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The regulation of cholinergic markers by exogenous nerve growth factor (NGF) and/or monosialoganglioside GM1 (GM1) treatment was examined in various brain areas of unilaterally decorticated rats, with particular emphasis on the basal forebrain system. Treatment of decorticated rats, i.c.v. via a minipump, with various doses of NGF or GM1 for a period of 7 days prevented the lesion-induced decline in nucleus basalis magnocellularis choline acetyltransferase (ChAT) activity in a dose-dependent manner. These treatments also stimulated cortical ChAT activity as well as high-affinity choline uptake. Cholinergic markers in other brain areas studied were unaffected by the lesion or these treatments, except in the striatum in which exogenous NGF but not GM1 caused dose-dependent increases in ChAT activity and high-affinity choline uptake. Glutamic acid decarboxylase activity was unaffected by the lesion or treatments in all brain areas studied. Cotreatment of lesioned rats with GM1 did not affect NGF potency but did increase its maximal efficacy in the nucleus basalis magnocellularis and cortex, but not in the striatum. Treatment of cortically lesioned rats with maximal doses of GM1 and/or NGF did not differentially alter "soluble" or "membrane bound" forms of ChAT. In addition, choline uptake kinetic parameters also were affected similarly by these agents as evidenced by augmented Vmax and unaltered Km values. Distinct effects of NGF and GM1 were observed, however, in regard to the delay possible in treatment time onset. No significant alteration in NGF receptor density or affinity were noted in the remaining cortex of GM1-treated decorticated rats, suggesting that GM1 interacts with an alternative facet of NGF signal transduction to potentiate NGF effects on cholinergic markers.

This study examined the behavioral effects of maximal doses of exogenous NGF and/or GM1 when given intracerebroventricularly to adult rats with unilateral cortical lesions. In addition, the long-term effects of these agents on choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD) activity, and choline uptake, as well as on ChAT and NGF receptor (p75NGFR) immunoreactivity in various brain regions, were also investigated. When retested in passive avoidance and Morris water maze tasks, 30 days postlesion (i.e., 2 weeks after termination of treatment), decorticated vehicle-treated rats showed retention and reacquisition deficits which were equally attenuated by NGF (6 micrograms/day, 14 days) or NGF + GM1 (750 micrograms/day, 14 days) treatment. By contrast, lesioned animals which received GM1 alone only showed improved reacquisition of the two tasks. After behavioral testing (52 days postlesion) lesioned vehicle-treated animals had decreased ChAT activity in the ipsilateral nucleus basalis magnocellularis (NBM) but not in other subcortical brain areas examined. Neuronal loss was observed only in the ventrolateral nucleus of the ipsilateral dorsal thalamus. However, using quantitative image analysis a significant shrinkage of ChAT immunoreactive (IR) and p75NGFR-IR NBM neurons as well as a decrease in their neuritic network was noted, particularly in the mid portion of the NBM. GM1 and NGF equally prevented these deficits in the NBM and, furthermore, enhanced ChAT activity and choline uptake in the remaining cortex ipsilateral to the lesion site. These alterations in NBM and cortical cholinergic markers were even more augmented in rats which received both NGF and GM1 treatment. By contrast, the noted NGF-induced increase in striatal ChAT activity was not further increased by concomitant GM1 treatment. GAD activity in all brain areas examined was unaltered by the lesion or any of the treatments and the apparent thalamic neuronal retrograde degeneration was not prevented by any of the treatments. It is concluded that GM1 or NGF treatment can distinctly affect performance of cortically lesioned rats in passive avoidance and Morris water maze tasks despite their equal ability to serve as long-term neuroprotective agents for the basalo-cortical cholinergic pathway.

