A. Contestabile

An in vivo model for studying function of brain tissue temporarily devoid of glial cell metabolism: the use of fluorocitrate.

Abstract: The effect of intrastriatal injection of fluorocitrate on amino acid pattern, cell enzyme markers, and ultrastruc‐tural appearance was investigated. A dose of 1 nmol of fluorocitrate resulted in temporarily decreased levels of glutamine, glutamate, and aspartate, whereas the level of alanine was increased. The glutamine level was severely reduced after 4 h but was reversed after 24 h. The activity of different cellular enzyme markers did not change markedly after this dose. Ultrastructural changes in glial cells were observed, concomitant with the biochemical changes. A dose of ≥2 nmol of fluorocitrate resulted in more marked and irreversible changes in amino acid levels. By 24–72 h after the injection of this dose, several marker enzyme activities decreased markedly. The ultrastructural changes affected the neurons as well as the glial cells and were not reversible. The use of microinjection of 1 nmol of fluorocitrate into the neostria‐tum of the rat to provide a model for studying transmitter amino acid metabolism in brain devoid of glial cell activity is discussed.

Topography of cholinergic and substance P pathways in the habenulo-interpeduncular system of the rat. An immunocytochemical and microchemical approach

The topography of cholinergic and substance P containing habenulo-interpeduncular projections has been studied in the rat. The research has been carried out by combining choline acetyltransferase and substance P immunohistochemistry to experimental lesions and biochemical assays in microdissected brain areas. In addition, computer-assisted image analysis has been performed in order to obtain quantification of immunohistochemical data. The results show that cholinergic and substance P containing neurons have a different localization in the medial habenula and project to essentially different areas of the interpeduncular nucleus. Cholinergic neurons are crowded in the ventral two-thirds of the medial habenula while substance P containing cells are exclusively localized in the dorsal part of the nucleus. In most parts of the interpeduncular nucleus, choline acetyltransferase and substance P containing fibres and terminals are similarly segregated and no overlapping is apparent except for the rostralmost and the caudalmost ends of the nucleus. Cholinergic activity is largely concentrated in the central core of the nucleus, while substance P is preferentially localized in the peripheral subnuclei of the interpeduncular nucleus. In addition, both substance P and choline acetyltransferase levels show peculiar regional variations along the rostrocaudal axis of the interpeduncular nucleus. The results of experimental lesions demonstrate that the substance P projection carried by each fasciculus retroflexus is prevailingly ipsilateral in the rostral part of the interpeduncular nucleus and becomes progressively bilateral as far as more caudal regions of the nucleus are reached. By contrast, the cholinergic projections carried by each fasciculus retroflexus intermingle more rapidly and only show a slight ipsilateral dominance in the interpeduncular nucleus. The results of the study are discussed with reference to previous anatomical and neurochemical data which, in several instances, had given rise to discrepant interpretations.

Structural, neurochemical and behavioural consequences of neonatal blockade of NMDA receptor through chronic treatment with CGP 39551 or MK-801

Recent evidence suggests that NMDA receptors may be involved in survival of neurons and establishment of correct connectivity during development. We have treated rat pups from postnatal day 1 to 22 with daily s.c. injections of a competitive (CGP 39551) and a non-competitive (MK-801) antagonist of the NMDA receptor. Body weight of treated rats was decreased by 50-65% at postnatal day 24 and by 25-32% at 70 days of age. Brain weight was decreased by 16-24% at both ages. Among the different brain regions, the cerebellum and striatum appeared more decreased in size than the cortex and hippocampus. Only few minor, and in some cases transient, differences were measured in the cerebellum, the hippocampus and the cortex for a battery of neurochemical markers related to cholinergic, GABAergic and glutamatergic transmission as well as to astrocyte and oligodendrocyte activity. When tested in actometric cages from postnatal days 28 to 60, treated rats exhibited a dramatic increase of spontaneous locomotor activity which was maximal in 28-day-old animals (380% and 250% of control values in CGP 39551 and MK-801 groups, respectively) and was still significant at 60 days of age. Therefore, long-lasting alteration of motor behaviour is obtained by the schedule of chronic treatment adopted for the present experiments. Our results suggest that blockade of NMDA receptors during the critical period of brain maturation may result in permanent alteration of neural circuits.

