Measuring Choline Acetyltransferase Research Articles

Hippocampal Lewy pathology and cholinergic dysfunction are associated with dementia in Parkinson's disease.

The neuropathological substrate of dementia in patients with Parkinson's disease is still under debate, particularly in patients with insufficient alternate neuropathology for other degenerative dementias. In patients with pure Lewy body Parkinson's disease, previous post-mortem studies have shown that dopaminergic and cholinergic regulatory projection systems degenerate, but the exact pathways that may explain the development of dementia in patients with Parkinson's disease remain unclear. Studies in rodents suggest that both the mesocorticolimbic dopaminergic and septohippocampal cholinergic pathways may functionally interact to regulate certain aspects of cognition, however, whether such an interaction occurs in humans is still poorly understood. In this study, we performed stereological analyses of the A9 and A10 dopaminergic neurons and Ch1, Ch2 and Ch4 cholinergic neurons located in the basal forebrain, along with an assessment of α-synuclein pathology in these regions and in the hippocampus of six demented and five non-demented patients with Parkinson's disease and five age-matched control individuals with no signs of neurological disease. Moreover, we measured choline acetyltransferase activity in the hippocampus and frontal cortex of eight demented and eight non-demented patients with Parkinson's disease, as well as in the same areas of eight age-matched controls. All patients with Parkinson's disease exhibited a similar 80-85% loss of pigmented A9 dopaminergic neurons, whereas patients with Parkinson's disease dementia presented an additional loss in the lateral part of A10 dopaminergic neurons as well as Ch4 nucleus basalis neurons. In contrast, medial A10 dopaminergic neurons and Ch1 and Ch2 cholinergic septal neurons were largely spared. Despite variable Ch4 cell loss, cortical but not hippocampal cholinergic activity was consistently reduced in all patients with Parkinson's disease, suggesting significant dysfunction in cortical cholinergic pathways before frank neuronal degeneration. Patients with Parkinson's disease dementia were differentiated by a significant reduction in hippocampal cholinergic activity, by a significant loss of non-pigmented lateral A10 dopaminergic neurons and Ch4 cholinergic neurons (30 and 55% cell loss, respectively, compared with neuronal preservation in control subjects), and by an increase in the severity of α-synuclein pathology in the basal forebrain and hippocampus. Overall, these results point to increasing α-synuclein deposition and hippocampal dysfunction in a setting of more widespread degeneration of cortical dopaminergic and cholinergic pathways as contributing to the dementia occurring in patients with pure Parkinson's disease. Furthermore, our findings support the concept that α-synuclein deposition is associated with significant neuronal dysfunction in the absence of frank neuronal loss in Parkinson's disease.

Krill phosphatidylserine improves learning and memory in Morris water maze in aged rats

The ameliorating effect of phosphatidylserine (PS) isolated from krill (KR-PS) on the learning and memory deficits associated with normal aging in rats was investigated, as compared with soybean PS (SOY-PS). Rats were orally administered with KR-PS (20, 50 mg kg − 1) and SOY-PS (50 mg kg − 1) daily, for 7 days, 30 min before behavioral assessment using the Morris water maze (MWM). Changes in the cholinergic system were examined by measuring choline acetyltransferase (ChAT) and acetylcholinesterase (AchE) immunoreactivity in the hippocampus. The daily administration of KR-PS produced a significant improvement in the escape latency for finding the platform in the MWM, as compared with SOY-PS. Consistent with the behavioral results, KR-PS treatments significantly alleviated age-associated losses of cholinergic immunoreactivity, and muscarinic acetylcholine receptor type 1 (mAChR-M1) and choline transporter (CHT) mRNA expression in the hippocampus. These findings demonstrate that KR-PS showed significant neuroprotective activity against the neuronal and cognitive impairments that occur with normal aging in rats; comparable results were obtained with SOY-PS. These data indicate that oral administration of PS derived from marine life could substitute for bovine cerebral cortex PS (BC-PS) as therapy for the improvement of diminished memory function in elderly people.