The effect of monosialoganglioside GM1 and/or nerve growth factor treatment on the cholinergic innervation of the rat cortex was studied using both light- and electron-microscopic techniques assisted by image analysis. Adult male Wistar rats were unilaterally decorticated and received continuous infusions, via minipump, of vehicle, GM1 (1.5 mg/day) and/or nerve growth factor (12 micrograms/day) into the cerebroventricular space. Treatments were initiated immediately post-lesion and ended after seven days. Thirty days post-lesion (i.e. 23 days after the end of drug administration) brains were processed for choline acetyltransferase immunocytochemistry for either light- or electron-microscopic analysis. At this time-point choline acetyltransferase-immunoreactive neurons in the ipsilateral nucleus basalis magnocellularis were significantly reduced in size especially in the mid portion of this nucleus, in lesion vehicle-treated rats. Moreover, decreases in choline acetyltransferase immunoreactive fibre length (ranging from 31 to 50%) and varicosity number (ranging from 26 to 39%) occurred in all cortical layers within a portion of the remaining cortex of these animals. Monosialoganglioside GM1 or nerve growth factor treatment equally attenuated deficits in nucleus basalis magnocellularis cell size and cortical choline acetyltransferase immunoreactive fibre length. However, nerve growth factor, but not monosialoganglioside GM1 treatment also increased choline acetyltransferase-immunoreactive varicosity number above control levels. In lesioned rats which received both nerve growth factor and the monosialoganglioside GM1, the mean cross-sectional area of nucleus basalis magnocellularis cholinergic neurons did not differ significantly from control values. By contrast, cortical choline acetyltransferase-immunoreactive fibre length and varicosity number were significantly increased above control values and that induced by nerve growth factor treatment alone. Quantitative electron-microscopic analysis showed that cholinergic boutons in cortical layer V were considerably shrunken in lesioned vehicle-treated rats and that GM1 treatment failed to significantly attenuate this deficit. However, exogenous nerve growth factor provoked a significant increase (35% above control values) in cortical cholinergic presynaptic terminal size which was even further augmented by concurrent GM1 treatment (69% above control values). This trophic factor-induced increase in bouton size was confirmed using serial electron microscopy and computer-assisted three-dimensional reconstruction of the cholinergic varicosities. The number of synaptic contacts in cortical layer V was also found to be significantly reduced (45% of control values) in lesioned vehicle-treated rats but was maintained at control levels by exogenous GM1 treatment. In addition, a significant increase (95% above control levels) in the number of choline acetyltransferase-immunoreactive boutons with synaptic differentiations was noted in lesioned nerve growth factor-treated rats.(ABSTRACT TRUNCATED AT 400 WORDS)

The cholinergic pathway ascending from the nucleus basalis magnocellularis (NBM) to the cortex has been implicated in several important higher brain functions such as learning and memory. Following infarction of the frontoparietal cortical area in the rat, a retrograde atrophy of cholinergic cell bodies and fiber networks occurs in the basalocortical cholinergic system. We have observed that neuronal atrophy in the NBM induced by this lesion can be prevented by intracerebroventricular administration of exogenous nerve growth factor (NGF) or the monosialoganglioside GM1. In addition, these agents can upregulate levels of cortical choline acetyltransferase (ChAT) activity in the remaining cortex adjacent to the lesion site. Furthermore, an enhancement in cortical high-affinity 3H-choline uptake and a sustained in vivo release of cortical acetylcholine (ACh) after K+ stimulation are also observed after the application of neurotrophic agents. Moreover, these biochemical changes in the cortex are accompanied by an anatomical remodeling of cortical ChAT-immunoreactive fibers and their synaptic boutons.

In this study light and EM quantitative analysis were used to examine whether exogenous nerve growth factor (NGF) could affect terminal fields and synaptic connections in the adult rat brain in vivo. Adult rats received, immediately after unilateral decortication, 2.5S NGF (12 micrograms/day) or vehicle intracerebroventricularly for 7 days. Thirty days after the lesion cholinergic fiber length was quantified, using image analysis, in the remaining cortical area adjacent to the lesion site in each animal. Rats that had received vehicle showed a significantly reduced cortical choline acetyl-transferase-immunoreactive fiber network in the remaining cortex when compared with control animals. By contrast, the network in lesioned rats that had received 2.5S NGF was not different from control animals. Furthermore, the number of cortical choline acetyltransferase-immunoreactive varicosities, which decreased in vehicle-treated lesioned rats, significantly increased above control in lesioned rats that had received 2.5S NGF. At the ultrastructural level, 30 days after the lesion, animals that had received vehicle showed shrunken cholinergic boutons in cortical layer V and fewer synapses compared with control animals. Exogenous NGF, administered to lesioned rats, increased to supernormal levels both size of cholinergic boutons and number of synaptic contacts. These parameters were unaltered in unlesioned rats treated with NGF. This study demonstrates that exogenous NGF can cause significant compensatory changes in terminal fields and synaptic connections in the adult fully differentiated central nervous system.

