Quinolinic acid induced neurodegeneration in the striatum: a combined in vivo and in vitro analysis of receptor changes and microglia activation.

PURPOSE: Huntington's disease (HD) is a progressive neurodegenerative disorder, which is characterised by prominent neuronal cell loss in the basal ganglia with motor and cognitive disturbances. One of the most well-studied pharmacological models of HD is produced by local injection in the rat brain striatum of the excitotoxin quinolinic acid (QA), which produces many of the distinctive features of this human neurodegenerative disorder. Here, we report a detailed analysis, obtained both in vivo and in vitro of this pharmacological model of HD. MATERIALS AND METHODS: By combining emission tomography (PET) with autoradiographic and immunocytochemical confocal laser techniques, we quantified in the QA-injected striatum the temporal behavior (from 1 to 60 days from the excitotoxic insult) of neuronal cell density and receptor availability (adenosine A(2A) and dopamine D(2) receptors) together with the degree of microglia activation. RESULTS: Both approaches showed a loss of adenosine A(2A) and dopamine D(2) receptors paralleled by an increase of microglial activation. CONCLUSION: This combined longitudinal analysis of the disease progression, which suggested an impairment of neurotransmission, neuronal integrity and a reversible activation of brain inflammatory processes, might represent a more quantitative approach to compare the differential effects of treatments in slowing down or reversing HD in rodent models with potential applications to human patients.

Advanced tracer techniques to monitor synaptic activity.

The two approaches presented here bypass postsynaptic receptors as indicators of quantal release, and thus they can provide information which is clearly distinct from that obtained with standard electrophysiological techniques. Indeed, the inherently variable responsiveness of the postsynaptic membrane makes it an unreliable indicator of presynaptic activity and this has fueled a lot of controversy, particularly in the area of synaptic plasticity. A major advantage of these two methods is their ability to detect changes at the single bouton level. This offers a lot of advantages including the possibility to study the functional role for exo-endocytosis but also plasticity against a background of great variability among a large number of synapses. The spatial resolving power of FM1-43 and anti-synaptotagmin antibodies may be valuable in future studies of spread of LTP between neighboring synapses and in the mapping the pattern of neuronal activity in complex networks of neurons.

Morphological organization of somatosensory cortex in Otx1(-/-) mice.

Knock-out Otx1 mice show brain hypoplasia, spontaneous epileptic seizures and abnormalities of the dorsal region of the neocortex. We investigated structural alterations in excitatory and inhibitory circuits in somatosensory cortex of Otx1(-/-) mice by immunocytochemistry using light, confocal and electron microscopy. Immunostaining for non-phosphorylated neurofilament SMI311 and subunit 1 of the NMDA receptor - used as markers of pyramidal neurons - showed reduced layer V pyramidal cells and ectopic pyramidal cells in layers II and III of the mutant cortex. Immunostaining for calcium-binding proteins calbindin, calretinin and parvalbumin - markers of non-overlapping types of GABAergic interneurons - showed no differences between wild-type and knock-out cortex for calbindin and calretinin neurons, while parvalbumin neurons were only patchily distributed in Otx1(-/-) cortex. The pattern of positivity of the GABAergic marker glutamic acid decarboxylase in Otx1(-/-) cortex was also altered and similar to that of parvalbumin. GABA transporter 1 immunoreactivity was greater in Otx1(-/-) than wild-type; quantitation of structures immunoreactive for this transporter in layer V showed that they were increased overall in Otx1(-/-) but the density of inhibitory terminals on pyramidal neurons in the same layer labeled with this transporter was similar to that in wild-type mice. No differences in the distribution or intensity of the glial markers GABA transporter 3 or glial fibrillary acidic protein were found. The defects found in the cortical GABAergic system of the Otx1(-/-) mouse can plausibly explain the cortical hyperexcitability that produces seizures in these animals.

Potentially epileptogenic dysfunction of cortical NMDA- and GABA-mediated neurotransmission in Otx1-/- mice.

Knockout Otx1 mice present a microcephalic phenotype mainly due to reduced deep neocortical layers and spontaneous recurrent seizures. We investigated the excitable properties of layer V pyramidal neurons in neocortical slices prepared from Otx1-/- mice and age-matched controls. The qualitative firing properties of the neurons of Otx1-/- mice were identical to those found in wild-type controls, but the proportion of intrinsically bursting (IB) neurons was significantly smaller. This is in line with the lack of the Otx1 gene contribution to the generation and differentiation of neurons destined for the deep neocortical layers, in which IB neurons are located selectively in wild-type rodents. The pyramidal neurons recorded in Otx1-/- mice responded to near-threshold electrical stimulation of the underlying white matter, with aberrant polysynaptic excitatory potentials often leading to late action potential generation. When the strength of the stimulus was increased, the great majority of the Otx1-/- neurons (78%) responded with a prominent biphasic inhibitory postsynaptic potential that was significantly larger than that observed in the wild-type mice, and was often followed by complex postinhibitory depolarizing events. Both late excitatory postsynaptic potentials and postinhibitory excitation were selectively suppressed by NMDA receptor antagonists, but not by AMPA antagonists. We conclude that the cortical abnormalities of Otx1-/- neocortex due to a selective loss of large projecting neurons lead to a complex rearrangement of local circuitry, which is characterized by an excess of N-methyl-d-aspartate-mediated polysynaptic excitation that is counteracted by GABA-mediated inhibition in only a limited range of stimulus intensity. Prominent postsynaptic inhibitory potentials may also act as a further pro-epileptogenic event by synchronizing abnormal excitatory potentials.

Synaptic properties of neocortical neurons in epileptic mice lacking the Otx1 gene.

PURPOSE: The murine homeobox-containing Otx gene is required for correct nervous system and sense organ development. Otx1-/1 mice obtained by replacing Otx with the lac Z gene show developmental abnormalities of the cerebellum, mesencephalon, and cerebral cortex associated with spontaneous epileptic seizures (1). The epileptogenic mechanisms accounting for these seizures were investigated by means of electrophysiological recordings made from neocortical slices. METHODS: The 400-microm slices were prepared from the somatosensory cortex of Otx1-/- and Otx1+/+ mice, and the current clamp intracellular recordings were obtained from layer V pyramidal neurons by means of pipettes containing K+ acetate 1.5 mol/L and biocytin 2% (pH 7.3). RESULTS: Synaptic responses could be evoked in the neocortical pyramidal neurons by electrically stimulating the underlying white matter. gamma-Aminobutyric acid A/B-mediated inhibitory postsynaptic potentials were more pronounced in the Otx1-/- than in the control pyramidal neurons from the earliest postnatal period; multisynaptic excitatory postsynaptic potentials were significantly more expressed in the Otx1-/- mice also at the end of the first postnatal month, when they were only rarely encountered in controls. CONCLUSION: Excessive excitatory amino acid-mediated synaptic driving may lead to a hyperexcitable condition that is responsible for the epileptic manifestations occurring in Otx1-/- mice. This excess of excitation is not counteracted by well-developed gamma-aminobutyric acid activity, which seems to be involved in the synchronization of cell discharges. Our ongoing and more extensive comparative analysis of the mutants and controls should help to clarify the way in which the putative rearrangement taking place in Otx1-/- neocortex may lead to the excitatory hyperinnervation of layer V pyramidal neurons.