GM1 ganglioside stimulates the regeneration of dopaminergic neurons in the central nervous system

In vivo differential pulse voltammetry was used for the detection of indoleamines during vertical electrode penetrations in rat first somatosensory cortex, for studying the laminar distribution of serotonin and/or its metabolites in that part of the cortex. The peak of current corresponding to 5-hydroxyindoles was maximum in the most superficial part of the cortex and diminished gradually in the deeper layers. These results suggest that the cortical serotonergic innervation is predominant in the superficial layers.     

Treatment with GM1 ganglioside increases rat spinal cord indole content

Chronic treatment with GM1 ganglioside apparently increases serotonin metabolism in the spinal cord of control and hemitransected rats. Dopamine metabolism is stimulated below a hemitransection with GM1 treatment. These observations are consistent with reports that GM1 promotes regrowth of neurons after experimental lesions of brain.     

The effect of GM1 ganglioside on cholinergic and serotoninergic systems in the rat hippocampus following partial denervation is dependent on the degree of fiber degeneration

The partial lesion paradigm of the dorsal hippocampal afferents in the rat was used as a model to study the effect of GM1 ganglioside treatment on recovery of neurotransmitter markers of the cholinergic and serotoninergic activity in various hippocampal regions. It was found that the enhancement of recovery of acetylcholinesterase, choline acetyltransferase and serotonin uptake by GM1 treatment (30 mg/kg i.m., daily), as studied on the 6th and 21st postlesion day, was dependent on the degree of fiber degeneration. The results may be interpreted in terms of the relationship between the action of GM1 and that of neuronotrophic factors whose release also depends on the extent of the fiber degeneration. These data indicate that GM1 elicits the recovery of biochemical parameters, or fails to, depending on the specificity of the trauma. The result may explain why, after certain brain lesions, GM1 does not promote functional recovery.     

The effect of GM1 ganglioside on coerulospinal, noradrenergic, adult neurons and on fetal monoaminergic neurons transplanted into the transected spinal cord of the adult rat

GM₁ ganglioside, thyroxine and hydrocortisone were tested for their ability to improve the survival and growth of fetal focus coeruleus noradrenergic neurons in the transected, adult spinal cord. GM₁ alone was also tested for its effect on fetal mesencephalic dopaminergic neurons implanted into a small dorsolateral cavity at the L2 region of the cord previously transected at the T9–T10 region. None of the substances tested had any measurable effect on either of the fetal implants. However, in the GM₁- and thyroxine-treated animals the somatic dendrites of the axotomized, noradrenergic, coerulospinal neurons appeared more robust, and more intensely fluorescent, compared to their appropriate controls. GM₁ also caused a pronounced sprouting of the axotomized monoaminergic (catecholaminergic and serotonergic) fibres in the rostral region of the cord adjacent to the transection site. All of the mesencephalic dopaminergic implants survived in both the GM₁-treated animals and their saline-injected controls. However, their development was apparently not influenced by GM₁. The results indicate that GM₁ and thyroxine can enhance those aspects of the reactive mechanisms of mature, axotomized, noradrenergic coerulospinal neurons that promote their regeneration. As such, GM₁ could become a useful tool in current attempts to foster the regeneration of damaged monoaminergic neurons in the mammalian CNS.     

Neurophysiological studies in GM1, gangliosidosis

Neurophysiological studies(EEG, ERG, VEP) have been carried out on 8 children with proven GM₁ gangliosidosis (3 of Type I and 5 of Type II). All the EEGs were abnormal showing an increasing amount of irregular slow activity as the disease progressed. Around 2 to 3 years of age, Type II patients often showed a fluctuating 4–5 c/s rhythmic activity especially prominent in the temporal regions. Paroxysmal activity was not a conspicuous feature in any of the patients. The ERG was normal in all cases but the VEP was variably altered. The EEG / ERG/ VEP findings in GM₁ gangliosidosis differ from those seen in most other neurometabolic disorders of childhood.