Brain nitric oxide and its dual role in neurodegeneration/neuroprotection: Understanding molecular mechanisms to devise drug approaches

Nitric oxide (NO) has been established as an important messenger molecule in various steps of brain physiology, from development to synaptic plasticity, learning and memory. However, NO has also been viewed as a major agent of neuropathology when, escaping controlled production it may directly or indirectly promote oxidative and nitrosative stress. The exact borderline between physiological, and therefore neuroprotective, and pathological, and therefore neurodegenerative, actions of NO is a matter of controversy among researchers in the field. This is reflected in the present status of drug research, that is focused on finding ways to block NO production, and therefore limit neuropathology, as well as on finding ways to increase NO availability and therefore elicit neuroprotection. As an unavoidable consequence, both classes of drugs are reported to have neurodegenerative or neuroprotective effects, depending on the models in which they are tested. Aim of the present paper is to provide the reader with a survey, as much complete as possible, on the main aspects of NO biology, from biochemistry and chemical reactivity to the molecular signals elicited in neural cells target of its neurodegenerative or neuroprotective action. In doing that, many controversial aspects related to basic biology and to neuropathology of NO are taken into account. In the final sections, main classes of drugs able to interfere with NO physiopathology are examined, in order to try to devise possible directions for future NO-based therapeutical strategies.

Kainic Acid Differentially Affects the Synaptosomal Release of Endogenous and Exogenous Amino Acidic Neurotransmitters

Abstract: Presynaptic actions of kainic acid have been tested on uptake and release mechanisms in synaptosome‐enriched preparations from rat hippocampus and goldfish brain. Kainic acid increased in a Ca2+‐dependent way the basal release of endogenous glutamate and aspartate from both synaptosomal preparations, with the maximum effect (40–80%) being reached at the highest concentration tested (1 mM). In addition, kainic acid potentiated, in an additive or synergic way, the release excitatory amino acids stimulated by high K+ concentrations. Kainic acid at 1 mM showed a completely opposite effect on the release of exogenously accumulated D‐[3H]aspartate. The drug, in fact, caused a marked inhibition of both the basal and the high K+‐stimulated release. Kainic acid at 0.1 mM had no clear‐cut effect, whereas at 0.01 mM it caused a small stimulation of the basal release. The present results suggest that kainic acid differentially affects two neurotransmitter pools that are not readily miscible in the synaptic terminals. The release from an endogenous, possibly vesiculate, pool of excitatory amino acids is stimulated, whereas the release from an exogenously accumulated, possibly cytoplasmic and carrier‐mediated, pool is inhibited or slightly stimulated, depending on the external concentration of kainic acid. Kainic acid, in addition, strongly inhibits the high‐affinity uptake of L‐glutamate and D‐aspartate in synaptic terminals. All these effects appear specific for excitatory amino acids, making it likely that they are mediated through specific recognition sites present on the membranes of glutamatergic and aspartatergic terminals. The relevance of the present findings to the mechanism of excitotoxicity of kainic acid is discussed.

Nitric oxide negatively regulates proliferation and promotes neuronal differentiation through N-Myc downregulation.

Nitric oxide (NO) has been found to act as an important negative regulator of cell proliferation in several systems. We report here that NO negatively regulates proliferation of neuronal cell precursors and promotes their differentiation by downregulating the oncogene N-Myc. We have studied this regulatory function of NO in neuroblastoma cell lines (SK-N-BE) and in primary cerebellar granule cell cultures. In a neuronal NO synthase (nNOS) overexpressing neuroblastoma cell line exposed to the differentiative action of retinoic acid, NO slowed down proliferation and accelerated differentiation towards a neuronal phenotype. This effect was accompanied by a parallel decrease of N-Myc expression. Similar results could be obtained in parental SK-N-BE cells by providing an exogenous source of NO. Pharmacological controls demonstrated that NOs regulatory actions on cell proliferation and N-Myc expression were mediated by cGMP as an intermediate messenger. Furthermore, NO was found to modulate the transcriptional activity of N-Myc gene promoter by acting on the E2F regulatory region, possibly through the control of Rb phosphorylation state, that we found to be negatively regulated by NO. In cerebellar granule cell cultures, NOS inhibition increased the division rate of neuronal precursors, in parallel with augmented N-Myc expression. Because a high N-Myc expression level is essential for neuroblastoma progression as well as for proliferation of neuronal precursors, its negative regulation by NO highlights a novel physiopathological function of this important messenger molecule.

Lithium restores neurogenesis in the subventricular zone of the Ts65Dn mouse, a model for Down syndrome.

Down syndrome (DS), a high‐incidence genetic pathology, involves brain hypoplasia and mental retardation. Emerging evidence suggests that reduced neurogenesis may be a major determinant of brain underdevelopment in DS. To establish whether it is possible to improve neurogenesis in DS, Ts65Dn mice—the most widely used model for DS—and euploid mice were treated with control or lithium chow for 1 month. During the last 3 days animals received one daily injection of 5‐bromo‐2‐deoxyuridine (BrdU)—a marker of proliferating cells—and were sacrificed 24u2003h after the last injection. Neurogenesis was examined in the subventricular zone (SVZ), a region that retains a neurogenic potential across life. We found that Ts65Dn mice had less (−40%) BrdU+ cells than euploid mice, indicating severe proliferation impairment. Treatment with lithium increased the number of Brdu+ cells in both euploid and Ts65Dn mice. In the latter the number of Brdu+ cells became similar to that of untreated euploid mice. Our study shows that lithium is able to restore cell proliferation in the SVZ of the Ts65Dn mouse and point at treatments with mood stabilizers as a potential tool to improve neurogenesis in patients with DS.

