Strychnine protection against excitotoxic cell death in primary cultures of rat cerebral cortex

Strychnine-insensitive glycine receptors are known to modulate the toxicity of excitatory amino acids via an allosteric action at the N-methyl-D-aspartate receptor complex. To elucidate whether strychnine-sensitive glycine receptors may also influence excitotoxicity, the effect of strychnine on the excitotoxic cell death was examined in primary cultures of the rat cerebral cortex. To exclude any interference at the N-methyl-D-aspartate receptor complex, cell death was evoked by kainic acid. The release of lactic dehydrogenase (LDH) into the culture medium was taken as a quantitative measure of cell death. Strychnine reduced the excitotoxic cell death in a concentration-dependent fashion. This finding indicates that glycine may modulate the vulnerability of cortical cells to excitotoxic insults not only via the strychnine-insensitive population of glycine receptors within the N-methyl-D-aspartate receptor-complex, but also via strychnine-sensitive receptor channels.     

Changes in histamine in the rat striatum following local injection of kainic acid

The influence of local administration of kainic acid on the metabolism of histamine in the striatum of the rat has been investigated. Intrastriatal injection of kainic acid (1 μg) resulted in a more than 100% increase of the striatal content of histamine, which persisted from two days after the lesion up to ten weeks. Using two independent methods this accumulation of histamine has been found to be associated with a neuronal compartment of the striatum: (a) the concentration of histamine in synaptosomes prepared from kainic acid lesioned striatum was higher than in synaptosomes derived from the untreated (contralateral) striatum; (b) electrolytic lesions of the medial forebrain bundle, in animals whose striatum had been lesioned with kainic acid, abolished the increase in histamine levels induced by the neurotoxin. Since the rate of disappearance of histamine from the tissue after inhibition of its synthesis by α-fluoromethylhistidine (20 mg/kg, i.p.) was reduced in the lesioned striatum, the kainic acid induced increase in striatal histamine level may be due to a decrease of histamine turnover.Specific binding of [³H]histamine and [³H]cimetidine was measured in the lesioned striatum, revealing a 40% and 20% decrease in binding sites, respectively, when compared with the untreated striatum; the affinity for either ligand was not changed in the lesioned tissue.The data demonstrate considerable neurochemical changes involving the neuronal histamine system in the striatum after destroying the interneurons and output neurons of this region with the convulsive neurotoxin kainic acid.     

Tissue remodeling following submassive hemorrhagic necrosis in rat livers induced by an intraperitoneal injection of dimethylnitrosamine

We examined regeneration and fibrosis in the necrotic areas of hepatic stellate cells (HSCs). Acute hepatic injury was induced in rats by administration of an intraperitoneal injection of high-dose dimethylnitrosamine (50 mg/kg body weight). Liver samples were obtained from rats 6, 12, 24, 36 h and 2, 3, 5, 7, 10, and 14 days after the injection. They were examined by light and electron microscopy and by immunohistochemical methods. Hemorrhagic necrosis became most prominent 36 h after treatment and extended into zones 3 and 2. In the submassive necrotic areas the sinusoidal structure was destroyed. No HSCs positive for alpha-smooth muscle actin or desmin were present. On day 5, when necrotic tissues were almost removed by infiltrating macrophages, HSCs strongly positive for alpha-smooth muscle actin and desmin appeared along the surface of the preserved parenchyma and migrated into the necrotic areas along the residual reticulin fibers. By day 14 most of the necrotic areas were almost completely replaced by the regeneration of hepatocytes and central to central (C-C) bridging fibrosis. Our results indicate that following submassive complete necrosis, HSCs in the preserved liver parenchyma have roles in the formation of sinusoidal wall for remodeling in necrotic areas via their activation, proliferation, and migration into the necrotic areas.     

Changes to synaptic ultrastructure in field CA1 of the rat hippocampus following intracerebroventricular injection of kainic acid

To assess the nature and extent of ultrastructural damage due to low unilateral intracerebroventricular doses of kainic acid, treated rats were killed at survival times from 8 h to 14 weeks. Degenerative changes in field CA1 of the hippocampus included dark profiles (often presynaptic), lucent areas enveloping axonic or dendritic elements, damaged myelin sheaths, and enlarged glial profiles. The effect of kainic acid ipsilaterally was maximal at three days but also apparent up to 14 weeks. Contralateral CA1 showed similar though less extensive abnormalities.

