore, exposure to AA may affect cholinergic neurons in the spinal cord. To test this hypothesis, mRNA expression and activity of choline acetyltransferase (ChAT) was measured in cultured spinal cord neurons treated with increasing concentrations (0.1-10 microm) of AA. Exposure to AA increased mRNA levels and activity of ChAT in dose- and time-dependent manners. The most marked effect of AA on ChAT expression was observed in spinal cord neurons treated with 10 microm AA for 1 h. To study the mechanisms associated with these effects, ChAT mRNA levels and activity were measured in cultured spinal cord neurons exposed to AA and inhibitors of protein kinase C (PKC), such as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dichloride (H-7) and chelerythrine. Inhibition of PKC completely prevented an AA-induced increase in ChAT expression. In addition, exposure of spinal cord neurons to phorbol-12-myristate-13-acetate (PMA), an activator of PKC, mimicked AA-induced stimulation of ChAT activity. The AA-mediated increase in ChAT mRNA levels and activity was also prevented by treatments with EGTA, indicating the role of calcium metabolism in induction of this enzyme. In contrast, treatments with 7-nitroindazole (7-NI, a specific inhibitor of neuronal nitric oxide synthase), sodium vanadate (NaV, a non-specific inhibitor of phosphatases), and N-acetyl-cysteine (NAC, an antioxidant) had no effect on AA-induced changes in ChAT activity. The protein synthesis inhibitor cycloheximide completely blocked AA-mediated increase in ChAT activity. These results indicate that the AA-evoked increase in ChAT activity in spinal cord neurons is mediated by PKC, presumably at the transcriptional level.

Estrogen-mediated regulation of cholinergic expression in basal forebrain neurons requires extracellular-signal-regulated kinase activity

Beyond the role estrogen plays in neuroendocrine feedback regulation involving hypothalamic neurons, other roles for estrogen in maintaining the function of CNS neurons remains poorly understood. Primary cultures of embryonic rat neurons together with radiometric assays were used to demonstrate how estrogen alters the cholinergic phenotype in basal forebrain by differentially regulating sodium-coupled high-affinity choline uptake and choline acetyltransferase activity. High-affinity choline uptake was significantly increased 37% in basal forebrain cholinergic neurons grown in the presence of a physiological dose of estrogen (5 nM) from 4 to 10 days in vitro whereas choline acetyltransferase activity was not significantly changed in the presence of 5 or 50 nM estrogen from 4 to 10 or 10 to 16 days in vitro. Newly-synthesized acetylcholine was significantly increased 35% following 6 days of estrogen treatment (10 days in vitro). These effects are in direct contrast to those found for nerve growth factor; that is, nerve growth factor can enhance the cholinergic phenotype through changes in choline acetyltransferase activity alone [J Neurochem 66 (1996) 804]. This is most surprising given that mitogen-activated protein kinase and extracellular-signal-regulated kinase1/2, kinases also activated in the signaling pathway of nerve growth factor, were found to participate in the estrogen-mediated changes in the cholinergic phenotype. Likewise, general improvement in the viability of the cultures treated with estrogen does not account for the effects of estrogen as determined by lactate dehydrogenase release and nerve growth factor-responsiveness. These findings provide evidence that estrogen enhances the differentiated phenotype in basal forebrain cholinergic neurons through second messenger signaling in a manner distinct from nerve growth factor and independent of improved survival.

Behavioral and neurochemical characterization of transgenic mice carrying the human presenilin-1 gene with or without the leucine-to-proline mutation at codon 235

Human presenilin-1 (PS1) mutations are associated with the incidence of familial Alzheimer’s disease. The present study evaluated the behavioral and neurochemical effects of the L235P mutation (substitution of leucine by proline at codon 235) of the human PS1 gene, which has been linked to a form of early-onset Alzheimer’s disease. Except for a significant increase in the production of β-amyloid-42, the mutant mice did not show any overt signs of Alzheimer-like neuropathology in the form of plaque formation, changes in choline acetyltransferase activity, or somatostatin content in the brain. Cognitive assays indicated that the mutation did not affect the acquisition or reversal of a spatial reference memory task in the water maze or performance on a spatial working memory task. In contrast, L235P PS1 transgenic mice exhibited a significant impairment in a test of spontaneous object recognition. This dissociation is suggestive of a preferential impairment of the extrahippocampal memory system and is consistent with what has been reported in another pathological mutation (substitution of leucine by valine at codon 286) of the PS1 gene.

