In vivo mitochondrial inhibition alters corticostriatal synaptic function and the modulatory effects of neurotrophins.

Experimental evidence has revealed the role of mitochondria in various aspects of neuronal physiology. Mitochondrial failure results in alterations that underlie the pathogeneses of many neurodegenerative disorders, such as Parkinson's disease, Alzheimer's disease, Huntington's disease (HD) and amyotrophic lateral sclerosis. The mitochondrial toxin 3-nitropropionic acid (3-NP) has been used to model failure; for example, systemic administration of 3-NP imitates the striatal degeneration that is exhibited in the postmortem tissue of patients afflicted with HD. We have demonstrated that low, sub-chronic doses of 3-NP are sufficient to initiate the damage to striatal neurons that is associated with changes in neurotrophin expression levels. However, the mechanisms underlying the alterations in neuronal activity and neurotransmission due to 3-NP-induced mitochondrial dysfunction remain to be elucidated. In this paper, we focus on how corticostriatal transmission and its modulation by neurotrophins are altered in vivo after 5 days of mitochondrial inhibition with 3-NP. Recordings of population spikes and a paired pulse (PP) stimulation protocol were used to document changes in corticostriatal synapses in 3-NP-treated brain slices. The corticostriatal synapses were modulated by neurotrophins but displayed differential amplitude increases in the presence of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), or neurotrophin-4/5 (NT-4/5) under control conditions. Neurotrophin-mediated synaptic modulation was decreased in slices from 3-NP-treated mice. The protein and mRNA levels of neurotrophins and their receptors were also modified in the 3-NP-treated tissue. Neuronal structural evaluation demonstrated that synaptic length and density were reduced in the 3-NP-treated mice, which partially explained the changes in the amplitudes of the synaptic field responses. Our results demonstrate that corticostriatal synapses are differentially modulated by neurotrophins and that this modulation is altered by mitochondrial failure. Mitochondrial dysfunction also affects neurotransmitter release in corticostriatal synapses, neurotrophin availability, dendritic arborization and the lengths of the striatal medium spiny neurons (MSNs).

3-Nitropropionic acid modifies neurotrophin mRNA expression in the mouse striatum: 18S-rRNA is a reliable control gene for studies of the striatum.

OBJECTIVE: The aim of the present study was to determine the changes in the mRNA levels of neurotrophins and their receptors in the striatal tissue of mice treated with 3-nitropropionic acid (3-NP). METHODS: At 1 and 48 h after the last drug administration, the mRNA expression of nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-4/5 as well as their receptors p75, TrkA, TrkB and TrkC, was evaluated using semi-quantitative (semi-Q) and real-time RT-PCR. ß-actin mRNA and ribosomal 18S (18S rRNA) were tested as internal controls. RESULTS: 3-NP treatment did not affect mRNA expression of all neurotrophins and their respective receptors equally. Also, differences in neurotrophin and receptor mRNA expression were observed between semi-Q and real-time RT-PCR. Real-time RT-PCR was more accurate in evaluating the mRNA expression of the neurotrophins than semi-Q, and 18S rRNA was more reliable than ß-actin as an internal control. CONCLUSION: Neurotrophins and their receptors expression is differentially affected by neuronal damage produced by inhibition of mitochondrial respiration with 3-NP treatment in low, sub-chronic doses in vivo.

Uncoupling oxidative/energy metabolism with low sub chronic doses of 3-nitropropionic acid or iodoacetate in vivo produces striatal cell damage.

A variety of evidence suggests that the failure of cellular metabolism is one of the underlying causes of neurodegenerative diseases. For example, the inhibition of mitochondrial function produces a pattern of cellular pathology in the striatum that resembles that seen in Huntington's disease. However, neurons can also generate ATP through the glycolytic pathway. Recent work has suggested a direct interaction between mutated huntingtin and a key enzyme in the glycolytic pathway, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Yet little work has been gone into examination of the cellular pathology that results from the inhibition of this alternative energy source. Therefore, the aim of the present study is to characterize the cellular pathology that results in the striatum of mice after treatment with a toxin (iodoacete, IOA) that compromises anaerobic metabolism. This striatal pathology is compared to that produced by a widely studied blocker of mitochondrial function (3-nitropropionic acid, 3-NP). We found that low doses of either toxin resulted in significant pathology in the mouse striatum. Signs of apoptosis were observed in both experimental groups, although apoptosis triggered by IOA treatment was independent from caspase-3 activation. Importantly, each toxin appears to produce cellular damage through distinct mechanisms; only 3-NP generated clear evidence of oxidative stress as well as inhibition of endogenous antioxidants. Understanding the distinct pathological fingerprints of cell loss produced by blockade of oxidative and anaerobic metabolisms may give us insights into neurodegenerative diseases.