Additive and synergistic effects of fetal nicotine and dexamethasone exposure on cholinergic synaptic function in adolescence and adulthood: Implications for the adverse consequences of maternal smoking and pharmacotherapy of preterm delivery

Maternal smoking contributes to preterm delivery; glucocorticoids are the consensus treatment for prematurity, thus producing fetal coexposure to nicotine and dexamethasone. We administered nicotine to pregnant rats throughout gestation at a dose (3 mg/kg/day) producing plasma levels typical of smokers. Later in gestation, animals received dexamethasone (0.2 mg/kg). We assessed developmental indices for acetylcholine (ACh) synaptic function throughout adolescence, young adulthood and later adulthood, evaluating brain regions possessing major ACh projections and cell bodies; we measured choline acetyltransferase activity, hemicholinium-3 binding to the presynaptic choline transporter and nicotinic ACh receptor binding. In general, nicotine and dexamethasone, alone or in combination, produced regionally-selective increases or decreases in choline acetyltransferase activity but larger, consistent elevations in hemicholinium-3 and nicotinic ACh receptor binding; the patterns were indicative of ACh synaptic hyperactivity. Superimposed on these overall effects, there were significant disparities in temporal and regional relationships among the different treatments, notably involving effects that emerged later in life, after a period of apparent normality. This indicates that nicotine and dexamethasone do not simply produce an initial ACh neuronal injury that then persists throughout the lifespan but rather, they alter the developmental trajectory of ACh function. Most importantly, the combined exposure to nicotine + dexamethasone elicited greater changes than either of the individual exposures, involving both additive and synergistic effects. Our results thus point to potentially worse neurobehavioral outcomes of the pharmacotherapy of preterm labor in the offspring of smokers.

NAALADase inhibition protects motor neurons against chronic glutamate toxicity

Glutamate toxicity is implicated in the pathogenesis of amyotrophic lateral sclerosis. The neuropeptide N-acetyl-aspartyl glutamate (NAAG) appears to function both as a storage form for glutamate and as a neuromodulator at glutamatergic synapses. N-acetylated-α-linked acidic dipeptidase (NAALADase; also termed glutamate carboxypeptidase II) yields N-acetyl aspartate (NAA) and glutamate. Prior studies have demonstrated NAALADase upregulation in motor cortex and increased NAAG, NAA and glutamate in cerebrospinal fluid from amyotrophic lateral sclerosis patients. The potent NAALADase inhibitor, 2-(phosphonomethyl)-pentanedioic acid (2-PMPA), was tested in an in vitro model of chronic glutamate-mediated motor neuron degeneration. Neuroprotection was determined (1) biochemically, by measuring choline acetyltransferase activity, (2) immunohistochemically, by counting neurofilament-H-positive motor neurons and (3) morphologically, with phase contrast microscopy. 2-PMPA (10 μM) had significant neuroprotective effects on motor neurons as evidenced by increased choline acetyltransferase activity, decreased motor neuron loss and improved gross morphology. Results suggest that NAALADase inhibitors protect against chronic glutamate-mediated motor neuron degeneration and may prove therapeutic towards amyotrophic lateral sclerosis.

Activation of the reticulothalamic cholinergic pathway by the major metabolites of aniracetam

The aim of the study was to further investigate the effects of aniracetam, a cognition enhancer, and its metabolites on the brain cholinergic system. We measured choline acetyltransferase activity and acetylcholine release using in vivo brain microdialysis in stroke-prone spontaneously hypertensive rats (SHRSP). The enzyme activity in the pons–midbrain and hippocampus, and basal acetylcholine release in the nucleus reticularis thalami were lower in SHRSP than in age-matched Wistar Kyoto rats, indicating central cholinergic deficits in SHRSP. Repeated treatment of aniracetam (50 mg/kg p.o.×11 for 6 days) preferentially increased the enzyme activity in the thalamus, whereas decreased it in the striatum. Among the metabolites of aniracetam, local perfusion of N-anisoyl-γ-aminobutyric acid (GABA, 0.1 and/or 1 μM) and p-anisic acid (1 μM) into the nucleus reticularis thalami, dorsal hippocampus and prefrontal cortex of SHRSP produced a significant but delayed increase of acetylcholine release. We failed, however, to find any effect of aniracetam itself. A direct injection of N-anisoyl-GABA (1 nmol) into the pedunculopontine tegmental nucleus of SHRSP enhanced the release in the nucleus reticularis thalami. Thus, these data prove that aniracetam can facilitate central cholinergic neurotransmission via both metabolites. Based on its pharmacokinetic profile, N-anisoyl-GABA may contribute to the clinical effects of aniracetam, mainly by acting on the reticulothalamic cholinergic pathway.