PKC is involved in arachidonic acid‐induced choline acetyltransferase activity in spinal cord neurons

Arachidonic acid (AA) may affect cholinergic neuronal circuits in spinal cord. To test this hypothesis, activity of choline acetyltransferase (ChAT) was studied in cultured spinal cord neurons treated with AA (0.1–10 mm). AA increased ChAT activity in dose‐ and time‐dependent manners. These effects were mimicked by exposure to PMA but inhibited by a coexposure to PKC inhibitors. AA‐mediated increase in ChAT activity also was prevented by treatments with EGTA, indicating the role of calcium. In contrast, cotreatments with 7‐nitroindazole, sodium vanadate, and N‐acetyl‐cysteine had no influence on AA‐induced changes in ChAT activity. These results indicate that the AA‐evoked increase in ChAT activity in spinal cord neurons is mediated by PKC, most likely, at the post‐transcriptional level.Acknowledgements: Supported by grants from KSCHIRT and Philip Morris Research Program.

Effects of gonadal hormone replacement on measures of basal forebrain cholinergic function

The effects of different hormone replacement regimens on basal forebrain cholinergic function were examined by measuring changes in choline acetyltransferase activity and high affinity choline uptake in adult, ovariectomized, rats. Increases in choline acetyltransferase activity were detected in the frontal cortex (20.1%) and olfactory bulbs (30.4%) following two weeks, but not four weeks, of repeated treatment with estrogen plus progesterone. Increases in high affinity choline uptake were detected in the frontal cortex (39.5–55.1%), hippocampus (34.9–48.9%), and olfactory bulbs (29.9%) after two weeks, but not four weeks, of either continuous estrogen administration, repeated progesterone administration, or repeated treatment with estrogen plus progesterone. Repeated administration of estradiol (2–25 μg/250 g body weight) for two or four weeks, and continuous estrogen administration for four weeks and six months, produced no significant changes in choline acetyltransferase activity or high affinity choline uptake in the hippocampus, frontal cortex or olfactory bulbs. Continuous estrogen administration for 13 months produced a significant decrease in high affinity choline uptake across all regions with the largest effect (−28.1%) detected in the hippocampus. The findings demonstrate that short-term treatment with estrogen and/or progesterone can significantly enhance cholinergic function within specific targets of the basal forebrain cholinergic projections. Most important is the fact that the effects varied considerably according to the manner and regimen of hormone replacement and did not persist with prolonged treatment. These findings could have important implications for the effective use of hormone replacement strategies in the prevention and treatment of Alzheimer’s disease and age-related cognitive decline in women.

Raloxifene and estradiol benzoate both fully restore hippocampal choline acetyltransferase activity in ovariectomized rats

Selective estrogen receptor modulators (SERMs) demonstrate tissue-specific estrogen receptor (ER) agonist or antagonist properties. Raloxifene, a prototypical SERM, has ER agonist properties in bone and on cholesterol metabolism but full antagonist properties in the uterus and breast. To characterize the ER agonist/antagonist profile of raloxifene in the brain, we have examined its effect on the activity of a known estrogen-responsive gene product, choline acetyltransferase (ChAT), in the hippocampus and other brain regions of 6-month-old ovariectomized (OVX) Sprague–Dawley rats. Three weeks post-ovariectomy, animals received estradiol benzoate (EB, 0.03 mg or 0.3 mg kg -1 day -1 for 3 or 10 days); raloxifene HCl (3.0 mg kg -1 day -1 for 3 or 10 days), tamoxifen (3.0 mg kg -1 day -1 for 10 days) or vehicle (20% CDX). As previously reported, ChAT activity decreased by approximately 20%–50% in the hippocampus of OVX compared with SHAM-operated control rats with no change in ChAT activity observed in the hypothalamus. Raloxifene or EB reversed the OVX-induced decrease in ChAT activity in the hippocampus but did not change ChAT activity in the hypothalamus. Animals that received combined EB (0.03 mg/kg) plus raloxifene (1 mg/kg) or tamoxifen alone (3.0 or 10 mg/kg) also showed increased hippocampal ChAT activity. Raloxifene failed to increase uterine weight and blocked the estrogen-induced increase in uterine weight, while another SERM, tamoxifen, increased uterine weight. These data demonstrate that raloxifene has estrogen-like properties on hippocampal ChAT activity in vivo, and suggest that benzothiophene SERMs may exert estrogen-like beneficial effects on cholinergic neurotransmission in brain without producing peripheral stimulation of breast or uterine tissue.

