MAB390-25UG Sigma-AldrichAnti-p75 LNGFR Antibody, Saporin conjugated, clone 192

Cholinergic neurons projecting from the medial septum to the hippocampus were lesioned with the selective neurotoxin 192 IgG-saporin. Injection of 300 ng of 192 IgG-saporin into the medial septum produced a 60% decrease in choline acetyltransferase activity. M1 muscarinic receptor function was examined by measuring enhancement of evoked release of norepinephrine from rat hippocampal slices by the M1 selective agonist McN-A-343. In hippocampal slices from rats which were lesioned with 192-saporin, the response to McN-A-343 was reduced compared to sham-operated controls. Pirenzepine binding demonstrated no change in M1 receptor number or affinity. However, the curve for displacement of pirenzepine by the muscarinic agonist oxotremorine-M was shifted to the right in hippocampal tissue from lesioned rats. This shift was identical to that produced by addition of the non-hydrolyzable GTP analogue GppNHp, which uncouples the M1 muscarinic receptor/G-protein complex. These results suggest that lesion of septal-hippocampal cholinergic inputs causes uncoupling of the M1 muscarinic receptor, decreasing responsiveness to stimulation. These findings are similar to reports of decreased M1 muscarinic receptor coupling to G-proteins and loss of function in Alzheimer's disease. The 192 IgG-saporin lesion may provide a viable animal model in which to study uncoupling of G-proteins and M1 muscarinic receptors.

The anatomical specificity of axon growth from fetal pig septal xenografts was studied by transplanting septal cells from E30-35 pig fetuses into cholinergic deafferented (192-IgG-saporin-infused) rats or into aged rats (> 18 months). Cell suspensions (100,000 cells/microl) were injected bilaterally into the dorsal and ventral hippocampus of immunosuppressed rats (10 mg/kg/day cyclosporine A). To assess axonal growth and synapse formation, acetylcholinesterase histochemistry, an antibody to choline acetyltransferase (ChAT), and three pig-positive/rat-negative antibodies: bovine 70kD neurofilament (NF70), human low-affinity NGF receptor (hNGFr), and human synaptobrevin (hSB) were used. In rats with surviving grafts at 6 months, NF70 axonal labeling was more extensive than either ChAT or hNGFr labeling. All three markers demonstrated graft axons extending selectively through the hippocampal CA fields and the molecular layer of the dentate gyrus. Graft axons did not extend into adjacent entorhinal cortex or neocortex. The distribution of pig hSB-positive synapses correlated with AChE-positive fiber outgrowth in to the host. Electron microscopic analysis of hSB-immunostained hippocampal sections revealed pig presynaptic terminals in contact with normal rat postsynaptic structures in the CA fields and the dentate gyrus. These data demonstrate target-appropriate growth of pig cholinergic axons and the formation of cross-species synapses in the deafferented or aged rat hippocampus.

In adult intact rat brain, leukemia inhibitory factor (LIF) mRNA has been found to be constitutively expressed in basal forebrain cholinergic neurons. To reveal a functional role of LIF in neurodegenerative events, the cellular expression pattern of LIF was determined by combining in situ hybridization and immunocytochemistry after specific and selective degeneration of basal forebrain cholinergic cells by a single intracerebroventricular application of the cholinergic immunotoxin 192IgG-saporin. Although basal forebrain cholinergic lesion resulted in a dramatic activation of micro- and astroglial cells at the lesion site, LIF mRNA expression was not detected in any of the lesion-induced activated glial cell types. As the cholinergic immunotoxin exerts its degenerative action by the ribosome-inactivating property of saporin, the lack of glial LIF induction might be due to the incapability of the dying cholinergic cell to form and release factors which induce LIF expression in activated glial cells.

The neuropeptide galanin is overexpressed in the basal forebrain in Alzheimer's disease (AD). In rats, galanin inhibits evoked hippocampal acetylcholine release and impairs performance on several memory tasks, including delayed nonmatching to position (DNMTP). Galanin(1-13)-Pro2-(Ala-Leu)2-Ala-NH2 (M40), a peptidergic galanin receptor ligand, has been shown to block galanin-induced impairment on DNMTP in rats. M40 injected alone, however, does not improve DNMTP choice accuracy deficits in rats with selective cholinergic immunotoxic lesions of the basal forebrain. The present experiments used a strategy of combining M40 with an M1 cholinergic agonist in rats lesioned with the cholinergic immunotoxin 192IgG-saporin. Coadministration of intraventricular M40 with intraperitoneal 3-(3-S-n-pentyl-1,2,5-thiadiazol-4-yl)-1,2,5, 6-tetrahydro-1-methylpyridine (TZTP), an M1 agonist, improved choice accuracy significantly more than a threshold dose of TZTP alone. These results suggest that a galanin antagonist may enhance the efficacy of cholinergic treatments for the cognitive deficits of AD.

This experiment examines the impact of the cholinergic input from the basal forebrain on the plasticity of the vibrissa-related somatosensory cortex. Newborn rat pups received intraventricular injections of the cholinergic immunotoxin IgG192-saporin, after bilateral removal of the C-line whisker follicles. Compared with saline-injected control animals, unilateral injections of 0.1 microg IgG192-saporin decreased the number of cholinergic neurons on the toxin injected side by 78% in the basal nucleus of Meynert and the vertical limb of the diagonal band of Broca, 80% in the magnocellular preoptic nucleus and the horizontal limb of the diagonal band of Broca, and 54% in the medial septal nucleus. Neuronal loss contralateral to the toxin was approximately half that on the ipsilateral side. The size of the C and D row barrels were compared from tangential sections through the barrel field. In control animals, D row barrels expanded into C row territory, giving a ratio of areas for D/C barrels of 2.03. Depletion of the cholinergic neurons reduced the expansion of D row barrels and hence decreased the D/C ratio, with a greater reduction on the toxin-treated side (1.43, P <0.005) compared with the contralateral side (1.64, P <0.05). This study implicates the basal forebrain cholinergic projection in somatosensory cortical plasticity.

