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.

Nerve growth factor (NGF) augments cortical and hippocampal cholinergic functioning after p75NGF receptor-mediated deafferentation but impairs inhibitory avoidance and induces fear-related behaviors.

Nerve growth factor (NGF) enhances cholinergic functioning in animals with a compromised cholinergic basal forebrain (CBF). Immunotoxic lesions targeting low-affinity NGF receptor (p75NGF receptor)-bearing CBF neurons provide a selective model for testing the effects of NGF on residual cholinergic neurons. Rats received PBS or the immunotoxin 192IgG-saporin (192Sap) intracerebroventricularly at two doses (1 or 2.7 microg) known to produce different degrees of cholinergic deficit. Seven weeks after lesioning, half of each group received either NGF or cytochrome c intracerebroventricularly for 7 weeks. The two doses of 192Sap produced 50 and 80% depletions of choline acetyltransferase (ChAT) activity in the neocortex and hippocampus. NGF produced the greatest increase in ChAT activity in controls, intermediate in low-lesioned, and smallest in highly lesioned animals. NGF-treated animals showed reduced weight gain, hyper-responsiveness to acoustic stimuli, and decreased inhibitory avoidance. Although general motor behavior was affected by neither 192Sap nor NGF in an open field task, highly lesioned rats took longer to reach the platform during water maze testing. Impaired spatial orientation in finding a hidden platform at the previously acquired position was mitigated by NGF. Hypertrophic changes of residual CBF neurons, Schwann cell hyperplasia, and aberrant axonal sprouting around the medulla were observed in NGF-treated animals only, independent of the preexisting lesion. Our results indicate that NGF has a limited capacity to enhance functioning of residual CBF neurons. More importantly, NGF augmented fear-related behaviors and adverse neuroproliferative changes that may restrict its therapeutic use.

Intraparenchymal infusions of 192 IgG-saporin: development of a method for selective and discrete lesioning of cholinergic basal forebrain nuclei.

The immunotoxin 192 IgG-saporin has a high degree of selectivity for cholinergic neurons within the basal forebrain (CBF). Intracerebroventricular delivery of 192 IgG-saporin results in a diffuse and massive depletion of choline acetyltransferase (ChAT) activity in projections of the CBF, and non-selective loss of Purkinje cells. To dissociate the basal-cortical and septo-hippocampal cholinergic systems and to minimize non-specific effects, we developed intraparenchymal parameters to deliver 192 IgG-saporin discretely to either the nucleus basalis magnocellularis (NBM) or the medial septum (MS). Intraparenchymal administration of the immunotoxin into the NBM or MS resulted in a dose-dependent depletion of ChAT activity in the corresponding projection areas and a concomitant loss of ChAT immunoreactive neurons in both nuclei. Both lesions were regionally restricted, having a minimal diffusion into adjacent CBF nuclei. Control infusions did not result in non-specific parenchymal damage. In addition, immunotoxic infusions had no effect on monoamine neurotransmitter systems. By optimizing the dosages for both CBF nuclei, we maximized ChAT depletion while minimizing diffusion into the adjacent CBF nuclei. This study delineated injection parameters enabling a selective dissociation of two cholinergic subpopulations in the basal forebrain for further functional characterization.

Changes in electrocortical power and coherence in response to the selective cholinergic immunotoxin 192 IgG-saporin.

Changes in brain electrical activity in response to cholinergic agonists, antagonists, or excitotoxic lesions of the basal forebrain may not be reflective entirely of changes in cholinergic tone, in so far as these interventions also involve noncholinergic neurons. We examined electrocortical activity in rats following bilateral intracerebroventricular administration of 192 IgG-saporin (1.8 microg/ventricle), a selective cholinergic immunotoxin directed to the low-affinity nerve growth factor receptor p75. The immunotoxin resulted in extensive loss of choline acetyl transferase (ChAT) activity in neocortex (80%-84%) and hippocampus (93%), with relative sparing of entorhinal-piriform cortex (42%) and amygdala (28%). Electrocortical activity demonstrated modest increases in 1- to 4-Hz power, decreases in 20- to 44-Hz power, and decreases in 4- to 8-Hz intra- and interhemispheric coherence. Rhythmic slow activity (RSA) occurred robustly in toxin-treated animals during voluntary movement and in response to physostigmine, with no significant differences seen in power and peak frequency in comparison with controls. Physostigmine significantly increased intrahemispheric coherence in lesioned and intact animals, with minor increases seen in interhemispheric coherence. Our study suggests that: (1) electrocortical changes in response to selective cholinergic deafferentation are more modest than those previously reported following excitotoxic lesions; (2) changes in cholinergic tone affect primarily brain electrical transmission within, in contrast to between hemispheres; and (3) a substantial cholinergic reserve remains following administration of 192 IgG-saporin, despite dramatic losses of ChAT in cortex and hippocampus. Persistence of a cholinergically modulated RSA suggests that such activity may be mediated through cholinergic neurons which, because they lack the p75 receptor, remain unaffected by the immunotoxin.