Cholinergic modulation of non-adrenergic, non-cholinergic relaxation in isolated, small coronary arteries from lambs.

The presence of cholinergic innervation of small coronary arteries in the lamb was investigated by measuring choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities and by performing in vitro experiments in a microvascular myograph to establish whether or not there is a cholinergic component in the response to electrical field stimulation (EFS). ChAT-specific activity was present in proximal coronary segments, but was significantly higher in small coronary arteries. AChE-positive ganglia and fibres were distributed within the adventitia and outer third of the media in proximal coronary segments, and dense perivascular nerve plexuses were observed in small coronary arteries. Acetylcholine induced contractions in all preparations examined and relaxations in 20% of the segments contracted with the thromboxane analogue U46619. EFS did not induce neurogenic contractions in lamb small coronary arteries. In the presence of the alpha-adrenoceptor antagonist, phentolamine, EFS caused frequency-dependent reproducible relaxations that were enhanced by the blocker of cholinergic transmission, botulinum neurotoxin. An inhibitor of AChE, physostigmine, had no significant effect on the relaxations caused by EFS, while both the muscarinic receptor antagonist, atropine, and the muscarinic M2-receptor antagonist, AFDX 116, enhanced these responses. Blockade of sympathetic neurotransmission with guanethidine or incubation with the P2-receptor antagonist, suramin, abolished the relaxations induced by EFS, whereas propranolol was without effect. Low-frequency EFS caused less relaxation in preparations activated by acetylcholine than in those contracted with U46619, while sensitivity and maximal relaxation induced by adenosine 5'-triphosphate (ATP) were not different in U46619- and acetylcholine-contracted arteries. The presence of the enzymes necessary for both biosynthesis and degradation of acetylcholine and the finding that blockers of cholinergic neurotransmission enhance EFS-induced relaxations suggest that small coronary arteries are cholinergically innervated.

Evidence suggesting that lateral parabrachial nucleus is responsible for enhanced medullary cholinergic activity in hypertension.

Cholinergic activities in the rostral ventrolateral medulla are enhanced in hypertensive animals, and enhanced cholinergic activity contributes to hypertension. Neurons in the lateral parabrachial nucleus and baroreceptors are suggested to be involved in mediation of acetylcholine release in the rostral ventrolateral medulla. To investigate the hypothesis that the lateral parabrachial nucleus is involved in the increased medullary cholinergic activity in hypertensive rats, we measured choline acetyltransferase activity in the rostral ventrolateral medulla, and examined effects of electrolytic lesioning of the lateral parabrachial nucleus in deoxycorticosterone acetate-salt hypertensive rats. We found that choline acetyl-transferase activity in the medullary region was increased in deoxycorticosterone acetate-salt hypertensive rats. Unilateral lesioning of the lateral parabrachial nucleus abolished the increase in choline acetyltransferase activity in the lesioned side of the medullary region of hypertensive rats, whereas such activity in the medullary region of control rats was unaffected by lesioning. Bilateral lesioning of the lateral parabrachial nucleus inhibited the development of hypertension in hypertensive rats. In contrast, baroreceptor denervation did not inhibit the increase in choline acetyltransferase activity in the medullary region of hypertensive rats. These results are compatible with the hypothesis that the lateral parabrachial nucleus area is involved in mediation of increased medullary cholinergic activity and thus plays a role in the development of hypertension.

Enhanced choline acetyltransferase activity does not explain the action of inhaled convulsants

Enhancement of choline acetyltransferase (ChAT) activity and increased intraneuronal acetylcholine (ACh) may explain the convulsant activity of some inhaled compounds. Enflurane, for example, enhances such activity. Accordingly, we measured choline acetyltransferase (ChAT) activity in rat cortical synaptosomes in the presence of two inhaled convulsants, flurothyl (CF3CH2OCH2CF3) and 1,2-dichlorohexafluorocyclobutane at partial pressures below and greatly exceeding those which produce convulsions in vivo. Neither agent changed the kinetic parameters, maximum velocity (vmax) or Michaelis constant (Km). The vmax for controls in the flurothyl series was 016 (0.06) nmol mg-1 min-1 and the Km was 0.23 (0.11) mmol litre-1. For the 1,2-dichlorohexafluorocyclobutane series of experiments the results for the controls were vmax 0.23 (0.10) nmol mg-1 min-1 and Km 0.20 (0.08) mmol litre-1. Modification of ChAT activity did not contribute to the excitatory effects of these agents.

