G. Toffano

Morphometrical and microdensitometrical studies on phenylethanolamine-N-methyltransferase- and neuropeptide Y-immunoreactive neurons in the rostral medulla oblongata of the adult and old male rat.

In the present paper the neuronal systems of the medulla oblongata containing phenylethanolamine-N-methyltransferase- and neuropeptide Y-like immunoreactivity have been characterized in adult (3-month-old) and old (24-month-old) male rats. The phenylethanolamine-N-methyltransferase and neuropeptide Y-immunoreactive neurons have been visualized by means of immunocytochemistry (peroxidase-antiperoxidase technique) and analysed in a quantitative fashion by means of morphometrical (phenylethanolamine-N-methyltransferase- and neuropeptide Y-immunoreactive cell groups) and microdensitometrical (phenylethanolamine-N-methyltransferase-immunoreactive cell groups) approaches developed on the IBAS II image analyser (Zeiss-Kontron). During aging there is (a) a reduction in the area covered by the phenylethanolamine-N-methyltransferase-immunoreactive neuropil for both the C1 and C2 adrenaline cell groups; (b) a reduction in the area covered by the phenylethanolamine-N-methyltransferase-immunoreactive cell bodies, which is highly significant only for the C2 cell group; (c) a decrease in the area covered by the phenylethanolamine-N-methyltransferase-positive cell cluster for both C1 and C2 cell groups; (d) a decrease in the degree of phenylethanolamine-N-methyltransferase immunoreactivity present in the C1 and C2 cell groups; (e) a decay of neuropeptide Y immunoreactivity in the C1 and C2 groups, while the C3 group is unaffected by aging as evaluated by number of phenylethanolamine-N-methyltransferase- and neuropeptide Y-immunoreactive cell body profiles. These results indicate heterogeneities in the responses of the adrenaline-neuropeptide Y cell groups to the aging process. The possible functional consequences of aging-induced changes in the cardiovascular adrenergic neurons are discussed, especially in relation to development of hypertension.

Transient forebrain ischemia produces multiple deficits in dopamine D1 transmission in the lateral neostriatum of the rat.

Striatal dopamine D1 transmission was studied in rats 7 days after transient (30 min) forebrain ischemia using the 4-vessel occlusion model. The striatal distribution of dopamine D1 ([3H]SCH 23390 binding sites) and D2 ([3H]sulpiride binding sites) receptors as well as the distribution of adenylate cyclase ( [3H]forskolin binding sites) and of the intracytoplasmic dopamine and cAMP-regulated phosphoprotein DARPP-32 related to D1 transmission were analyzed. While the distribution of D2 receptors was unaffected 7 days after the ischemic insult, all the other markers showed a patchy disappearance in the dorsolateral part of the neostriatum. These findings underline the existence of selective multiple deficits in D1 transmission after transient forebrain ischemia in rat striatum.

Chronic ganglioside treatment counteracts the biochemical signs of dopamine receptor supersensitivity induced by chronic haloperidol treatment

Chronic ganglioside treatment (10 mg/kg, i.p.) using the molecular species with only one neuroaminic acid residue (GM1) given together with haloperidol (0.3 and 5 mg/kg, i.p.) once daily in male rats, counteracted the haloperidol-induced increase in the number of [3H]spiperone binding sites in striatal membranes when the low dose of haloperidol, but not the high dose, was administered. The present results therefore indicate that chronic GM1 treatment can partially counteract the increase in the number of dopamine receptors having a high affinity for neuroleptics (D2 type) induced by chronic haloperidol treatment in striatal membranes, and therefore may also partially counteract the development of neuroleptic-induced dopamine receptor supersensitivity.

Effects of neurotoxic and mechanical lesions of the mesostriatal dopamine pathway on striatal polyamine levels in the rat: Modulation by chronic ganglioside GM1 treatment

In male rats, partial hemitransections but not 6-hydroxydopamine (6-OHDA)-induced lesions of the mesostriatal dopamine (DA) pathway produce after 7 days a marked and a modest increase of striatal putrescine and spermidine levels, respectively, on the lesioned side. Following chronic ganglioside GM1 treatment of partially hemitransected rats, an increase of striatal polyamine levels was observed also on the intact side. It is suggested that retrograde cell body changes produced by hemitransection may induce striatal ornithine decarboxylase activity and in this way increase striatal putrescine levels, favoring regenerative mechanisms. The increase of striatal polyamine levels by GM1 treatment on the intact side of both 6-OHDA and mechanically lesioned rats compared with intact unoperated rats may also reflect an increased synthesis of striatal polyamines.