These observations suggest that, despite rapid synaptic replacement (Nadler et al., Brain Res.191, 387–403, 1980), long-term electrophysiological abnormalities (Cornish and Wheal, Neuroscience 28, 563–571, 1989) may stem not only from inappropriate reactive synaptogenesis but also from a continuing state of neuronal degeneration.     

Peroxynitrite generated in the rat spinal cord induces apoptotic cell death and activates caspase-3

We previously demonstrated that the peroxynitrite concentration increases after impact spinal cord injury. This study tests whether spinal cord injury-elevated peroxynitrite induces apoptotic cell death. Peroxynitrite was generated at the concentration and duration produced by spinal cord injury by administering S-morpholinosydnonimine through a microdialysis fiber into the gray matter of the rat spinal cord. Fragmented DNA was visualized by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling. Transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling-positive neurons were quantitated by counting the transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling and neuron-specific enolase double-stained neurons along the fiber track in the sections removed at 6, 12, 24 and 48 h post-peroxynitrite exposure. Peroxynitrite significantly increased transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling-positive neurons at all time points examined (P< or =0.001) compared with artificial cerebrospinal fluid controls (Two-way analysis of variance followed by Tukey test), peaking at 24 h post-exposure. Electron microscopic observation of characteristic features of apoptosis confirmed peroxynitrite-induced neuronal apoptosis. Total transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling-positive cells were counted in areas near and 0.2 mm away from the fiber track. The counts both peaked at 24 h with no significant difference between the two areas. However, at 6 and 12 h post-exposure the counts were significantly higher near than away from the fiber track (P=0.03 and P=0.007 respectively, paired t test). Immunohistochemical staining indicates caspase-3 was activated by peroxynitrite; this activation peaked at 6 h post-exposure, suggesting that activation of caspase-3 might be an early event in the apoptotic cell death cascade. We conclude that 1) peroxynitrite generated in the cord at the level produced by spinal cord injury induces neuronal apoptosis, indicating a role for peroxynitrite in secondary spinal cord injury; 2) caspase activation might be involved in peroxynitrite-induced neuronal apoptosis; 3) therefore removal of peroxynitrite should reduce secondary cell death after spinal cord injury.     

Modelling cognitive dysfunctions with bilateral injections of ibotenic acid into the rat entorhinal cortex

Neurodegenerative diseases, traumatic brain injury and stroke are likely to result in cognitive dysfunctioning. Animal models are needed in which these deficits and recovery of the affected functions can be investigated. In the present study, the entorhinal area was chosen as the target for lesioning and for assessing the lesion-induced deficits in the Morris water maze. The entorhinal cortex is regarded as an interface between the hippocampus and neocortex. Deafferentiating the hippocampus through entorhinal lesions impairs spatial learning. The effects of lesions, induced by either electrocoagulation (experiment 1) or ibotenate excitotoxicity (experiment 2), on spatial orientation behaviour were investigated. Water maze performance after unilateral or bilateral ibotenate injections into the entorhinal cortex was studied in the third experiment. In an additional study, the replicability of the spatial learning deficit after lesions induced by bilateral injections of ibotenic acid into the entorhinal cortex was assessed by comparing the results of nine experiments. We found that spatial learning was impaired after bilateral lesions aimed at the entorhinal cortex. The electrolytic lesion technique produced a relatively large sham effect, whereas the excitotoxic lesioning method did not. Unilateral injections of ibotenic acid into the entorhinal cortex did not affect spatial navigation. The ibotenate-induced lesions replicably produced deficits in the Morris tasks. The degree of the induced spatial learning impairments and the effects on the rate of acquisition during training, however, differed between experiments. This result suggests that the fundamental biological diversity between shipments of rats can account for variation in the effects of parahippocampal damage on spatial learning even in highly standardized experimental set-ups.Rats lesioned by bilateral injections of ibotenic acid into the entorhinal cortex provide an interesting and reliable model for investigating cognitive dysfunctions in neurodegenerative diseases, stroke or traumatic brain injury.     

Increased [3H]kainic acid binding in the prefrontal cortex in schizophrenia

[3H]Kainic acid binding sites were measured post mortem in the putamen and prefrontal cortex areas from 10 control subjects and 12 schizophrenic patients. A 25-50% increase in [3H]kainic acid binding was observed in the medial frontal (Brodmann areas 9, 10 and 46) and eye-movement areas (8), but not in the other regions of schizophrenic brains. No significant correlation between the binding and either age at death, storage of the brains, duration of illness or neuroleptics-free period was observed. These findings suggest that a dysfunction of cortical excitatory amino acidergic transmission may be involved in schizophrenia.     

