Cerebro-protective effects of ENA713, a novel acetylcholinesterase inhibitor, in closed head injury in the rat

Focal ischemic brain damage and diffuse brain swelling occur in severe cases of traumatic head injury. Ischemia decreases brain acetylcholine (ACh) levels and head trauma upregulates acetylcholinesterase (AChE) in experimental animal models. The present study determined whether a brain-selective AChE inhibitor, ENA713, given once, up to 2 h after closed head injury (CHI) could reduce the vasogenic edema and accelerate recovery from neurological deficits induced by the injury in rats. ENA713 1–5 mg/kg produced a dose-related inhibition of AChE ranging from 40–85% in the cortex and hippocampus. Doses of 1, 2 and 5 mg/kg, significantly reduced the motor and neurological deficits and speeded recovery, as indicated by measurements made 7 and 14 days after injury. The two larger doses were still effective when injected 1 or 2 h after CHI. The acceleration by ENA713 of recovery of motor function was independent of its reduction in body temperature and was prevented by the simultaneous injection of mecamylamine (2.5 mg/kg), but not by scopolamine (0.2 or 1 mg/kg). Edema in the contused hemisphere (24 h after injury) and disruption of the blood brain barrier (4 h after injury) were significantly reduced (about 50%) by doses of 2 and 5 mg/kg, but not by 1 mg/kg. The data support the hypothesis that ENA713 exerts a neuroprotective effect in brain injury by preventing the decrease in cholinergic activity in cerebral vessels and in neurones.     

Systemic administration of GM1 ganglioside increases choline acetyltransferase activity in the brain of aged rats

In the brain of aged rats (22–24 months old) choline acetyltransferase (ChAT) activity in striatum and frontal cortex is lower than in young rats (4–5 months old). In contrast, ChAT activity in the hippocampus is similar in the two groups. Treating old animals with GM1 ganglioside, 30 mg/kg ip, for 30 or 45 days enhances ChAT activity in the striatum and frontal cortex, but has no effect on activity in the hippocampus. ChAT activity remains elevated in the striatum and frontal cortex for 15 days after discontinuing treatment with GM1.     

Dose-dependent CSF acetylcholinesterase inhibition by SDZ ENA 713 in Alzheimer's disease

Introduction - This study evaluates the activity of SDZ ENA 713, a centrally-selective acetylcholinesterase (AChE) inhibitor, in the cerebral spinal fluid (CSF) of patients with Alzheimer's disease (AD), and its relationship to central and peripheral pharmacokinetic parameters. Methods - Eighteen AD patients were enrolled in this open-label, multiple-dose study. Patients were titrated in 1 mg bid/week increments to target doses of 1, 2, 3, 4, 5, or 6 mg bid SDZ ENA 713. After patients had been maintained at their target dose for at least 3 days, continuous CSF samples were obtained via a lumbar catheter for 12.5 h, beginning 0.5 h prior to the final dose of SDZ ENA 713. Results - Dose-dependent inhibition of CSF AChE was significantly correlated (P<0.05) with plasma drug and metabolite concentrations. The 6 mg bid treatment group showed a maximum mean inhibition of 62% at 5.6 h post-dose. Conclusion - Rapid, sustained, dose-dependent inhibition of CSF AChE suggests that SDZ ENA 713 has therapeutic potential in AD patients.     

Neuroprotective efficacy of curcumin in arsenic induced cholinergic dysfunctions in rats

Our recent studies have shown that curcumin protects arsenic induced neurotoxicity by modulating oxidative stress, neurotransmitter levels and dopaminergic system in rats. As chronic exposure to arsenic has been associated with cognitive deficits in humans, the present study has been carried out to implore the neuroprotective potential of curcumin in arsenic induced cholinergic dysfunctions in rats. Rats treated with arsenic (sodium arsenite, 20mg/kg body weight, p.o., 28 days) exhibited a significant decrease in the learning activity, assessed by passive avoidance response associated with decreased binding of (3)H-QNB, known to label muscarinic-cholinergic receptors in hippocampus (54%) and frontal cortex (27%) as compared to controls. Decrease in the activity of acetylcholinesterase in hippocampus (46%) and frontal cortex (33%), staining of Nissl body, immunoreactivity of choline acetyltransferase (ChAT) and expression of ChAT protein in hippocampal region was also observed in arsenic treated rats as compared to controls. Simultaneous treatment with arsenic and curcumin (100mg/kg body weight, p.o., 28 days) increased learning and memory performance associated with increased binding of (3)H-QNB in hippocampus (54%), frontal cortex (25%) and activity of acetylcholinesterase in hippocampus (41%) and frontal cortex (29%) as compared to arsenic treated rats. Increase in the expression of ChAT protein, immunoreactivity of ChAT and staining of Nissl body in hippocampal region was also observed in rats simultaneously treated with arsenic and curcumin as compared to those treated with arsenic alone. The results of the present study suggest that curcumin significantly modulates arsenic induced cholinergic dysfunctions in brain and also exhibits neuroprotective efficacy of curcumin.     