Differential expression of neurotrophins in postnatal C57BL/6 mice striatum.

Neurotrophin expression in early stages of development is crucial for brain assembly and function. In particular, postnatal expression of neurotrophins has not been well documented in the neostriatum and in general neurotrophins or their receptor mRNA's are normally reported, but not protein expression. In the present study, immunocytochemical expression of BDNF, NT-3 and NT-4/5 was characterized in striatal tissue of C57BL/6 mice at postnatal days 10(th )(P10), 21(st) (P21), 42(nd) (P42) and 80(th) (P80). We found that the expression of BDNF diminished along the postnatal time course we evaluated, while staining for NT-4 increased up to age P42 and remained constant, thereafter in the cell's soma. In contrast, NT-3 was first expressed in the neostriatal bundles and later on, in neostriatal cell somas. These results provide information about differences in the spatial and temporal expression of each neurotrophin in the neostriatum during the first 80(th) postnatal days. RT-PCR procedures were also carried out to further determine whether protein levels of neurotrophins observed in the neostriatum were under control of gene expression. All neurotrophin mRNAs were expressed and only mRNA(BDNF) was reduced during the postnatal evaluated days. Differences in temporal expression of neurotrophins may be related to the heterochronic development of neostriatal cell populations, but also with the specificity of each neurotrophin modulating different neuronal targets.

Somatostatinergic modulation of firing pattern and calcium-activated potassium currents in medium spiny neostriatal neurons.

Somatostatin is synthesized and released by aspiny GABAergic interneurons of the neostriatum, some of them identified as low threshold spike generating neurons (LTS-interneurons). These neurons make synaptic contacts with spiny neostriatal projection neurons. However, very few somatostatin actions on projection neurons have been described. The present work reports that somatostatin modulates the Ca(2+) activated K(+) currents (K(Ca) currents) expressed by projection cells. These actions contribute in designing the firing pattern of the spiny projection neuron; which is the output of the neostriatum. Small conductance (SK) and large conductance (BK) K(Ca) currents represent between 30% and 50% of the sustained outward current in spiny cells. Somatostatin reduces SK-type K(+) currents and at the same time enhances BK-type K(+) currents. This dual effect enhances the fast component of the after hyperpolarizing potential while reducing the slow component. Somatostatin then modifies the firing pattern of spiny neurons which changed from a tonic regular pattern to an interrupted "stuttering"-like pattern. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) tissue expression analysis of dorsal striatal somatostatinergic receptors (SSTR) mRNA revealed that all five SSTR mRNAs are present. However, single cell RT-PCR profiling suggests that the most probable receptor in charge of this modulation is the SSTR2 receptor. Interestingly, aspiny interneurons may exhibit a "stuttering"-like firing pattern. Therefore, somatostatin actions appear to be the entrainment of projection neurons to the rhythms generated by some interneurons. Somatostatin is then capable of modifying the processing and output of the neostriatum.

Blockade of N-methyl-D-aspartate receptors in the insular cortex disrupts taste aversion and spatial memory formation.