Age-related changes in learning and memory and cholinergic neuronal function in senescence accelerated mice (SAM)

The senescence-accelerated mouse (SAM) has been established as a murine model of accelerated aging. We investigated learning ability and memory in various tasks in a SAM strain, SAMPITA, and in a control strain of SAMR1TA at the ages of 20, 30 and 40 weeks. We also measured choline acetyltransferase (ChAT) and cholinesterase (ChE) activity in the brains of these mice at the same ages. In a Y-maze task, in which short-term memory can be examined, there was no difference in learning ability between SAMP1TA and SAMR1TA at any age. Ability in latent learning and passive-avoidance tasks was less in SAMP1TA at 30 weeks of age than in age-matched SAMR1TA. The level of ChAT activity in the striatum of SAMP1TA was lower, than that of SAMR1TA at the ages of 20 and 30 weeks. At the ages of 40 and 50 weeks, ChE activity in the striatum of SAMP1TA was lower than that of SAMR1TA. These results suggest that SAMP1TA has a deficit, with cholinergic neuronal dysfunction, in learning ability and memory, as shown by impairment of performance in latent learning and long-term memory, but not in short-term memory.

6,7-Dinitroquinoxaline-2,3-dione but not MK-801 exerts a protective effect against kainic acid neurotoxicity in the goldfish retina

Recent findings indicated that the excitotoxicity of glutamate analogues was prevented in the mammalian nervous system by N-methyl- d-aspartate (NMDA) antagonists. The neurodegenerative effects of kainic acid, and the putative protection of MK-801 and 6,7-dinitroquinoxaline-2,3-dione (DNQX), were investigated by morphological studies showing the toxicity of kainic acid to the neurons of the inner nuclear layer, and measuring choline acetyltransferase and glutamate decarboxylase activities in the retina. In addition, the proliferation of Miller retinal cells was assumed as an index of neuronal degeneration and was quantified by counting glial fibrillary acidic protein immunopositive cells. Our observations suggest that the non-NMDA receptor antagonist DNQX exerted a protective effect on goldfish retinal neurons, while MK-801 did not prevent the neurotoxicity induced by kainic acid in the goldfish retina. This finding is in agreement with previous work on kainic acid toxicity in the goldfish optic tectum.

Behavioural and neurochemical effects of superior cervical ganglionectomy in rats with septo-hippocampal lesions.

This longitudinal study, extending over 12 months, assessed the behavioural and biochemical effects of hippocampal sympathetic ingrowth (HSI) into the partially denervated hippocampus. Male Long-Evans rats received fimbria-fornix lesions (FIFO) or sham operations at 90 days of age. At the same time half of the rats from each group sustained bilateral ablation of the superior cervical ganglia (SCGX). A battery of behavioural tests, measuring spontaneous alternation, activity in the open field and home cage, and radial-maze performance, were employed, starting after one very short (16 days) and one extended (216 days) post-operative delay. Neurochemical analyses measuring choline acetyltransferase (ChAT) activity, high-affinity choline (HACU) and noradrenaline uptake by hippocampal synaptosomes (HANU), hippocampal noradrenaline ([NA]), serotonin ([5-HT]) and 5-hydroxyindoleacetic acid ([5-HIAA]) concentrations were carried out in a dorsal, a "middle" and a ventral region of the hippocampus. Lesion of the FIFO induced a significant and enduring deficit in radial-maze performance, in addition to a persistent locomotor hyperactivity. ChAT and HACU were significantly depleted in all three regions of the hippocampus at 12 months, and these deficits were negatively correlated with maze performance. SCGX in the presence of the FIFO lesion significantly reduced [NA] in the middle region of the hippocampus, as compared to SCGX rats, and contributed to a restoration of lesion-induced depletions in [5-HT] and [5-HIAA] in the middle and ventral hippocampal regions, whilst failing to elicit any behavioural changes at either time point. It is concluded that if lesion-induced HSI indeed occurred, as is suggested by neurochemical evidence, it had no effect upon the observed behavioural deficits elicited by transection of the FIFO in the rat.