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Sommario Sono stati condotti studi neurofisiologici(EEG, ERG, PEV) in 8 bambini con accertato GM₁ gangliosidiosi (3 di tipo I e 5 di tipo II). Tutti gli EEG sono risultati anormali dimostrando un aumento di attività lenta e irregolare col procedere della malattia. I pazienti del gruppo II presentano, attorno ai 2–3 anni di età, un'attività fluttuante e ritmica a 4–5 c/s prevalente soprattutto nelle regioni temporali. L'attività parossistica non risultava continua in alcun caso. L'ERG era normale in tutti i casi e la PEV era variamente alterata. In sintesi i rilievi EEG, ERG, PEV nella GM₁ gangliosidiosi differiscono da quelli che si riscontrano nella maggioranza delle malattie neurometaboliche infantili.

     

Ganglio-N-tetraosylceramide (Asialo GM1), an antigen common to the brain and immune system: its localization in myelin

Anti-brain antisera are known to contain several interesting antibodies showing immunological cross-reactions between the nervous and hematopoietic systems, suggesting the existence of antigens common to the two systems (common antigens). One of these is the glycosphingolipid asialo GM1 (ganglio-N-tetraosylceramide, GgOse4Cer), but its location in the nervous system is unknown. Therefore, the location of the brain antigen that reacts with anti-asialo GM1 antibody was examined. On immunohistochemical treatment of sections of mouse brain with purified anti-asialo GM1 IgG, the white matter was stained specifically. Because myelin is a major component of the white matter, neutral glycosphingolipids were prepared from the myelin fraction. On thin-layer chromatography, myelin glycosphingolipids gave a band with the same mobility as asialo GM1. This band was also stained immunochemically by the peroxidase anti-peroxidase procedure using anti-asialo GM1 IgG. The glycosphingolipid of this band gave the molar ratio of sugars expected for asialo GM1. From these results it was concluded that the glycosphingolipid asialo GM1 exists in myelin.     

Interaction between nerve growth factor and GM1 monosialoganglioside in preventing cortical choline acetyltransferase and high affinity choline uptake decrease after lesion of the nucleus basalis

Monosialoganglioside GM₁ and nerve growth factor (NGF) were administered alone or concomitantly to adult male rats with a unilateral ibotenic acid lesion of the nucleus basalis magnocellularis (NBM). High-affinity choline uptake (HACU) rate and choline acetyltransferase (ChAT) activity were measured, 4 and 21 days after surgery, respectively, in the frontal and parietal cortices of both hemispheres. A 33–34% decrease in HACU rate and a 43-39% decrease in ChAT activity was found in the ipsilateral cortices 4 and 21 days, respectively, after the lesion. If the lesioned rats received NGF (10 μg i.c.v.) twice a week or daily administrations of GM₁ (30 mg/kg, i.p.), beginning immediately after surgery the decrease in HACU rate and ChAT activity was smaller. If NGF and GM, were given concomitantly no decrease in HACU rate and ChAT activity was detected in the lesioned hemisphere and a slight increase occurred in the contralateral hemisphere. However, after the concurrent administration of NGF (10 μg i.c.v.) and the inactive dose of GM₁ 10 mg/kg i.p. no decrease in HACU and ChAT activity was also found in the lesioned rats. The latter finding indicates a potentiation by GM₁ of NGF effects on the cholinergic neurons of the IBM. The two drugs may either antagonize the neurotoxic effects of ibotenic acid or stimulate a compensatory activity in the remaining neurons.     

Effect of an NMDA receptor antagonist and a ganglioside GM1 derivative upon ethanol-induced modification of parameters of oxidative stress in several brain regions

Dietary administration of ethanol to rats for 2 weeks was able to depress levels of glutathione (GSH) and Cu/Zn superoxide dismutase (SOD) in several brain regions. This was indicative of the generation of excess levels of reactive oxygen in treated animals. The potentially protective effect of both an NMDA receptor blocker (MK-801) and an internally esterified derivative of ganglioside GM₁ (AGF₂) upon ethanol-induced changes in these indices of oxidative stress, was studied. Both of these agents are reported to have neuroprotective properties, but neither was able to prevent ethanol-induced reduction of GSH and SOD levels in any brain area studied. In fact, both agents depressed SOD and GSH levels in midbrain independently of ethanol. MK-801 had a pronounced pro-oxidant potential, and when administered in combination with ethanol, GSH and SOD were reduced in midbrain and striatum to levels below those obtained with either agent alone. The pro-oxidant properties of ethanol may thus act independently of its actions upon the NMDA receptor. The protective properties of NMDA receptor inhibitors or gangliosides cannot be attributed to any antioxidant effect.     