Changes in choline acetyltransferase activity and distribution following incomplete cervical spinal cord injury in the rat

Incomplete cervical spinal cord injuries were produced in rats by placing 10g or 20g weight on exposed dura at the C6 level for 5min (Mild or Moderate injury). These two degrees of the injury resulted in initial motor functional deficits, followed by recoveries. In this study, changes in choline acetyltransferase activity and distribution following the incomplete cervical cord injuries were investigated using radioenzyme assay, and fluorescence microphotometry. We demonstrated that mild injury led to a transient decrease of choline acetyltransferase activity in the compressed spinal cord segment, but showed almost no histologic change at two days after injury. Although a low level of choline acetyltransferase immunofluorescence was found in the ventrolateral anterior horn at two days after injury, it recovered completely by one week after injury. These findings suggest that there was a strong correlation between the transient motor functional deficit and the decrease in choline acetyltransferase activity following mild injury.Moderate injury resulted in a persistent low level of choline acetyltransferase activity in the compressed spinal cord segment accompanied by a striking loss of gray matter. On the other hand, at seven, 14 and 28 days after injury, over-expressions of choline acetyltransferase activity were found in the neighboring spinal cord segments located both rostral and caudal to the injury, which showed no histologic change. In addition, excessively high levels of choline acetyltransferase immunofluorescence were found in the ventrolateral anterior horn of these segments. A strong correlation was found between the motor functional recovery and the late, excessive high levels of choline acetyltransferase activity in the neighboring regions.These results suggest that cholinergic neurons, especially spinal motor neurons may play an important role in the motor functional recovery following incomplete cervical spinal cord injury.

Age-dependent effect of AF64A on cholinergic activity in the septo-hippocampal pathway of the rat brain: decreased responsiveness in aged rats

Our previous studies have demonstrated that intracerebroventricular (icv) administration of low doses of ethylcholine mustard aziridinium (AF64A), up to 1.0 nmol/side, induces a reversible cholinergic deficit in the hippocampus, paralleled by a compensatory transient increase in choline acetyltransferase (ChAT) activity in the septum [El Tamer, A., Corey, J., Wülfert, E. and Hanin, I., Neuropharmacology, 31 (1992) 397–402]. In the present study we have addressed the question as to whether this effect might differ in old rats. AF64A (0.5 nmol/side) icv administered to three groups of rats aged 4, 12 and 22 months, respectively, induced a reduction of ChAT activity in the hippocampus to the same extent (−26%, −30% and −29.6%; P < 0.01) by 7 days post-icv injection. Acetylcholinesterase (AChE) was decreased to a similar extent in the 4 and 12 month old rats (−22% and −29%; P < 0.01), respectively, but remained unchanged in the 22 month old group. Whereas AChE activity remained unchanged in the septum in all three groups of rats, ChAT activity was increased significantly (+20% and +20.8%; P < 0.05 versus corresponding control group) in the 4 and 12 month old groups, respectively. No change in ChAT activity was measured in the septum of the 22 months old group. By 14 days post-icv injection of AF64A, ChAT and AChE activities were back to normal in all three groups and in both brain regions studied. These results demonstrate that a difference in AF64A's effect does exist between the 22 month old group and the youngest group. This might reflect a possible age-dependent change in the ability of the cholinergic system to respond to the cholinotoxicity of AF64A, as well as in the potential of the cell bodies, at the septal level, to respond to such an insult by a compensatory mechanism such as increasing ChAT activity.