Effect of nerve growth factor and GM1 ganglioside on the recovery of cholinergic neurons after a lesion of the nucleus basalis in aging rats

Summary A unilateral ibotenic acid lesion was placed in the nucleus basalis magnocellularis of 3- and 18-month-old rats. In the lesioned aging rats, the number of choline acetyltransferase-immunoreactive neurons of the nucleus basalis magnocellularis was markedly reduced in the ipsilateral side and to a lesser extent in the contralateral side. Twenty-one days after the lesion, the activity of choline acetyltransferase in the ipsilateral cortex was reduced by 40% in both groups of rats and by 24% in the contralateral frontal cortex of the aging rats. Intracerebroventricular administration of nerve growth factor (10 g, twice a week) to aging lesioned rats for 3 weeks after surgery resulted in a complete recovery in the number of choline acetyltransferase-immunoreactive neurons in the nucleus basalis of both sides, and choline acetyltransferase activity in the contralateral cortex, with little effect on the ipsilateral cortex. No potentiation was seen after the concurrent administration of GM1 ganglioside and nerve growth factor. Complete recovery in cortical choline acetyltransferase activity was only observed in the lesioned rats treated with nerve growth factor for 1 week before and 3 weeks after lesioning. Nerve growth factor treatment, both after the lesion, and before and after the lesion, improved the passive avoidance performance disrupted by the lesion. In young lesioned rats daily intraperitoneal administration of GM1 (30 mg/kg) for 21 days after surgery promoted both the recovery of choline acetyltransferase activity and passive avoidance performance. In aging rats GM1, even at a dose twice as large, failed to reverse the biochemical and morphological deficits and behavioral impairment induced by the lesion. Only when GM1 administration was started 3 days before the lesion, were a complete recovery in choline acetyltransferase activity in the contralateral cortex and a partial recovery in the ipsilateral cortex obtained.Our results indicate that nerve growth factor and, to some extent, GM1 facilitate the recovery of the cholinergic neurons after a lesion of the nucleus basalis in aging rats, but their efficacy is reduced. The lower efficacy of GM1 as compared to NGF might be due to the different routes of administration used.     

Modifications in the cortical regional distribution of choline acetyltransferase, somatostatin and somatostatin binding sites in the normal rat and following lesion of the nucleus basalis

The regional distribution of choline acetyltransferase activity, somatostatin levels and 125I-CGP 23996 (a somatostatin agonist analog) specific binding sites in 10 separate zones of the cerebral cortex was analyzed. The study was performed in normal rats as well as 15 days after unilateral excitotoxic lesion of the nucleus basalis. A significant correlation was found in the controls between the regional distribution of choline acetyltransferase activity and somatostatin concentrations, both most highly concentrated in the piriform and entorhinal cortex. In contrast, the regional density of 125I-CGP 23996 binding sites correlated neither with choline acetyltransferase activity nor with somatostatin levels. Unilateral lesions of the basal forebrain decreased choline acetyltransferase activity in the frontal and parietal cortex, while 125I-CGP 23996 binding decreased in frontal and occipital regions. No decrease in somatostatin content was observed. The results suggest that, in rats, cortical somatostatin receptors could be associated with cholinergic afferents from the nucleus basalis in the frontal cortex only.     