The present experiments examined the effects of direct intracortical microinjections of the N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonovaleric acid directly into the insular cortex of rats, before or immediately after training of conditioned taste aversion and the water maze spatial learning task. In the first series of experiments animals received bilateral injections of 2-amino-5-phosphonovaleric acid prior to taste aversion conditioning or spatial training. A strong disruptive effect was found in the acquisition of training tasks. To determine the possible involvement of N-methyl-D-aspartate receptors in the early post-training processes taking place in the cortex during both learning paradigms, in a second series of experiments, animals received bilateral 2-amino-5-phosphonovaleric acid microinjections 30, 60 or 120 min after the acquisition trial, and 15 min before the retention test. For spatial learning successive treatments were independently done either starting at the onset of the asymptotic phase of the learning curve, 0, 30 or 120 min after finishing the training session, as well as 15 min before the retention test trial. The conditioned taste aversion task remained sensitive to N-methyl-D-aspartate blockade during a period of at least 2 h after the first presentation of the gustatory stimulus, while in the case of the spatial learning task, a gradually decreasing effect was observed from the onset of the asymptotic phase onwards. Taken together, these results provide direct evidence for N-methyl-D-aspartate receptor involvement in cortical regulation of memory formation. Furthermore, our results suggest that in the same cortical region, a different time-course for the activation of N-methyl-D-aspartate-dependent mechanisms occurs during the early formation of cortically mediated memories, depending on the particular behavioural task.

3-Alpha-chloro-imperialine, a potent blocker of cholinergic presynaptic modulation of glutamatergic afferents in the rat neostriatum.

Cortico-thalamic glutamatergic afferents control neuronal activity in the neostriatum. Cholinergic interneurons modulate the activity of medium spiny neurons through both pre- and post-synaptic actions via the activation of muscarinic receptors. The muscarinic pre-synaptic modulation was analyzed electrophysiologically. The transmitter release, induced by 4-AP, was studied and the block of paired pulse facilitation (PPF) by different muscarinic receptor antagonists was analyzed. The GABA(A) antagonist bicuculline isolated the glutamatergic transmission. Muscarinic agonists decreased the frequency of random synaptic potentials induced by 4-AP in about 60% of the cases without changes in input resistance (RN) of the post-synaptic neuron or in the mean amplitude of the synaptic events; indicating a presynaptic action. The administration of both 1 microM carbachol or 20 nM muscarine increased PPF. Muscarinic receptor antagonists blocked this action with a potency order: 3-alpha-chloroimperialine > 4-DAMP>>AFDX-116 > or = gallamine >> pirenzepine. The IC50's for the first three antagonists were (nM): 0.65, 1.1, and 3.0. Their respective Hill coefficients were: 1.9, 1.4, and 1.3. 3-alpha-Chloroimperialine reduced the PPF almost completely. The M3 and the M2 muscarinic receptor antagonists 4-DAMP and AFDX-116, given at saturating concentrations, consistently blocked only a part of the PPF but had additive effects when given together. These data are consistent with the existence of both M2 and M3 muscarinic receptors in striatal glutamatergic afferents.

Effects of a Ginkgo biloba extract on two models of cortical hemiplegia in rats.

To screen drugs potentially useful in the pharmacological treatment of subjects with brain lesions, we studied the effects of chronic (7 and 30 days) treatments with a Ginkgo biloba extract (EGb761-IPSEN; EGb) in two animal models of cortical hemiplegia: one induced by motor cortex aspiration and another using a reversible inactivation of the motor cortex through chronic, localized infusion of y-aminobutyric acid (GABA), via osmotic minipumps. The elevated beam test was used in water-deprived animals trained to drink saccharin-sweetened solutions (with or without EGb) and to perform to criteria before the surgical procedures. From the day after surgery, the rats were administered 100 mg/kg of EGb daily for 7 or 30 days. In all groups with motor impairment in which the extract was administered, a faster and more complete recovery was observed, which was significantly different from that of rats which received only saccharin solutions. The salutary effect of EGb was more marked in ablation-induced hemiplegia than in the GABA-treated group. In the former injury model, EGb-treated animals had smaller ventricular diameters than non-treated rats. No differences concerning sensory deficits were detected among groups. EGb was also acutely administered during the epileptic syndrome that follows interruption of chronic GABA infusions (the GABA-withdrawal syndrome). No anticonvulsant effects of EGb were observed. These results suggest a potential use of EGb in brain-injured patients as this product shows little toxicity in animals and man after chronic administration. The active principles among terpenes (ginkgolides, bilobalides and flavonolheterosides present in the EGb) and the mechanisms for this beneficial effects remain to be elucidated.