Recent research suggests that the basal forebrain cholinergic neurons innervating the cortex play a role in attentional functions in both primates and rodents. Among the cortical targets of these projections in primates is the posterior parietal cortex (PPC), a region shown to be critically involved in the regulation of attention. Recent anatomical studies have defined a cortical region in the rat that may be homologous to the PPC of primates. In the present study, cholinergic innervation of the PPC was depleted by intracortical infusion of the immunotoxin 192 IgG-saporin. Control and lesioned rats were then tested in two associative learning paradigms designed to increase attentional processing of conditioned stimuli (CSs). In one experiment, attention was manipulated by shifting a predictive relation between a light CS and another CS to a less predictive relation. Unlike control rats, lesioned rats failed to increase attention when the predictive relation was modified. In a second experiment, attentional processing of a tone CS was increased when its introduction during training coincided with a change in the value of the unconditioned stimulus, a phenomenon referred to as unblocking. Unlike control rats, lesioned rats failed to exhibit unblocking. In both paradigms, lesioned rats conditioned normally when the training procedures did not encourage increased attentional processing. These findings, across different behavioral paradigms and stimulus modalities, provide converging evidence that intact cholinergic innervation of the PPC is important for changes in attention that can increase the processing of certain cues.

Lesion models in the rat were used to examine the effects of removing innervation of the hippocampal formation on glutamate receptor binding in that system. Bilateral aspiration of the entorhinal cortex was used to remove the cortical innervation of the hippocampal formation and the dentate gyrus. The subcortical input to the hippocampus from cholinergic neurons of the basal forebrain was lesioned by microinjection of the immunotoxin 192 IgG-saporin into the medial septum and vertical limb of diagonal band. After a 30-day postlesion survival, the effects of these lesions on N-methyl-D-aspartate-displaceable [3H]glutamate and [3H]kainate binding in the hippocampus were quantified using in vitro autoradiography. The bilateral entorhinal lesion induced a sprouting response in the dentate gyrus, measured by an increase in the width of [3H]kainate binding. It also induced an increase in the density of [3H]kainate binding in CA3 stratum lucidum and an increase in N-methyl-D-aspartate binding throughout the hippocampus proper and the dentate gyrus. The selective lesion of cholinergic septal input did not have any effect on hippocampal [3H]kainate binding and induced only a moderate decrease in N-methyl-D-aspartate binding that was not statistically reliable. The entorhinal and cholinergic lesions were used as in vivo models of the degeneration of hippocampal input that occurs in normal aging and Alzheimer's disease. The results from the present lesion study suggest that some, but not all, of the effects on hippocampal [3H]kainate and N-methyl-D-aspartate binding induced by the lesions are consistent with the status of binding to these receptors in aging and Alzheimer's disease. Consistent with the effects of aging and Alzheimer's disease is an altered topography of [3H]kainate binding after entorhinal cortex lesion and a modest decline in N-methyl-D-aspartate binding after lesions of the cholinergic septal input to the hippocampus.

Pathological processing of the amyloid precursor protein (APP) is assumed to be responsible for the amyloid deposits in Alzheimer-diseased brain tissue, but the physiological function of this protein in the brain is still unclear. The aim of this study is to reveal whether the expression of different splicing variants of APP transcripts in distinct brain regions is driven by postnatal maturation and/or regulated by cortical cholinergic transmission, applying quantitative in situ hybridization histochemistry using 35S-labeled oligonucleotides as specific probes to differentiate between APP isoforms. In cortical brain regions, the expression of both APP695 and APP751 is high at birth and exhibits nearly adult levels. The developmental expression pattern of cortical APP695 displays a peak value around postnatal day 10, while the age-related expression of APP751 demonstrates peak values on postnatal days 10 and 25, with the highest steady state levels of APP751 mRNA on day 25. During early development, the cortical laminar distribution of the APP695, but not APP751, mRNA transiently changes from a more homogeneous distribution at birth to a pronounced laminar pattern with higher mRNA levels in cortical layer III/IV detectable at the age of 4 days and persisting until postnatal day 10. The distinct age-related changes in cortical APP695 and APP751 mRNA levels reflect the functional alterations during early brain maturation and suggest that APP695 might play a role in establishing the mature connectional pattern between neurons, whereas APP751 could play a role in controlling cellular growth and synaptogenesis. Lesion of basal forebrain cholinergic system by the selective cholinergic immunotoxin 192IgG-saporin resulted in decreased levels of APP695 but not APP751 and APP770 transcripts by about 15-20% in some cortical (cingulate, frontal, parietal, piriform cortex), hippocampal regions (CA1, dentate gyrus), and basal forebrain nuclei (medial septum, vertical limb of diagonal band), detectable not earlier than 30 days after lesion and persisting until 90 days postlesion, suggesting that the nearly complete loss of cortical cholinergic input does not have any significant impact on the expression of APP mRNA isoforms in cholinoceptive cortical target regions.