Deficit in selective and divided attention associated with cholinergic basal forebrain immunotoxic lesion produced by 192-saporin; motoric/sensory deficit associated with Purkinje cell immunotoxic lesion produced by OX7-saporin.

The immunotoxin 192-saporin, infused intracerebroventricularly into rats, destroys cholinergic neurons in the basal forebrain nuclei. Doses required for complete cholinergic loss also kill some Purkinje cells. The immunotoxin OX7-saporin, when infused intraventricularly into rats, destroys Purkinje cells in a pattern similar to that produced by 192-saporin, without affecting cholinergic neurons. Thus, we used OX7-saporin to distinguish behavioral effects of 192-saporin due to cerebellar damage versus those due to cholinergic cell loss. Three doses of 192-saporin (1.6, 2.6, and 3.3 micrograms/rat) were chosen along with a dose of OX7-saporin (2.0 micrograms/rat) that produced Purkinje loss equivalent to the two highest doses of 192-saporin. Groups of Fischer-344 rats were trained in the multiple choice reaction time task and retested with more complex tasks after lesioning. They were also tested in the water maze, passive avoidance, acoustic startle, and open field. The OX7-saporin group exhibited changes in many tests suggesting hypermotility and sensory deficits. The 192-saporin groups differed from the OX7-saporin group when they displayed deficits in multiple choice reaction time tasks in which novel challenges were introduced, including sessions with a noise distractor, shortened and lengthened intertrial intervals, and use of nine instead of five sources of light stimulus. The 192-saporin groups showed no impairment in the other tasks. The cholinergic basal forebrain lesion may mask some of the effects of cerebellar damage up to a threshold after which effects of Purkinje cell loss predominate when 192-saporin is administered intraventricularly.

Selective immunotoxin-induced cholinergic deafferentation alters blood flow distribution in the cerebral cortex.

Adult rats received intracerebroventricular (i.c.v.) administration of either phosphate buffer (PBS) or 192 IgG-saporin (Toxin), 3.6 micrograms rat-1, a cholinergic immunotoxin. Six to eight weeks later, the animals received a continuous intravenous (i.v.) infusion of either physostigmine (4.2 micrograms kg-1 min-1) or saline, followed by measurement of cerebral cortical blood flow (CBF) with the autoradiographic Iodo-14C-antipyrine methodology in four groups of animals: Toxin i.c.v.+saline i.v. (n=9), Toxin i.c.v.+physostigmine i.v. (n=6), PBS i.c.v.+saline i.v. (n=6) and PBS i.c.v. +physostigmine i.v. (n=6). Choline acetyltransferase activity (ChAT) was assessed with Fonnum's method in samples of cortical tissue adjacent to the sites of CBF measurement. ChAT decreased in all regions of the Toxin groups when compared to PBS (% decrease: hippocampus=93%, neocortex=80-84%, entorhinal-piriform cortex=42%, amygdala=28%). CBF decreased globally in Toxin+SAL, most severely in posterior parietal and temporal regions (24-40% decrease from PBS+saline). Physostigmine enhanced CBF predominantly in these same areas both in PBS and Toxin animals although to a lesser extent in the latter. Our results demonstrate the importance of cholinergic mechanisms in the control of CBF. The similarity between the topography of CBF decrease following administration of the immunotoxin to that observed in Alzheimer's disease suggests that the CBF pattern observed in this disease may be the result of cholinergic deafferentation.

Lesions of the cholinergic nuclei in the rat basal forebrain: excitotoxins vs. an immunotoxin.

Infusion of the excitotoxins, ibotenic acid, quisqualic acid, or AMPA, into the medial septal nucleus, diagonal band, and the nucleus basalis magnocellularis of rats produced less cholinergic cell loss as assessed by choline acetyltransferase activity in the projection fields, cortex and hippocampus, than that obtainable by intraventricular administration of the immunotoxin, 192 IgG-saporin. All excitotoxins produced reductions in tissue levels of some monoamines, while no decreases were found for the immunotoxin. All toxins produced acquisitional impairment in the hidden platform water maze. This behavioral deficit was slightly greater for the excitotoxic-lesioned rats than for those given 192 IgG-saporin at a dose which produced ChAT depletions similar to the most potent excitotoxin (AMPA). This supports the idea that some of the behavioral effects produced by excitotoxic lesions are due to the cholinergic basal forebrain lesion and some are due to noncholinergic damage.