AF64A-induced cytotoxicity and changes in choline acetyltransferase activity in the LA-N-2 neuroblastoma cell line are modulated by choline and hemicholinium-3

The cholinergic neurotoxin AF64A (ethylcholine aziridinium) has been used to selectively destroy the cholinergic system. Due to its structural similarity to choline, this compound may be selectively taken up by the cholinergic terminal via the high-affinity choline transport (HAChT) system to produce persistent and selective cholinergic deficits. The mechanism by which it exerts its cholinotoxicity remains to be elucidated. We have examined the effects of AF64A in the human neuroblastoma cell line, LA-N-2, which has an intact sodium-coupled choline uptake system, and is capable of synthesizing acetylcholine (ACh). AF64A (25, 50 and 100 μM) produced dose-dependent increases in cell kill as measured by colony formation assay. The addition of increasing concentrations (10 −5, 10 −4 and 10 −3 M) of choline and hemicholinium-3 (HC-3) protected the cells from the cytotoxic effects of AF64A. At the same doses, AF64A also decreased choline acetyltransferase (ChAT) activity. In the presence of the highest concentration of choline or HC-3 (10 −3 M) which produced complete protection against AF64A's cytotoxicity in the colony formation assay, ChAT activity was restored to control values. These results demonstrate that agents that utilize (i.e., choline) or inhibit (i.e., HC-3) the choline uptake system prevented AF64A-induced cytotoxicity and decreases in ChAT activity, in a manner similar to that which has been observed in chick and rat primary cholinergic cultures in vitro. The LA-N-2 neuroblastoma cell line thus serves well as an in vitro model of the cholinergic neuron and provides a useful system to study the mode of cholinotoxicity induced by AF64A.

A radiometric microassay for choline acetyltransferase. Some observations on the spinal cord of the chicken embryo

This paper describes cation-exchange methods for separating acetyl[3H] coenzyme A from [acetyl-3H]choline. Blanks for the routine method were approximately 0.05% of the substrate radioactivity; product recoveries were approximately 97%. The cation-exchange method was more efficient than the standard methods using either anion-exchange chromatography or periodide precipitation. The cation-exchange method was also more specific than either of the other two standard methods for estimating choline acetyltransferase (ChAT) activity. ChAT activity was detected in the chicken lumbar spinal cord on embryonic day (E) 2 1/4 with the cation-exchange method. This developmental stage is about 6 hours before the final mitosis of any neuroblast in the ventral horn. Total ChAT activity per lumbar spinal cord increased more than 10,000-fold between E 3 and E 18. Changes in ChAT activity in the lumbar spinal cord following limb-bud extirpation appeared to mirror (with a phase lag) the changes in the number of motoneurons in the lateral motor column.

Age-related changes in brainstem auditory neurotransmitters: Measures of GABA and acetylcholine function

This study was designed to determine if there are age-related alterations in the bio-synthetic enzyme glutamic acid decarboxylase (GAD), the degradative enzyme GABA-transaminase (GABA-T), and the uptake system for GABA in the central nucleus of the inferior colliculus (CIC), the cochlear nucleus (CN), and/or nuclei of the lateral lemniscus (NLL) of Fischer-344 rats. For purposes of comparison, the cholinergic neuronal system was studied in parallel in young adult (3–7 months), mature (15–17 months) and aged (24–26 months) rats. In young adults GAD activity was highest in the CIC (219 nmol/mg protein/h; N = 5), intermediate in NLL (82 nmol/mg protein/h), and lowest in CN (34 nmol/mg protein/h). Choline acetyltransferase (ChAT) activity was highest in NLL and CN, and approximately 35–40% lower in CIC. A more uniform pattern was observed with GABA-T activity. Reductions in GAD activity were seen in the CIC of mature (-31%) and aged (-30%) rats that were not graded with age when compared to young adult, P < 0.05 ( N = 5). This effect was regionally selective, since the CN did not show any loss of GAD or ChAT activity. The neurotransmitter selectivity of this deficit in CIC is supported by the non-parallel changes in ChAT activity (-22%, aged vs. mature, P < 0.05) that occurred after the changes in GAD activity. In contrast to the loss of GABAergic biosynthetic capacity in aged CIC, high affinity uptake processes (K d and V max) for 14C-GABA and 3H- d-aspartate were not significantly altered ( P > 0.05). Similar to the CIC, the NLL showed remarkable age-related deficits, but these deficits were more substantial for the cholinergic system (ChAT activity: -56% aged vs. young adult. P < 0.05; GAD activity: -35% aged vs. mature). None of the areas examined showed a significant loss of GABA-T activity with aging. These data suggest: 1) Age-related loss of GABA-mediated inhibition in the CIC of Fischer-344 rats is not attributable to changes in uptake or degradation of GABA, but may be related loss of biosynthetic capacity (i.e. activity or quantity) of the GAD present; 2) processing centers of the central auditory pathway (i.e. CIC and NLL), but not necessarily primary (i.e. CN) integrativc nuclei, demonstrate selective, age-related neurochemical deficits; and 3) age-related neurochemical changes in central auditory structures may contribute substantially to the abnormal perception of signals in noise and loss of speech discrimination observed in neural presbycusis.