In vivo neurochemical effects of the acetylcholinesterase inhibitor ENA713 in rat hippocampus

Oral ENA713 (0.5, 1.5 and 4.5 mg/kg), an acetylcholinesterase inhibitor (AChEI), dose-dependently enhanced extracellular acetylcholine concentrations in the hippocampus of freely moving rats. This effect was paralleled by changes in both noradrenergic and dopaminergic transmission. In particular, ENA713 significantly decreased noradrenaline concentrations, whereas it significantly increased homovanillic acid levels, without affecting dopamine concentrations. Neither serotonin nor gamma-aminobutyric acid levels were modified by ENA713. These findings extend the neurochemical profile of ENA713 and suggest that it could be useful for the treatment of Alzheimer-type dementia which is associated with multiple neurotransmitter abnormalities in the brain.     

The effects of d-cycloserine, a partial agonist at the glycine binding site, on spatial learning and working memory in scopolamine-treated rats

Summary The present study investigated the effect of d-cycloserine, a partial agonist at the glycine binding site on NMDA receptor complex, on the performance of scopolamine-treated adult rats in a water maze task assessing spatial learning and in a delayed non-matching to position task assessing working memory in a spatial context. In the spatial learning task, scopolamine (0.4 mg/kg, i.p.) impaired acquisition (increased escape latency and distance) and increased swimming speed of rats. D-cycloserine (1.0 mg/kg, i.p.) reversed the deficits in acquisition performance but not the increases in behavioral activity. In the working memory task, scopolamine (0.2 mg/kg, i.p.) produced deficits on nonmnemonic rather than on mnemonic performance factors; scopolamine delay-independently decreased the percent correct responses and reduced behavioral activity of rats. D-cycloserine (1.0, 3.0 and 10 mg/kg, i.p.) did not reverse these performance deficits. When administered alone, the moderate to higher doses of d-cycloserine had no effects on working memory but the lower dose produced slight deficits in mnemonic performance factors; the 1.0 mg/kg dose delay-dependently decreased the percent correct responses without affecting behavioral activity of rats. In the water maze task, d-cycloserine had no effects on acquisition performance or behavioral activity of rats. These results suggest that acute, systemic administration of d-cycloserine does not improve spatial learning or working memory. However, at appropriate doses this agent may be efficacious in disease states of central cholinergic hypofunction since 1.0 mg/kg d-cycloserine was able to reverse the scopolamine-induced deficits in acquisition.     

Enhancement of spatial learning in F344 rats by physical activity and related learning-associated alterations in hippocampal and cortical cholinergic functioning.

The effects of physical activity on spatial memory performance and associated cholinergic function were examined in F344 rats. Cholinergic analysis included resting and depolarization-induced activation of high-affinity choline uptake and muscarinic receptor binding in the hippocampus, parietal cortex and frontal cortex. Rats that were physically trained, using chronic treadmill running, demonstrated significantly enhanced performance on the spatial learning task, both in second trial latency and first and second trial proximity ratio scores (P less than 0.002). Concomitant with enhanced behavioral performance were neurochemical changes of a reduction in hippocampal high-affinity choline uptake, an upregulation of muscarinic receptor density, and an increase in high-affinity choline uptake 24 h after spatial memory testing (P less than 0.05). Spatial memory tested rats demonstrated enhanced depolarization-induced activation of high-affinity choline uptake (P less than 0.001). Rats that were yoked for swim time to spatial memory tested rats did not show any spatial learning-induced alterations in high affinity choline uptake. These spatial learning- and physical activity-induced cholinergic alterations were observed only in the hippocampus, not in the parietal or frontal cortex. These data indicate that the chronic running-induced alterations in hippocampal high-affinity choline uptake and upregulation of muscarinic receptor density, in combination with enhancement of high-affinity choline uptake related to spatial learning, may contribute to the enhanced spatial learning performance of chronic-run rats.     