192 immunoglobulin G-saporin produces graded behavioral and biochemical changes accompanying the loss of cholinergic neurons of the basal forebrain and cerebellar Purkinje cells.

Immunolesions of the cholinergic basal forebrain were produced in rats using various intraventricular doses of the immunotoxin 192 immunoglobulin G-saporin: 0.34, 1.34, 2.0, 2.7 and 4.0 micrograms/rat. A battery of behavioral tests, chosen on the basis of reported sensitivity to conventional medial septal or nucleus basalis lesions, was administered. Dose-dependent impairments were found in acquisition, spatial acuity and working memory in the water maze. Dose-dependent hyperactivity in the open field and in swimming speed was observed. The highest dose group (4.0 micrograms) exhibited motoric disturbances which were particularly apparent in swimming and in clinging to an inclined screen. Response and habituation to acoustic startle were diminished in the three higher dose groups. Histological results from acetylcholinesterase and low-affinity nerve growth factor receptor staining showed that the lesion was selective for cholinergic neurons bearing p75 nerve growth factor receptors in the basal forebrain nuclei. However, some Purkinje cells in the superficial layers of the cerebellum were also destroyed at the higher doses of immunotoxin. The activity of choline acetyltransferase, used as a marker of cholinergic deafferentation in regions innervated by the basal forebrain nuclei, was decreased with increasing doses to a plateau level of about 90% (average depletion) for the two highest dose groups. These two groups were the only ones to exhibit consistent and severe behavioral impairments on all behavioral tests performed. Thus, for a relatively selective cholinergic basal forebrain lesion, almost a 90% reduction in choline acetyltransferase activity is needed to produce substantial behavioral deficits. It appears that either a considerable safety factor exists or robust compensatory mechanisms can ameliorate behavioral deficits from a major, but incomplete loss of cholinergic basal forebrain innervation.

The behavioral effects of heptylphysostigmine on rats lesioned in the nucleus basalis.

The time course and dose dependence of acetylcholinesterase inhibition in three regions of brain were measured for the long-acting physostigmine derivative, heptylphysostigmine (HP). Behavioral studies were performed on rats lesioned with ibotenic acid in the nucleus basalis magnocellularis (nBM) using doses of HP that inhibit cholinesterase activity 20%, 40%, and 60% 2 h after injection. Spatially-cued learning and memory were tested in the water maze. Lesioned animals that received higher doses of HP showed a trend towards improvement in the acquisition of this task, but this was not statistically significant. Swimming speed was reduced in the group receiving the highest dose of HP in comparison with a lower dose. The acoustic startle response was diminished in all groups given HP in comparison with both lesioned and sham-lesioned saline-injected controls. Open field activity was slightly enhanced by the presence of the nBM lesion. HP reduced the hyperactivity in a dose-dependent manner. Deficiencies in limb strength or coordination were not detected. These results suggest that HP may decrease spontaneous, stressed, or reflexive activity, although an effect on the spatial learning deficit produced by an ibotenic acid lesion of the nucleus basalis was not detected.

Behavioral and biochemical consequences of combined lesions of the medial septum/diagonal band and nucleus basalis in the rat when ibotenic acid, quisqualic acid, and AMPA are used.

Combined lesions in the medial septum/diagonal band and nucleus basalis magnocellularis (NBM) in rats were produced using three excitotoxins, ibotenate (Ibo), quisqualate (Quis), and AMPA. Reductions in choline acetyltransferase (ChAT) activity differed in the cortical regions for the three toxins (AMPA > Quis > Ibo), but were fairly similar in the hippocampus. ChAT activities were not reduced in the globus pallidus, but AMPA reduced ChAT in the amygdala. Lesions with all three toxins produced similar decrements in hippocampal and posterior cortical serotonin levels. A small reduction in posterior cortical norepinephrine was detected for Quis and Ibo lesions. Spatial memory impairments were found for all three toxin groups compared with controls in acquisition, platform reversal, and a spatial probe in the water maze. The learning deficit was greatest with the Quis lesion and equivalent for the Ibo and AMPA lesions. There was no deficit in single trial passive avoidance retention for the Ibo and AMPA groups. The AMPA group was slower than controls on both training and retention trials to enter the dark compartment. This group also showed a tendency to hypoactivity as measured in an open-field test. Excitotoxic infusions into medial septum/diagonal band and NBM produced spatial mnemonic deficits which do not parallel reductions in overall ChAT activity and do not resemble the profile of behavioral changes previously reported for NBM lesions alone using these toxins.

