L. Garofalo

Potentiation of nerve growth factor-induced alterations in cholinergic fibre length and presynaptic terminal size in cortex of lesioned rats by the monosialoganglioside GM1

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)

Striatal and cortical acetylcholine release in vivo in rats with unilateral decortication: effects of treatment with monosialoganglioside GM1.

Striatal and cortical extracellular acetylcholine (ACh) and choline (Ch) levels were determined in samples collected under in vivo conditions using microdialysis in normal (naive) and decorticated rats treated with saline or the monoganglioside GM1. ACh and Ch were assayed using a sensitive high performance liquid chromatography technique coupled to a postcolumn reactor with immobilized enzymes. Picomole amounts of ACh could be measured in the presence of an acetylcholinesterase inhibitor (neostigmine) in the microdialysis perfusion medium (striatal ACh = 0.2-0.4 microM; cortical ACh = 0.03-0.04 microM). Ch was detected both in the presence and in the absence of neostigmine (striatal Ch = 0.5-0.6 microM; cortical Ch = 0.6-2 microM). ACh, but not Ch, was strongly stimulated by 100 mM of KCl included in the perfusion medium. Decortication produced by devascularization did not significantly modify the cortical or striatal basal levels of ACh. However, in the cortex, KCl produced a higher ACh stimulation in the GM1-treated than in the saline-treated decorticated or naive rats. The present results indicate that GM1-treatment increases the ability of cortical cholinergic terminals to release ACh and support the idea that trophic factors such as monoganglioside GM1 can promote recovery following injuries of the central nervous system.

Derivatives of ganglioside GM1 as neuronotrophic agents: comparison of in vivo and in vitro effects

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.

Trophic factor effects on cholinergic innervation in the cerebral cortex of the adult rat brain

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-affinity3H-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.

Microencapsulated monosialoganglioside GM1: physical properties and in vivo effects

The prevention of the decrease of choline acetyltransferase (ChAT) enzymatic activity was achieved by applying GM1 in an animal model for studying retrograde degenerations of cholinergic neurons. Devascularizing lesions of the rat cortex led to a significant decrease in activity of ChAT in the nucleus basalis magnocellularis (NBM), but this decrease was effectively prevented by GM1 administration either centrally or locally in a microencapsulated form. Compared with the relatively large dose of GM1 which has to be given when the drug is administered. i.p. microencapsulated GM1 applied locally and directly over the lesioned cortical surface seems to be effective in much lower doses.

Effects of microencapsulated monosialoganglioside GM1 on cholinergic neurons

The preparation, physical characterization and effects of microcapsules containing the monosialoganglioside GM1 in an in vivo rat model are described herewith. Several preparations of microcapsules were obtained differing in physical and chemical properties. Human serum albumin (HSA) microcapsules with or without GM1 are spherical in shape, have a consistent particle size (8-10 microns in diameter) and are devoid of large pores. In agreement with our previous work, we now provide further evidence that GM1 can prevent shrinkage and the decrease of choline acetyltransferase activity in the nucleus basalis magnocellularis (NBM) of the rat following a unilateral cortical lesion. In the present study we examined the effect of microencapsulated GM1 in this in vivo rat model. Local application of HSA-microencapsulated GM1 (in doses comparable to those obtained by i.c.v. administration) onto the surface of the lesioned cortex prevents both the biochemical and morphological degenerative changes in the NBM of rats with unilateral devascularizing cortical lesions. The results from these studies show that microencapsulated GM1 can be applied successfully and a prolonged controlled release of this drug obtained, thus avoiding surgical implantation of a cannula.