Differential changes in rat cholinergic parameters subsequent to immunotoxic lesion of the basal forebrain nuclei

The degree of lesion produced by 192 IgG-saporin relative to controls was compared using three independent methods. Microdialyzed acetylcholine (ACh), choline acetyltransferase (ChAT) activity, and the rate of ACh synthesis were compared in the frontal cortex and hippocampus. Microdialysis of rats was performed 1 and 15 weeks post-lesion. In week 16, the rats were sacrificed after an injection of deuterated choline (Ch) for determination of the rate of ACh synthesis. ChAT activity was determined at the same timepoints in a separate set of rats. At 1 week, ChAT activity and microdialyzed ACh showed similar degrees of depletion. At 15 weeks, microdialyzed ACh was significantly lower than the synthesis rate in cortex, but not in hippocampus. A small increase in ChAT activity between 1 and 15 weeks was found in the cortex, but not hippocampus. In the hippocampus, however, the rate of ACh synthesis was significantly greater than ChAT activity. This was true for two doses of immunotoxin; the greater compensation occurring with the lesser lesion. Microdialyzed ACh levels were not different from the other measures in hippocampus. Residual cholinergic terminals in the hippocampus, but not frontal cortex, compensate for a selective cholinergic lesion by increasing the rate of synthesis and may thereby alleviate hippocampus-dependent behavioral deficits.     

Somatostatin, neuropeptide Y, GABA and cholinergic enzymes in brain of pentylenetetrazol-kindled rats

We studied the effect of pentylenetetrazol (PTZ)-induced kindling (35 mg/kg, i.p., daily) on somatostatin-like immunoreactivity (SOM) with special attention to the duration of changes (rats were sacrificed either 10 days or 4 months after the development of kindling) and to transmitters or modulators related to somatostatin (neuropeptide Y (NPY), GABA, choline acetyltransferase (ChAT), acetylcholinesterase (AchE]. In rats sacrificed 10 days after the last kindled seizure, SOM was elevated in frontal cortex and striatum (p less than 0.01); NPY was elevated in frontal cortex, striatum and hippocampus (p less than 0.05) of kindled or prekindled rats (i.e., rats which were treated daily with PTZ but did not express three consecutive generalized seizures). ChAT activity was slightly decreased (p less than 0.05) in cortex. GABA levels and AchE activity were unchanged in kindled cortex. In rats sacrificed 4 months after the development of kindling none of the parameters analyzed differed from controls. The present study suggests that the cortical and striatal neurons containing SOM/NPY are affected by PTZ-kindling. The cortical cholinergic system is affected to a much smaller extent. The neuropeptide changes are not persistent, as is the lowered seizure threshold, so they are probably not involved in the maintainance of the latter.     

Chronic brain cytochrome oxidase inhibition selectively alters hippocampal cholinergic innervation and impairs memory: prevention by ladostigil

A 25-35% reduction of brain cytochrome oxidase (COx) activity found in Alzheimer's disease (AD) could contribute to neuronal dysfunction and cognitive impairment. The present study replicated the reduction in brain COx activity in rats by administering sodium azide (NaN(3)) for 4 weeks via Alzet minipumps at the rate of 1 mg/kg/h, and determined its effect on hippocampal cholinergic transmission, spatial and episodic memory. NaN(3) caused a selective reduction in choline acetyltransferase (ChAT) immunoreactivity in the diagonal band, a major source of cholinergic input to the hippocampus and cingulate cortex, without altering the number of cholinergic neurons. NaN(3) also induced a significant increase in vesicular acetylcholine transporter (VAChT)-immunoreactive varicosities, GAP-43 in the subgranular layer and of transferrin receptors (TfR) in the hilus of the dentate gyrus. These neurochemical changes were associated with impairment in spatial learning in the Morris water maze and in episodic memory in the object recognition test. Chronic treatment with ladostigil, a novel cholinesterase and monoamine oxidase inhibitor, prevented the decrease in ChAT in the diagonal band, the compensatory increase in synaptic plasticity and TfR and the memory deficits without restoring COx activity. Ladostigil had no significant effect on ChAT activity, synaptic plasticity or TfR in control rats. Ladostigil may have a beneficial effect on cognitive deficits in AD patients that have a reduction in cortical COx activity and cholinergic hypofunction.     

Counteractive effects of a partial (sabcomeline) and a full (RS86) muscarinic receptor agonist on deficits in radial maze performance induced by S-AMPA lesions of the basal forebrain and medial septal area.

