Susan A. Greenfield

Synaptic connections between pars compacta and pars reticulata neurones: electrophysiological evidence for functional modules within the substantia nigra

Intracellular recordings were performed in vitro from both pars compacta and pars reticulata neurones of the substantia nigra, and their postsynaptic responses to electrical stimulation within the nucleus were analysed. Intracellular staining by biocytin was used to reveal the morphology and location of the recorded neurone and its position and distance to the stimulating electrode. Inhibitory postsynaptic potentials in pars compacta neurones were evoked exclusively from a specific region of stimulation within the pars reticulata; this field could be mapped out as a cone-shaped region surrounding the apical dendrite of the cell and perpendicular to the plane of the pars compacta. Furthermore, hemitransection, prior to the experiments which eliminated the most likely source of extrinsic inhibition (the GABAergic striatonigral pathway) affected neither the generation of inhibitory postsynaptic potentials nor its topographic pattern during pars reticulata stimulation. In contrast to the response of dopaminergic pars compacta neurones, pars reticulata neurones responded to stimulation over wide areas of substantia nigra, without any clear site-specific selectivity. It is concluded that within the substantia nigra, dopaminergic neurones are arranged in functional modules such that most inhibition is derived from a highly local circuit with the collaterals of adjacent pars reticulata cells. This module is intrinsic to the substantia nigra and may represent the basic functional unit of the nucleus.

Parkinson's disease, Alzheimer's disease and motor neurone disease: Identifying a common mechanism

Although Alzheimers disease, Parkinsons disease, and motor neurone disease are distinct disorders, there could be a common neurodegenerative mechanism that characterises the death of selective neurone populations in each case. We propose that this mechanism could be an aberrantly activated, developmental process involving a non-classical, non-enzymatic action of acetylcholinesterase mediated via a short linear motif near the C-terminal end of the molecule. Since this motif has a highly conserved homology with part of the amyloid precursor protein, it may be particularly attractive as a target for novel therapeutic strategies in neurodegeneration.

Neurotoxic and neurotrophic effects of chronic N-methyl-d-aspartate exposure upon mesencephalic dopaminergic neurons in organotypic culture

Current theories regarding the mechanisms of degeneration of dopaminergic nigrostriatal neurons in Parkinsons disease suggest a pivotal role for excitotoxicity. In this study, the effects of chronic exposure of rat ventral mesencephalic slice cultures to the excititoxin N-methyl-D-aspartate, were investigated. Chronic (18 day) exposure to N-methyl-D-aspartate produced widely varying, dose-dependent effects. High doses (100 mu M) caused a pronounced toxicity upon tyrosine hydroxylase-positive neurons, with the surviving neurons possessing shrunken somata and stunted neurites: co-administration of the N-methyl-D-aspartate receptor antagonist MK-801, inhibited N-methyl-D-aspartate-induced toxicity. In contrast, exposure to a low concentration of N-methyl-D-aspartate (0.1 mu M), stimulated the outgrowth of tyrosine hydroxydase-positive neurites from the culture; this effect was abolished by MK-801. Chronic application of glutamate had similar, though not as pronounced, growth-promoting actions. However, the concentration of glutamate required was 1000 times that of N-methyl-D-aspartate, due to the presence ot high-affinity glutamate transport mechanisms. Cultures exposed to a submicromolar concentration of N-methyl-D-aspartate exhibited a significant resistance to subsequent exposure to a lethal (300 mu M) concentration of the toxin. It would thus appear that N-methyl-D-aspartate may have both trophic and toxic actions upon dopaminergic neurons in culture. Moreover, the ability of low doses of N-methyl-D-aspartate to protect neurons in this critical brain region may be of relevance to future attempts to arrest the degeneration associated with Parkinsons disease. The putative mechanisms of these phenomena are discussed.

The influence of target and non-target brain regions on the development of mid-brain dopaminergic neurons in organotypic slice culture.