Energy metabolism in rat brain structures after injections of kainic acid into the frontal cortex

We studied behavioral reactions of rats after injection of subconvulsive dose of kainic acid into the frontal cortex and mitochondrial respiration in the hippocampus and frontal and temporal cortex 17–20 days after administration of kainic acid. Retention of acquired habit and the dynamics of its extinction in experimental rats were close to those in the control group. Changes in mitochondrial function were observed only in the region of kainic acid injection: activation of phosphorylating respiration during oxidation of succinate. Presumably, the detected activation of energy metabolism in the frontal cortex indicates functional restructuring in mitochondria, aimed at compensation of disorders caused by the neurotoxin. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 11, pp. 510–513, November, 2007     

Intrahippocampal injection of kainic acid produces significant pyramidal cell loss in neonatal rats

Previous reports have indicated that pyramidal cells in the developing rat hippocampal formation are not destroyed by intraventricular or intraperitoneal administration of kainic acid. We examined the neurotoxic properties of kainic acid and ibotenic acid following intrahippocampal injection in neonatal rats and found significant pyramidal cell death following injection of 1.0 microgram kainic acid in 6, 7 and 9-day-old pups. At doses 2.5 or five times this amount, significant pyramidal cell loss was obtained in 5-day-old rats as well. The susceptibility of pyramidal neurons to kainic acid increased as a function of age. The developing hippocampus was considerably more vulnerable to ibotenic acid compared with kainic acid, in contrast to the order of potency reported in adult rats. The increased sensitivity of CA3 pyramidal cells parallels the development of the mossy fiber innervation to the dendrites of these cells supporting the twofold mechanism suggested by Coyle for kainic acid neurotoxicity; that is, a direct cytotoxic action via postsynaptic receptors as well as increased sensitivity due to the presence of excitatory inputs.     

Adrenergic facilitation of GABAergic transmission in rat entorhinal cortex

Whereas the entorhinal cortex (EC) receives noradrenergic innervations from the locus coeruleus of the pons and expresses adrenergic receptors, the function of norepinephrine (NE) in the EC is still elusive. We examined the effects of NE on GABA(A) receptor-mediated synaptic transmission in the superficial layers of the EC. Application of NE dose-dependently increased the frequency and amplitude of spontaneous inhibitory postsynaptic currents (IPSCs) recorded from the principal neurons in layer II/III through activation of alpha(1) adrenergic receptors. NE increased the frequency and not the amplitude of miniature IPSCs (mIPSCs) recorded in the presence of TTX, suggesting that NE increases presynaptic GABA release with no effects on postsynaptic GABA(A) receptors. Application of Ca(2+) channel blockers (Cd(2+) and Ni(2+)), omission of Ca(2+) in the extracellular solution, or replacement of extracellular Na(+) with N-methyl-D-glucamine (NMDG) failed to alter NE-induced increase in mIPSC frequency, suggesting that Ca(2+) influx through voltage-gated Ca(2+) or other cationic channels is not required. Application of BAPTA-AM, thapsigargin, and ryanodine did not change NE-induced increase in mIPSC frequency, suggesting that Ca(2+) release from intracellular stores is not necessary for NE-induced increase in GABA release. Whereas alpha(1) receptors are coupled to G(q/11) resulting in activation of the phospholipase C (PLC) pathway, NE-mediated facilitation of GABAergic transmission was independent of PLC, protein kinase C, and tyrosine kinase activities. Our results suggest that NE-mediated facilitation of GABAergic function contributes to its antiepileptic effects in the EC.     

Transient increase of contraversive saccade parameters following kainic acid injection in the periparabigeminal area of guinea pig

Summary Anatomical and electrophysiological data have shown that, in the guinea pig as well as in the cat, the nucleus prepositus hypoglossi gives rise to a disynaptic ascending projection to the superior colliculus via the peri-parabigeminal area in the mesencephalon. The functional role of this indirect pathway in the generation of eye movements has been studied by pharmacologically interfering at the mesencephalic level and by examining the induced effects on two differently elicited saccades in the alert guinea pig. A small iontophoretic injection of kainic acid induces a transient increase of the spontaneous saccadic activity in the contraversive direction leading to a pseudo-nystagmus. Both the amplitude and the mean velocity of the contraversive saccades are greater than before the injection. A similar enhancement also affects the parameters of the contraversive saccades induced by trunk rotations. These results suggest that peri-parabigeminal neurones exert an excitatory influence on their target cells in the ipsilateral superior colliculus which is responsible for contraversive rapid eye movements.     

