Activation of group II metabotropic glutamate receptors blocks zinc release from hippocampal mossy fibers

BACKGROUND: The hippocampal CA3 area contains large amounts of vesicular zinc in the mossy fiber terminals which is released during synaptic activity, depending on presynaptic calcium. Another characteristic of these synapses is the presynaptic localization of high concentrations of group II metabotropic glutamate receptors, specifically activated by DCG-IV. Previous work has shown that DCG-IV affects only mossy fiber-evoked responses but not the signals from associational-commissural afferents, blocking mossy fiber synaptic transmission. Since zinc is released from mossy fibers even for single stimuli and it is generally assumed to be co-released with glutamate, the aim of the work was to investigate the effect of DCG-IV on mossy fiber zinc signals. RESULTS: Studies were performed using the membrane-permeant fluorescent zinc probe TSQ, and indicate that DCG-IV almost completely abolishes mossy fiber zinc changes as it does with synaptic transmission. CONCLUSIONS: Zinc signaling is regulated by the activation of type II metabotropic receptors, as it has been previously shown for glutamate, further supporting the corelease of glutamate and zinc from mossy fibers.

Neurogenesis induced by seizures in the dentate gyrus is not related to mossy fiber sprouting but is age dependent in developing rats / A neurogênese induzida por crises no giro denteado não está relacionada ao brotamento de fibras musgosas, mas é dependente da idade, em ratos durante o desenvolvimento

Neurogenesis in the dentate gyrus (DG) has attracted attention since abnormal supragranular mossy fiber sprouting occurs in the same region, in temporal lobe epilepsy. Thus, we submitted developing rats to pilocarpine-induced status epilepticus (SE) to study the relationship between neurogenesis and mossy fiber sprouting. Groups were submitted to SE at: I-P9, II-P7, P8 and P9, III-P17 e IV-P21. Neurogenesis was quantified using BrdU protocol and confirmed through double staining, using neuronal pentraxin. Other animals were monitored by video system until P120 and their brain was studied (Timm and Nissl staining). The neurogenesis at P17 (p=0.007) and P21 (p=0.006) were increased. However, only P21 group showed recurrent seizures and the mossy fiber sprouting in the same region, during adult life, while P17 did not. Thus, our results suggest that neurogenesis is not related to mossy fiber sprouting neither to recurrent spontaneous seizures in pilocarpine model.

A neurogênese no giro dentado tem atraído atenção já que ela ocorre na mesma região do hipocampo que o brotamento das fibras musgosas, na epilepsia do lobo temporal. Assim, submetemos ratos em desenvolvimento ao status epilepticus induzido (SE) por pilocarpine. Grupos foram submetidos em I-P9, II-P7, P8, P9; III-P17 e IV-P21. A neurogênese foi observada usando o protocolo do BrdU e confirmada por dupla marcação com pentraxina neuronal. Outros animais foram monitorados até P120 e seus cérebros analisados (Nissl e Timm). A neurogênese nos grupos P17 (p=0,007) e P21 (p=0,006) aumentaram. Entretanto, o P21 apresentou crises espontâneas e brotamento de fibras musgosas, na mesma região onde ocorreu a neurogênese, enquanto o grupo P17 apresentou somente aumento na neurogênese. Assim, nossos resultados sugerem que o fenômeno da neurogênese não está relacionado com o brotamento de fibras musgosas nem com o aparecimento de crises espontâneas e recorrentes no modelo da pilocarpina.

Protein tyrosine kinase inhibitors modify kainic acid-induced epileptiform activity and mossy fiber sprouting but do not protect against limbic cell death