Selective reduction of glucocorticoid receptor immunoreactivity in the hippocampal formation and central amygdaloid nucleus of the aged rat

Hippocampal cell loss during aging has been related to the toxic effects of corticosterone on this cell population. It is not known which receptor mediates corticosterone cytotoxicity. At least two types of receptors for corticosterone have been recognized in the rat brain, type I (corticosterone preferring receptor, CR) and type II (glucocorticoid receptor, GR). In the present study the possible changes in GR immunoreactivity (IR) in various tel- and diencephalic regions of the aged rat have been investigated using immunocytochemistry coupled with computer-assisted image analysis. Male Sprague-Dawley rats of 3, 12 and 24 months of age were used (n = 5/group). A selective decrease of GR-IR was observed in the CA1 hippocampal field and central amygdaloid nucleus of the 24-month-old with respect to both 3- and 12-month-old rats. While in the former region GR-IR decrease was paralleled by a decrease of IR field area, no age-related decrease of GR-IR profile number was detected in central amygdaloid nucleus. A significant decrease of GR-IR and IR field area was also observed in the dentate gyrus of 24- vs 12-month-old rats but not vs 3-month-old rats. The analysis of adjacent sections stained with Cresyl violet showed a pattern of age-related changes (decrease of neuronal profiles in CA1 field pyramidal layer and dentate gyrus granular layer, and no change in the central amygdaloid nucleus of aged rats) which paralleled the observed changes in GR-IR in the same areas. This study provides evidence that GR are selectively decreased in the hippocampal formation and in the central amygdaloid nucleus of the aged rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular and synaptic alterations in the aging brain

The morphological and functional impairments observed in the aging brain are discussed in the framework of theoretical concepts, such as the existence of different modalities of intercellular communication and of specific trophic features in the central nervous system. The relevance of changes at the cellular level (disappearance of neuronal cell bodies and proliferation of astroglial cells) and at the synaptic level (alterations in neurotransmitter and receptor levels) is discussed. Two, non-mutually exclusive hypotheses are advanced to explain the frequent absence of correlation between neuropathological findings and functional deficits in aged patients. According to the first, the physiological reshaping of brain circuits during aging may lead to “wrong” readjustments of neural networks (e.g. due to less effective endogenous and exogenous orienting signals) causing minor morphological alterations but marked functional deficits. The second hypothesis maintains that the absence of correlation between neuropathological and functional deficits is due to the impairment of restricted neuronal populations (“pacemaker and command neurons”) which play a special role in the hierarchical organization of neuronal networks. These neurons (inter alia, peptidergic neurons) may also be involved in volume transmission (diffusion of electrical and chemical signals in the extracellular fluid to reach distant targets). Moreover, the relevance of glial cells, not only as regulators of the extracellular medium but also on the basis of their trophic links with neurons, is considered. Finally, the interplay between trophic factors and therapeutical experience for the maintenance and/or recovery of an impaired function in elderly patients is discussed. (Aging 2: 5–25, 1990)

Regeneration in the Central Nervous System: Concepts and Facts

In many years, based on Cajal’s statement “… nerve paths are fixed, ended, immutable. Everything may die, nothing may be regenerated…” (Ramon y Cajal 1928) it has been assumed that the central nervous system (CNS) is unable to regenerate a lesioned pathway. Current opinion is in many instances the opposite (Liu and Chambers 1958; Fuxe et al. 1974; Cotman 1978; Bjorklund and Stenevi 1979). There is now a general belief in possible regeneration in the CNS, even if clearcut results remain scarce (Finger and Almli 1985; Berry 1985). In fact, in some cases, functional recovery after a CNS lesion has been observed and correlated with a morphological readjustment of the neural circuitry. However, a cause-effect link between morphological and functional recovery has not yet been provided, at least not beyond any doubt (Finger and Almli 1985). Since, as a general rule, for both invertebrates and vertebrates, there is little or no further generation of nerve cells in the mature brain, the morphological recovery observed after a lesion must rely on the ability of neurons to form new processes and new contacts (Raisman and Field 1973; Goldberg 1980; Veraa and Grafstein 1981). In fact, neuronal connections are in a dynamic state (morphological plasticity), since also in physiological conditions in the mature brain they are subjected to continuous remodelling (Bjorklund and Stenevi 1979; Cotman et al. 1981; Cotman and Nieto-Sampedro 1984).

Siagoside selectively attenuates morphological and functional striatal impairments induced by transient forebrain ischemia in rats.