Neuronal sources of theta rhythm in the entorhinal cortex of the rat

The discharge patterns and layer distribution of entorhinal cortex (EC) units were investigated in paralysed and locally anesthetized rats injected with physostigmine in order to induce theta (theta) rhythm. Entorhinal unit activity and field potentials were recorded simultaneously with the same micropipette. Hippocampal CA1 theta rhythm was used as reference. Statistical analysis included auto- and cross-correlations and interval histograms. Results showed: a. the existence of rhythmic and non-rhythmic cells, both tending to fire in a constant phase relationship with theta rhythm; b. in all EC subdivisions, most rhythmic cells were located in superficial cell layers (II-III); c. on the average, rhythmic cells from the medial EC fired synchronously; d. non-rhythmic cells tended also to fire synchronously but with an opposite phase relationship with respect to rhythmic neurons. Although a complex organization in the rhythmicity of EC units is revealed, it is concluded that the neuronal sources of theta activity in the EC are located in superficial cell layers, and it is strongly suggested that the EC output through the perforant path may rhythmically modulate the discharge pattern of hippocampal pyramidal and dentate granule cells.     

Neuronal sources of theta rhythm in the entorhinal cortex of the rat

Summary The laminar distribution of theta () field potentials in the entorhinal cortex (EC) was investigated in paralysed and locally anesthetized rats injected with physostigmine in order to induce rhythm. Electrode penetrations through the medial, intermediate and lateral subdivisions of the EC showed in all cases: 1. the presence of rhythm from layer VI to layer III approximately in phase with CA1 rhythm; 2. an amplitude minimum between the outer third of layer III and the inner half of layer I; and 3. a phase-reversed rhythm in layers II-I with an amplitude maximum in the outer half of layer I. Results indicate the existence of neuronal sources of rhythm in the EC.     

Striatal ganglioside levels in the rat following kainic acid lesions: Comparison with Huntington's disease

Summary Ganglioside and DNA levels were estimated in the striatum of rats 10 days and 6 weeks after lesioning by intrastriatal injection of kainic acid. There was a moderate, 21–24% decrease of the ganglioside concentration per unit protein on the side of the lesion, which can be ascribed to the loss of the intrinsic striatal neurons following the injection of kainic acid. On the other hand, there was a 131 and 60% increase of DNA per unit protein in the kainate injected side 10 days and 6 weeks after the lesions, respectively; these changes apparently reflected the gliotic reaction brought about by the neurotoxin. Qualitatively similar findings — a decrease of ganglioside and an increase of DNA levels per unit protein — were also found in the brain of patients with Huntington's disease; however, as compared with the corresponding control material, the decrease of the ganglioside concentration was more pronounced in the striatum of Huntington's disease (by 38% in the caudate nucleus and by 46% in the putamen) than in the kainate lesioned rat striatum. This difference could be due to the different proportions of the intrinsic and extrinsic neuronal plasma membranes in the striatum of the two species; however, the possibility of a more generalized affection of neuronal plasma membranes in Huntington's disease may also be envisaged.The work was supported by Austrian Science Research Fund, Project No S-25     

Relationships between stress protein induction and NMDA-mediated neuronal death in the entorhinal cortex

The entorhinal cortex (EC) appears to be one of the earliest regions to express cellular pathology in aging and Alzheimer's disease. The relationships between cellular stress protein responses and the temporal and spatial aspects of cell death induced by N-methyl-D-aspartate (NMDA) was investigated in this anatomical region. Low doses of NMDA were infused stereotactically into the medial EC of the rat. At intervals starting from 0.5 h up to 7 days after a 1.25-l EC infusion of 15 mM NMDA, 30 mM NMDA, or saline, the expression of ubiquitin (Ub), 72-kDa heat shock protein (HSP 72), and c-Fos was determined in relation to neuronal death. Volumes of entorhinal Ub- and HSP 72-like immunoreactivity peaked between 18 and 48 h after either 15 or 30 mM NMDA infusions. After 15 mM NMDA infusions, maximal volumes of HSP 72- and Ub-like immunoreactivity in the EC at 48 h were similar to the subsequent maximal volume of neuronal loss in the EC seen after 96 hours. After infusion of 30 mM NMDA, the final EC volume of neuronal loss seen at 7 days after NMDA corresponded to 70–80% of the maximal HSP-Ub stress protein response seen at 2 days, implying that a population of HSP 72- and Ub-immunopositive cells survived the NMDA insult. C-Fos expression as determined by immunoreactivity for the nuclear phosphoprotein (Fos) indicated neuronal activation at NMDA infusion sites, in the perirhinal cortex, hippocampus, and other sites throughout the injected hemisphere. In the EC, c-Fos immunoreactivity returned to baseline levels by 8 h, well before the dramatic increases in HSP 72 and Ub volumes. Our results demonstrate that HSP 72 and Ub expression in vivo precedes and correlates with, but does not necessarily lead to, neuronal death following glutamate receptor-mediated toxicity in the EC.     