Cell Cycle Elongation Impairs Proliferation of Cerebellar Granule Cell Precursors in the Ts65Dn Mouse, an Animal Model for Down Syndrome

Mental retardation, the hallmark of Down syndrome (DS), has been attributed to the reduced number of neurons populating the DS brain. The Ts65Dn mouse model of DS displays several anomalies analogous to those in individuals with DS, including neurogenesis impairment. The goal of the current study was to determine whether cell cycle alterations underlie neurogenesis impairment in the cerebellum of the Ts65Dn mouse and to identify the molecular mechanisms responsible for this defect. In neonatal (2‐day old) Ts65Dn mice, cerebellar granule cell precursors exhibited a reduced proliferation rate (−40%) and a notable elongation (+45%) of the cell cycle. Alteration of cell cycle rate was due to elongation of the G2 and G1 phases. Microarray screening of cell cycle regulatory genes showed that Ts65Dn mice had a decreased expression of Cyclin B1 and Skp2, two key regulators of G2/M and G1/S transition. Results point at cell cycle elongation as major determinant of neurogenesis reduction in the cerebellum of Ts65Dn mice and suggest that this defect is specifically linked to an altered expression of two cell‐cycle regulatory genes, Cyclin B1 and Skp2. These findings may establish the basis for a therapeutic approach aimed at restoring neurogenesis in the DS brain.

Choline acetyltransferase activity at different ages in brain of Ts65Dn mice, an animal model for Down's syndrome and related neurodegenerative diseases

Ts65Dn mice, trisomic for a portion of chromosome 16 segmentally homologous to human chromosome 21, are an animal model for Downs syndrome and related neurodegenerative diseases, such as dementia of the Alzheimer type. In these mice, cognitive deficits and alterations in number of basal forebrain cholinergic neurons have been described. We have measured in Ts65Dn mice the catalytic activity of the cholinergic marker, choline acetyltransferase (ChAT), as well as the activity of the acetylcholine‐degrading enzyme acetylcholinesterase (AChE), in the hippocampus and in cortical targets of basal forebrain cholinergic neurons. In mice aged 10u2003months, ChAT activity was significantly higher in Ts65Dn mice, compared to 2N animals, in the hippocampus, olfactory bulb, olfactory cortex, pre‐frontal cortex, but not in other neocortical regions. At 19u2003months of age, on the other hand, no differences in ChAT activity were found. Thus, alterations of ChAT activity in these forebrain areas seem to recapitulate those recently described in patients scored as cases of mild cognitive impairment or mild Alzheimers disease. Other neurochemical markers putatively associated with the disease progression, such as those implicating astrocytic hyperactivity and overproduction of amyloid precursor protein family, were preferentially found altered in some brain regions at the oldest age examined (19u2003months).

The Place of Choline Acetyltransferase Activity Measurement in the “Cholinergic Hypothesis” of Neurodegenerative Diseases

The so-called “cholinergic hypothesis” assumes that degenerative dysfunction of the cholinergic system originating in the basal forebrain and innervating several cortical regions and the hippocampus, is related to memory impairment and neurodegeneration found in several forms of dementia and in brain aging. Biochemical methods measuring the activity of the key enzyme for acetylcholine synthesis, choline acetyltransferase, have been used for many years as a reliable marker of the integrity or the damage of the cholinergic pathways. Stereologic counting of the basal forebrain cholinergic cell bodies, has been additionally used to assess neurodegenerative changes of the forebrain cholinergic system. While initially believed to mark relatively early stages of disease, cholinergic dysfunction is at present considered to occur in advanced dementia of Alzheimer’s type, while its involvement in mild and prodromal stages of the disease has been questioned. The issue is relevant to better understand the neuropathological basis of the diseases, but it is also of primary importance for therapy. During the last few years, indeed, cholinergic replacement therapies, mainly based on the use of acetylcholinesterase inhibitors to increase synaptic availability of acetylcholine, have been exploited on the assumption that they could ameliorate the progression of the dementia from its initial stages. In the present paper, we review data from human studies, as well as from animal models of Alzheimer’s and Down’s diseases, focusing on different ways to evaluate cholinergic dysfunction, also in relation to the time point at which these dysfunctions can be demonstrated, and on some discrepancy arising from the use of different methodological approaches. The reviewed literature, as well as some recent data from our laboratories on a mouse model of Down’s syndrome, stress the importance of performing biochemical evaluation of choline acetyltransferase activity to assess cholinergic dysfunction both in humans and in animal models.