Retrograde changes in the nucleus basalis of the rat, caused by cortical damage, are prevented by exogenous ganglioside GM1

In rats with extensive unilateral cortical damage, retrograde effects upon the cholinergic cells of the basal nucleus were observed. Cells of the basal nucleus stained immunocytochemically for choline acetyltransferase were shrunken and choline acetyltransferase enzymatic activity in that region was reduced. Both these effects could be prevented by the administration of the ganglioside GM1.     

Effects of ganglioside treatment in rats with a lesion of the cholinergic forebrain nuclei

The effects of GM1 ganglioside (30 mg/kg i.p.) administration for 22 days on choline acetyltransferase (ChAT) activity and noradrenaline (NA) levels in the cerebral cortex and on the acquisition of active and passive avoidance-conditioned responses were investigated in both sham-operated rats and in rats with a unilateral electrolytic lesion of the magnocellular forebrain nuclei (MFN). A statistically significant ChAT decrease in cortical areas ipsilateral to the lesion was found in saline-treated lesioned rats. In the lesioned GM1-treated rats, ChAT activity was only reduced in the frontoparietal areas and was significantly increased in the ipsilateral parietooccipital areas as well as in both contralateral regions. NA levels in the cortex were neither significantly affected by the lesion nor by GM1 treatment. The lesion impaired the acquisition of active and passive conditioned avoidance responses. GM1 treatment improved acquisition of the active avoidance response in the lesioned rats as indicated by a larger number of avoidances and a smaller number of escape failures during training in comparison with saline treatment. Ganglioside had no effect on the passive avoidance responses. These results demonstrate that GM1 administration facilitates the recovery of the cortical cholinergic system and of behavioral responses impaired by an electrolytic lesion of the cholinergic forebrain nuclei.     

The role of dopamine in the maintenance and breakdown of D1/D2 synergism

The neurochemical factors involved in the maintenance and breakdown of dopamine D₁/D₂ receptor synergism were investigated by giving rats various pharmacological treatments that diminish the ability of dopamine to interact with its D₁ and/or D₂ receptors. Following these treatments, rats were observed for the expression of stereotyped motor behavior in response to independent stimulation of D₁ or D₂ receptors. Independent D₂-mediated responses were observed: (a) 2 h after the last of three daily reserpine (1 mg/kg) injections, (b) 48 h after bilateral 6-hydroxydopamine (6-OHDA) lesions of the mesostriatal pathways, (c) 24 h after a concentrated 48-h regimen (one injection/6 h) of eticlopride (0.5 mg/kg) or eticlopride + SCH 23390 (0.5 mg each), and (d) 2 h after a concentrated 48-h regimen (one injection/6 h) of α-methyl-p-tyrosine (αMPT; 100 mg/kg), but not after control treatments or a concentrated regimen of SCH 23390 alone. By contrast, independent D₁-mediated responses were observed only after three daily reserpine injections or 48 h after bilateral 6-OHDA lesions. Independent D₁-mediated stereotypy was not observed under control conditions or following a concentrated 48-h regimen of (a) SCH 23390 or eticlopride (0.5 mg/kg each) alone or in combination, (b) a high dose of SCH 23390 (1.0 mg/kg), (c) αMPT (100 mg/kg), or (d) αMPT (100 mg/kg)+SCH 23390 (1.0 mg/kg). Reserpine, bilateral 6-OHDA, and αMPT treatments produced striatal dopamine depletions of 96%, 92%, and 71%, respectively. These data indicate that the breakdown in D₁/D₂ synergism consists of two components: (a) D₁ independence from the controlling influence of D₂ receptors, and (b) D₂ independence from the controlling influence of D₁ receptors. The interaction of synaptic DA with its D₂ receptors plays a major role in determining whether these receptors can function independently of D₁ receptors, whereas reduced DA-D₁ activity alone appears insufficient to elicit D₁ independence.     