After S-AMPA (8.0 mM) 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 and relatively stable impairment in long-term reference and short-term working memory in both spatial (place) and associative (cue) radial maze tasks. Treatment with four doses of the partial agonist at the M1 cholinergic muscarinic receptor, sabcomeline (formerly known as SB 202026: 0.01-0.156 mg/kg), substantially reduced working and reference memory errors in both tasks in lesioned rats, in a mainly dose-related manner. These effects were more consistent than those found with the direct muscarinic agonist RS86 (0.05-0.781 mg/kg). The performance of non-lesioned controls was largely unaffected by either treatment. These findings are consistent with previous evidence for cholinergic participation in the radial maze deficits induced by excitotoxic lesions to the forebrain cholinergic projection system. They show that with a relatively selective lesion, which respectively, reduced choline acetyltransferase activity to 36.5 and 22.5% of control level in frontal and dorsolateral cortex, and to 61.8 and 69.2% of control level in dorsal and ventral hippocampus, lesioned rats were responsive to pharmacological treatments aimed to enhance cholinergic function by full or partial agonist activity at M1 receptors. Findings that nicotine (0.1 mg/kg) also reduced radial maze errors in the lesioned animals supports the suggestion that lesion-induced deficits in radial maze performance were amenable to improvement by cholinergic receptor manipulation. However, given the potential adverse side effects of full receptor agonists, which nonselectively target cholinergic receptors throughout the organism, the functional efficacy of sabcomeline, which shows regional selectivity for the central M1 receptor subtypes, suggests that deleterious effects of cholinergic depletion on cognition can be counteracted without incurring the risk of unwanted side effects.     

NGF Deprivation of Adult Rat Brain Results in Cholinergic Hypofunction and Selective Impairments in Spatial Learning

Cholinergic hypofunction has often been correlated with a variety of behavioural impairments. In the present study, adult Wistar rats were intraventricularly infused with antibodies to nerve growth factor (anti-NGF) to examine the effects on cholinergic neurons of the basal forebrain, and on behavioural performance. Immunocytochemical techniques indicated that chronically infused anti-NGF penetrates into the basal forebrain, cortex, striatum, corpus callosum and hippocampus, confirming previous findings after a single injection. Treatment with anti-NGF for 1 or 2 weeks resulted in a significant decrease of 27-33% in density of choline acetyltransferase immunostaining of the cholinergic cell bodies in the medial septum and vertical diagonal band, and a 26% reduction in choline acetyltransferase enzyme activity in the septal area. An array of spatial learning Morris water maze tasks was used to distinguish between acquisition skills and the flexible use of learned information in novel tests. Rats subjected to the spatial learning paradigm received anti-NGF infusion for 2 weeks prior to and for another 2 weeks during the behavioural testing. The anti-NGF-treated animals were found to be no different from those receiving control serum in the Morris water maze acquisition task, either in the latency to find the platform or in the time spent searching in the training quadrant when the platform was removed. However, in consecutive extinction trials, anti-NGF rats continued to search in the empty training quadrant, suggesting the occurrence of perseveration; control rats expanded their search over other areas of the pool. This inflexibility of the anti-NGF rats was also evident from their difficulty in learning to find a relocated platform in the reversal task. Finally, the anti-NGF-treated animals demonstrated hyperactivity in the open field, resembling in this respect the behaviour of animals after septal and fimbria fornix lesions, and during pharmacological cholinergic blockade. While these data support a role for NGF in cholinergic function and spatial learning behaviour, they indicate that deficits in the latter, e.g. those demonstrated in impaired aged rats, may not be attributable selectively to deficits in the function of NGF-sensitive cholinergic neurons of the basal forebrain.     

Glutamate receptor binding in the frontal cortex and dorsal striatum of aged rats with impaired attentional set-shifting

Aged Long-Evans rats exhibit deficits in attentional set shifting, an aspect of executive function, relative to adult rats. Impairments in set shifting and spatial learning are uncorrelated in aged rats, indicating a possible dissociation of the effects of ageing in prefrontal versus hippocampal systems. Ionotropic glutamate receptor binding was assessed using an in vitro autoradiography method in young and aged rats. The rats had been tested on a set-shifting task that measured attentional set shifts and reversal learning, as well as in a spatial learning task in the Morris water maze. [3H]Kainate, [3H]AMPA and NMDA-displaceable [3H]glutamate receptor binding were quantified in orbital cortex, cingulate cortex, medial frontal cortex, dorsolateral and dorsomedial striatum. Age-related decreases in [3H]kainate binding were apparent in all regions measured. Similarly, NMDA-displaceable [3H]glutamate binding was decreased in the aged rats in all the regions measured except for the medial frontal area where no age effects were observed. [3H]AMPA receptor binding was preserved with age in all the regions measured. Lower levels of [3H]kainate binding in the cingulate cortex were significantly correlated with poorer set-shifting performance, whereas higher levels of NMDA binding in the dorsomedial striatum were correlated with poorer set-shifting performance. There were no significant correlations between the levels of ionotropic glutamate receptors and performance in the reversal task or spatial learning in the Morris water maze. These results indicate that age-related behavioural deficits in attentional set shifting are selectively associated with neurobiological alterations in the cingulate cortex and dorsomedial striatum.     