2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline protects against both AMPA and kainate-induced lesions in rat striatum in vivo

In the present work we have tested the neuroprotective effect of 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX) on the excitotoxic damage induced by the injection of several glutamate receptor agonists into the rat striatum. NBQX was co-injected with each of the agonists studied (1 μl) in the striatum and damage was assessed by the determination of both glutamate decar☐ylase and choline acetyltransferase activities in striatal homogenates, five days after the lesion. Additionally, animals were transcardially perfused with 0.9% saline/4% paraformaldehyde and brain coronal sections were stained with Cresyl Violet for histological analysis. Our results show that NBQX (25 nmol) did not protect against the damage induced by the intrastriatal injection of 200 nmol quinolinic acid monitored by either choline acetyltransferase or glutamate decar☐ylase activity. In contrast, the same concentration of NBQX partially protected against 200 nmol N-methyl- d-aspartate induced damage; this protection was more notable as detected by changes in choline acetyltransferase activity. When non- N-methyl- d-aspartate receptor agonists were used as excitotoxins, coinjection of NBQX (25 nmol) resulted in a notable protection against both α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA, 40 nmol) and kainate (10 nmol) induced neurodegeneration. At this concentration, protection was slightly better in AMPA-injected animals (71% protection averaged from choline acetyltransferase and glutamate decar☐ylase enzyme activities) as compared to kainate-injected animals (47.5% protection). When a higher concentration of NBQX was tested (40 nmol) the protection against kainate improved to 65% while that against AMPA remained constant (64% protection). Quantitative analysis of damaged cells in Cresyl Violet-stained sections corroborated the protective effect of NBQX against neuronal damage mediated bynon- N-methyl- d-aspartate receptors. p ]It is concluded that NBQX equally protects against AMPA- and kainate-induced lesions in the striatum in vivo and that non- N-methyl- d-aspartate receptor antagonists might be useful as protectors against neuronal damage produced by excess activation of glutamate receptors.

Asymmetrical changes in the choline acetyltransferase activity in the preoptic-anterior hypothalamic area during the oestrous cycle of the rat.

There is evidence that the cholinergic system modulates, in a circadian and asymmetric way, the neural activity involved in the regulation of ovulation. In the present study we measured choline acetyltransferase (ChAT) activity during the oestrous cycle in both sides of the preoptic-anterior hypothalamic area (POA-AHA) of the rat. The right side of POA-AHA showed significant changes in ChAT activity during the oestrous cycle. The activity of the enzyme was higher in the right side on the day of oestrous (47.3 +/- 3.2 nmol mg-1 protein 1 h-1 vs 25.8 +/- 2.4, p < 0.05), whilst on the second day of dioestrous the activity was higher in the left one (30.6 +/- 3.4 vs 20.0 +/- 1.5). The differences in the activity of ChAT observed support the idea of the existence of asymmetry in the POA-AHA cholinergic system which varies during the oestrous cycle.

Altered levels and splicing of the amyloid precursor protein in the adult rat hippocampus after treatment with DMSO or retinoic acid

Alzheimer's disease and cognitive impairment in rats has been associated with an increase in the percentage of amyloid precursor protein (APP) containing the KPI domain. It has recently been reported that retinoic acid (RA) is capable of increasing the levels and altering the splicing ratio of APP in cultured SH-SY5Y cells. The effects of peripherally administered RA (64 or 640 μg/kg; i.p.; q.d.) on the abundance of APP, the ratio of the three major isoforms, and the relative abundance of nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) were determined by rtPCR in the hippocampus of aged rats. Corresponding changes in choline acetyltransferase (ChAT) activity were also measured. Vehicle (DMSO) treated rats exhibited a 2× ( P < 0.01) increase in total APP and an 8× ( P < 0.001) decrease in the cyclophilin transcript. In addition, DMSO increased the percentage of APP 695 from 89% in saline treated rats to 94%. Treatment of RA in DMSO decreased the accumulation of total APP relative to cyclophilin at both the low (6.4×; P < 0.01) and high (8×; P < 0.05) dosages when compared to DMSO treated rats. Furthermore, the level of APP-695 decreased to 82% with low dosage of RA and 75% at high dosage of the total APP transcripts. No significant change in either NGF, NT-3, or BDNF transcripts were observed following low or high dosage RA administration relative to cyclophilin RNA nor was a change in ChAT activity detected at either of the dosages tested. These results demonstrate that both DMSO and RA are capable of altering APP accumulation and splicing in distinct manners.