Late low magnesium-induced epileptiform activity in rat entorhinal cortex slices becomes insensitive to the anticonvulsant valproic acid

We investigated time-dependent changes in low magnesium-induced epileptiform activity in combined rat entorhinal cortex/hippocampal slices with extracellular recording techniques. While in area CA3 short interictal discharges are generated without any major changes in activity during prolonged recording periods, initial tonic clonic ictaform events in the entorhinal cortex may change with time. We observed often a transition into a state of recurrent tonic activity without any clonic afterdischarges. Alternatively, seizures could stay in the clonic discharge mode for the rest of the experiment. These different seizure states were not equally affected by the anticonvulsant valproic acid. While the early clonic tonic discharges in the entorhinal cortex and the interictal like activity in area CA3 were effectively suppressed by valproic acid (VPA) the late recurrent tonic seizure discharge state was unaffected by the drug. It was, however, still sensitive to the N-methyl-D-aspartate (NMDA) receptor antagonist 2-aminophosphonovalerate. These findings point to seizure-induced changes in neuronal interaction in rat entorhinal cortex.     

Olfactory bulbectomy induces neuronal rearrangement in the entorhinal cortex in the rat

In humans, depression has been associated with disturbances in olfactory circuitry. Symptoms of depression can be mimicked in animals after olfactory bulbectomy (OBX). Animal models of depression-like behavior produce similar neuronal rearrangements in various brain regions as seen in patients affected by depression. We have recently observed that OBX produces neuronal hypotrophy in the piriform cortex (PirC) and CA1 hippocampus as well as decreased adult cell proliferation in the dentate gyrus (DG) of hippocampus. Thus we further evaluated the effects of OBX in neuronal arborization and spine density in brain regions involved in the control of circadian circle, emotion and memory processing such as the prefrontal cortex (PFC), nucleus accumbens (NAcc), infralimbic cortex (ILC), orbitolateral cortex (OLC) and entorhinal cortex (EC). Our present results show that along with severe behavioral deficits observed in these animals, OBX considerably decreased dendritic branching and the total dendritic length in the EC, a major interface of the hippocampus and neocortical regions. The remaining cortices and NAcc were not affected by OBX. Thus, we propose that the lack of input from the olfactory bulbs resulted in serial neuronal rearrangements in the PirC, EC, and hippocampus leading, at least partially, to behavioral deficits in emotion and memory processes.     

A new immunosuppressant, FTY720, induces bcl-2-associated apoptotic cell death in human lymphocytes.

FTY720 is a unique immunosuppressive drug produced by modification of a metabolite from Isaria sinclairii. In vitro treatment of human mononuclear cells with FTY720 resulted in a dose-dependent reduction of cell viability. These treated cells demonstrated characteristic DNA ladder formation on agarose gel electrophoresis. Jurkat cells transfected with human bcl-2 gene were resistant to FTY720; their neo type was susceptible to the drug. A rapid acceleration of cell death in human mononuclear cells was seen as early as 2 hr after incubation with FTY720. The intracellular Bax protein increased remarkably 1 hr after the culture; it markedly decreased in the surviving cells at 2 and 3 hr. Coincidental to the Bax decrease. Bcl-2 progressively decreased beginning 2 hr after the culture. Thus, the ratio of Bcl-2 to Bax was decreased by the enhanced expression of Bax immediately after FTY720-treatment, resulting in rapid cell death acceleration. The surviving cells (FTY720-resistant cells) at 2 and 3 hr after culture showed a similar ratio of Bcl-2 to Bax as was observed in the control cells. These results suggest that FTY720 displays bcl-2-associated apoptotic cell death in human mononuclear cells.