Sustained unilateral blockade of rat striatal M1 muscarinic receptors with m1-toxin1 in vivo

m1-Toxin1 is a trace component of the venom of the green mamba that antagonizes M₁ muscarinic receptors specifically and irreversibly in vitro. It was injected into the right caudatoputamen of Wistar rats to occlude M₁ receptors specifically for several days (‘M₁ knockdown’), and to begin to establish the unknown effects of striatal M₁-neurotransmission on movement. The extent and duration of M₁-blockade were evaluated with receptor binding assays and light microscopic autoradiography, using 1–2 nM [³H]pirenzepine to assess unoccupied M₁ receptors. An injection of 27 pmol m1-toxin1 blocked almost all of the M₁ receptors in one caudatoputamen (4 pmol), followed by their slow recovery to supranormal levels (115%) during a week. During maximum M₁-blockade, the binding of 1 nM [³H]N-methylscopolamine to striatal membranes was reduced by only half, indicating no blockade of M₄ receptors, which comprise 51% of striatal muscarinic receptors. It is concluded that m1-toxin1 can produce acute, focal, selective, unilateral and long-lasting M₁-blockade for investigations of the effects of M₁-neurotransmission in vivo. Rats with extensive unilateral striatal M₁-blockade appeared normal, and did not show unilateral deficits in spontaneous forearm use, in contrast to rats with unilateral 6-hydroxydopamine lesions of the substantia nigra. Since M₁-blockade should decrease the activity of both striatonigral and striatopallidal projection neurons, and the relative activity of these neurons is believed to control movement, the results suggest that a balanced decrease in the activity of these neurons on one side of the brain does not produce a right/left imbalance in spontaneous movement.     

D1 and D2 receptor modulation in rat striatum and nucleus accumbens after subchronic and chronic haloperidol treatment

The antipsychotic effects of neuroleptic drugs are believed to be achieved by chronic blockade of dopaminergic transmission in the limbic system. Nevertheless, the effects of chronic (3-12 months) haloperidol administration on the dopaminergic transmission in the nucleus accumbens of rodents remains poorly understood. Studies of spontaneous locomotor activity (SLA), a behavioral measure related to limbic dopamine transmission, and of dopamine D2 receptor density in the nucleus accumbens after chronic oral haloperidol treatment have yielded conflicting results. We evaluated these indices after 8 months of parenteral administration of haloperidol decanoate. We report here that, after 8 months of parenteral treatment, SLA stays significantly decreased and D2 receptors in the nucleus accumbens exhibit the same up-regulation as in the striatum (about 50%). These results fail to support the notion of a different pattern of D2 receptor adaptation to neuroleptic treatment between the nucleus accumbens and the striatum. In contrast, dopamine D1 receptors were found to be unaffected in the nucleus accumbens but decreased in the striatum by 22% after 8 months of treatment. This observation could be relevant to the pathogenesis of tardive dyskinesia.     

Topographical distributions of 32K and 48K cAMP-regulated phosphoproteins: Relationships to dopamine and serotonin innervations in striatum, substantia nigra and cerebral cortex