The histopathological, behavioral and neurochemical effects of intraventricular injection of ethylcholine mustard aziridinium (AF64A) in the neonatal rat.

This study investigated the histopathological, behavioral and neurochemical effects of bilateral injection of 2.0, 0.5 and 0.1 nmol/ventricle ethylcholine aziridinium (AF64A) on postnatal day (PND) 2. The rats showed a significant, but non-dose-related reduction of choline acetyltransferase (ChAT) in the hippocampus but not the cerebral cortex or the caudate nucleus when sacrificed on PND 16. No effect on ChAT was found in any region at PND 58. The group given 2 nmol/ventricle were hyperactive and showed a deficit in spatial learning when tested on the Morris water maze at PND 38-43. No such differences were observed for the rats injected with 0.1 or 0.5 nmol/ventricle AF64A. This spatial learning impairment in the 2 nmol group was associated with non-specific tissue damage seen only in animals from this group that were sacrificed at PND 40. This tissue damage was most evident in the left medial frontal cortex, the caudate nuclei and the anterior dorsal hippocampus.     

Effect of Venezuelan equine encephalomyelitis virus infection on brain choline acetyltransferase and acetylcholinesterase activities

Mice and rats infected with the Venezuelan equine encephalomyelitis virus showed a significant decrease in the choline acetyltransferase activity of caudate nucleus, hypothalamus, midbrain, hippocampus and frontal cortex. Acetylcholinesterase activity was not affected in any of the same brain regions analyzed. In surviving rats no alterations were observed in the activities of choline acetyltransferase, tyrosine hydroxylase, and glutamate decar☐ylase 3 months after the infection.     

Place navigation in rats is impaired by lesions of medial septum and diagonal band but not nucleus basalis magnocellularis

The role of forebrain cholinergic projections in place navigation learning was assessed in two experiments. Following surgery, rats were required to learn the spatial location of an underwater platform on the basis of distal room cues. Bilateral injections of ibotenic acid into the nucleus basalis magnocellularis depleted choline acetyltransferase (ChAT) from the anterior and temporoparietal cortex but not the hippocampus. Separate histological studies confirmed the accuracy of the lesions and demonstrated a marked loss of cortical acetylcholinesterase. These rats subsequently showed no deficits in spatial learning or memory. In a second experiment, bilateral lesions of the vertical limb of the diagonal band of Broca and medial septum depleted ChAT from the hippocampus and posterior cortex but not the anterior cortex. Histological studies confirmed the accuracy of the lesion and showed a pronounced loss of acetylcholinesterase from the hippocampus. These rats were deficient in spatial learning and showed reduced spatial bias during transfer tests. The data are discussed in the light of the hypothesis that the cholinergic innervation of the hippocampus plays a key role in spatial reference memory processes involved in place navigation.     

Prenatal choline supplementation increases NGF levels in the hippocampus and frontal cortex of young and adult rats

Female Sprague-Dawley rats received approximately 300 mg/kg per day of choline chloride through their drinking water on days 11 of pregnancy through birth and the level of nerve growth factor (NGF) in the hippocampus and frontal cortex of their male offspring was measured at 20 and 90 days of age. Prenatal choline supplementation caused significant increases in hippocampal NGF levels at 20 and 90 days of age, while levels of NGF in the frontal cortex were elevated in choline-supplemented rats at 20 days of age, but not 90 days of age. These results suggest that increases in NGF levels during development or adulthood may be one mechanism underlying improvements in spatial and temporal memory of adult rats exposed to elevated levels of choline chloride perinatally.     

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

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