Lack of long-term effects after beta-amyloid protein injections in rat brain.

Rat beta(1-42) peptide (beta/A4) or phosphate buffered saline (PBS) was bilaterally injected into the hippocampus (HIP) or the lateral ventricle (ICV) of 3-month-old Fischer-344 rats. Fifteen months later, the animal's ability to learn a spatial memory task was tested using the Morris water maze. Acquisition of the task was impaired by the bilateral injection of either peptide or PBS into the hippocampus. Hippocampal-injected animals showed an increased average latency to find the platform by approximately 6 s (p < 0.05). However, injection of rat beta-peptide into the hippocampus or lateral ventricles failed to induce behavioral impairment when compared to vehicle injected controls. Retention of this task was not significantly impaired in any group. The spatial acuity test, a trial without the platform, revealed that both groups of animals that received hippocampal injections were impaired, spending 23% less time in the target quadrant compared to ICV-injected animals (p < 0.005). Hippocampal ChAT activity was decreased in beta/A4-injected animals but not significantly (p < 0.06). beta/A4-immunoreactivity was detected at the bottom of the needle track and the adjacent parenchyma of beta/A4 hippocampal-injected animals after 16 months. However, long-term in vivo deposition of beta/A4 in both regions did not result in an upregulation of hippocampal amyloid precursor protein (APP) expression and there was no qualitative neuronal loss in the hippocampus.(ABSTRACT TRUNCATED AT 250 WORDS)

Time course of cholinergic and monoaminergic changes in rat brain after immunolesioning with 192 IgG-saporin.

192 IgG-saporin, an immunotoxin targeted at the low affinity NGF receptor, was infused into the lateral ventricle of rat brain. Three days and one week post lesion, choline acetyltransferase activity was markedly decreased in cortex, hippocampus, olfactory bulbs, and septum (brain regions innervated by the cholinergic neurons of the basal forebrain) with no change in cerebellum, striatum or pons. Measurement of monoamine levels revealed increases in HVA, DOPAC and dopamine, primarily in the olfactory bulbs at the 28-day time point only, suggesting a compensation for cholinergic inactivity. High levels of basal forebrain cholinergic lesioning can be obtained with this immunotoxin with minimal or no effects on monoaminergic or other cholinergic systems.

Global in vivo replacement of choline by N-aminodeanol. Testing a hypothesis about progressive degenerative dementia: I. Dynamics of choline replacement.

Severe disruption of certain cholinergic pathways is a characteristic feature of Alzheimer's disease. Attempts to establish animal models by interfering with cholinergic function have not been very successful. We now present data which show a substantial and progressive replacement of free and phospholipid-bound choline by the novel choline isostere N-amino-N,N-dimethylaminoethanol during its dietary administration in place of choline. Free choline in blood fell to approximately 20% of controls after 10 to 30 days on diet. Phospholipid-bound choline in plasma was reduced to less than 15%, and in erythrocytes to about 22%. After 120 days of diet free and bound choline were reduced in most tissues to approximately 30% of controls. Only liver retained more than 80% of free choline. Acetylcholine was decreased to 33 to 50% of control. Total true and false transmitter in experimental animals was in all tissues less that acetylcholine in controls, suggesting that muscarinic transmission would be impaired. Moderate reduction of choline acetyltransferase activity was seen in striatum and myenteric plexus, and of QNB-binding in hippocampus, striatum and myenteric plexus.

G-protein linked polyphosphoinositide phospholipase C activity in cell membranes from clonally derived and ras-transformed Madin-Darby canine kidney cells.

Membranes prepared from clone D1 of Madin-Darby canine kidney (MDCK) cells contain activity that can be attributed to Gp, a guanine nucleotide binding protein linked to phosphatidylinositol 4,5-bisphosphate dependent phospholipase C. Polyphosphoinositides are produced by addition of GTP, nonhydrolyzable GTP analogs, or fluoroaluminate. This production is inhibited by guanosine 5'-(beta-thiodiphosphate). While Ca2+ at 1 microM or more can generate high yields of inositol phosphates, guanine nucleotide activation of Gp can potentiate this Ca2(+)-dependent yield at resting levels of the cation. Membranes from cells expressing large amounts of ras-p21 exhibit small differences in guanine nucleotide induced polyphosphoinositide quantities. The greatest difference between normal and ras membranes was seen with AlF4- incubation. Of the three inositol phosphates measured, only the inositol bisphosphate yield was greatly increased in ras membranes compared with membranes from both parental and the D-1 clone of MDCK cells. From these data, we conclude that the presence of ras-p21 may affect production of polyphosphoinositides in MDCK cell membranes by some means other than direct participation in phospholipase C activation.