Reduced choline acetyltransferase activity and muscarinic M1 receptor levels in aged Fisher 344 rat brains did not parallel their respective mRNA levels

Differences in the acetylcholine (ACh)-mediated neuronal system of the brain between aged and young rats were studied by measuring choline acetyltransferase (ChAT) activity, muscarinic M1 receptor (M1-R) and their respective mRNA levels. In aged rats, ChAT activity and the M1-R level were significantly reduced in the cerebral cortex, hippocampus and striatum compared with that in young rats. On the other hand, there was no difference in the ChAT mRNA level in the striatum and the basal forebrain, or the M1-R mRNA level in the cerebral cortex, hippocampus and striatum between aged and young rats. The effects of chronic administration of bifemelane (4-(2-benzylphenoxy)-N-methylbutylamine hydrochloride), which is used for the treatment of sequelae of cerebrovascular diseases, were also evaluated. In aged rats chronically administered bifemelane, the ChAT activity recovered to the level in the young rats in the cerebral cortex and hippocampus, and the M1-R level recovered completely in the cerebral cortex, hippocampus and striatum. However, the ChAT mRNA level and the M1-R mRNA level were not affected by bifemelane administration. Thus, the decreases and recoveries in ChAT activity and M1-R level did not parallel the changes in their respective mRNAs. These results suggest that the age-related impairments in ACh-mediated neuronal system are considered to be caused primarily by disorders of post-transcriptional events.

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.

Development of plasticity of brain function with repeated trainings and passage of time after basal forebrain lesions in rats.

Basal forebrain (BF) lesion-induced amnesia in rats is widely used as an animal model of Alzheimer's disease (AD). To study the plasticity of brain function in BF-lesioned rats, we examined the effects of repeated trainings and the passage of time after the lesion on learning ability 3 weeks and 3 months after BF-lesions with ibotenic acid, using an eight-arm radial maze and passive avoidance tasks, and measured choline acetyltransferase (ChAT) activity. Both time and re-training played important roles in the recovery of the ability to learn, as measured with the eight-arm radial maze task, but not the passive avoidance task. In contrast, ChAT activity in the frontal cortex, which was low 3 weeks after the lesion, still low 3 months after lesion, even though the ability to learn had recovered. Recovery of the ability to learn can be attributed to undamaged cholinergic neurons, or to other neuronal systems, or to both. This animal model can be used to demonstrate the plasticity of brain function.

Action of picolinic acid and structurally related pyridine carboxylic acids on quinolinic acid-induced cortical cholinergic damage

Picolinic acid, a pyridine monocarboxylic acid derived from tryptophan metabolism by the kynurenine pathway, was shown to block cortical cholinergic neurotoxicity induced by quinolinic acid (QUIN), a pyridine dicarboxylic acid yielded by the same pathway. This study examined the specificity of the anti-toxic effect of picolinic acid by comparing its effect with several structurally related mono- and dicarboxylic acids, and by evaluating its potential to influence cholinergic neurotoxicity produced by kainic, ibotenic and quisqualic acid. Picolinic acid or related agents were injected alone or in combination with a fixed dose of QUIN into the right nucleus basalis magnocellularis (nbM) of rats anesthetized with halothane. Cholinergic neurotoxicity was assessed 7 days post injection by measuring choline acetyltransferase (ChAT) activity in the frontoparietal cortex on the injected and uninjected side. In picolinate experiments, the staining of nbM neurons by acetylcholinesterase (AChE) histochemistry was also examined. Focal injections of QUIN depleted cortical ChAT activity and staining of AChE in the nbM. Co-injection of picolinic acid with QUIN attenuated the decline in these two cholinergic neuron markers. Isonicotinate (4-pyridine monocarboxylate), but not nicotinate (3-pyridine monocarboxylate), significantly attenuated QUIN's effect on cortical ChAT activity. Among several dicarboxylic acids examined, only dipicolinate (2,6-pyridine dicarboxylate) showed activity. It produced a modest QUIN-like effect, but in co-injection experiments it attenuated the QUIN-induced decrease in cortical ChAT. When co-injected into the nbM with a neurotoxic dose of kainic, ibotenic or quisqualic acid which produced a neurotoxic response comparable to that of QUIN, picolinic acid attenuated kainic acid-induced neurotoxicity, but not ibotenate or quisqualate neurotoxicity. In contrast, kynurenic acid, which blocked QUIN's action, blocked the neurotoxicity of both kainate and ibotenate. The results suggest that actions of picolinic acid against QUIN show certain selectivity in regard to the chemical structure of picolinic acid. In addition, picolinic acid has selectivity against excitotoxins acting via different mechanisms. Its ability to influence only those excitotoxins which require an intact glutamatergic afferent input for their neurotoxic effect suggests that the protective effect of picolinic acid may involve an interaction with this input.