Intrahippocampal administration of kainic acid (KA) induces synaptic release of neurotrophins, mainly brain-derived neurotrophic factor, which contributes to the acute neuronal excitation produced by the toxin. Two protein tyrosine kinase inhibitors, herbimycin A and K252a, were administered intracerebroventricularly, in a single dose, to attenuate neurotrophin signaling during the acute effects of KA, and their role in epileptogenesis was evaluated in adult, male Wistar rats weighing 250-300 g. The latency for the first Racine stage V seizure was 90 ± 8 min in saline controls (N = 4) which increased to 369 ± 71 and 322 ± 63 min in animals receiving herbimycin A (1.74 nmol, N = 4) and K252a (10 pmol, N = 4), respectively. Behavioral alterations were accompanied by diminished duration of EEG paroxysms in herbimycin A- and K252a-treated animals. Notwithstanding the reduction in seizure severity, cell death (60-90 percent of cell loss in KA-treated animals) in limbic regions was unchanged by herbimycin A and K252a. However, aberrant mossy fiber sprouting was significantly reduced in the ipsilateral dorsal hippocampus of K252a-treated animals. In this model of temporal lobe epilepsy, both protein kinase inhibitors diminished the acute epileptic activity triggered by KA and the ensuing morphological alterations in the dentate gyrus without diminishing cell loss. Our current data indicating that K252a, but not herbimycin, has an influence over KA-induced mossy fiber sprouting further suggest that protein tyrosine kinase receptors are not the only factors which control this plasticity. Further experiments are necessary to elucidate the exact signaling systems associated with this K252a effect.

Effect of the zinc chelator N, N, N',N'- tetrakis (2-pyridylmethyl)ethylenediamine (TPEN) on hippocampal mossy fiber calcium signals and on synaptic transmission

An important pool of chelatable zinc is present in the synaptic vesicles of mossy fiber terminals from hippocampal CA3 area, being zinc released following single or repetitive electrical stimulation. Previous studies have suggested different synaptic roles for released mossy fiber zinc, including the inhibition of presynaptic calcium and of postsynaptic N-methyl-D-aspartate (NMDA) and gamma amino-butiric acid (GABA A) receptors. The effect of endogenously released zinc on mossy fiber long-term potentiation (LTP) induction also is not yet established. We have investigated the effect of the permeant zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) on mossy fiber calcium and on synaptic transmission, before and during the application of LTP-inducing stimulation. We have found, using the calcium indicator Fura-2, that single and tetanically-evoked mossy fiber calcium signals are both enhanced in the presence of 20 ìM TPEN, while the single field potentials are unaffected. As expected, no effect was observed on the single calcium signals or field potentials obtained at the CA3-CA1 synapses, from the CA1 area, which has a lower concentration of vesicular zinc. These results support the idea that at the hippocampal mossy fiber synapses, released zinc inhibits presynaptic calcium mechanisms. A higher concentration of TPEN (100 ìM) significantly reduced mossy fiber synaptic transmission but did not prevent the induction of mossy fiber LTP, suggesting that zinc is not required for the formation of this form of LTP.

Role of gamma-aminobutyric acid B (GABA B) receptors in the regulation of kainic acid-induced cell death in mouse hippocampus

Kainic acid (KA) is well-known as an excitatory, neurotoxic substance. In mice, KA administered intracerebroventricularly (i.c.v.) lead to morphological damage of hippocampus expecially concentrated on the CA3 pyramidal neurons. In the present study, the possible role of gamma-aminobutyric acid B (GABA B) receptors in hippocampal cell death induced by KA (0.1 microgram) administered i.c.v. was examined. 5-Aminovaleric acid (5-AV; GABA B receptors antagonist, 20 microgram) reduced KA-induced CA3 pyramidal cell death. KA increased the phosphorylated extracellular signal-regulated kinase (p-ERK) and Ca2+ /calmodulin-dependent protein kinase II (p-CaMK II) immunoreactivities (IRs) 30 min after KA treatment, and c-Fos, c-Jun IR 2 h, and glial fibrillary acidic protein (GFAP), complement receptor type 3 (OX-42) IR 1 day in hippocampal area in KA-injected mice. 5-AV attenuated KA-induced p-CaMK II, GFAP and OX-42 IR in the hippocampal CA3 region. These results suggest that p-CaMK II may play as an important regulator on hippocampal cell death induced by KA administered i.c.v. in mice. Activated astrocytes, which was presented by GFAP IR, and activated microglia, which was presented by the OX-42 IR, may be a good indicator for measuring the cell death in hippocampal regions by KA excitotoxicity. Furthermore, it showed that GABA B receptors appear to be involved in hippocampal CA3 pyramidal cell death induced by KA administered i.c.v. in mice.