Exogenously administered gangliosides have been shown to behave as neuronotrophic/neuritogenic agents in a variety of cell culture systems and animal models, but it is not known whether they operate by the same mechanism in vivo and in vitro. To probe this question we have employed two derivatives of GM1 lacking the negative charge: the methyl ester (GM1-CH3) and the NaBH4 reduction product of the latter (GM1-OH) in which the carboxyl group is replaced by a primary alcohol. Both derivatives proved to be as neuritogenic as GM1 in 3 cell culture systems: neuro-2A cels, PC12 cells and explanted dorsal root ganglia. However, GM1-OH proved ineffective when applied to two animal models involving reduction of cholinergic markers in: (a) hippocampus following lesion of the lateral fimbria and (b) nucleus basalis magnocellularis following cortical lesion; GM1-CH3 showed marginal activity in (a) but more in (b), possibly owing to slow hydrolysis to GM1 which was highly active in both animal models. These results indicate the necessity of a negative change on the ganglioside molecule for in vivo but not in vitro activity and point to different mechanisms for the trophic effects of exogenous gangliosides.

Cholinergic neurons of the forebrain respond trophically to nerve growth factor (NGF) in some experimental circumstances. The cholinergic cell system of the nucleus basalis magnocellularis (NBM) which projects to the cortex shows signs of cellular degeneration following limited devascularizing cortical lesions, while no apparent damage is observed in the remaining ipsilateral cortex. These cholinergic cells possess receptors for NGF and the administration of this peptide into the cerebroventricular space prevents cell shrinkage and loss of activity of the biosynthetic enzyme for acetylcholine, choline acetyltransferase (ChAT). Analogous trophic responses can be elicited in this system with the application of the sialoganglioside GM1. In addition, GM1 can increase the effects of NGF on ChAT activity in lesioned neurons of the NBM-to-cortex model system described above. This cooperative interaction is observed even when ineffective doses of GM1 are administered. Furthermore, an interaction between these two putative neurotrophic substances has been noted over other cholinergic parameters such as cortical high affinity choline uptake (HACU). These studies confirm the idea that trophic factors can be utilized to rescue degenerating neurons of the CNS and, in addition, lend support to the concept that gangliosides can facilitate actions of endogenously produced trophic factors.

The effects of nerve growth factor beta (beta-NGF) and ganglioside GM1 on forebrain cholinergic neurons were examined in vivo and in vitro. Following unilateral decortication of rats, GM1 (5 mg/kg per day) administered intracerebroventricularly could protect forebrain cholinergic neurons of the nucleus basalis magnocellularis from retrograde degeneration in a manner comparable to beta-NGF. Administered in combination with beta-NGF, GM1 produced a significant increase in choline acetyltransferase activity in the nucleus basalis magnocellularis and remaining cortex ipsilateral to the lesion. Concentrations of GM1 that were ineffective when administered alone in this lesion model, when given with beta-NGF, potentiated beta-NGF effects in both of the above brain areas. In dissociated septal cells in vitro, an increase in choline acetyltransferase activity was noted at beta-NGF concentrations as low as 0.1 pM and reached a plateau at 1 nM. A moderate (up to 35%) stimulation of choline acetyltransferase activity was observed with 10 microM GM1. The application of beta-NGF in combination with 10 microM GM1 or 0.1 microM GM1, a concentration that is ineffective in these cultures, produced a much greater increase in choline acetyltransferase activity than did beta-NGF alone. These observations support the idea that exogenously applied gangliosides can elicit trophic responses in cholinergic neurons of the central nervous system. That GM1 increases and even potentiates beta-NGF effects suggests that some of the trophic actions of this compound may be mediated through endogenous trophic factors.