The development and regeneration of rat dopaminergic neurons of the ventral mesencephalon was studied in organotypic slice cultures. Single ventral mesencephalon cultures and co-cultures of ventral mesencephalon with striatum (a target region) or cerebellum (a non-target region) were prepared from postnatal day 1 Wistar rats. Cultures were processed for tyrosine hydroxylase and glial fibrillary acidic protein immunoreactivity, at two day intervals, for an overall incubation period of 20 days. Analysis of these cultures revealed that the striatal target tissue, exerted neither a trophic nor a tropic influence on the tyrosine hydroxylase immunoreactive neurons. In both single and co-cultures, tyrosine hydroxylase immunoreactive neurites projected radially from the ventral mesencephalon slice. However, in striatal co-cultures, tyrosine hydroxylase immunoreactive neurites were seen penetrating the striatal slice, whereas in cerebellar co-cultures no tyrosine hydroxylase immunoreactive neurites entered the cerebellar tissue. Glial fibrillary acidic protein positive cells actively migrated from the tissue sections, however tyrosine hydroxylase immunoreactive neurite outgrowth was not guided by these glial cells. Tyrosine hydroxylase immunoreactive neurites terminated once they had penetrated the striatal slice. This retardation of neurite growth by a target region could be important in establishing and reinforcing synaptic connections in the developing nigro-striatal pathway.

Astroglia up-regulate transcription and secretion of 'readthrough' acetylcholinesterase following oxidative stress.

Novel and diverse functions of glial cells are currently the focus of much attention [A. Volterra and J. Meldolesi (2005)Nature Rev. 6, 626–640]. Here we present evidence that rat astroglia release acetylcholinesterase (AChE) as part of their response to hypoxic damage. Exposure of astroglia to tert‐butyl hydroperoxide, and hence oxidative stress, subsequently leads to a switching in mRNA from the classical membrane‐bound T‐AChE to a preferential increase in the splice variant for a soluble form, R‐AChE, This change in expression is reflected in increased perinuclear and reduced cytoplasmic AChE staining of the insulted glial cells, with a concomitant and marked increase in extracellular secretion that peaks at 1 h post‐treatment. An analogous increase in R‐AChE, over a similar time scale, occurs in response to psychological stress [D. Kaufer et al. (1998)Nature 93, 373–377], as well as to head injury and stroke [E. Shohami et al. (1999)J. Neurotrauma 6, 365–76]. The data presented here suggest that glial cells may be key chemical intermediaries in such situations and, perhaps more generally in pathological conditions involving oxidative stress, such as neurodegeneration.

Bioactivity of a peptide derived from acetylcholinesterase: electrophysiological characterization in guinea-pig hippocampus.

Acetylcholinesterase is well known to have noncholinergic functions. Only recently, however, has the salient part been identified of the molecule responsible for these nonclassical actions, a peptide of 14 amino acids towards the C‐terminus of acetylcholinesterase. The aim of this study was to test the bioactivity of this ‘acetylcholinesterase‐peptide’ using intracellular recordings in guinea‐pig hippocampal slices. In the presence of tetrodotoxin, acetylcholinesterase‐peptide alone affected neither the membrane potential nor the input resistance of CA1 neurons; however, a modulatory action was observed, as a concentration‐dependent decrease of N‐methyl‐d‐aspartic acid‐induced depolarization. When calcium potentials were elicited by a depolarizing current pulse, application of acetylcholinesterase‐peptide increased or reduced the degree of calcium spike firing in, respectively, the presence or absence of the N‐methyl‐d‐aspartic acid antagonist d(–)‐2‐amino‐5‐phosphonopentanoic acid. In contrast, a peptide derived from the equivalent region of butyrylcholinesterase, which also hydrolyses acetylcholine, had no effect. In conclusion, acetylcholinesterase‐peptide has a selective bioactivity in the hippocampus; it could thus offer new ways of targeting mechanisms of calcium‐induced neurotoxicity.

Involvement of the NMDA Receptor in a Non–cholinergic Action of Acetylcholinesterase in Guinea–pig Substantia Nigra Pars Compacta Neurons

Evidence is accumulating that a soluble, secretory form of acetylcholinesterase may have novel, non–cholinergic functions in certain brain regions, such as the substantia nigra. In this study, application of human recombinant acetylcholinesterase (rhAChE) to pars compacta neurons in the rostral substantia nigra resulted in a sustained hyperpolarization that was not only mimicked by application of N‐methyl–D–aSpartate (NMDA) but also blocked by the NMDA receptor antagonists MK801 and 2–amino–5phosphonopentanoic acid. Neither the rhAChE–nor the NMDA–induced hyperpolarization was seen when the calcium chelator BAPTA was injected into the neuron; hence the effect is mediated by accumulation of intracellular calcium. This intracellular calcium appears sufficient to compromise neuronal metabolism since the rhAChE–induced hyperpolarization was reversed by application of the K–ATP channel antagonist tolbutamide. Butyrylcholinesterase, a protein of similar molecular weight to acetylcholinesterase, which also hydrolyses acetylcholine, had no effect whatsoever. The results suggest that, independent of its normal catalytic function, acetylcholinesterase can act via the NMDA receptor complex to enhance calcium entry into nigral neurons and jeopardize cell metabolism. This non–classical action of acetylcholinesterase might thus be an important factor in the mechanisms underlying parkinsonian neurodegeneration.