Excitotoxic lesions of rat basal forebrain: Differential effects on choline acetyltransferase in the cortex and amygdala

Previous studies have shown that basal forebrain lesions using different excitotoxins produce similar decreases in cortical choline acetyltransferase, but differential effects on memory. However, basal forebrain cholinergic neurons send efferents to the amygdala and cortex. The present studies compared the effects of several excitotoxins on choline acetyltransferase levels in both of these structures. Lesions of the basal forebrain were made in rats by infusing different doses of either α-amine-3-hydroxy-5-methyl-4-isoxazole propionic acid, ibotenic acid, quisqualic acid, quinolinic acid or N- methyl- d-aspartic acid and measuring choline acetyltransferase seven days later. All of the excitotoxins exerted a differential response on cholinergic neurons of the basal forebrain projecting to the cortex or amygdala. Quinolinic acid was a more potent neurotoxin to cholinergic neurons innervating the amygdala than those projecting to the cortex. In contrast, quisqualic acid and α-amine-3-hydroxy-5-methyl-4-isoxazole were more potent neurotoxins to the cortical projection. α-Amine-3-hydroxy-5-methyl-4-isozazole propionic acid was the most potent excitotoxin for destroying cholinergic neurons innervating either the cortex or amygdala. A parallel neurotoxic response was obtained in the cortex and amygdala following infusion of ibotenic acid or N- methyl- d-aspartic acid with little selectivity for choline acetyltransferase depletion in the cortex or amygdala. Histological analysis of the injection site revealed that acetylcholinesterase-positive neurons were destroyed by the excitotoxins in a dose-dependent manner. Excitotoxins (ibotenic acid, quinolinic acid, N- methyl- d-aspartic acid) that produce the greatest impairments in memory were found to produce the greatest depletion of choline acetyltransferase in the amygdala. These results might suggest that cholinergic neurons projecting to the amygdala play an important role in memory.

Effects of septal and/or raphe cell suspension grafts on hippocampal choline acetyltransferase activity, high affinity synaptosomal uptake of choline and serotonin, and behavior in rats with extensive septohippocampal lesions

3t 31 days of age, Long-Evans female rats sustained aspirative lesions of the septohippocampal pathways and, 14 days later, received intrahippocampal suspension grafts prepared from the region including the medial septum and the diagonal band of Broca (Group S, n = 11), from the region including the mesencephalic raphe (Group R, n = 11) or from both regions together (Group S + R, n = 11). Sham-operated (Group Sham, n = 9) and lesion-only (Group Les, n = 11) rats served as non-grafted controls. Seven Sham, 7 Les and 8 rats from each transplant group were tested for home cage activity (6 months after grafting) and radial maze performance (between 7.5 and 8.5 months post-grafting). One month after completion of behavioral testing, the dorsal hippocampi of these rats were prepared for measuring choline acetyltransferase (ChAT) activity and high affinity synaptosomal uptake of both [ 3H]choline and [ 3H]serotonin. The remaining rats were used for histological verifications on brain sections stained for acetylcholinesterase (AChE). The lesions increased locomotor activitu, impaired radial maze learning and, in the dorsal hippocampus, reduced AChE positive staining, decreased ChAT activity (−73%) as well as high affinity uptake of both choline (−81%) and serotonin (−82%). Neither type of transplant produced any significant behavioral recovery. However, septal transplants increased hippocampal AChE positivity, restored ChAT activity and enhanced choline uptake to 116% and 70% of the values found in sham-operated rats, respectively; they had no significant effect on uptake of serotonin. Transplants from the raphe region had weak affects on hippocampal AChE positivity, increased both the ChAT activity and the choline uptake to 70% ad 38% of sham-operated rats, respectively, and produced an (over)compensation of the serotonin uptake which reached 324% of the values found in sham-operated rats. The co-transplantation of both regions resulted in restoration of ChAT activity (113% of sham-operated rats values), choline uptake (83% of sham-operated rats) and serotonin uptake (129% of sham-operated rats). Our neurochemical data show that after extensive denervation of the hippocampus, intrahippocampal grafts of fetal neurons may foster a neurotransmitter-specific recovery which depends upon the anatomical origin of the grafted cells; a graft rich in serotonergic neurons overcompensates the serotonergic deficit, a graft irich in cholinergic neurons attenuates the cholinergic deficit, whereas a mixture of both types of grafts produces recovery from both types of deficits. Thereby, both the feasibility and the interest of the co-grafting technique are confirmed. However, our behavioral data also suggest that separate or combined attenuation of the lesion-induced cholinergic and serotonergic deficits in the dorsal hippocampus does not seem to be a sufficient condition to induce attenuation of the lesion-induced behavioral deficits (hyperactivity, impaired memory).