The topographical distribution of a cAMP-regulated phosphoprotein of an apparent molecular weight (M_r) of 32,000 (32K) was examined in homogenates prepared from microdiscs punched out from serial frozen slices of the striatum. The amount of this phosphoprotein progressively diminished from the rostral to the caudal part of the striatum as did both the dopamine-innervation and the dopamine (D₁)-sensitive adenylate cyclase. After kainic acid lesion of the rostral part of the striatum, the 32K phosphoprotein disappeared in this area and we observed a 48% decrease in the amount of 32K phosphoprotein found in the substantia nigra. 6-Hydroxydopamine lesions of the nigro-striatal dopaminergic pathway did not affect the 32K phosphoprotein either in striatum or substantia nigra. These results suggest that in the nigro-striatal pathway, the 32K phosphoprotein is closely associated with dopaminoceptive neurons containing D₁ receptors. In the cerebral cortex the association of 32K phosphoprotein with dopaminoceptive neurons is more questionable since we did not find a higher density of this phosphoprotein in areas containing a high amount of D₁ receptor (frontal cerebral cortex) than in areas containing a low amount of D₁ receptor (parietal cerebral cortex). In the course of this study we found another cAMP-regulated phosphoprotein of an M_r of 48,000 (48K). The amount of this phosphoprotein increased progressively from the rostral to the caudal part of the striatum, a pattern of distribution close to that of serotonin terminals. This protein was also present in the substantia nigra. Kainic acid lesioning of the rostral part of the striatum did not affect the amount of the 48K phosphoprotein within the substantia nigra. 6-Hydroxydopamine lesions of the substantia nigra also did not alter the amount of 48K phosphoprotein in this brain region.     

Selective unilateral inactivation of striatal D1 and D2 dopamine receptor subtypes by EEDQ: turning behavior elicited by D2 dopamine receptor agonists

The unilateral intrastriatal injection of the irreversible dopamine (DA) receptor blockerN-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) induces a marked decrease in the density of D₁ (-48%) and D₂ (-51%) DA receptors available for binding to [³H]SCH 23390 and [³H]raclopride, respectively. A challenge dose of the D₂ agonist LY 171555 (1 mg/kg, i.p., 24 h after EEDQ) causes intensive ipsiversive circling behavior, whereas the selective D₁ agonist SKF 38393 (20 mg/kg, i.p., 24 h after EEDQ) is unable to induce rotations. The density of D₁ and D₂ DA receptors returns to basal levels by 7 days after the intrastriatal infusion of EEDQ. This biochemical recovery is associated with a progressive decrease in the number of rotations elicited by a challenge dose of LY 171555, suggesting the EEDQ does not cause any relevant neuronal damage. A selective inactivation of striatal D₁ or D₂ DA receptors can be obtained by injecting EEDQ 30 min after the administration of the D₂ antagonist raclopride (20 mg/kg, i.p.) or of the D₁ antagonist SCH 23390 (2 mg/kg, s.c.), respectively. The intensity of the circling behavior induced by LY 171555 24 h after EEDQ in animals with a selective inactivation of D₂ DA receptors is similar to that found in rats in which both D₁ and D₂ DA receptors have been inactivated. In contrast, LY 171555 does not cause rotations when the density of D₁ DA receptors is selectively decreased by EEDQ in rats pretreated with raclopride. These results indicate that the imbalance in striatal D₂ receptors, but not in D₁ receptors, is a critical factor for the expression of the motor effects elicited by LY 171555 in EEDQ-treated rats.     

Possible role of dopamine D1 receptor in the behavioural supersensitivity to dopamine agonists induced by chronic treatment with antidepressants

The effect of chronic treatment with antidepressants (ADs) on the behavioral responses to LY 171555, a selective D₂ receptor agonist, SKF 38393, a selective D₁ receptor agonist, and B-HT 920, a selective DA autoreceptor agonist, was studied in rats. In normal rats small, intermediate and high doses of LY 171555 produced hypomotility, hyperactivity and stereotypies, respectively. Chronic but not acute pretreatment with imipramine (IMI) greatly potentiated the motor stimulant effect of LY 171555, but failed to modify its stereotypic and sedative effect. The potentiation of the motor stimulant effect of LY 171555 was observed also after chronic, but not acute, treatment with desmethylimipramine (DMI), mianserin (MIA) or repeated electroconvulsive shock (ECS). Chronic treatment with IMI failed to modify the effect of SKF 38393 (motor stimulation, grooming and penile erection), but reversed the sedative effect of B-HT 920 into a motor stimulant response. The motor stimulant response to LY 171555 in IMI-pretreated animals was suppressed byl-sulpiride, a D₂ antagonist, and by a combination of reserpine with α-methyltyrosine (α-MT), but it was only partially antagonized by high doses of SCH 23390, a selective D₁ antagonist. The results indicate that chronic treatment with ADs potentiates the behavioural responses mediated by the stimulation of postsynaptic D₂ receptors in the mesolimbic system and suggest that this behavioural supersensitivity is due to enhanced neurotransmission at the D₁ receptor level.     