5-HT1B RECEPTOR REGULATION OF SEROTONIN (5-HT) RELEASE BY ENDOGENOUS 5-HT IN THE SUBSTANTIA NIGRA

Axonal release of serotonin (5-hydroxytryptamine, 5-HT) in the CNS is typically regulated by presynaptic 5-HT autoreceptors. Release of 5-HT in substantia nigra pars reticulata (SNr), a principal output from the basal ganglia, has seemed an interesting exception to this rule. The SNr receives one of the highest densities of 5-HT innervation in mammalian brain and yet negative feedback regulation of axonal 5-HT release by endogenous 5-HT has not been identified here. We explored whether we could identify autoregulation of 5-HT release by 5-HT(1B) receptors in rat SNr slices using fast-scan cyclic voltammetry at carbon-fiber microelectrodes to detect 5-HT release evoked by discrete stimuli (50 Hz, 20 pulses) paired over short intervals (1-10 s) within which any autoreceptor control should occur. Evoked 5-HT release exhibited short-term depression after an initial stimulus that recovered by 10 s. Antagonists for 5-HT(1B) receptors, isamoltane (1 microM) or SB 224-289 (1 microM), did not modify release during a stimulus train, but rather, they modestly relieved depression of subsequent release evoked after a short delay (< or =2 s). Release was not modified by antagonists for GABA (picrotoxin, 100 microM, saclofen, 50 microM) or histamine-H(3) (thioperamide, 10 microM) receptors. These data indicate that 5-HT release can activate a 5-HT(1B)-receptor autoinhibition of subsequent release, which is mediated directly via 5-HT axons and not via GABAergic or histaminergic inputs. These data reveal that 5-HT release in SNr is not devoid of autoreceptor regulation by endogenous 5-HT, but rather is under modest control which only weakly limits 5-HT signaling.

Dopamine is released spontaneously from developing midbrain neurons in organotypic culture.

While neuronal activity is important in CNS development, little is known of the behaviour of the actual neurotransmitters released during this period. None the less, indirect evidence has suggested that the neurotransmitter dopamine actually has a morphogenic role. This study is the first attempt to monitor directly and in real-time, the release of dopamine from midbrain neurons developing as an isolated organotypic slice culture. The observed release of dopamine was both spontaneous and synchronized and occurred with an average periodicity that is two orders of magnitude longer than the characteristic neuronal discharge activity of midbrain dopamine cells. Moreover, elevations in the extracellular concentrations of dopamine were markedly more prolonged in these and other developing systems than in axon terminal regions in mature striatum in which dopaminergic innervation is fully established. Thus, dopamine may have an action in developing circuits over spatial and temporal scales that vastly exceed those in mature, synaptic-like transmission.

Uptake of acetylcholinesterase by neurons in the substantia nigra.

It is known that acetylcholinesterase is secreted by the dopaminergic neurons of the substantia nigra and has a subsequent action independent of cholinergic transmission. Although non‐cholinergic actions of this protein have been demonstrated, the subsequent fate of acetylcholinesterase is unknown. One possibility is that acetylcholinesterase is taken up following secretion into the extracellular space. This hypothesis has been tested in vivo, in both conscious and anaesthetized guinea‐pigs. Exogenous acetylcholinesterase (2–20 pM) was infused via a push‐pull cannula implanted into either the substantia nigra or the surrounding extranigral regions; the amount subsequently recovered in the perfusate was then compared with control values. Only when the push‐pull cannulae were implanted in the substantia nigra was there a marked decrease in the amount of acetylcholinesterase recovered; this selective retention was abolished when the perfusion medium was cooled to 4°C or when the experiment was performed post mortem. Direct visualization of immunocytochemically identified nigral dopaminergic cells revealed co‐localized deposits of labelled, exogenous acetylcholinesterase. Moreover, when exogenous acetylcholinesterase was boiled to prevent detection by the assay system and to eliminate any classical enzymatic action, an enhancement in perfusate levels of endogenous acetylcholinesterase was observed from nigral but not from extranigral sites, indicating that endogenous and exogenous acetylcholinesterases were in competition. These results suggest that, within the substantia nigra, secreted acetylcholinesterase may be subject to a temperature‐ and energy‐dependent uptake mechanism.