Influence of Gangliosides and Nerve Growth Factor on the Plasticity of Forebrain Cholinergic Neurons

Neurons from the medial septal nucleus and nucleus of the vertical limb of the diagonal band of Broca provide an important cholinergic input to the hippocampus (Lewis and Shute, 1967; Oderfeld-Nowak et al, 1974; Meibach and Siegel, 1977). The cortex receives a widespread distribution of cholinergic fibres, the majority of which seem to originate from the nucleus basalis magnocellularis (NBM) (Johnston et al, 1981; Fibiger, 1982; Cuello and Sofroniew, 1984). From immunohistochemical studies it would appear that a topographic representation exists for this projection (Mesulam et al, 1986; Ingham et al, 1985). These fibers represent approximately 70% of the total cholinergic component of the cortex (Lehman et al, 1982), the remainder deriving from local circuit neurons (Sofroniew et al, 1982; Johnston et al, 1981). This participation of forebrain cholinergic neurons has been emphasized in recent years since a decrease in choline acetyltransferase (ChAT) activity has been reported to occur in the cortex and in the NBM of patients with Alzheimer’s disease Bowen et al, 1983; Davies and Maloney, 1976; Perry et al, 1977; Rossor et al, 1982; Sims et al, 1983). Furthermore, a reduced number of cells in the latter area has been reported as a feature of Alzheimer’s disease (Whitehouse et al, 1982). On the basis of

Chapter 26 Cooperative effects of gangliosides on trophic factor-induced neuronal cell recovery and synaptogenesis: studies in rodents and subhuman primates

Publisher Summary To better estimate the potential therapeutic value of gangliosides or neurotrophins in central nervous system (CNS) trauma or degenerative processes, this chapter presents the rodent experimental model, involving loss of a portion of the cortical cholinergic network of nucleus basalis magnocellularis (nbm) projections in subhuman primates. A similar cholinergic atrophic reaction was elicited in primates ( Cercopithecus aethiops ) and the neurotrophic therapy was equally effective in such animals. The details of these investigations are also discussed in this chapter. The studies applying putative neurotrophic agents (NGF) in the cholinergic basolo-cortical lesion model suggested that these substances might induce an important re-arrangement of the cholinergic innervations. The chapter also investigates the incidence of CUT-IR varicosities in animals as these are sites for storage of acetylcholine as well as the enzyme involved in its synthesis, and are where physiological release of the neurotransmitter occurs. This study was accomplished by using a image analysis system combined with immunohistochemistry, except that 24 fields per animal were scanned at higher magnification. The program employed was designed to recognize immunoreactive elements, falling within the size range of nerve varicosities, along fibers.

Chapter 32 Injury and repair of central cholinergic neurons

Publisher Summary This chapter discusses the injury and repair of the central cholinergic neurons. Trophic responses to β - nerve growth factor (NGF) by centrally located cholinergic neurons are well substantiated for septal-hippocampal and basalis-cortical projections. From the cellular and subcellular analysis of NGFR immunoreactive sites, it can be assumed that those responses are receptor mediated. The sequence of events that follow this receptor-ligand interaction may include induction of specific genes and second messenger systems, which result in the translation and/or modification of specific proteins involved in trophic responses. The interaction of gangliosides with β -NGF over central cholinergic neurons could occur at any of those levels. The possibility that this interaction occurs at the level of the cell membrane is considered in the chapter. Exogenous gangliosides are known to be incorporated into the neural cell membranes and immobilized GM1 is capable of binding β -NGF with low affinity. Therefore, it is possible that gangliosides could provide additional binding sites for growth factors or modify the state of the growth factor receptor.

Effects of Nerve Growth Factor and Monosialoganglioside GM1 on Forebrain Cholinergic Neurones

The relevance of cortical cholinergic innervation has attracted great interest since severe losses of choline acetyltransferase (ChAT) enzymatic activity and cell loss in the nucleus basalis of Meynert were demonstrated in the cerebral cortex of patients with Alzheimer’s disease (Coyle et al. 1983, Collerton 1986). In rats with unilateral cortical lesion specific biochemical and morphological retrograde changes occur in the nucleus basalis magnocellularis (NBM). Thirty days following devascularizing lesion of the cortex choline acetyl transferase (ChAT) activity in the NBM decreased significantly from control values while no significant change was noted in the neurochemical marker for GABAergic function glutamic acid decarboxylase (GAD).