Differential development of cholinergic nerve terminal markers in rat brain regions: implications for nerve terminal density, impulse activity and specific gene expression

During critical developmental periods, cholinergic activity plays a key role in programming the development of target cells. In the current study, ontogeny of cholinergic terminals and their activity were contrasted in 4 brain regions of the fetal and neonatal rat using choline acetyltransferase activity, which is unresponsive to changes in impulse flow, and [3H]hemicholinium-3 binding, which labels the high-affinity choline transporter that upregulates in response to increased neuronal stimulation. In all 4 regions (cerebral cortex, midbrain + brainstem, striatum, hippocampus) choline acetyltransferase activity increased markedly from late gestation through young adulthood, but generally did so in parallel with the expansion of total membrane protein, reflective of axonal outgrowth and synaptic proliferation. In contrast, [3H]hemicholinium-3 binding was extremely high in late gestation and immediately after birth, declined in the first postnatal week and then rose again into young adulthood. The ontogenetic changes reflected alterations primarily in the number of binding sites (Bmax) and not in binding affinity. Only the latter phase of development of [3H]hemicholinium-3 binding corresponded to the ontogenetic changes in choline acetyltransferase activity; in the hippocampus, there were disparities even in young adulthood, where [3H]hemicholinium-3 binding showed a spike of activity centered around the 5th to 6th postnatal week, whereas choline acetyltransferase did not. Correction of binding for membrane protein development did not eliminate any of the major differences in developmental patterns between the two markers. These results suggest that development of the choline transporter binding site is regulated independently of the outgrowth of the bulk of cholinergic nerve terminals.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of hypoxia on neurotransmitter phenotype of forebrain cholinergic neurons

The effect of hypoxia on the neurotransmitter phenotype of rat forebrain cholinergic neurons was analyzed using a dissociated fetal rat culture system. The aims of this study were to examine the feasibility of using choline acetyltransferase (ChAT) activity as a measure of cell injury and/or recovery, to measure the time course of hypoxic effects on ChAT activity, to determine how changes in ChAT activity at 48 h post-injury relate to microscopic changes and LDH release into the medium during that time, and finally to explore the possible mechanisms of hypoxic injury in this model. At exposure to 0.5-1.5% O2 there was a time-dependent decrease in ChAT activity when cells were harvested 48 h after exposure. Forty-eight hours after 8-9 h hypoxic exposure ChAT activity was 50-60% that of controls without any alteration in morphology of neurons. An 8 h exposure to hypoxic conditions caused a post-exposure time-dependent decrease in ChAT activity to 20% of control level at 72 h. Thereafter there was spontaneous recovery of phenotype to 60% of control which remained stable between 5 and 7 days post-exposure. Loss of neurotransmitter phenotype was not well correlated with other measures of cytotoxicity including morphological changes and LDH release. The loss of phenotype observed with hypoxia was mimicked by glutamate and kainate but not by NMDA. Consistent with these observations, neither APV nor AP3 significantly altered the effect of hypoxia on forebrain cholinergic neurons, while the addition of APV and CNQX in combination protected the phenotype of these neurons only if there was 50% or less loss of phenotype following hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

Increase in nerve growth factor-like immunoreactivity and decrease in choline acetyltransferase following contusive spinal cord injury

We have previously described a graded spinal cord injury model in the rat. Mild contusive injury results in an initially severe functional deficit that is attenuated over time to reveal the mild chronic deficits that characterize this injury. In this study, we have shown that mild contusive injury also results in a significant decrease in choline acetyltransferase (ChAT) activity during the first week after injury. At 1 week ChAT activity is maximally reduced at the site of the contusion and is also significantly lowered throughout the spinal cord. ChAT activity then rebounds during the following 3 weeks, partially at the injury site where there is considerable loss of gray and white matter, and completely in rostral and caudal cord segments. The rebound in ChAT activity is temporally associated with the partial recovery of function. Further, the changes in ChAT activity after injury are mirrored by changes in nerve growth factor-like immunoreactivity (NGF-LI) as determined by a specific two-site ELISA. NGF-LI increases significantly after injury, reaching a maximum at 7 days after contusion and at the injury site. However, levels of NGF-LI are also significantly increased throughout the spinal cord. NGF-LI then decreases at 2 and 4 weeks as ChAT activity rebounds. Further experiments will be needed to examine the possibility of a role for NGF in promoting the recovery of function after spinal cord injury.