Transient forebrain ischemia induced in rats by the four-vessel occlusion method is known to produce severe neural damage in the hippocampus and striatum and a behavioral syndrome the major symptom of which is a working memory deficit. Recent evidence suggests that monosialogangliosides can ameliorate postischemic symptoms. Our purpose was to study the effect of siagoside, the inner ester of GM1 ganglioside, on some behavioral and morphological impairments induced by four-vessel occlusion in rats. Methods Rats were injected daily with 5 mg/kg i.p. siagoside starting 4 hours after the cerebral ischemia. After 14 days the rats were tested for working memory in a water T maze or scored for apomorphine-induced stereotypy. The rats were killed 21 days after the cerebral ischemia. Histological and computer-assisted morphometric analyses were performed on cresyl violet-stained brain sections, which were graded according to a neuropathologic score, and on sections stained with a monoclonal antiserum against dopamine and cyclic adenosine-3′,5′-monophosphate-regulated phosphoprotein, a marker for striatal dopaminoceptive neurons. Results Siagoside treatment reduced the stereotypy score induced by low doses of apomorphine and the extent of striatal lesions but did not affect the working memory deficit or the extent of hippocampal lesions. Conclusion Daily siagoside treatment after acute cerebral ischemia attenuates some morphological and functional deficits related to striatal damage. These effects can be interpreted as a selective protective action on striatal neural populations or as a modulatory action on neural systems involved in striatal control. These data are consistent with preliminary clinical reports showing that monosialogangliosides enhance motor recovery after acute ischemic stroke.

Aspects of neural plasticity in the central nervous system—IV. Chemical anatomical studies on the aging brain

Some effects of aging processes on the neurochemical features of central transmitter-identified neuronal populations have been investigated by means of immunocytochemistry and receptor autoradiographic techniques coupled with image analysis. A selective decrease of tyrosine hydroxylase immunoreactivity in the ventrolateral region of the arcuate nucleus in aged rats was observed. The level and turnover (recovery after irreversible blockade of monoamine receptors with the peptide coupling agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline) of ?2-adrenergic ([(3)H]paraaminoclonidine binding) and D2 dopamine ([(3)H]spiperone binding) receptors were reduced in most regions of the rat brain. Peptide receptors showed a more complex pattern of change, since while ? opiate receptors (preferentially labeled with [(3)H]etorphine binding) were reduced in the old animals, ? opiate ([(3)H]DSTLenkephalin binding) receptors were affected only in certain areas. The effect of irreversible blockade of monoamine receptors on ? and ? opiate receptors was also studied in young adult and aged rats. A ? but not ? opiate receptor up-regulation was observed after monoamine receptor blockade in the young adult animals. This effect was greatly reduced in the n. caudatus-putamen, n. accumbens and tuberculum olfactorium of the old animals.

New Evidence for the Morphofunctional Recovery of Striatal Function by Ganglioside GM1 Treatment Following a Partial Hemitransection of Rats. Studies on Dopamine Neurons and Protein Phosphorylation

In previous studies it has been shown that chronic treatment with GM1 can increase the survival of dopamine (DA) cell bodies with their dendrites in substantia nigra following a partial unilateral hemitransection of rats (Agnati et al., 1983a; 1984; Fuxe and Agnati, 1984; Toffano et al., 1983; 1984a). This in turn may be responsible for the enhancement of collateral sprouting observed from remaining striatal DA nerve terminals, leading to recovery of dopaminergic synaptic function in the striatum. This action has been found to be specific and not related to antiinflammatory effects of ganglioside GM1, since neither treatment with dexamethazone nor treatment with acetylsalicylic acid has been able to produce any trophic action on the nigral DA nerve cell bodies following a partial hemitransection in rats (Agnati et al., 1984). Recently we have also analyzed the effects of ganglioside GM1 treatment in unilateral partially hemitransected rats on striatal energy metabolism using the radioactive deoxyglycose technique and on striatal blood flow using radiolabelled iodoantipyrine as tracer (Agnati et al., 1985a). The results showed that GM1 can counteract the imbalance in striatal energy metabolism and in striatal blood flow found between the striatum of the lesioned and unlesioned side. This action may be related to excitatory effects of GM1 on the lesioned side and to inhibitory effects of GM1 on the unlesioned side. These results underline the evidence obtained in previous functional studies indicating that the metabolism of a striatum undergoing degenerative and regenerative changes can be at least partially restored following chronic GM1 treatment (Agnati et al., 1985a).