Spontaneous regeneration of free muscarinic receptor after alkylation by BM 123. I. Recovery in vivo and in cell culture.

The recovery of rat muscarinic receptor number from the effects of a specific alkylating ligand, N-[4-(2-chloroethylmethylamino)-2-butynyl]-2-pyrrolidone (BM 123), in three tissues is presented as an exponential function of time. No significant difference was found in the recovery rate constants derived from analysis of recovery time courses in corpus striatum, cerebral cortex and ileal longitudinal muscle. The single rate constant (0.021/hr) was also independent of amount and duration of BM 123 dose. Additional analysis of agonist-defined high and low affinity subsites in cortex revealed that recovery of these populations also followed similar time courses although the alkylation proceeds more slowly for the high affinity sites. The rate constant for recovery of both subsites was 0.029/hr. Recovery from BM 123 alkylation occurred in NG108-15 neuroblastoma X glioma cells. The presence of cycloheximide in the recovery medium did not significantly inhibit this recovery process in the clonal cell line, suggesting that de novo receptor synthesis is unnecessary for regeneration of unalkylated receptors.

Spontaneous regeneration of free muscarinic receptor after alkylation by BM 123. II. Recovery in broken cell preparations.

Muscarinic receptors from rat cerebral cortex were found to recover from alkylation by the specific ligand N-[-(2-chloroethyl-methylamino)-2-butynyl]-2-pyrrolidone (BM 123) without the benefit of cellular integrity, i.e., in washed membranes incubated at 37 degrees C for relatively long times (days). Unalkylated receptor number increased in alkylated preparations whether viewed as femtomoles per milligram of protein or as a percentage of the atropine-protected control. The rate of recovery was independent of pH over the range 6 to 9 and it was slowed but not eliminated by incubation at 4 degrees C. Muscarinic receptor recovery from BM 123 was linearly dependent on the concentration of alkylated receptors, indicating first-order kinetics and yielding a rate constant 0.026/hr. Of the other chloroethylamines examined for evidence of in vitro muscarinic receptor recovery from alkylation, only one, phenoxybenzamine, exhibited irreversible receptor blockade under these conditions. Each of the others showed different rates for regeneration of free receptors.

A model hypocholinergic syndrome produced by a false choline analog, N-aminodeanol.

N-aminodeanol is an analog of choline that serves as a less effective substrate in all of its known enzymatic and transport mechanisms. It was utilized to test the hypothesis that the selective vulnerability of cholinergic neurones in Alzheimer's disease is due to competition for the available choline between pathways for acetylcholine and phospholipid synthesis. Rats placed on a choline free diet containing an equivalent amount of N-aminodeanol develop a model hypocholinergic state comprising hyperreactivity, hyperalgesia, aggressive behavior and a deficit in learning and memory. These effects are associated with a progressive replacement of free and lipid-bound choline and acetylcholine with N-aminodeanol and its corresponding esters. Choline acetyltransferase is reduced in some brain regions, suggesting a loss of cholinergic neurones. We propose that this represents a potentially useful animal model of Alzheimer's disease which deserves further investigation.

Behavioral and physiological effects associated with changes in muscarinic receptors following administration of an irreversible cholinergic agonist (BM 123).

Previous work in our laboratory has shown that the aziridinium ion of BM 123 (N-[4(2-chloroethylmethylamino)-2-butynyl]-2 pyrrolidone) is a potent and selective muscarinic agonist and binds irreversibly to muscarinic receptors (mAChR). The present series of experiments was designed to study the effects of BM 123 on behavioral and physiological variables known to be sensitive to manipulations of the cholinergic neurotransmitter system. BM 123 was injected into the tail vein of Sprague-Dawley rats, reducing mAChR to approximately 10% of normal as judged by [3H](-)QNB binding. Oxotremorine was injected IV for purposes of comparison. Behavioral and physiological variables were measured daily for 26 days. Physiological variables (e.g., tremor, chromodacryorrhea, salivation, and temperature) showed effects in less than 5 min after injection and returned to their pretreatment baselines within minutes. Nociceptive thresholds, dependent on sensory-perceptual processes, showed peak changes of approximately +230% and returned to normal within hours. Motoric responses, i.e., drinking and general activity, recovered in 3-4 days. Learned responses and those requiring temporal discrimination took 8-11 days to recover and were the only responses paralleling the return of the mAChRs to their normal levels. Changes elicited by oxotremorine recovered more rapidly than those elicited by BM 123. The results suggest that the different variables measured are dependent on different densities of functional receptors. Implications for a theoretical model are discussed.