Effects of cholinergic-rich neural grafts on radial maze performance of rats after excitotoxic lesions of the forebrain cholinergic projection system—I. Amelioration of cognitive deficits by transplants into cortex and hippocampus but not into basal forebrain

After ibotenate (10.0 mg/ml) lesions to the nucleus basalis and medial septal regions, at the source of the cortical and hippocampal branches of the forebrain cholinergic projection system, rats displayed long-lasting stable impairment in reference and working memory in both spatial (place) and associative (cue) radial maze tasks. Cell suspension transplants of cholinergic-rich fetal basal forebrain tissue dissected at embryonic day 15 substantially improved all aspects of radial maze performance to a comparable degree whether sited in cortex, hippocampus, or both regions of the host brain. No additive effects were obtained with grafts in both terminal regions, but total graft volume, assessed stereologically, showed a significant negative correlation with error scores. Rats with behaviourally effective grafts, like controls, were disrupted in the place task when tested in dim light which obscured extra-maze spatial cues. Lesioned rats were not affected by change in lighting. Grafts of cholinergic-poor fetal hippocampal tissue did not improve radial maze performance; neither did grafts of cholinergic-rich tissue placed within the host basal forebrain lesion sites. In rats with cholinergic-rich terminal grafts, cortical and hippocampal choline acetyltransferase activity was restored to control level, commensurate with site of transplant, whereas it was significantly reduced in lesioned animals and those with functionally ineffective grafts. The indiscriminate error pattern and insensitivity to changes in lighting shown by lesioned rats suggested that lesioning primarily disrupted attention rather than short- or long-term spatial or associative memory processes. Since rats with cholinergic-rich grafts showed both reduced errors and recovery of stimulus control, the data indicated that grafts affected information processing, rather than changes in motor or motivational processes. Changes in choline acetyltransferase activity and the behavioural efficacy of cholinergic-rich grafts are consistent with the involvement of acetylcholine in the behavioural deficits and recovery displayed by lesioned and grafted groups, but do not rule out contributions from other factors. The equipotency of grafts within each terminal region suggests also that there may be a considerable degree of functional cooperation between the two branches of the forebrain cholinergic projection system. Functional recovery may involve local, nonspecific synaptic or paracrine mechanisms within the target regions, since grafts were efficacious only when placed in the terminal areas, but not when sited homotopically in the basal forebrain, indicating that they did not achieve any functionally significant structural repair to the host brain at that site.

Nerve growth factor levels and choline acetyltransferase activity in the brain of aged rats with spatial memory impairments

Nerve growth factor (NGF) and choline acetyltransferase (ChAT) activity levels were measured in 7 different brain regions in young (3-month-old) and aged (2-years-old) female Sprague-Dawley rats. Prior to analysis the spatial learning ability of the aged rats was assessed in the Morris' water maze test. In the aged rats a significant, 15–30%, increase in NGF levels was observed in 4 regions (septum, cortex, olfactory bulb and cerebellum), whereas the levels in hippocampus, striatum and the brainstem were similar to those of the young rats. The NGF changes did not correlate with the behavioral performance within the aged group. Minor 15–30%, changes in ChAT activity were observed in striatum, brainstem and cerebellum, but these changes did not correlate with the changes in NGF levels in any region. The results indicate that brain NGF levels are maintained at a normal or supranormal levels in rats with severe learning and memory impairments. Ther results, therefore, do not support the view that the marked atrophy and cell loss in the forebrain cholinergic system that is known to occur in the behaviorally impaired aged rats is caused by a reduced availability of NGF in the cholinergic target areas. The results also indicate that the slightly increased levels of NGF are not sufficient to prevent the age-dependent atrophy of cholinergic neurons, although they might be important for the stimulation of compensatory functional changes in a situation where the system is undergoing progressive degeneration.