Plasminogen activators in the neuromuscular system of the wobbler mutant mouse

Wobbler, the neurological mutant mouse, carries an autosomal recessive gene (wr) and has been characterized as a model of lower motoneuron disorders with associated muscle atrophy, denervation and reinnervation. During normal murine neuromuscular development a decrease in muscle plasminogen activator (PA) activity accompanies synapse maturation. In contrast, experimental denervation in adult mice leads to an increase in muscle PA activity. The purpose of the present study was to determine the possible involvement of PAs in the denervation/ reinnervation phenomena and motoneuron degeneration that characterize the wobbler mutant mouse. We determined the degree of innervation and its characteristics in wobbler mice by measuring choline acetyltransferase (ChAT) activity. We measured ChAT in the spinal cord as well as in two different muscles known to be differentially affected, biceps brachii and gastrocnemius. We found a sharp decrease of ChAT activity in both muscles but not in spinal cord extracts. We estimated the extent of sprouting by the silver/chilinesterase stain. Motoneuron terminal sprouting, not detected in normal animals, was present in 40% of the neuromuscular junctions in wobbler mice. We estimated specific PA activities in biceps brachii and gastrocnemius muscle extracts, as well as spinal cord extracts, using both an amidolytic assay and fibrin zymography. Increased PA, predominantly urokinase-PA (uPA), was observed in wobbler mouse muscle. A greater uPA was detected in biceps brachii muscle than in gastrocnemius muscle, which is less impaired by the mutation. There was no change in spinal cord PA, although tissue type PA (tPA) is the predominant PA type there. The present study demonstrates an activation of a specific serine protease, urokinase PA, in wobbler mouse muscle and may support the hypothesis of its involvement in the pathogenesis of lower motoneuron diseases.

Development of the basal forebrain cholinergic system: Phenotype expression prior to target innervation

We measured choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities in the rat to determine the time course of development, maturity, and senescence of ChAT activity. Tissue was obtained from Sprague-Dawley rats ranging in age from embryonic day 14 through 23 months. Seven regions were examined, including the magnocellular preoptic/substantia innominata region, frontal cortex, medial septal region, hippocampus, diagonal band, and medial and lateral striatum. ChAT and AChE activities were first detected as early as E18 in the medial septum, diagonal band and magnocellular preoptic area, all regions of cholinergic cell bodies. Enzyme activity subsequently developed in terminal fields of these cholinergic perikarya (hippocampus and frontal cortex) as well as in the striatum. For all regions, enzyme activity rose during the first four postnatal weeks. This increase in enzyme activity was transient and, in most instances, decreases were observed between postnatal days 30 and 60. Most dramatic were the decreases in enzyme activity in the magnocellular preoptic/substantia innominata and diagonal band regions. Age-related declines also occurred in the frontal cortex, hippocampus, magnocellular preoptic/substantia innominata region, and the striatum. Cholinergic systems undergo dynamic changes especially during development and adulthood.

Trimethyltin increases choline acetyltransferase in rat hippocampus

The environmental neurotoxin trimethyltin (TMT) destroys parts of the hippocampal formation as well as the entorhinal cortex but leaves the septal cholinergic projection to the hippocampus and dentate gyrus intact. In this study we measured choline acetyltransferase (ChAT) activity in micropunch samples of the dentate gyrus, the CA1 region of Ammon's horn, and the caudate-putamen as a measure of density of cholinergic innervation in control rats and rats exposed to 7 mg/kg TMT by means of gastric intubation. Three months after the rats were exposed to a single dose of TMT both the dentate gyrus and CA1 demonstrated significantly higher ChAT activity in TMT-exposed rats than in control rats. No differences were found between groups for the caudate-putamen samples. These results support the hypothesis that exposure to TMT causes reactive synaptogenesis in the cholinergic septohippocampal system.