Interactive effects of stimulation of D1 and D2 dopamine receptors on fos-like immunoreactivity in the normosensitive rat striatum

Systemic administration of the selective, full, D₁ dopamine agonist A-77636 [(1R,3S)3-(1′-adamantyl)-1-aminomethyl-3,4-dihydro-5,6-dihydroxy-1H-2-benzopyranhydrochloride] (0.36–2.9 mg/kg) led to a dose-dependent induction of Fos-like immunoreactivity (FLI) in the striatum. Quantitative analysis of the sections indicated that immunoreactive cells were more numerous in the medial than the lateral striatum and, within these regions, appeared to be randomly distributed. The staining produced by A-77636 could be abolished by pretreatment with the selective D₁ antagonist SCH-23390. The selective D₂ dopamine agonist quinpirole (3 mg/kg) had no effect on striatal FLI when given by itself, but markedly potentiated the weak striatal staining produced by low doses of A-77636. When combined with the highest dose of A-77636, which produced substantial staining by itself, quinpirole produced an increase in the number of immunoreactive cells seen in the lateral striatum but actually decreased the number present in the medial striatum. Statistical analysis of the distribution of immunoreactive cells demonstrated that, in both regions, quinpirole converted the relatively homogeneous staining seen after A-77636 alone into a markedly patchy pattern. These findings indicate that stimulation of D₂ receptors produces both stimulatory and inhibitory effects on the D₁-mediated expression of Fos in the striatum and that the interaction between D₁ and D₂ receptor stimulation must, therefore, be more complex than the simple synergism suggested by previous studies.     

Sex differences in GABAAergic system in rat substantia nigra pars reticulata

The substantia nigra pars reticulata (SNR) is involved in the control of movement disorders including seizures through its GABAergic neurons. Microinfusions of muscimol (a GABA(A) receptor agonist) produce specific effects on seizures depending on sex, infusion site (SNR(anterior) or SNR(posterior)) and age. To assess whether these effects are due to sex differences in GABAergic indices within the SNR we analyzed the expression of alpha(1) subunit mRNA of the GABA(A) receptor and the levels of GABA immunoreactivity (IR) of male and female rats at postnatal day 15 (PN15) and PN30. In each age, within the same SNR region, expression of alpha(1) subunit mRNA and intensity of GABA IR per neuron was higher in females compared to males. At PN15, in both sexes, there were no regional differences in expression of alpha(1) subunit mRNA and intensity of GABA IR. However, at PN30 in both sexes, expression of alpha(1) subunit mRNA and intensity of GABA IR per cell was higher in SNR(anterior) than in SNR(posterior). These results demonstrate that expression of alpha(1) subunit mRNA for GABA(A) receptor and levels of GABA IR in the SNR are sex- and site-specific, which may contribute to sex-, regional- and age-related differences in the expression of movement disorders and seizures.     

Dopamine D2 receptor agents, but not dopamine D1, modify brain glucose metabolism

The effects of recently described selective dopamine D₁ and D₂ agonists and antagonists on brain glucose metabolism were studied using the 2-[¹⁴C]deoxyglucose autoradiographic technique. The administration of LY-141865 or YM-09151-2, which behave as a specific D₂ agonist and antagonist respectively, modified brain glucose metabolism in a manner similar to that previously described for more classical dopaminergic agents, such as apomorphine and haloperidol. In contrast, the administration of SKF 38393 or SCH 23390, a specific D₁ agonist and antagonist respectively, was not followed by significant modifications of brain glucose metabolism in any of the brain regions studied. These results indicate that D₂ but not D₁ dopamine receptors are involved in the regulation of local brain glucose metabolism.